Amino Acid Antagonisms

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An amino acid antagonism may be defined as a deleterious interaction between structurally similar amino acids. This category of adverse effects was devised to accommodate the unique and separate effects of lysine and leucine in the rat. Demonstrations of antagonisms have been extended to farm animals (D'Mello and Lewis, 1970a,b,c; Papet et al., 1988a). In addition, it is now recognized that antagonisms may be precipitated by a wide range of analogues occurring naturally in crop plants as non-protein amino acids. In most cases, the action of these analogues is targeted at the metabolism and utilization of specific structurally related essential amino acids.

Branched-chain amino acid antagonisms

Interest in the antagonisms involving the branched-chain amino acids (BCAA) has been sustained by the knowledge that maize byproducts, sorghum, and blood meal contain disproportionate quantities of these amino acids. In addition, maintenance requirements of animals for BCAA may be influenced by these antagonisms.


Following initial demonstrations of leucine-induced antagonisms in the rat, much evidence has emerged to confirm the specificity and complexity of interactions among BCAA in the chick and turkey poult. In one study, D'Mello and Lewis (1970b) showed that excess dietary leucine permitted the growth response of chicks to the first-limiting amino acid, methionine, only in the presence of supplementary isoleucine. The specificity of the leucine-isoleucine antagonism was thus established for the first time. However, other results led D'Mello and Lewis (1970b) to conclude that the leucine-valine interaction was relatively more potent. This conclusion was based on growth and plasma amino acid data (Table 7.3). The addition of excess leucine to a diet equally but marginally limiting in isoleucine and valine precipitated a severe growth depression in young chicks. Valine supplementation reversed this effect but isoleucine addition failed to elicit a response. Indeed, a combination of excess leucine and supplementary isoleucine impaired growth even further and precipitated a marked fall in plasma valine concentrations. In contrast, circulating levels of isoleucine remained undisturbed

Table 7.3. Branched-chain amino acid antagonisms in the young chick: effects of excess dietary leucine and supplements of valine and isoleucine on growth and plasma amino acid concentrations. (Adapted from D'Mello and Lewis, 1970b.)

Daily weight Plasma amino acid concentrations (p.mol 100 ml"1)

Diet chick 1) Val lie Leu Gly Lys Arg









Basal + Val








Basal + lie








Basal + Val + Ile








Basal + Leu








Basal + Leu + Val








Basal + Leu + lie








Basal + Leu + Val + Ile








aBasal diet marginally deficient in Val and lie.

aBasal diet marginally deficient in Val and lie.

following individual or combined additions of leucine and valine. The efficacy of valine was further confirmed by its ability to reduce high plasma levels of leucine, whereas isoleucine addition was ineffective in this respect. The sensitivity of valine to leucine antagonism and its exacerbation by isoleucine was confirmed in a later study with chicks (D'Mello and Lewis, 1970c). Excess dietary leucine depressed plasma valine concentrations to the greatest extent when added with isoleucine. This reduction is of particular significance since dietary valine was set at adequate levels.

The specificity of the leucine-valine antagonism was further exemplified in studies with turkey poults fed diets supplemented with graded combinations of leucine and valine (D'Mello, 1975). Both amino acids accumulated in plasma following supplementation, but the extent of valine accretion was reduced as dietary levels of leucine increased. However, valine supplementation failed to suppress accumulation of leucine in plasma.


Despite the primacy of the leucine-valine antagonism, it is possible to devise dietary conditions demonstrating enhanced sensitivity of isoleucine in BCAA interactions. D'Mello (1974) showed that a diet supplemented with a small excess of leucine depressed chick growth, which was only par tially alleviated with valine additions. Examination of the plasma amino acid data indicated that circulating levels of Isoleucine had declined most severely on combined addition of leucine with valine. The complexity of BCAA interactions is further illustrated by the response of laying pullets to excess dietary leucine (Bray, 1970). Egg production and egg yield were reduced by this excess but were restored to satisfactory levels only-after isoleucine and valine were added in combination. Failure to recognize the complexity of these interactions may account for the lack of effect of Isoleucine alone in alleviating a leucine-induced antagonism in pigs (Oestemer et ai, 1973). Inspection of the plasma data, however, indicated moderate decreases in the circulating levels of both isoleucine and valine, thus implying that combined supplements of these amino acids might have been more effective. However, in lactating sows, independent increases in litter weaning weights and changes in milk composition from valine and Isoleucine supplements were assumed to imply separate modes of action for these amino acids in milk synthesis (Richert et ai, 1997).


