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The Journal of Nutrition

6th Amino Acid Assessment Workshop

Nutritional Consequences of Interspecies

Differences in Arginine and

Lysine Metabolism

13

Ronald O. Ball,46* Kristine L. Urschel,4 and Paul B. Pencharz46

4Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada T6G 2P5; 5The Research

Institute, The Hospital for Sick Children, Toronto, ON, Canada M5G 1X8; and the 6Departments of Paediatrics and Nutritional Science,

University of Toronto, Toronto, ON, Canada M5G 1X8

Abstract

Differences in lysine and arginine requirements among various species such as omnivores (humans, pigs, rats, dogs),

carnivores (cats), herbivores (rabbits, horses), ruminants (cattle), poultry, and fish, are covered in detail in this article.

Although lysine is classified as an indispensable amino acid across species, the classification of arginine as either an

indispensable or dispensable amino acid is more ambiguous because of differences among species in rates of de novo

arginine synthesis. Because lysine is most often the limiting amino acid in the diet, its requirement has been extensively

studied. By use of the ideal protein concept, the requirements of the other indispensable amino acids can be extrapolated

from the lysine requirement. The successful use of this concept in pigs is compared with potential application of the ideal

protein concept in humans. The current dietary arginine requirement varies widely among species, with ruminants,

rabbits, and rats having relatively low requirements and carnivores, fish, and poultry having high requirements.

Interspecies differences in metabolic arginine utilization and reasons for different rates of de novo arginine synthesis are

reviewed in detail, as these are the primary determinants of the dietary arginine requirement. There is presently no dietary

requirement for humans of any age, although this needs to be reassessed, particularly in neonates. A thorough

understanding of the factors contributing to the lysine and arginine requirements in different species will be useful in our

understanding of human amino acid requirements. J. Nutr. 137: 1626S1641S, 2007.

Lysine is an indispensable dietary amino acid for all vertebrates.For arginine, the extent to which it is a dietary indispensableamino acid varies among, and within, species because of avariable capacity for endogenous arginine synthesis. The lysinerequirement in all species is primarily influenced by the re-quirement for protein synthesis, with obligatory oxidation andcarnitine synthesis accounting for the remainder. The argininerequirement, however, is influenced by many factors in addition

to protein synthesis, including rates of de novo synthesis andoxidation, its use for urea cycle function in ureotelic species, andsynthesis of additional metabolites. These interspecies differ-ences in lysine and especially arginine metabolism are importantto recognize and understand because of the resulting implica-tions for nutritionists. In addition, there is a well-describedantagonism that can occur between lysine and arginine in somespecies, where excessive intakes of one of these amino acids willadversely affect the metabolism of the other amino acid,increasing its requirement. The objectives of the present articleare to discuss interspecies differences in lysine and argininemetabolism and the resulting nutritional implications. Datafrom representative species of the following categories arecompared and contrasted: omnivores (humans, dogs, rats, andpigs), carnivores (cats), nonruminant herbivores (rabbits, horses),ruminant herbivores (cattle), poultry, and fish.

Definition of the scope and terms used in this article

The discussion in this article focuses primarily on interspeciesdifferences in metabolism during growth because this is wherethe most data are available. Dietary requirements at mainte-nance are also discussed, particularly in species that spend themajority of their lifespan at maintenance, specifically humans,dogs, and cats. In the case of dietary indispensable amino acids,

e.g., lysine, the dietary amino acid requirement is equivalent to

1 Published in a supplement to The Journal of Nutrition. Presented at the

conference The Sixth Workshop on the Assessment of Adequate and Safe

Intake of Dietary Amino Acids held November 67, 2006 in Budapest. The

conference was sponsored by the International Council on Amino Acid Science

(ICAAS). The organizing committee for the workshop was David H. Baker,

Dennis M. Bier, Luc A. Cynober, Yuzo Hayashi, Motoni Kadowaki, Sidney M.

Morris, Jr., and Andrew G. Renwick. The Guest Editors for the supplement were

David H. Baker, Dennis M. Bier, Luc A. Cynober, Motoni Kadowaki, Sidney M.

Morris, Jr., and Andrew G. Renwick. Disclosures: all Editors and members of the

organizing committee received travel support from ICAAS to attend the

workshop and an honorarium for organizing the meeting.2 Author disclosures: R. O. Ball, receives partial salary support from Alberta Pork,

and travel expense to attend the meeting was paid for by the ICAAS; K. L.

Urschel, supported by a Natural Sciences and Engineering Research Council of

Canada PGSD Scholarship; P. B. Pencharz, no conflicts of interest.3 Supported by grants from the Natural Sciences and Engineering Research

Council of Canada and the Alberta Pork Producers Development Corporation.

* To whom correspondence should be addressed. E-mail: [email protected].

1626S 0022-3166/07 $8.00 2007 American Society for Nutrition.

atCAPESConsortiumonSeptember20,2013

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the metabolic amino acid requirement. In this article, the use ofthe term metabolic amino acid requirement represents theobligatory amino acid demand defined by Millward (1), whichrepresents the amount of amino acid needed to support netprotein synthesis, obligatory levels of oxidation, and thesynthesis of nonprotein products, including carnitine for lysine,and creatine, nitric oxide, polyamines, and urea for arginine.Therefore, there is a metabolic, but not necessarily a dietary,requirement for all amino acids. This point is emphasizedbecause it is particularly important with regard to arginine

metabolism and variation in the dietary requirement bothamong and within species.

The criteria used to define amino acid requirements varyamong species and make comparisons more difficult. The newdietary reference intake system in humans uses the estimatedaverage requirement (EAR; the median requirement for thenutrient),7 the recommended dietary allowance (RDA; nutrientintake level that meets the requirements of ;97% of thepopulation), or adequate intake (AI; intake that appears to beadequate when not enough information is available to calculatean RDA) to express amino acid requirements (2). In mostanimals, the requirements provided by the National ResearchCouncil (NRC) publications are mean population requirements,

which is roughly equivalent to the EAR in humans. The recentlyreleased dog and cat requirements (3) use a system of expressingrequirements that more closely resembles the human RDA,provides a recommended allowance that is greater than theminimum requirement, takes into account the fact that nitrogenbalance is relatively insensitive in adult animals and can occurover a range of intakes, and considers that the bioavailabilities ofamino acids vary among feed ingredients. The bioavailability ofdietary amino acids is considered in most animal species [seeNutrient Requirements of Swine (4) for examples of applicationof amino acid bioavailability] except humans. These differencesin expression of dietary requirements are often a source ofconfusion and misinterpretation when species are compared

because many readers are not aware of these differences.Finally, the metabolic uses and specifics of metabolism oflysine and arginine have been thoroughly discussed, includingother reviews in the present Supplement. Therefore, the speciesdifferences are discussed with the assumption that the reader hasan understanding of the details of lysine and arginine metabolism.

Interspecies differences in lysine metabolism

and requirements

Lysine is a dietary indispensable amino acid in all species thathave been studied. It is usually the most limiting dietary aminoacid for body protein synthesis, which explains why there is sucha vast quantity of literature, compared with other amino acids,on lysine requirements in different species. In growing animals,

the lysine requirement accounts for 3.6% (broiler breeders, 36wk) to 6.1% (rats) of the recommended crude protein (CP)intake (5,6). Human requirements during growth are also withinthis range with the exception of the newborn, where a lysine AImay be slightly higher at 7.0% of the AI of protein (2). Duringmaintenance, the lysine requirement (as a percentage of the CP

intake requirement) varies from 1.7% in cats (3) to 4.8% inhumans (2).

Because lysine is the first limiting amino acid in most grain-and cereal-based animal diets, it also defines the protein intakerequired to meet the animals amino acid requirements. Theprotein requirement published for most species is thereforeactually the protein intake required to satisfy the dietary need forlysine. Comparisons among species show that, on a CP basis, therequirements for lysine are remarkably similar. Implications ofthis are discussed below.

The importance of lysine as the first limiting amino acid hasbeen used in the development of the ideal protein concept: theexpression of the requirement of all the amino acids relative tothe lysine requirement. The concept of ideal protein is that thereis an optimal pattern of dietary amino acids that corresponds tothe amino acid requirements of the animal. This is a fundamen-tal concept in animal nutrition and has been found to apply to allspecies in which it has been tested. In growing mammals, theideal amino acid pattern determined experimentally has beenfound to be similar to the amino acid patterns in milk proteinand body tissue protein within that species. The primary bodyprotein is muscle, and muscle is structurally, biochemically, andcompositionally similar across species (although there are many

types of muscle, when the amino acid composition of all themuscle in the mammalian body is compared, the values arestrikingly similar across species), including nonmammalianspecies (Table 1) (2,4,7,8). Although there are some differencesamong species, there are many more striking similarities. This isone of the reasons that the lysine requirement expressed permass CP and the ideal amino acid pattern are also similar acrossmany mammalian species.

