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British Poultry Science

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Amino acid interactions in chick

nutrition
J. P. F. D'mello

a b

& D. Lewis

Department of Applied Biochemistry and Nutrition, University of

Nottingham, School of Agriculture, Sutton Bonington, Loughborough
b

Department of Agricultural Biochemistry, The East of Scotland

College of Agriculture, West Mains Road, Edinburgh, 9
Published online: 08 Nov 2007.

To cite this article: J. P. F. D'mello & D. Lewis (1970) Amino acid interactions in chick nutrition,
British Poultry Science, 11:3, 299-311
To link to this article: http://dx.doi.org/10.1080/00071667008415820

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Br. Poult. Sci., I I : 299-311. 1970

Oliver & Boyd: printed in Great Britain

AMINO ACID INTERACTIONS IN CHICK

NUTRITION
I. THE INTERRELATIONSHIP BETWEEN LYSINE
AND ARGININE
J. P. F. D'MELLO1 AND D. LEWIS

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Department of Applied Biochemistry and Nutrition,

University of Nottingham, School of Agriculture, Sutton Bonington, Loughborough
Received for publication 13th October 1969

SYNOPSIS

A series of experiments has been conducted to investigate the specificity of

the interaction between lysine and arginine in chick nutrition, in the light of the
concept of agent and target advanced by Lewis (1965). Diets were designed in which
the level of methionine, tryptophan, histidine, or threonine was appreciably
inadequate, while the arginine concentration in each diet was marginally satisfactory.
Excess lysine was added to these diets in a standard sequence.
The profound ill-effects induced by excess lysine were, in all experiments,
alleviated only by arginine and not by the amino acids originally limiting in the
control diets. The findings support the existence of an unique relationship between
lysine and arginine.
INTRODUCTION

Although there is a considerable body of evidence to suggest the existence of

an interrelationship between lysine and arginine in chick nutrition (O'Dell, Laerdal,
Jeffay and Savage, 1958; Jones, 1961, 1962, 1964; Smith and Lewis, 1966) the
degree and extent of specificity of this interaction has remained largely unresolved.
Lewis (1965) has proposed a concept of agent and target to account for this relationship: the agent is the amino acid which precipitates the ill-effects on growth when
added in excess in the diet (in this case lysine). The target is the amino acid which
must be added to counteract the phenomenon (in this instance, arginine).
The specificity of the interaction between lysine and arginine has been examined
in this study in relation to the concept of interacting pairs advanced by Lewis (1965).
In particular, the ability of arginine to alleviate the ill-effects exerted by excess
lysine was considered under varying conditions of dietary amino acid balance.
MATERIALS AND METHODS

Housing and management of experimental animals

The experiments were carried out using sexed Cobb broiler chicks (White
Cornish x White Rock).
1
Present address: Department of Agricultural Biochemistry, The East of Scotland College of Agriculture,
West Mains Road, Edinburgh, 9.

299

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300

J. P. F. D MELLO AND D. LEWIS

The chicks were housed in cages in a flat-roofed windowless, insulated room

built within a larger building. The air temperature was thermostatically
controlled.
Lighting was provided by means of three 100W white bulbs placed at equal
intervals along the centre of the ceiling. Ventilation was controlled by means of
an electric extractor fan.
Sixteen blocks of 6 cages were housed in the room; each cage being 38 cm long,
30-5 cm wide and 25-4 cm high. The detachable fronts of the cages consisted of
vertical galvanised metal bars placed 3-2 cm apart. A metal barrier which could
be moved up or down these bars held the food and water containers in position.
Beneath the floor of each cage was a polythene droppings tray.
At the beginning of an experiment, 8-d-old cockerels were placed in each cage.
During the first week of life of the chicks the temperature was maintained at 32 C,
and was subsequently reduced by 2-5 C each week. Ventilation during the first
week was by seepage only. Additional ventilation in the following weeks was effected
by means of the extractor fan. Lighting was provided continuously for 22 h each
day with the dark break occurring from 02.00 h to 04.00 h.
The chicks were offered food and water ad libitum. During the first week, the
chicks received a commercial-type starter diet. At the end of this period, the
excessively light and heavy birds were discarded. Those animals within the weight
range of 90 to 120 g were selected at random using tables of random numbers
(Fisher and Yates, 1963). The number of birds in each cage was reduced to four;
weighed quantities of the experimental diets were then offered. The animals were
weighed individually at the end of each experiment2 weeks after introduction to
the experimental diets.
Parameters of observations