From studies principally with the rat, Harper et ai (1984) attributed the leucine-induced changes in plasma levels of isoleucine and valine to increased oxidation of these two amino acids, having discounted any effects emanating from competition for intestinal or renal transport. Limited studies with the chick support this view. Thus, Calvert et al. (1982) demonstrated that excess leucine failed to influence excretion of KC-labelled isoleucine or valine, but markedly increased oxidation of these amino acids as indicated by enhanced in uiuo output of 14C02. The catabolism of BCAA is initiated by a reversible aminotransferase reaction (Fig. 4.13). The branched-chain keto acids (BCKA) so formed then undergo irreversible oxidative decarboxylation to yield acyl-CoA compounds which are degraded further in a series of reactions analogous to those involved in fatty acid oxidation. Harper et al. (1984) suggested that enhanced BCKA oxidation might account for the depletion of plasma isoleucine and valine pools in animals fed excess leucine. Studies with preruminant lambs support this view in that marked reductions in plasma concentrations of keto acids derived from isoleucine and valine were recorded in response to excess intake of leucine (Papet et al., 1988a). Subsequently, Papet et al. (1988b) observed that excess leucine increased activities of aminotransferases in the liver and jejunum and also activated BCKA dehydrogenase in the jejunum of lambs.

Excess BCAA may, additionally, induce depletion of brain pools of other amino acids, particularly those which are the precursors of the neurotransmitters. In this regard, Harrison and D'Mello (1986) showed that excesses of the three BCAA reduced brain concentrations of noradrenaline, dopamine and 5-hydroxy-tryptamine in the chick and that levels of these neurotransmitters were restored by dietary supplementation with their precursors, phenyalalanine and tryptophan. The significance of these results awaits elucidation. Nevertheless, it is generally conceded that changes in brain metabolism of amino acids and neurotransmitters may be associated with alterations in food intake and feeding behaviour (Leung and Rogers, 1987). Consistent with this concept has been the observation that a substantial element of the adverse effects of excess leucine arises from the reduction in food intake (Calvert et al., 1982;

Papet et al., 1988a) which subjugates effects emanating from oxidative catabolism of isoleucine and valine.

The lysine-arginine antagonism

The considerable variation in the arginine requirements of the chick has provided the impetus for extensive and sustained investigations on the lysine-arginine antagonism. In addition, a number of feedstuffs contain adverse ratios of lysine relative to arginine. Indications of a potent antagonism between lysine and arginine in the chick dates back to the studies by Jones (1961) on the toxicity of lysine. Since then considerable evidence has emerged to identify features such as specificity, reciprocity and mechanisms of action in this antagonism.


The unique specificity of this antagonism was tested in several experiments by D'Mello and Lewis (1970a) who designed basal diets which were first-limiting in methionine, tryptophan, histidine or threonine, with arginine marginally deficient. Addition of excess lysine to each of these diets precipitated a severe growth depression in chicks which, in every case, was reversed by arginine supplementation and not by the amino acid originally deficient in the basal diet. A selection of the results relating to the threonine-deficient diet is shown in Table 7.4, which further illustrates the specific effect of lysine in reducing plasma levels of arginine. In contrast, circulating levels of threonine were unaffected by the precipitation of this antagonism. Evidence of specificity was also provided by Nesheim (1968) in studies with two strains of chicks differing substantially in their requirements for arginine. Chicks with a high arginine requirement were less able to tolerate dietary excesses of lysine than chicks with a low requirement for arginine. However, a number of factors can affect the severity of the lysine-arginine antagonism. Excess chloride augments the adverse effects, whereas alkaline salts of monovalent mineral cations reduce or eliminate the potency of this antagonism.