This concept raises the very interesting question of whetherthe experimentally determined human amino acid requirementsare similar to the pattern shared by other mammals. Table 2contains the dietary recommendations for humans from therecent DRI (2), and Table 3 contains a calculation of these data

as amino acid ratio to lysine. Table 4 contains the amino acidratios to lysine for swine (4). The similarities are quite striking,as are some of the differences. Baker (9) previously discussed thedifferences and similarities in amino acid requirements, relativeto the ratios to lysine, between adult humans and 44-kg pigs.Our comparisons are based on NRC values for swine (4),whereas Baker (9) used the maintenance data for pigs fromHeger et al. (10,11); the conclusions drawn in both cases are thesame. Although swine are used in this example, comparison toother species would yield similar conclusions.

Tables 14 demonstrate that the ideal protein concept, basedon amino acid ratios to lysine, also appears to hold for humannutrition. The differences between humans and animals in theratios of some amino acids to lysine suggest that some amino

acids may require reevaluation in humans. These amino acidsshould be studied again, either to confirm or to improve theestimates or to determine what is different about their metab-olism in humans compared with animals (9). For example, theisoleucine requirement of humans has not been experimentallydetermined, and its current requirement was calculated based onits proportion to the other branched-chain amino acids relativeto their requirement for protein synthesis (2); direct measure-ment of the isoleucine requirement is required. The estimatedmaintenance requirement of the sulfur amino acids (SAA) inhumans, which is based on many experiments (12), is similar tothat for growing swine but lower than that determined formature swine. Either there is something different about human

utilization of SAA compared with animals or the estimates in

7 Abbreviations used: AI, adequate intake; ASL, argininosuccinate lyase; ASS,

argininosuccinate synthetase; CP, crude protein; CPS I, carbamoyl phosphate

synthetase I; CPS III, carbamoyl phosphate synthetase III; DRI, dietary reference

intake; EAR, estimated average requirement; NRC, National Research Council;

OAT, ornithine aminotransferase; OTC, ornithine transcarbamoylase; P5C,

pyrroline-5-carboxylate; RDA, recommended dietary allowance; SAA, sulfur

amino acids.

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either swine or humans require reevaluation. The estimatedmaintenance requirement for threonine in humans is againsimilar to that for growing swine but much lower than thatmeasured for maintenance in mature swine. A possible clue tothese differences is the time period during which the require-ments for these amino acids have been measured. In the animalstudies the diets are generally fed for long periods (weeks tomonths) (4), whereas most of the human studies were conductedover relatively short time periods (,1 wk) (2,12). SAA andthreonine may require longer periods to fully adapt to changes inintake because of their complex metabolism. Additional re-search appears to be required.

Although the primary use of dietary lysine is for body proteinsynthesis, the metabolic requirement for lysine also includes

carnitine synthesis and obligatory oxidation. The most impor-tant role of carnitine is in transporting long-chain fatty acids intothe mitochondria for b-oxidation and subsequent energyproduction via the citric acid cycle. Carnitine biosynthesisinvolves the methylation of protein-bound lysine, after whichthe carnitine moiety is released from the protein [as recentlyreviewed by Vaz and Wanders (13)]. Although carnitine servesan important metabolic role, data from rats indicate that ,1%of dietary lysine is actually converted to carnitine when lysine isnot limiting in the diet (14,15). However, this study also found

that with a diet limiting in lysine, there was a decline inextrahepatic levels of carnitine (14); therefore, although carni-tine synthesis uses only a small amount of dietary lysine, it isinfluenced by dietary lysine intake. Because carnitine is notfound in vegetarian diets and is lower in milk and eggs than inmeat (13), humans consuming vegan and lactoovovegetariandiets had lower levels of circulating carnitine than omnivoroushumans (16). Furthermore, the humans consuming vegetarian-type diets also had carnitine levels that were very highlycorrelated to plasma lysine concentrations, suggesting thatwhen carnitine is not provided in the diet, there is a greaterdependence on endogenous carnitine synthesis from lysine.Therefore, in domestic animals and humans consuming primar-ily grain-based diets, the importance of dietary lysine intake for

carnitine synthesis cannot be overlooked.The obligatory oxidation of dietary lysine, an important

component of the lysine requirement, responds to deficiencydifferently than other amino acids. A study by Moehn et al. (17)in growing pigs found that even when the dietary intake of lysinewas well below (6090%) its requirement for maximum proteinsynthesis, the rate of lysine oxidation was relatively stable untillysine intake was 60% or less of the required intake (17). This iscontrary to the results observed for other dietary indispensableamino acids, whose degradation/oxidation declines, often to the

TABLE 2 Amino acid requirements of humans1

EAR for children aged

13 y, mgkg21d21EAR for children aged

13 y, mg/g protein2EAR for adults,

mgkg21d21EAR for adults,

mg/g protein3

Lysine 45 52 31 47

Histidine 16 18 11 17

Isoleucine 22 25 15 23

Leucine 48 55 34 52

Valine 28 32 19 29

Methionine 1 cysteine 22 25 15 23

Phenylalanine 1 tyrosine 41 47 27 41

Threonine 24 28 16 24

Tryptophan 6 7 4 6

1 All data from the Institute of Medicine (2).2 Calculated by dividing the estimated average daily requirement by the estimated average protein requirement (0.87 gkg21d21).3

Calculated by dividing the estimated average daily requirement by the estimated average protein requirement (0.66 gkg21d21

).

TABLE 1 Comparison of body amino acid composition of different species1

Human2 Pig2,3 Rat2 Calf2 Chick2 Fish4

Lysine 72 (100) 75 (100) 77 (100) 69 (100) 71 (100) 95 (100)

Arginine 77 (107) 69 (105) 73 (95) 75 (109) 67 (94) 62 (65)

Histidine 26 (36) 28 (45) 30 (39) 27 (39) 19 (27) 32 (34)

Isoleucine 35 (49) 38 (50) 39 (51) 30 (43) 41 (58) 45 (47)

Leucine 75 (104) 72 (109) 85 (110) 74 (107) 68 (96) 86 (91)

Valine 47 (65) 52 (69) 52 (68) 42 (61) 61 (86) 54 (57)

Methionine1

cysteine 35 (49)

5

34

6

(45) 20 (26)

7

18 (26)

7

18 (25)

7

32 (34)Phenylalanine 1 tyrosine 70 (97) 74 (103) 77 (100) 66 (96) 66 (93) 80 (84)

Threonine 41 (57) 37 (58) 43 (56) 43 (62) 42 (59) 49 (52)

Tryptophan 12 (17)5 86 (10) NR8 NR8 NR8 12 (13)

1 Values are in milligrams per gram total amino acids, and the values in parentheses represent the content as a percentage of the lysine content. With the exception of the pig, the

percentage of lysine content values were calculated by dividing each amino acids content by the lysine content and multiplying by 100.2 Values in milligrams per gram total amino acid for humans, pigs, rats, calves, and chicks from Davis et al. (7), unless otherwise noted.3 Values for the content as a percentage of lysine content for pigs taken from the National Research Council (4).4 Values for fish calculated based on values provided in Walton et al. (8) and reflect the amino acid composition of cod muscle protein.5 The methionine 1 cysteine and tryptophan content of human protein taken from the Institute of Medicine (2).6 The methionine 1 cysteine and tryptophan content of muscle calculated by multiplying the percentage of lysine content value by the lysine content of protein.7 Values for rat, calf, and chick are only the methionine content of muscle; cysteine values were not available.8 Values not reported.

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basal or obligatory rate, when intake is below requirement (18).We have recently demonstrated in vitro (19,20) that lysineoxidation is relatively constant under a variety of conditions andalso occurs in several different tissues of the pig, including aphysiologically significant level of oxidation in the intestine. Theimplication of these results for nutritionists is that a slight

decrease in lysine intake below requirement is not readilycompensated for by decreasing oxidation. Therefore, proteinsynthesis by the animal is relatively more sensitive to a deficiencyof lysine than of many other amino acids, where a decrease inintake below requirement results in a decrease in catabolism ofthe amino acid. So far, these results have been observed only inpigs and poultry (21), and research in other species is required todetermine whether this is a unique or widespread phenomenon.