Live-weight gain. The main parameter of observation used in these studies was
the assessment of live-weight gain. This choice was based on the premise that there
is good correlation between nitrogen gain and live-weight gain in the growing animal.
Unless the composition of the chick in terms of the proportions of fat, lean or bone
becomes important, live-weight gain remains the most convenient index of adequacy
of a dietary regime. In these experiments, gain in weight was expressed as gain
per animal per day (g/d), and represented a mean of four replicate groups of four
animals per replicate.
Food consumption and efficiency of food conversion. Food intake was measured at

times coincident with the measurements of growth or of nitrogen retention. The

efficiency of food conversion was expressed as g gain/g of food ingested.
Nitrogen retention. The growth data in some experiments were supported by
relevant observations on nitrogen-retention during the last 3 d of each experiment.
On the 18th d of an experiment the plastic droppings tray beneath each cage was
cleaned; 450 ml of o-i N H 2 SO 4 were placed in each tray. At the same time during
each of the 3 subsequent days, the contents of each tray were transferred quantitatively into a Waring blender and homogenised to an even consistency after diluting
to an appropriate volume. A 15 ml sample of the homogenate was placed directly
into a Kjeldahl flask and the nitrogen determined. Relevant food consumption
records were also kept during each of the 3 d of the nitrogen balance period. Samples

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INTERRELATIONSHIP BETWEEN LYSINE AND ARGININE

301

of the diets were analysed for nitrogen by the Kjeldahl procedure. The results
were expressed as g of nitrogen retained/g of nitrogen ingested.
Plasma amino acid status. At the termination of each experiment food was withdrawn for 10 h. The animals were re-offered the appropriate diets for 2 h, after
which they were subjected to another fast of 2 h duration. Blood was then withdrawn
by cardiac puncture from one chick from each replicate chosen at random, and
collected in a 10 ml centrifuge tube lined with sodium oxalate (BDH, Analar grade).
After centrifugation the plasma was decanted, the supernatants from each replicate
of each treatment were pooled and stored at 5 C until deproteinisation with
absolute alcohol. This was used in preference to other methods since pH of the
resultant sample remained unaffected by this procedure arid consequently offered
no interference in the subsequent amino acid analysis. The protein-free plasma
filtrate was dried under vacuum in a rotary evaporator and taken up in 2 ml of a
solution of 10 per cent sucrose in o-i N HC1: 1 ml of this sample was quantitatively
analysed for amino acids by ion-exchange chromatography using the Technicon
automatic amino acid analyser. The value for each amino acid was expressed as
JU, moles/100 ml of plasma.
Experimental design and statistical analysis of results

All experiments were conducted in the form of a randomised block design.

Four replicates of each treatment were used, with the treatments randomised within
each replicate group to offset effects of temperature variation within the room
housing the chicks.
Statistical evaluation of the significance of the results was performed on all
growth, efficiency of food conversion, and nitrogen retention data. The results of
each experiment have been illustrated in tables of means, the significance being
indicated by the standard error of means (S.E.M.) and the least difference between
means significant at the 5 per cent level of probability (L.S.D.).
The plasma amino acid data have not been subjected to statistical analysis
since determinations were of necessity limited to one sample per treatment. In
most instances, trends were observed which confirmed gross observations and the
conclusions drawn from such observations. In no cases were conclusions based
entirely upon the strength of evidence given by plasma amino acid data.
Experimental diets