Table 7.4. Specificity of the lysine-arginine antagonism in the young chick: effects of excess dietary lysine on growth and plasma amino acid concentrations. (Adapted from D'Mello and Lewis, 1970a.)

Daily weight Plasma amino acid concentrations (pimol 100 ml"1)

Table 7.4. Specificity of the lysine-arginine antagonism in the young chick: effects of excess dietary lysine on growth and plasma amino acid concentrations. (Adapted from D'Mello and Lewis, 1970a.)

Daily weight Plasma amino acid concentrations (pimol 100 ml"1)


aa"1KW chick-1)















Basal + Arg








Basal +Thr








Basal + Arg + Thr








Basal + Lys








Basal + Lys + Arg








Basal + Lys + Thr








Basal + Lys + Arg + Thr








aBasal diet first-limiting in Thr and second-limiting in Arg.

aBasal diet first-limiting in Thr and second-limiting in Arg.

Effects in mammals

Although the lysine-arginine antagonism has been demonstrated in rats fed casein diets (Jones et al., 1966), its existence in the pig has been refuted (Edmonds and Baker, 1987). Relatively large excesses (35 g kg-1 diet) of lysine were required to reduce food intake and efficiency of food utilization. This level of lysine failed to influence arginase activity in any of the tissues examined. Excess lysine depressed plasma arginine but did not affect its concentration in liver, kidney or muscle. On the basis of this evidence, Edmonds and Baker (1987) attributed the adverse effects of excess lysine in the pig to an amino acid imbalance rather than to a specific antagonism.


D'Mello and Lewis (1970c) showed that excess arginine depressed growth of chicks fed a lysine-deficient diet, an effect which was reversed by supplementary lysine. However, the specificity and metabolic basis of this effect remain unresolved. There is some evidence that alterations in arginine:lysine ratios may be beneficial in heat stress in broilers (Brake et al., 1998; Balnave and Brake, 1999). In the pig, excess arginine is considered to precipitate its adverse effects through an imbalancing action rather than through a genuine antagonism (Anderson et al., 1984).


By virtue of their uricotelism, avian species are unable to synthesize arginine and are particularly sensitive to the lysine-arginine antagonism. The most significant factor in the avian manifestation of this antagonism is the enhanced activity of kidney arginase which results in increased catabolism of arginine (see Fig. 4.2). If arginase activity is suppressed by the use of a specific inhibitor, then the severity of the antagonism is also attenuated. A second factor is the depression in food intake, presumably arising from lysine-induced disruption of brain uptake and metabolism of other amino acids and their biogenic amines. It should be noted, however, that in this antagonism, the depression in growth precedes the reduction in food intake (D'Mello and Lewis, 1971). Secondary mechanisms include enhanced urinary excretion of arginine and inhibition of hepatic transamidinase activity (Fig. 4.8) with consequent reduction in endogenous synthesis of creatine (D'Mello, 1994).

Antagonisms induced by non-protein amino acids

A wide array of amino acids occurring naturally in unconjugated forms in plants are capable of precipitating adverse effects in animals. The presence of these non-protein amino acids in economically important species of legumes and brassicae has thwarted attempts to maximize utilization of these plants as sources of food for farm livestock. Non-protein amino acids may occur in all parts of the plant, but the seed is normally the most concentrated source (D'Mello, 1991). In many instances these compounds bear structural analogy with the nutritionally important amino acids or their neurotransmitter derivatives active in the central nervous system of animals. Consequently, manifestations of deleterious effects range from reductions in food intake and nutrient utilization to profound neurological disorders and even death (Table 7.5).


The aromatic amino acid mimosine contributes significantly to the toxicity of the ubiquitous tropical forage legume, Leucaena leucocephala. Mimosine may be regarded as a structural analogue of tyrosine and its neurotransmitter derivatives, dopamine and noradrenaline. However, the effects on brain metabolism of these biogenic amines have yet to be confirmed, and evidence that tyrosine may reverse the deleterious effects of mimosine is equivocal (D'Mello, 1994). The adverse properties of mimosine are extensive and include disruption of reproductive function, teratogenic effects, loss of hair and wool and even death. Similar effects may be induced by feeding Leucaena to cattle and sheep. Thus the defleecing effects in sheep may be precipitated by administration of pure mimosine or by feeding Leucaena forage.