Interspecies differences in arginine metabolism

and dietary requirements

The metabolic arginine requirement is primarily influenced bydemand for protein deposition, as for all amino acids. However,different species have different capacities for endogenous argi-nine synthesis, and therefore, the dietary arginine requirementvaries widely among species during growth, ranging from 1.4%of CP intake in 100-kg swine (4) to over 5% in young broilerchicken (5) and Pacific salmon (22). Therefore, the effects ofdietary arginine intake, from deficient to excess, on endogenoussynthesis are also highly species dependent. A representation ofthe metabolic requirement for arginine and its partitioning intodietary contribution and endogenous synthesis is shown in

Figure 1. The metabolic requirement is the total use of argininefor all functions, net of recycling. This representation demon-strates the relation between maximum and minimum rates ofendogenous synthesis and dietary requirement. This representa-tion assumes that there is a minimum obligatory arginine syn-thesis rate, although this has not been measured or looked for in

most species. This minimum also includes the probable contri-bution of arginine synthesized by intestinal microflora andabsorbed by the animal, which also needs to be quantified. Thedifference between metabolic requirement and the rate of max-imum endogenous synthesis corresponds to the minimum die-tary arginine intake that is required. This is the intake of arginineidentified in most requirement experiments. However, addi-tional dietary intake of arginine will spare the utilization ofenergy and nitrogen for arginine synthesis by the amount thatequals the difference between the maximum and minimum en-dogenous synthesis rates. We studied the metabolic response toarginine intake in piglets over the range from deficient to morethan adequate and showed that there was a slow but steady

decline in plasma ammonia with intakes greater than theminimal dietary requirement (23). Another consideration is

TABLE 3 Amino acid requirements of humans expressed as a percentage of lysine requirement1

EAR for children aged

13 y, mgkg21d21EAR for children aged

13 y, mg/g protein

EAR for adults,

mgkg21d21EAR for adults,

mg/g protein

Lysine 100 100 100 100



Leucine 107 106 110 110

Valine 62 62 61 62

Methionine1

cysteine 49 48 48 49Phenylalanine 1 tyrosine 91 90 87 87


Tryptophan 13 13 13 13

1 All values calculated by dividing the values from Table 2 by the respective lysine requirement.

TABLE 4 Comparison of the amino acid ratios in ideal proteinfor maintenance, protein accretion, and milkproduction in swine to the amino acid compositionof swine tissue1

Maintenance

Protein

accretion

Milk

synthesis

Body

tissue

Lysine 100 100 100 100



Leucine 70 102 115 109

Valine 67 68 85 69

Methionine/cysteine 123 55 45 45

Phenylalanine/tyrosine 121 93 112 103


Tryptophan 26 18 18 10

1

Table adapted from the National Research Council (4).

FIGURE 1 Partitioning of the metabolic and dietary arginine

requirement in animals.



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that in many species, such as piglets as a example, there is aminimum arginine intake for survival (24) that is less than theminimum dietary requirement. For example, enterally fed pigletsreceiving 0.20 gkg21d21 of arginine were able to survive, butthey had elevated plasma ammonia as well as urea concentra-tions and low plasma arginine concentrations despite maximumrates of endogenous arginine synthesis (24). Therefore, tonormalize all of these parameters, the minimum dietary argininerequirement must be greater than the minimum argininerequirement for survival. Investigation in clinical and disease

states of possible metabolic advantages to increasing arginineintake above the minimum dietary requirement is worthconsidering. Quantification of all these levels in the differentspecies would aid greatly in completing our understanding ofarginine metabolism.

Figure 2 shows a variety of possible changes in metabolicfunction with varying dietary arginine intake from deficient toexcess, relative to the dietary requirement to optimize proteinsynthesis. These metabolic functions could include synthesis ofurea, nitric oxide, and creatine. There are many possible slopesand inflections in response; to simplify the figure they are onlyshown as 2-phase linear responses, but the actual shapes of theresponses could be nonlinear and not show any inflection. The

shapes of the responses of these nonprotein arginine functionsare very important, but they have not been well described. Mostexperiments only use a very small number of treatments andtherefore provide data only at specific points, without knowingwhere these are relative to minimal or maximal response inmetabolic function. We recommend that future experimentsconsider the advantages for interpretation of including sufficientdata points to describe the entire response pattern.

Species differences in the mechanism of nitrogenous wasteexcretion have significant impacts on the metabolic and dietaryarginine requirements. For example, only ureotelic animalsrequire arginine for urea cycle function, and hyperammonemiais the metabolic consequence of insufficient dietary arginine

intake in these species (2527). However, species such as birdsand fish use arginine-independent forms of nitrogen wasteexcretion, and dietary arginine deficiency primarily reduces

growth rate but does not cause hyperammonemia. The metabolicability to deal with excess dietary consumption does not seem tobe related to the method of nitrogen excretion but rather to thedietary protein requirement and the overall capacity to metab-olize excess protein. Carnivorous species, for example, have highdietary arginine requirements but can readily degrade and excretevery large quantities of amino nitrogen with no deleteriouseffects, whereas omnivorous animals usually have a lower dietaryrequirement, as a result of higher endogenous synthesis, and lesscapacity to degrade excess protein. Many of these differences are

described in detail below.

Arginine requirements and metabolism in pigs. Arginine isconsidered a conditionally indispensable amino acid in pigs.There is a dietary requirement in the neonate, but in the healthyadult, endogenous synthesis is adequate to meet all metabolicrequirements (28). However, there is recent experimental evi-dence that in some physiological instances arginine supplemen-tation may be beneficial. Arginine intake from sows milk, whichhas been estimated at 0.42 gkg21d21 in wk-old piglets (29), issimilar to the estimated dietary arginine requirement of 3- to 5-kgsuckling piglets (0.35 gkg21d21) (4). However, the factorialestimate of the metabolic arginine requirement of wk-old piglets

is 1.1 gkg21

d21

(29), and the arginine requirement of parenter-ally fed piglets, where de novo arginine synthesis is very low(25,30), is ;1.2 gkg21d21 (23). Therefore, to meet the entiremetabolic arginine requirement, there must be substantial re-liance on de novo arginine synthesis in suckling piglets. This wasconfirmed in neonatal piglets given an injectionof gabaculline,aninhibitor of ornithine amino transferase (OAT; E.C. 2.6.1.13),where there were sharp declines in plasma arginine, citrulline,and ornithine concentrations and an increase in plasma ammoniaconcentration (31). A study by our group (24) found that themaximal rate of arginine synthesis, determined in enterally fedpiglets receiving the lowest intake of dietary arginine that couldbe given without inducing hyperammonemia (0.20 gkg21d21),

was 0.68g

kg

21

d

21

. Adding this to the estimated intake in sowsmilk (24) gives a value not different from the estimated totalmetabolic requirement of arginine (0.42 1 0.68 1.1gkg21d21). Despite the fact that dietary intake and de novosynthesis appear to be adequate to meet the metabolic argininerequirement in young piglets, piglet growth was reported to beenhanced by feeding a milk replacer with additional arginine(32). Therefore, endogenous arginine synthesis and normal milkintake may not be sufficient to meet the total metabolicrequirement for arginine in piglets. In this arginine supplemen-tation study, however, the arginine intake in the control diet(;0.50 gkg21d21) was based on the authors measured argininecontent in sows milk, which wasreported as 7.69 g/kg drymatter(32). However, in a separate study, the ileal digestible arginine

content of sows milk on a dry matter basis was found to besubstantially greater at 12.4 g/kg (33). It is therefore possible thatthe reason for the growth response to the supplemental argininein the previous study reflected an abnormally low arginine intakein the milk received by the control group of piglets (32).Additional research on arginine content of sows milk andpotential benefit of supplementation in neonates is required toconfirm these results. Supplementation of the gestation diet ofsows with ;1% arginine was recently shown to increase thenumber of piglets born alive by upward of;2 piglets (34,35),presumably through improved placental transfer of nutrients as aresult of the role of arginine as a precursor for nitric oxide andpolyamines. The implications of these data with respect to

neonatal and gestating humans should be considered. Additional

FIGURE 2 Metabolic responses to dietary arginine intake relative to

changes in protein synthesis. Solid lines: 3 possible response patterns

to arginine intake for arginine use in other metabolic functions (i.e.,

nitric oxide synthesis), based on the assumption that they demon-

strate an inflection at the same intake as protein synthesis. Dashed

lines: Other possible response patterns to arginine intake for arginine

use in other metabolic functions, where the arginine intake required

for the maximum response in arginine use is different from the

requirement necessary for maximum protein synthesis. Many com-

binations of different slopes and inflection points exist as described in

the text. In addition, some responses, such as plasma ammonia and

urea concentrations (see text), decrease with increasing arginine intake.

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discussion may be found in the section on Implications ofInterspecies Comparisons for Human Nutrition.