The diets were composed of conventional ingredients, and were offered in a

ground form. An average of about 20 per cent crude protein (N x 6-25) was maintained in all experimental diets. Energy status of the diets within each experiment
was constant at around 3100 kcal (metabolisable energy)/kg. It was ensured that
the basal diets used supplied all known essential nutrients other than those under
study, at a level optimal for chick growth, by following the recommendations of the
Agricultural Research Council (1963) and the National Research Council (1966)
with judiciously selected margins of safety. The values of amino acid content of
ingredients used in the preparation of the diets were those described by Lewis,
Smith and Payne (1963). The determination of the precise amino acid composition
of the ingredients or of the basal diets was considered unnecessary in the present

302

J. P. F. D MELLO AND D. LEWIS

study since any deficiencies of amino acids were in every instance tested by appropriate dietary supplementation. The various supplements of amino acids were
effected by means of pure crystalline amino acids; arginine-HCl, tyrosine, lysineHC1, histidine-HCl and leucine were supplied in the L form. Valine, methionine,
threonine, tryptophan and isoleucine were provided in the DL form: the isomers of
these amino acids can be utilised by the chick (Fell, Wilkinson and Watts, 1959).
In several experiments, the desired degree of deficiency of an amino acid could
not be achieved when all the protein was derived from an intact source. In such
instances a variable proportion of the protein ranging from 16 per cent to 18 per
cent was supplied by conventional ingredients, the remainder being provided in the
form of synthetic essential amino acids and glutamic acid.
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TABLE I

Composition of the mineral and vitamin supplement, expressed in terms

of the final concentration contributed to the diet

Minerals
Calcium
0-85 per cent
Phosphorus
0-30 per cent
Sodium
o-ia per cent
Chlorine
0-18 per cent.
Manganese
70 mg/kg
Zinc
50 mg/kg
Iron
20 mg/kg
Copper
2 mg/kg
Iodine
1 mg/kg
Cobalt
o-i mg/kg
Molybdenum
2 mg/kg
Selenium
o-i mg/kg

Vitamins, etc.
Vitamin A
6000 i.u./kg
Vitamin D 3
1500 i.u./kg
Vitamin E
4 mg/kg
Choline chloride
500 mg/kg
Menadione sodium bisulphite
5 mg/kg
Calcium pantothenate
10 mg/kg
Riboflavin
4 mg/kg
Folic acid
2 mg/kg
Vitamin BXJ
o-oi mg/kg
Nicotinic acid
20 mg/kg
Procaine penicillin
10 mg/kg
Pancoxin (coccidiostat)
0-0125 P er cent
B.H.T. (antioxidant)
0-0125 P er c e n t

Adequate supply of minerals, vitamins, antibiotics, coccidiostat and antioxidants were ensured by the inclusion in the diets of a mineral and vitamin
supplement (Table 1). Unidentified growth factors were provided by the incorporation in all diets of 3 per cent dried whey.
The ingredients and amino acid composition of the basal diets are listed in
Table 2. Since adverse effects of amino acids are more readily observed in diets
low in protein or lacking in individual essential amino acids, most diets in the present
study were designed to be deficient in one or two essential amino acids.
The experimental programme was conducted in two phases: in experiments
1 and 2, the occurrence of the interaction between lysine and arginine was examined;
experiments 3-6 were concerned with examining the specificity of this interaction.
The basal diet used in experiment 1 was considered to be marginally adequate in
arginine at 1-20 per cent of the diet, and fully adequate in all other essential constituents. This was verified by the addition of arginine to the basal diet; other
treatments constituted the addition of two levels of lysine in excess of its requirements
(o-6 per cent and o-8 per cent of the diet of lysine). The aim of this experiment was
to examine the consequences of excess lysine intake.
Experiment 2 was designed to investigate the efficacy of arginine in alleviating
the stress induced by excess dietary lysine by following growth and plasma amino
acid patterns. A basal diet calculated to be inadequate in arginine (0-87 per cent