Manifestations of Leucaena toxicity are determined by geographical differences in rumen microbial ecology and are critically dependent on the rate and extent of bacterial breakdown of mimosine (see Fig. 4.5). Following degradation, 3-hydroxy-4(lH)-pyridone (3,4-DHP) is synthesized; this itself is capable of causing loss of appetite, goitre and reductions of blood thyroxine concentrations (Jones, 1985). Another goitrogen and isomer, 2,3-DHP may also be synthesized in the rumen. Thus the association of mimosine with tyrosine metabolism appears to be mediated indirectly via the two forms of

Table 7.5. Distribution and adverse effects of some non-protein amino acids. (Adapted from D'Mello, 1991.)

Amino acid


Concentration (9 kg"1 dry weight)

Adverse effects

Aromatic Mimosine

Analogues of sulphur amino acids

Se-methylselenocysteine Selenocystathionine Selenomethionine J

S-methylcysteine sulphoxide Bmssica

Leucaena leucocephala


Arginine analogues Canavanine

Indospicine Homoarginine

Canavalia ensiformis Gliricidia sepium Robinia pseudoacacia Indigofera spica ta Indigofera spica ta

Lathyrus cicera

25-51 (seed) 40 (seed) 98 (seed) 9 (seed) 20 (seed)

Loss of wool; teratogenic offsets, organ damage; death

'Blind staggers'; death

Haemolytic anaemia; loss of appetite; reduced milk yield; organ damage; death

Reduced growth and nitrogen retention

Teratogenic effects; liver damage

Reduced growth and food intake

DHP. Some rumen bacteria are capable of detoxifying both forms of DHP. Despite these reactions, considerable quantities of mimosine and 3,4-DHP may escape rumen degradation and other derivatives may also be excreted. Ruminants in Australia, the USA and Kenya lack the requisite bacteria involved in the detoxification of the two DHP isomers and, consequently, succumb to their goitrogenic effects if high intakes of Leucaena are maintained over a protracted period of time. On the other hand, in certain other regions where Leucaena is indigenous (Central America) or is naturalized (Hawaii and Indonesia), ruminants possess the full complement of bacteria that are required for DHP degradation, which accounts for the absence of Leucaena toxicity in these countries (Jones, 1985). However, the transfer of DHP-degrading bacteria to cattle in Australia has been achieved with complete success. Inoculated animals grazing Leucaena show markedly higher live-weight gains and serum thyroxine concentrations than untreated Leucaena-fed controls (Quirk et al., 1988). Dosed cattle rapidly reduce urinary DHP excretion despite a doubling of Leucaena intake. The isolation of active DHP-degrad-ing bacteria from the faeces of dosed animals implies that treatment of just a few animals may provide sufficient inoculum for the entire herd. This technique thus offers a viable strategy for maximizing utilization of Leucaena with the added benefit of improved cattle performance.

Despite its toxicity, mimosine may have a role to play in the removal of fibre from Angora goats (Reis et al., 1999). However, further research is necessary to develop a convenient means of mimosine delivery to the goats in a manner that maximizes fibre yield and avoids toxicity.

Analogues of sulphur-containing amino acids

Striking analogues of the sulphur-containing amino acids exist naturally in plants (D'Mello, 1991), particularly in those species where the sulphur atom is replaced by selenium. The debilitating disorders associated with these selenoamino acids are manifestations of acute selenium poisoning.