In growing pigs, the dietary arginine requirement decreases asthe pigs get older and larger, with 4-kg piglets requiring 2.4% oftheir CP intake as arginine and 100-kg pigs requiring only 1.4%of CP as arginine (4). Arginine is considered a dispensable aminoacid following puberty and during gestation in breeding femalesbased on nitrogen retention, urinary urea, citrate and orotateexcretion, and plasma urea concentrations (28). When the ar-ginine requirement of pigs over 20 kg, per kilogram per day, is

plotted against whole-body protein accretion, also per kilogramper day, there is a decrease in the dietary arginine requirementwith increased pig size despite the large increase in rate ofprotein accretion (Fig. 3). Although endogenous arginine syn-thesis has been measured in wk-old (;2.5-kg) piglets (24,36,37),it has not been measured for any other age of pig. Therefore,there is no definitive evidence whether the decreasing dietaryarginine requirement results from an increase in endogenousarginine synthesis or a decrease in the metabolic arginine re-quirement. However, based on Figure 3, an increase in endog-enous arginine synthesis probably explains the decrease indietary requirement with weight and age, despite the increases inprotein accretion.

Much research has been conducted in young pigs relating tothe enzymes, sites of synthesis and precursors of arginine syn-thesis. In wk-old piglets, the intestine has measurable activity ofall enzymes needed for arginine synthesis from either proline orglutamine/glutamate (38,39), and arginine synthesis occurs invitro (38,39). Arginase activity is very low in the intestine ofsuckling piglets (40,41); thus, the intestine does not reduce thedietary arginine contribution to whole-body arginine status.Following weaning there is an increase in arginase activity (40,41), and a decrease in the ability of the porcine enterocytes tosynthesize arginine from proline in vitro (39). Ornithine andcitrulline production from proline in isolated enterocytes in vitrois greater in 28-d-old weaned vs. 7-d-old suckling piglets (39).

Thus, in weaned pigs, similar to in rodents, the citrulline releasedby the small intestine is believed to be used by the kidney tosynthesize arginine. The kidney has measurable in vitro activityof both argininosuccinate synthetase (ASS; E.C. 6.3.4.5) andargininosuccinate lyase (ASL; E.C. 4.3.2.1) in all ages of piglets(40), although to our knowledge, the quantitative importance ofrenal metabolism in arginine synthesis and the effect of piglet agehave not been investigated in vivo.

The enzymes necessary for glutamate, glutamine, and prolineto be precursors for arginine synthesis have been detected invitro (38,39), although in vivo research by our group has shownthat proline is the major arginine precursor in wk-old piglets andnot glutamine/glutamate (24,25). A diet containing ample amountsof glutamate, but not proline, was unable to prevent hyperam-monemia in either enterally or parenterally fed piglets receivingan arginine-free diet (25). Furthermore, when a radioactiveglutamate isotope was intragastrically infused into enterally fedpiglets receiving either an arginine-deficient or generous diet,

none of the label was recovered in arginine (24). The enzymealanine aminotransferase (E.C. 2.6.1.2), an enzyme that can con-vert glutamate and pyruvate to alanine and a-ketoglutarate, waspresent in the intestinal mucosa of piglets of all ages (42), and thesmall intestinal mucosa was also able to oxidize a-ketoglutarate.Furthermore, in enterally fed piglets, only 5% of an intragastri-cally administered glutamate isotope appeared in the portalblood, and 50% of this dietary glutamate was oxidized to CO2(43). This explains why glutamate was a poor arginine precursorin enterally fed piglets. We have also shown in vivo that inwk-old enterally fed piglets citrulline synthesis is limiting towhole-body arginine synthesis because citrulline addition was aseffective as arginine addition, whereas ornithine or proline were

not (37). Both citrulline and arginine addition to the arginine-deficient diet spared the use of proline for arginine synthesis(37), demonstrating in vivo that the partitioning of proline usefor arginine synthesis, vs. other activities, is dependent on thedemand for endogenous arginine synthesis. Metabolism in grow-ing pigs is similar to that in the young pig; in growing pigsreceiving an arginine-deficient diet, citrulline was more effectivethan ornithine at promoting pig growth and efficiency (44).

To verify the enzyme and in vitro work, we investigated thesites of arginine synthesis in wk-old piglets and found that, invivo, 4060% of whole-body arginine synthesis occurred withfirst-pass intestinal metabolism, regardless of arginine intake(24). First-pass hepatic metabolism does not contributeto whole-

body arginine synthesis in the neonate (36). The remaining 4060% of whole-body arginine synthesis was from the metabolismof circulating precursors (36),and we have experimental evidenceshowing that the intestinal metabolism of arterial proline may beanother majorcontributorto whole-body arginine synthesis (30).From ourin vivo isotope data, it wasnot possible to conclude thatfirst-pass intestinal metabolism or the intestinal metabolism ofcirculating precursors were responsible for the entire conversionof proline to arginine, only that they were necessary for theconversion. Intestinal metabolism is critical in the neonate forornithine synthesis from proline (39,45). However, it is possiblethat renal or extraintestinal metabolism may also be involved inthe remainder of the arginine synthetic pathway, even in wk-oldpiglets. This requires experimental confirmation.

Arginine requirements and metabolism in rats. Much of thepioneering research relating to arginine and urea cycle metab-olism was conducted in rodents, and many review articlessummarize the results of rodent research (4649); however,there are a few instances and some information that is differentor unique in arginine metabolism and requirements in ratscompared with other species. When rat nutrient requirements,including amino acids, are compared with those of other species,it must always be considered that rats practice coprophagy(50,51). Coprophagy provides an unknown amount of micro-bial amino acid to the diet, with the effect that most dietarystudies in rats produce an underestimate of actual dietary

requirement.

FIGURE 3 The relation among dietary arginine requirement, pig

body weight, and the rate of protein accretion in pigs from 20 to 100

kg. Protein accretion (g/d) was calculated using protein accretion

[0.47666 1 0.02147*body weight 1 0.00023758*(body weight)2 1

0.000000713*(body weight)3

] 3 127.5 (4).



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Arginine is classified as an indispensable amino acid for ratsduring growth (52) but not for adult rats (53). However, in adultrats formerly subjected to protein malnutrition, increasing incre-ments of dietary arginine resulted in a positive linear responsewith regard to weight gain and nitrogen retention and a negativelinear response for urinary orotate (54); therefore, in some casesarginine may be indispensable for mature rats. Although thisresearch has implications for clinical treatment of humans, thiseffect has not been tested, to our knowledge, in humans or otherspecies.

The dietary arginine requirement for growing rats is based ona crude protein intake of 15% (6). As in other species, thearginine requirement for tissue accretion and normal levels ofmetabolite excretion in rats increases as the protein content ofthe diet increases (55). This is likely because of arginines role inthe urea cycle and the fact that increasing levels of dietaryprotein have been shown to increase the activity of urea cycleenzymes (56,57). Therefore, the protein content of the diet,particularly the excess amino acid intake, must also be takeninto account when determining if arginine intake is adequate.

Growing rats receiving an arginine-free or arginine-deficientdiet have elevated urinary orotate, citrate, and urea excretion(55,58,59) and decreased nitrogen retention (58,60). It is of

interest, however, that improvements in nitrogen retention, inresponse to supplemental arginine, appear to occur at a lowerintake of arginine than changes in the metabolite (such as citrate,urea, and orotic acid) excretions (59). Furthermore, in adult,mature, female nonpregnant rats, although there was no weightchange in response to feeding an arginine-free diet, there was an;27-fold increase in orotate excretion and an 8-fold increase incitrate excretion relative to rats receiving a control, arginine-containing, diet (60). We have shown in piglets that there was abreakpoint in plasma urea and ammonia with increasing arginineintake but that these continued to decline at a rate significantlydifferent from zero until the entire metabolic requirement (1.2gkg21d21) was provided by the diet (23). Based on these

findings, it is clearly inappropriate to define arginine as dispens-able or indispensable based on weight gain andnitrogen retentionalone because metabolic perturbations may continue to existevenwhen growth and/or nitrogen balance is apparently maximized.