Another analogue, S-methylcysteine sulphoxide (SMCO) occurs in forage and root brassica crops. The presence of this amino acid constitutes a significant deterrent to the exploitation of these crops as fodder for ruminant animals. The adverse effects of SMCO occur after its metabolism by rumen bacteria to dimethyl disulphide (Smith, 1980). A severe haemolytic anaemia appears within 1-3 weeks in animals fed mainly or exclusively on brassica forage. Initial overt indications of the disorder include loss of appetite and reduced milk production, whereas internal changes include the appearance of retractile, stainable granules (Heinz-Ehrlich bodies) within the erythrocytes and reduced blood haemoglobin concentrations. Extensive organ damage is an accompanying feature of this condition, with the liver becoming swollen, pale and necrotic. Critical daily intakes of SMCO range from 15 to 19 g kg-1 body weight irrespective of the source of the amino acid. Surviving animals continuing to graze the crop may make spontaneous but incomplete recovery with further fluctuations in blood haemoglobin concentrations. Withdrawal of the forage usually results in the restoration of normal blood composition within 3-4 weeks.

Analogues of arginine

Of the three analogues of arginine (Table 7.5), canavanine is more widely distributed and present in higher concentrations in leguminous seeds. Canavanine contributes significantly to the toxicity of Canavalia ensiformis (jack bean, JB) for young chicks. Adverse effects may also arise through the synthesis of canaline, a structural analogue of ornithine, by the action of arginase on canavanine. The mammalian metabolism of canavanine corresponds with that of arginine in the urea cycle (see Fig. 4.4). Since this cycle is non-functional in avian species, they are unable to synthesize arginine and, consequently, readily succumb to the adverse effects of canavanine in jack beans (D'Mello et al., 1989). As shown in Table 7.6, chicks fed JB, autoclaved to denature potent lectins, grew at reduced rates and utilized food and dietary N less efficiently than control animals. Canavanine appeared in the serum of JB-fed

Table 7.6. Whole-animal and metabolic responses of chicks fed a control diet or autoclaved jack bean (JB) diets containing canavanine.3 (Adapted from D'Mello eta!., 1989.)

Efficiency Efficiency of food of nitrogen

Table 7.6. Whole-animal and metabolic responses of chicks fed a control diet or autoclaved jack bean (JB) diets containing canavanine.3 (Adapted from D'Mello eta!., 1989.)

Efficiency Efficiency of food of nitrogen





(g gain g 1

(g N retained




dry matter

g 1 N




(g chick-1)



(mg I"1)

(mg I"1)







JB basal






JB basal + Lys






JB basal + Arg






JB basal + Lys + Arg






aCanavanine content of JB basal diet: 3.7 g kg 1 diet dry matter.

aCanavanine content of JB basal diet: 3.7 g kg 1 diet dry matter.

chicks and, in addition, serum urea concentrations exceeded control values. Lysine supplementation aggravated the effects on growth and on food intake and utilization. In contrast, arginine supplementation enhanced weight gain and food intake. These results support the existence of a canavanine-arginine antagonism analogous to that between lysine and arginine. Similarities exist in several respects. Thus in both antagonisms, arginine requirements and urea excretion are enhanced, although the relative proportions of this additional urea arising from canavanine and arginine remain to be established. The failure of supplementary arginine to substantially reduce circulating levels of the respective antagonists is a feature common to both interactions. Creatine supplementation markedly improved the effi-of dry matter and N utilization in chicks fed JB (D'Mello et al., 1990). It is noteworthy that Austic and Nesheim (1972) also reported improvements in food utilization efficiency with creatine supplementation in the lysine-arginine antagonism. However, differences between the two interactions are also apparent in that arginine enhanced efficiencies of utilization of food and N in chicks fed excess lysine but not in those fed diets containing canavanine. Homoarginine (Table 7.5) exacerbated the effects of canavanine-induced toxicity in chicks fed JB diets, serving to illustrate the diversity of interactions among the analogues and antagonists of arginine (D'Mello, unpublished).

Neurotoxic amino acids

Neurotoxic non-protein amino acids occur in the form of the structurally related lathyro-gens, p-(N)-oxalyl amino alanine and <x,y-diaminobutyric acid. Their occurrence in certain legume seeds has been associated with the condition of neurolathyrism in humans, but well-defined neurotoxic effects have also been observed on administration of the pure forms of these amino acids to animals (D'Mello, 1991). There is currently some interest in the use of Lathyrus sativus and Vicia satiua grains for poultry, but the occurrence of neurolathyrogenic amino acids in these seeds may represent a primary limiting factor. Ruminants may be able to degrade neurolathyrogens as shown by the absence of toxicity in wether lambs on feeding Lathyrus sylvestris hay with a relatively high diaminobutyric acid concentration of 12 g kg-1 dry weight (Forster et al., 1991).