The capacity of growing rats to endogenously synthesizearginine for use in tissue accretion was demonstrated over 70 yago (61), when it was discovered that the amount of arginineaccreted in tissue was 2- to 3-fold greater than the amountprovided in the diet. However, to our knowledge, the actual invivo arginine synthesis rate has never been quantified in ratsunder various conditions. Although the total metabolic argininerequirement does not appear to have been studied in rats, it isclearly greater than the dietary requirement (2.9% of CP) (6), asillustrated by the fact that a diet containing 1% arginine (over

twice the dietary recommendation) was still not enough tosupport optimal rat growth in the absence of endogenous syn-thesis (62). The rat was the main experimental animal modelused to define the intestinal-renal axis of endogenous argininesynthesis in weaned mammals. The perfused rat intestine showsa substantial uptake of luminal and circulating glutamine andglutamate (63) and a release of citrulline (63). The intestine wasshown to be the primary organ responsible for citrulline pro-duction (64,65) but was unable to metabolize the citrulline toother metabolites (64). The kidney was identified as the organresponsible for the majority of whole-body arginine synthesis invivo in several different perfusion (64), mass balance (66), andligation (67) studies. The release of arginine by the kidney was

directly related to citrulline uptake by the kidney (66); therefore,

if circulating citrulline concentrations were increased, there wasa subsequent increase in renal arginine release (66). In rats,unlike piglets (24), arginine intake did not affect the renal releaseof arginine (68).

The endogenous synthesis of citrulline by the rat intestineappears to be critical for the growth of rats. Citrulline, unlikearginine (69) and ornithine (70), was not extensively metabo-lized by the liver or the intestine (64); therefore, supplementalcitrulline provided in an arginine-free diet fed to rats resulted inan increase in circulating citrulline and arginine concentrations

(70). Moreover, when the intestinal activity of ornithine trans-carbamoylase (OTC; E.C. 2.1.3.3) was inhibited, using aninfusion of a glycyl-glycyl derivative ofd-N-(phophonacetyl)-L-ornithine, the rats lost weight or gained less than when OTCactivity was present (62). The growth retardation was onlypartially reversed by the addition of 1% arginine to the caseinhydrosylate diet and was completely reversed by adding 1%citrulline (62). The main implication of this is that, in rats, theendogenously synthesized arginine is the primary source ofarginine used for growth, whereas the dietary arginine seems tobe used mainly as a precursor for intestinal ornithine or citrullineformation. The partitioning of the metabolism of endogenouslysynthesized vs. dietary arginine in other species still requires

investigation. Interestingly, ornithine addition to an arginine-deficient growing rat diet supported the same growth rates andplasma ammonia and urea concentrations as either citrulline orarginine addition (62,70), but plasma arginine concentrationswere still lower than if either citrulline or arginine were added tothe diet (70). Therefore, in growing, postweaning rats, if there isthe capacity for endogenous arginine synthesis, then growth andurea cycle function can be maintained by the addition of orni-thine, citrulline, or arginine to an arginine-free diet (62,70,71).This is in contrast to other species, where citrulline addition isclearly advantageous compared with ornithine addition. Theimportance of proline as an arginine precursor and its ability tospare a portion of the dietary arginine requirement has not been

investigated in rats.

Arginine metabolism and requirements in cats. Cats wereunable to synthesize enough ornithine, and therefore citrulline,to satisfy their metabolic arginine requirement, resulting in therapid onset of hyperammonemia when arginine-free diets werefed (26,72). On a per-kilogram body weight basis, the smallintestinal mucosa of cats only had 5% of the pyrroline-5-carboxylate (P5C) synthase (E.C. number not assigned) activity(73) of the rat intestinal mucosa. Furthermore, the activity ofOAT per gram mucosa in cats was only 23% of the activity inrats (73). Perfusion studies in rats showed that the primary siteof citrulline synthesis was the small intestine (63,64), and studiesin pigs found that P5C synthase activity is exclusively small

intestinal and OAT activity is primarily small intestinal (74). Thevery low intestinal activities of these enzymes in cats (73) resultsin very low de novo synthesis of ornithine, and subsequentlycitrulline, compared with other species. These differences inenzymes explains why arginine is an indispensable amino acid incats of all ages. In addition to not being able to synthesizeornithine from glutamate, cats also have low intestinal alanineaminotransferase activity compared with rats (;58%) (73).Therefore, unlike other mammals, cats cannot tolerate diets thatare high in glutamate (75), likely because of an inability toconvert glutamate nitrogen to alanine nitrogen.

Compared with other species, cats also have very low renalarginine synthesis from citrulline: 86 mmol/d (76) vs. over 350

mmol/d in rats fed a standard diet (66). The low plasma citrulline

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concentrations in cats (.10 mmol/L) (76) compared with rats,rabbits, or pigs (60120 mmol/L) (44,76), explains the limitedamount of renal arginine synthesis from citrulline in cats (76).For example, when kittens were fed an arginine-free diet withsupplemental citrulline, plasma citrulline concentrations were;250 mmol/L, and plasma arginine concentrations were notdifferent from those in kittens receiving an arginine-sufficientdiet (77). These observations demonstrate an increase in renalarginine synthesis with increased plasma citrulline concentration(77), which is in agreement with rat research where the plasma

citrulline concentration increased 3.9 times and the rate of renalarginine release increased 3.4 times (66). Therefore, in cats theprimary limitation in de novo arginine synthesis is the low levelsof intestinal enzymes needed for ornithine synthesis, whichlimits citrulline formation and circulating citrulline concentra-tions and subsequently renal arginine synthesis.

Cats must, as a result of lack of de novo synthesis, rely ontheir diet to meet the arginine requirement, which ranges from0.54 gkg21d21 (4.3% of CP) in kittens to 0.12 gkg21d21

(3.9% of CP) in adult cats (3). The implication of this is that thedietary arginine requirement of the cat also closely represents thetotal metabolic requirement: the amount of arginine used forprotein synthesis, support of urea cycle function, for the syn-

thesis of other metabolites including creatine, polyamines, andnitric oxide (4,27), and for basal arginine catabolism. This isin contrast to other species where de novo synthesis providessignificant, and often variable, proportions of the total meta-bolic requirement. This also means that, in comparison to othernoncarnivorous ureotelic species, cats have a very high dietaryarginine requirement. The high protein intake of the cat requiresa high rate of nitrogen excretion and thus also contributes to thehigh dietary requirement for arginine. In the cat, the hepaticactivities of most enzymes involved in amino acid degradationare constitutively high and unaffected by diet (78), relative toother species, meaning that the urea cycle must function at ahigh rate to detoxify the resulting ammonia. The effect of

arginine intake on hepatic or renal arginase activity has not beenquantified; however, in cats, unlike in rats (56), the hepaticactivity of arginase was not affected by dietary protein intake(78). However, the hepatic activities of the urea cycle enzymes(OTC, ASS plus ASL, and arginase) in the liver of cats were 2550% of the activities in the livers of rats fed a 90% protein diet(56,78) and therefore may more closely resemble the hepaticactivity in rats receiving;4050% protein diet. Because proteinintake does not affect urea cycle enzyme activities in cats,arginine may need to be provided at a greater percentage of thetotal crude protein intake in cats receiving a low dietary proteinintake compared with those fed a high-protein diet, to maintainthe high basal level of urea cycle function. With high proteinintakes, however, it is likely that arginine intake from the diet

(assuming the diet has a balanced amino acid composition) willbe adequate to support urea cycle function and that rate of ureacycle function would be similar to that of other mammals re-ceiving a high-protein diet.

Although proline is a major precursor for arginine synthesis(10), to the best of our knowledge, the expression of prolineoxidase (E.C. 1.5.99.8) has not been investigated in the cat. Wepredict that even if there is measurable activity of this enzyme infeline tissues, proline is unlikely to make a large contribution towhole-body arginine synthesis in cats because of the low OATactivity relative to other species.

Arginine requirements and metabolism in dogs. Both

growing (27,79) and adult (80) dogs have dietary requirements

for arginine (0.36 gkg21d21 in young puppies and 0.06gkg21d21 in mature dogs; 3.5% of CP in both ages of dogs)(3) that are intermediate to the requirements of the cat and therat. Unlike cats, dogs are not obligate carnivores, and this maypartly explain the lower arginine requirements in dogs than cats.To the best of our knowledge, the activities of the urea cycle/arginine synthetic enzymes have not been studied in dogs. Theactivities of these enzymes, particularly P5C synthase, have beensuggested by others (81) to be intermediate between those of thecat and the rat, thereby explaining why their arginine require-

ment is also intermediate. This requires experimental confirma-tion.

Arginine requirements and metabolism in rabbits and

horses. Dietary arginine is necessary in growing rabbits formaximum growth and feed efficiency (82,83), and estimates ofthe arginine requirement are between 0.6 (82) and 1.23%(83) ofdiet. In comparison to other species, there has been little researchon arginine requirements and metabolism in rabbits. Factors be-lieved to affect the arginine requirement in rabbits included thecrude protein content of the diet (increasing arginine requirementwith increasing protein intake) (83) and the dietary concentra-tions of indispensable amino acids (increased arginine require-

ment when higher levels of indispensable amino acids are fed,even if diets were isonitrogenous) (82). The requirement of 0.60%of diet was based on growth, feed efficiency, [U-14C]arginineoxidation, and serum arginine concentrations (82). There wereno effects of arginine intake on the plasma urea concentrations,urine creatinine excretion, the hepatic arginase activity, or therenal glycine transamidinase (E.C. 2.1.4.1) (82). Even whenrabbits received an arginine-free diet for 7 d, they continued togrow, although at only ;10% of the rate of those receiving anarginine-adequate diet (82). The authors did not observe anysigns of hyperammonemia as a result of feeding the arginine-freediet, and they concluded that most aspects of arginine metab-olism in growing rabbits were similar to those of the growing rat

(82). They also concluded that de novo synthesis of arginineappeared adequate to meet the metabolic demands for arginine(82). However, the ability of the young growing rabbit to sustaingrowth in the absence of dietary arginine could also be the resultof microbial synthesis of arginine in the ceca, subsequent ex-cretion, and then intake of the microbial arginine via coproph-agy. This has been recently described for microbially synthesizedlysine in rabbits (84).

The rabbit must, at this time, be considered representative ofother nonruminant herbivores because, to the best of our knowl-edge, there has been little or no research conducted in othernonruminant herbivores, such as horses. However, there may beunique aspects of arginine metabolism in horses. For example,mature horse milk has a high concentration of arginine (on a

wt:wt total amino acid basis); compared with other mammalianspecies only feline milk has more arginine than mares milk (85).Unlike other mammals, the arginine content of mares milkincreases 29% from colostrum to mature milk, whereas thearginine content of the milk in other mammals either decreasesorremains unchanged (86). Arginine metabolism in horses is a clearopportunity for further investigation.

Arginine metabolism and requirements in ruminants.

There are no amino acid requirements published for ruminantssuch as growing beef (87) or dairy (88) cattle because it is as-sumed that suckling, preruminant offspring obtain enoughamino acids from milk (or milk replacer formula) (89), and

that once the rumen is developed, ruminants receive enough



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amino acids from the combination of diet and rumenal microbialamino acid synthesis. However, in a study that determined thelysine requirement of preruminant calves and then used the ratioof lysine to the other indispensable amino acids in calf bodyprotein to estimate the amino acid requirements for the otheramino acids, arginine intake was found to be only ;60% of theestimated metabolic requirement (89). Furthermore, in milkreplacer-fed calves, weaning weight and plasma arginine con-centrations were greater when calves received an arginine-supplemented formula (90). The administration of arginine into

the abomasum (similar to the stomach) of growing calves inconjunction with ammonium acetate improved nitrogen reten-tion and lowered plasma ammonia concentrations relative toammonium acetate administration alone (91). Therefore, similarto other young mammals, arginine should probably be reclas-sified as a conditionally indispensable amino acid for youngruminants. The estimated dietary arginine requirement of 8.5 g/din a 500-kg cow (0.17 gkg21d21) (89) is very low comparedwith the dietary requirements of other growing mammals(;0.300.50 gkg21d21, as discussed previously). However, be-cause this requirement was based only on amino acid compo-sition of body protein, it does not reflect the entire need forarginine to support urea cycle function; therefore, the require-

ment may be underestimated. The ruminant liver has detectableactivity of all of the urea cycle enzymes (92), and cattle have afunctional arginine-dependent urea cycle. Interestingly, however,unlike other mammalian species, an increase in arginine intakedid not affect hepatic arginase activity (91). Ruminants exten-sively utilize nitrogen recycling; blood urea is secreted into thegastrointestinal tract, where it is used by the rumen microflorato resynthesize amino acids (93). Therefore, to ensure maximalamino acid efficiency in ruminants, a functional urea cycle, andtherefore adequate whole-body arginine status, is critical to main-tain normal nitrogen metabolism. These are examples, amongothers, of the many aspects of arginine, urea, and nitrogen metab-olism that are unique to ruminants.

Arginine requirements and metabolism in chicks. Thedietary essentiality of arginine in the diet of chicks was firstdemonstrated in the 1930s (94,95). Further investigation re-vealed that arginine was a dietary indispensable amino acid inchicks because of the lack of detectable mitochondrial carbam-oyl phosphate synthetase I (CPS I; E.C. 6.3.4.16) in all tissuesinvestigated, including the liver, kidney, pancreas, and spleen(96). Although chick kidney, but not liver, has detectableactivity of the enzymes OTC, ASS, and ASL, in comparison torats (56), the renal activities of these enzymes in the chick arelow. These low renal ASS and ASL activities have physiologicalsignificance, however, because citrulline addition to an argininedeficient diet was shown to be equally effective at promoting

chick growth as arginine addition (97). The ability of citrullineto spare the arginine requirement in chicks was further con-firmed when label from [ureido-14C]citrulline was found inchick body protein as arginine (98). Neither ornithine (95,9799) nor bicarbonate (98) was a precursor for arginine, sup-porting the enzymatic data finding no detectable mitochondrialCPS I activity (96).

Unlike mammals, the metabolic arginine requirement ofchicks does not include the support of urea cycle function. Themain nitrogenous waste product in birds is uric acid, and itsformation is not arginine-dependent. Because the urea cycle isnot necessary for nitrogenous waste excretion, this is believed tobe the reason for the almost complete lack of urea cycle enzymes

(which are also arginine synthetic) in the chick liver (96).

Dietary arginine in chicks is therefore used only for proteinsynthesis and the synthesis of metabolically important moleculessuch as creatine. Creatine concentrations are related to arginineintake and availability in chicks (100102). Therefore, althoughonly a small amount of dietary arginine is used for creatinesynthesis, adequate arginine intake is important for the synthesisof creatine.

Chicks have the enzymatic capacity for arginine degradationvia arginase (E.C. 3.5.3.1) activity. However, unlike mammals,the arginase activity of the kidney is ;30 times higher than that

in the liver (96). Despite the fact that there is no functional ureacycle in chicks, chicks do excrete some urea, and this is a mea-sure of arginine degradation via arginase. In laying hens, renalarginase activity and urea excretion were both increased by in-creasing arginine or protein intake (103), and urea productionwas related to plasma arginine concentration (103). Therefore,similar to other animals, arginine concentrations are regulatedby arginine degradation in chickens. However, although arginineadministration increased urea excretion in hens, the same wasnot observed with ornithine infusion (103), further demonstrat-ing that ornithine is an ineffective arginine precursor in poultry.Studies in chicks showed that when renal arginase activity wasaltered by either changing dietary factors (increasing either ly-

sine, arginine, or tyrosine content of the diet) or using strains ofbirds that were selected to have different levels of renal arginaseexpression, as arginase activity increased, there was also anincrease in the arginine requirement (104). Therefore, the die-tary requirement for arginine in chicks represents the metabolicarginine requirement for protein and metabolite synthesis plusthat required to replace the arginine that is degraded by renalarginase.

The chick requirement for dietary arginine, on both a dietaryconcentration (1.101.25 g/100 g) and a percentage of CP basis(5.45.5%), is among the highest of any of the species studieddespite not requiring arginine for urea cycle function. This highdietary requirement results from 1) lack of endogenous synthesis,

2) high rate of protein deposition because of thevery rapid growthrate of meat-type chickens (;50gBWto2kgBWin6wk),and 3)the metabolic interaction between dietary lysine and arginine(discussed below). In comparison to most other indispensableamino acids, excess dietary arginine (up to 4 g/100 g of the diet)was well tolerated in young chicks, and only a small reduction inweight gain (;9%) relative to a control diet was observed (105).

Arginine requirements and metabolism in fish. Arginine isa dietary indispensable amino acid in fish. The activities of theurea cycle enzymes in various types of fish and the differences inurea metabolism among fish species have been reviewed (106).Therefore, this discussion focuses mainly on arginine and ureametabolism in rainbow trout, in comparison to mammals and

chicks.Similar to chicks, the limitation in arginine synthesis in fish

is the synthesis of carbamoyl phosphate. Unlike in mammals,the enzyme that catalyzes the synthesis of carbamoyl phosphatein fish is carbamoyl phosphate synthetase III (CPS III; E.C.6.3.5.5). Similar to CPS I, CPS III is mitochondrial and requiresN-acetylglutamate as a cofactor, but instead of ammonia as thesubstrate, CPS III requires glutamine (107). Studies in rainbowtrout have shown that there is no hepatic activity of CPS III andvery low hepatic OTC activity (108). The muscle of trout has thegreatest activity of CPS III (108); however, in comparison to theactivity of CPS I in rat liver (56), its activity is very low (300mmol of product formedg liverh21 in rats vs. 0.0144 mmol of

product formedg muscleh21

in rainbow trout), and thus there is

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a very limited capacity for carbamoyl phosphate synthesis andsubsequently de novo arginine synthesis in fish. There is de-tectable activity in tissues (specifically liver, kidney, muscle, andintestine) of rainbow trout of all of the other enzymes involvedin arginine synthesis (108,109). Isotopic evidence indicates thattrout are capable of some de novo arginine synthesis; injection of[1-14C]ornithine resulted in ;10% of the label being recoveredin arginine (109). There must be carbamoyl phosphate forma-tion in order for ornithine to be used for arginine synthesis.Therefore, although the activity of CPS III may be low (108), it is

of physiological relevance. However, de novo arginine synthesisis not sufficient to sustain optimal trout growth; addition ofan equimolar amount of either arginine or citrulline to a low-arginine basal diet resulted in greater rates of gain, better feedefficiency, and higher plasma arginine concentrations than theaddition of either glutamate or ornithine (109).