There is overwhelming evidence to indicate that the adverse effects of the non-protein amino acids are mediated via diverse mechanisms. These are summarized in Table 7.7 and discussed at length by D'Mello (1991). The multimodal action of these amino acids is exemplified by the mechanisms proposed for canavanine. The increased urea output in chicks fed JB diets containing canavanine may reflect enhanced arginase activity in the

Table 7.7. Diverse mechanisms underlying the adverse effects of selected non-protein amino acids.

Amino acid

Biochemical changes



Enhanced arginase activity

Increased arginine catabolism

Decreased activity of ornithine

Reduced polyamine synthesis

decarboxylase following synthesis

of canaline

Competition with lysine and

Reduced intestinal absorption of

arginine for transport

lysine and arginine

Inhibition of transamidinase

Reduced creatine synthesis


Synthesis of aberrant proteins

Enhanced protein turnover3

Inhibition of nitric oxide synthesis

Impaired immunocompetence3; reduced

food intake3


Reduced synthesis of high-

Reduced wool strength3

tyrosine proteins

Reduced DNA synthesis

Inhibition of wool biosynthesis

Complex formation with pyridoxal


phosphate-dependent enzymes

S-methyl cysteine

Blockage of sulphydryl groups

Inactivation of key proteins



kidney. Such an increase might lead to an inadvertent loss of arginine in a manner analogous to that observed in the lysine-arginine antagonism. Canavanine administration to rats induces substantial increases in serum and urinary concentrations of ornithine. Hepatic activity of ornithine decarboxylase is reduced by a factor of five in chicks fed JB diets containing canavanine (D'Mello, 1993). These effects may be attributed to the synthesis of canaline, which forms a covalent complex with pyridoxal phosphate, thereby inhibiting activities of enzymes such as ornithine decarboxylase which require the vitamin as a cofac-tor. Ornithine decarboxylase is a key enzyme in the synthesis of the polyamines (see Fig. 4.3) involved in the regulation of cell growth and differentiation. Canavanine may also act by competing with arginine and lysine for transport across membranes. Another focal point for toxic action may reside in the ability of canavanine to act, like lysine, by inhibiting transamidinase activity, resulting in reduced creatine synthesis. In addition, canavanine may replace arginine during protein synthesis leading to the formation of aberrant proteins with modified functional properties. However, the evidence is equivocal and D'Mello (1991) proposed that canavanyl proteins may be degraded as rapidly as they are formed leading to enhanced overall protein turnover rates, a feature which might contribute to the poor N retention efficiencies of chicks fed JB diets (Table 7.6). Canavanine may also inhibit nitric oxide synthesis which would impair immunocompetence and reduce food intake. Canavanine is now recognized to be a potent inhibitor of food intake in pigs (Enneking et al., 1993).

Despite the economic importance of Leucaena, information concerning the mechanism of action of mimosine remains fragmentary (Table 7.7). Consistent with the action of several structural analogues, synthesis of high-tyrosine protein components of wool is reduced in sheep given intravenous infusions of mimosine (Frenkel et al., 1975). The activities of several pyridoxal phosphate-dependent enzymes may be inhibited through the ability of mimosine to form complexes with the vitamin moiety. In addition, both mimosine and 3,4-DHP are known to cause inhibition of wool follicle DNA synthesis in uitro (Ward and Harris, 1976).

The mode of action of SMCO requires elucidation, although it is acknowledged that its derivative, dimethyl disulphide, inactivates key proteins through blockage of sulphydryl groups. The reaction with reduced glutathione, a key factor in the protection of red blood cells from oxidative injury, represents one mechanism for SMCO toxicity. Brassica-fed ruminants appear to compensate for inac-tivation of proteins by increasing synthesis of growth hormone and thyroxine which, in turn, stimulate production of replacement proteins (Barry et ai, 1985).

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