Many types of fish produce urea, but, as in the chick, this islargely believed to be caused by the degradation of dietaryarginine (106,110). Arginase activity, primarily mitochondrial(8,108), has been detected in the liver, kidney, muscle, andintestine of rainbow trout (108,109), with the kidney, followedby the liver, having the greatest activity of arginase per gramtissue (108,109). In rainbow trout, hepatic arginase activity was

not affected by arginine intake (8); however, in turbot fish, therewas a strong correlation between arginine intake and urea pro-duction, which the authors concluded was a result of increasinghepatic arginase activity (110). The relation between arginineintake and whole-body arginine degradation via arginase activity,therefore, may be species-specific in fish. The extrahepatic con-tribution to arginine degradation in fish requires further investi-gation.

The main nitrogenous excretory product in fish is ammonia,which can diffuse through the gills into the aquatic environment[as outlined by Wright and Land (106)]. Therefore, unlike mam-mals, the metabolic and dietary requirement for arginine in fishdoes not include nitrogenous waste excretion. The dietary

arginine requirement has been determined in many species of fish(8,111117) using growth, feed efficiency, and plasma or serumarginine concentrations as the endpoint measurements. Onestudy also used arginine oxidation (8). The importance of ar-ginine for the synthesis of other metabolic products, to the bestof our knowledge, has not been studied in fish. However, the lowestimated maintenance requirements for arginine (calculated tobe;10% or less of the arginine requirement for gain) (110,113),suggest that protein synthesis for growth is the primary use forarginine in fish.

Despite differences in the arginine biosynthetic enzymes indifferent species of fish (106), the arginine requirement of fish,1.201.50 g/100 g of diet and 3.73.9 g/100 g of CP, is relativelysimilar among fish species, with the exception of the Pacific

salmon (2.04 and 5.4, respectively) This similarity suggests thatthe observed differences in de novo arginine synthesis among fishspecies may not be nutritionally important under normal con-ditions in any of these species of fish.

The high arginine requirements in fish, expressed as grams per100 g of diet, are comparable to the requirements in the chick,another nonureotelic species. These similarities probably existbecause protein synthesis is the major contributor to the dietaryarginine requirement in both species. Amino acid composition ofmuscle protein, specifically with regard to arginine, is also similaramong these species (Table 1).

General observations on arginine metabolism across

species. The research on arginine metabolism in all of the

species covered in this article raises many interesting points fordiscussion. First, it is commonly accepted that it is the ASS stepof the urea cycle that is limiting for hepatic synthesis of arginineand therefore urea synthesis (56,118). However, in all speciesstudied, citrulline was a more effective arginine precursor thaneither ornithine or proline (37,44,70,77,79,97,98), which showsthat it is citrulline formation and not the conversion of citrullineto arginine that is the limiting step for endogenous argininesynthesis. The reason for the limitation in citrulline synthesisvaries among species depending on the enzymes present. For

example, the limitation in chicks and fish is because they do notexcrete urea as their nitrogenous waste and lack mitochondrialCPS I activity (96,108), whereas in carnivores the reason is lowP5C synthase and OAT activity (73). In other mammals, thereason that citrulline formation is limiting to arginine synthesishas not been conclusively determined, but recent research inneonatal piglets has suggested that it may be N-acetylglutamatesynthesis, which in turn limits carbamoyl phosphate synthesis(119). Furthermore, these comparisons indicate that ASS andASL enzyme function in arginine synthesis is fairly ubiquitousand similar across many species, including mammals, birds, andfish.

In piglets, proline is the major dietary precursor for endoge-

nous arginine synthesis (24), and therefore, it is reasonable toassume that proline partially spares the dietary arginine require-ment. In piglets receiving an adequate arginine diet, or anarginine-deficient diet supplemented with citrulline, the use ofproline for arginine synthesis was reduced, showing that argininecan partially spare the proline requirement (37). Experimentalconfirmation is still required in other species to determine theability of proline and arginine to spare the dietary requirement ofthe other amino acid.

Arginine deficiency symptoms are most severe in carnivores,with severe hyperammonemia occurring after a single arginine-free meal in cats (26,77), followed by omnivores, and are leastsevere in herbivores, with rabbits continuing to grow on an

arginine-free diet, albeit at a reduced rate (82). Omnivoresdisplay a wide range of deficiencysymptoms, with dogs appearingmore sensitive to arginine deficiency than rats (27,55,5860,80).Deficiency symptoms were also more severe in young vs. oldergrowing mammals (25,27,44,80). The order of sensitivity toarginine deficiencyfollows approximately the same pattern as thedietary arginine requirements on the bases of grams per 100 g ofdiet, grams per kilogram per day, and grams per 100 g of CP. Thespecies with virtually no capacity for endogenous arginine syn-thesis, fish and chicks, have the highest dietary arginine require-ments despite not requiring arginine for urea cycle function.Therefore, the capacity for endogenous arginine synthesis is themajor determinant of the dietary arginine requirement. Thisimplies that all future research on arginine requirements and

metabolism, regardless of species, should include a measurementof in vivo endogenous synthesis rate.

Because arginine requirements are highest in species withouturea cycle function, this clearly shows that the major metabolicuse of arginine is for protein synthesis. In piglets, it has beenestimated that ;70% of the daily arginine use is for proteinsynthesis (29). Therefore, although the other metabolic functionsof arginine are critical for growth and health, protein synthesis isstill the primary component of the metabolic arginine require-ment.

The potential contribution to the animal of microbial syn-thesis of arginine in the small intestine, cecum, and largeintestine does not appear to have been investigated thoroughly.

In the young rabbit this source of arginine may be part of the



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reason that complete dietary deficiency does not result in thedrastic response observed in other monogastric species. Rabbitsand rodents may acquire significant intake of amino acids fromcoprophagy, and this is seldom considered when estimatingdietary requirements. In contrast, there is evidence that micro-bially derived lysine may be used by the animal (120) in theabsence of coprophagy. Therefore, additional research on mi-crobial contribution of amino acids is required with all species.

To date, very little research has specifically been conducted,in any of the species discussed, to examine the effects of

superphysiological intakes of arginine. In rats, for example, thedaily intraperitoneal administration of 3.5 g/kg of body weightarginine induced an experimental form of pancreatitis (121).The effect of high arginine intake relative to dietary require-ments in pigs is discussed in more detail below under lysine-arginine antagonism; however, it appears that the main effect ofhigh arginine intake, relative to the amino acid profile, is areduction in feed intake to compensate for the amino acid im-balance (122125). In neonatal piglets, the twice-daily supple-mentation of arginine resulted in a decrease in plasma arginineand histidine concentrations relative to piglets receiving eitherno supplement, water, or alanine administration (119), whichcould have the negative implications of decreasing weight gain

because of a limitation, in particular, in lysine from lysine-arginine antagonism. However, when the arginine was added tothe milk-replacer and not given as a twice-daily bolus dose, therewas no effect of arginine intake on plasma concentrations ofeither histidine or lysine, and piglets receiving the supplementalarginine grew better (32). In cattle, an increase in CP intake isassociated with an increase in rumen ammonia concentrations asa result of microbial metabolism (126). Extremely high arginineintake in cattle may have effects, with regards to ammoniaproduction, compared with an extremely high intake of otheramino acids, because the microbial degradation of the arginine,which contains 4 nitrogen atoms, in the rumen would result in agreater rate and quantity of ammonia production than from the

degradation of other amino acids. In species such as fish (110)and chicks (103), tissue arginase expression was generally in-duced by increasing arginine intake. Therefore, high arginineintake increases arginine degradation and urea excretion andproduces no adverse effects. The limit to this adaptive degrada-tion does not appear to have been defined in all species. Intakesof 4 to 5 times the dietary requirement have been used inrequirement experiments, apparently without adverse effectsbased on lack of comments by the authors. Although futureresearch is warranted to elucidate species-specific differencesregarding the effects of excess arginine intake, it appears thatarginine toxicity is unlikely to be a concern in any of the speciesexamined.

Implications of interspecies comparisons for human nu-trition.

Arginine metabolism and requirements in humans.

There are presently no dietary recommendations for humans forarginine during either growth or maintenance. There is a cleardietary arginine requirement for growth in all major experi-mental species; humans appear to be the exception. In light ofthe animal data summarized in this article, this position shouldbe reconsidered, especially in neonates, and based on research inrats (88) during recovery from trauma or malnutrition at allages. Arginine was concluded to be dispensable for neonates andchildren based on nitrogen retention and growth data (127,128);however, studies in rats have clearly showed that there can be

metabolic aberrations related to inadequate arginine intake,

even when growth is unaffected (59). Premature infants may beparticularly susceptible to arginine deficiency (129), and poorarginine status has been associated with the onset of neonataldiseases including necrotizing enterocolitis (NEC) (130,131)and persistent pulmonary hypertension of the neonate (PPHN)(132,133). Supplemental arginine has been suggested to beprotective against both NEC and PPHN (134136). Further-more, when intravenously fed infants with hyperammonemiawere given additional arginine, the symptoms of hyperammo-nemia were alleviated (137,138). Together these observations

suggest that even the combination of endogenous arginine syn-thesis and normal dietary arginine intake may not be enough foroptimal neonatal health, particularly when health is alreadycompromised by prematurity or intestinal disease. The questionremains: what are the metabolic and dietary arginine require-ments for neonates and growing children? Based on the recentresearch on arginine supplementation for suckling piglets (32), itshould be considered whether arginine supplementation wouldbe beneficial for preterm or low-birth-weight or normal-weightinfants. Based on recent research in gestating sows (33), argininesupplementation may be a way to increase birth weight ofinfants with intrauterine growth retardation. Despite the im-portant clinical implications relating to arginine intake in human

neonates, there is presently no recommended adequate intakefor arginine (2). Other young mammals clearly have a dietaryrequirement for arginine, which varies with a number of con-ditions. Therefore, it is particularly important that endogenoussynthesis rates and metabolic/dietary requirements in humanneonates and children be determined.

Although healthy adult males do not appear to be adverselyaffected by prolonged feeding of an arginine-free diet (139142),in part because of the ability to decrease arginine catabolism(140), arginine metabolism is altered by pathological conditionssuch as sepsis (143), burn trauma (144), and end-stage renaldisease (145), and therefore both metabolic and dietary argininerequirements must be investigated under each of these condi-

tions. The metabolic requirement for arginine in adult humansfollowing major trauma or surgery should also be determinedbecause it is important for optimal clinical treatment (146,147).

The dietary arginine requirements for adult animals of specieswithout appreciable amounts of endogenous arginine synthesis,specifically cats, fish, and chicks, may provide some insight intometabolic arginine requirements for humans. This is an exampleof an amino acid where application of the ideal protein conceptcould be used to recommend an adequate intake until experi-mentaldata areavailable.Neither chicksnor fishhave an argininerequirement for urea cycle function, and dietary arginine is usedprimarily for protein synthesis. Therefore, their dietary argininerequirement may be comparable to the human arginine require-ment for protein synthesis when corrected for the interspecies

differences in rates of protein accretion. Cats appearto synthesizenegligible amounts of arginine (76) but otherwise use arginine inthe same basic manner as humans. Thus, their dietary argininerequirement could also be used as an initial estimate of the totalmetabolic arginine requirement in humans.

The closest approximation of the human neonatal argininerequirement is probably the pig. The pig is well recognized as anappropriate model to study human amino acid metabolism(148), and the amino acid content of porcine tissue is verysimilar to that of human tissue (7). Clearly these speculationsrequire experimental confirmation, but the animal work hasprovided an excellent basis for the design of future studies inhumans. Based on what has been learned from research in

animals, the following must be considered when measuring

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arginine requirements in human populations: 1) the maximumrate of endogenous arginine synthesis under specific physiolog-ical conditions (i.e., illness, growth, pregnancy, lactation); 2) themetabolic arginine requirement for each physiological condi-tion; 3) the shape of the response in metabolic function withintake varying from deficient to more than adequate; and 4) thebioavailabilty of dietary arginine and proline and the extent towhich proline spares the dietary arginine requirement. Indetermining the ability of proline to spare arginine, the differ-ence in molecular weights between proline and arginine must be

recognized and taken into account in the calculations, similar tothe process used to calculate cysteine sparing of methioninerequirement (149152). We have fully described how to calcu-late sparing capacity in our review of cysteine and methioninerequirements (12).

Interspecies differences in the metabolic interaction

between lysine and arginine

Amino acid antagonism vs. an amino acid imbalance must beclearly defined before it is possible to discuss the antagonismbetween lysine and arginine and its nutritional implications indifferent species. Both amino acid antagonisms and imbalances

can cause reductions in rates of weight gain in growing animals.However, with an amino acid antagonism there is an interfer-ence in the metabolism of 1 amino acid caused by the intake ofthe other amino acid (153). Therefore, with an amino acidantagonism the negative effects on body weight are not onlycaused by a reduction in feed intake, which is also observedduring cases of a dietary amino acid deficiency and imbalance,but by other effects on metabolism and utilization of the an-tagonized amino acid (153). Unlike the case of an imbalance, theeffects of an amino acid antagonism can be reversed only bysupplementing the diet with the amino acid that is being antag-onized and not by supplementing the diet with the limitingamino acid(s) (153). The differences between amino acid antag-

onism and imbalance are illustrated in Figure 4.The potential for metabolic antagonism between lysine andarginine has been studied in many different species, includingcats (154,155), chicks (100,156160), dogs (161), fish (116,117), pigs (122124,162), and rats (163). Young, growing

animals are most susceptible to imbalances in amino acidintakes, including amino acid antagonism (153). To the best ofour knowledge lysine-arginine antagonism has not been studiedin humans. However, given the commercial availability of aminoacids and trends toward personal supplementation by humans,this possibility should be investigated.

Of the species studied, chicks (100,156160), dogs (161), andrats (163) displayed evidence of a lysine-arginine antagonism.For chicks, antagonism was observed when the lysine content ofthe diet was;23.5% (157160) or when there was a lysine-to-

arginine ratio of 2.22.6:1 (156). In rats, when lysine content ofthe diet was .2.8%, there was a decrease in growth rate (163),whereas in dogs, the antagonism of lysine on arginine was notobserved until lysine content was 4.9%, and not at either 1.9%or 2.9% (161). In all 3 species, the addition of arginine to thediet containing growth-depressing amounts of lysine resulted inincreased growth and improved gain to feed ratios (156,161,163), substantiating an antagonistic relation of lysine onarginine metabolism.

The exact mechanisms for the lysine-arginine metabolism inchicks, dogs, and rats have not been conclusively elucidated.Lysine and arginine share and compete at both intestinal andrenal transporters (164). However, in all 3 species there was no

evidence to support that it was competition for intestinal ab-sorption that was causing the observed antagonism (158,161,163). A deleterious effect on the digestion of arginine-containingprotein by excess lysine was also eliminated; activities of pan-creatic enzymes were not associated with differences in growthin rats (163), and diets containing free amino acids were equallygrowth-depressing for chicks as diets containing a similar aminoacid composition as a casein protein (157). In chicks (157) anddogs (161), but not rats (163), there appears to be competitionbetween lysine and arginine for renal reabsorption when growth-depressing intakes of lysine are fed. Thus, urinary arginineexcretion was higher than when the control diet was fed(157,161). In chicks, renal arginase (E.C. 3.5.3.1) activity was

markedly increased by excess lysine intake (157), which wouldcause increased arginine catabolism and thus explain the an-tagonism in chicks. However, increased arginase activity is notthe only cause of the antagonism because this response is notapparent until after 24 d of feeding of the growth-depressing

FIGURE 4 Amino acid imbalance vs.

amino acid antagonism. A diet with methio-

nine as its limiting amino acid and excess

amounts of threonine added, creating an

amino acid imbalance (A). The effects of the

amino acid imbalance in A are alleviated by

adding the limiting amino acid, methionine, to

the diet (B). Adding lysine to a diet that is

limiting in methionine also has an adverse

effect on daily gain because of an effect on

arginine metabolism, which is an amino acid

antagonism. Adding methionine to this diet

will alleviate the limitation of methionine on

protein synthesis but will not counteract the

antagonism induced by the excess lysine

intakes (C). The effects of the amino acid

antagonism in C can be alleviated by adding

arginine, the amino acid that was antago-

nized by the lysine, to the diet, although

methionine is still the limiting amino acid (D).



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