INTERRELATIONSHIP BETWEEN LYSINE AND ARGININE

3O3

of the diet) was employed. The treatments included: basal diet alone; basal +
arginine; basal + lysine; basal + lysine + arginine.
The remaining experiments (3-6) were conducted with the aim of examining
the specificity of the lysine-arginine interaction; all were variations on a common
theme. A basal diet limiting in an essential amino acid other than arginine was
prepared. A standard sequence of treatments to this control diet followed, involving
the testing of the basal diet to ensure the designed sequence of limitation of the
essential amino acids; other treatments constituted the addition of excess lysine to
the basal diet and subsequent testing to investigate which amino acid was effective
in alleviating the consequent growth retardation.

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TABLE 2

Experiments j-6: Ingredients and composition of basal diets. {Composition is expressed as percentage of air-dry diets.
Amino acid composition derivedfrom values described by Lewis et al. (1963), and includes amino acids added as supplements)
Experimental diets

Maize meal
Wheat meal
Soyabean meal
Ground nut meal
Maize gluten meal
Dried whey
Minerals and vitamins
Fat (Mazola)
Essential amino acid mixture plus
glutamic acid
Amino acid content
Arginine
Glycine
Histidine
Leucine
Isoleucine
Lysine
Methionine+cystine
Phenylalanine + tyrosine
Threonine
Tryptophan
Valine
Nx6-25 (determined)

64-0

25-0
43-o

70-5

25-0
43-o

20-0
50-0

8-5
u-5

3'5

5-o

3'5
21-0

3-0

3-0

3-0

2I-O
3-0

...
68-5
16-o
...
...

2-5

2-5

2-5

25-5

1-20
1-22
o-43

9-0

2-5

2-5

2-O

5-o

2-5
3-0

4-0

...

5-o

5-0

0-87
1-23
o-43
2-45

o-88
1-22
0-30
1-44
o-86

0-85
1-22
0-40
1-63
o-86

2-O
...

...

7-5

I-8I

0-87
1-23
o-43
2-45

0-84

-93

0-90
1-22
0-48
2-35
0-87

I'll

i-n

I'lO

I'll

I'lO

I'lO

o-86
1-58
0-84
0-28
1-03
20-03

0*91
i-68
0-83
0-24
0-96
20-06

0-58
1-84
o-86
0-25

0-91
i-68
0-83
0-15
0-96
20-06

0-89
'57
0-82
0-26
o-99
19-94

0-89
i-59
o-55
0-25
0-97
20-00

I'OI

20-08

-93

In the first of these experiments (experiment 3), the basal diet was prepared
first limiting in the sulphur amino acids (0-58 per cent of the diet) and second limiting
in arginine (0-90 per cent of the diet). All other essential amino acids were provided
at adequate concentrations, including lysine (at I-IO per cent of the diet). Eight
treatments followed: control alone; basal + arginine; basal + methionine; basal +
arginine + methionine; basal + excess lysine; basal + excess lysine + arginine; basal
+ excess lysine + methionine; basal 4- excess lysine + arginine + methionine.
The basal diet employed in experiment 4 was designed to be first limiting in
tryptophan (0-15 per cent of the diet) and marginally inadequate in arginine (0-87
per cent of the diet). A similar sequence of treatments to experiment 3 was imposed
on this control diet, the supplements of methionine being replaced by additions of
tryptophan.

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304

J. P. F. D'MELLO AND D. LEWIS

Experiment 5 was conducted in order to provide additional evidence of a specific

interaction between lysine and arginine, by following the fate of amino acids in
plasma. The basal diet was designed to be first limiting in histidine (0-30 per cent
of the diet) and second limiting in arginine (o-88 per cent of the diet). The treatments followed the standard pattern: basal diet alone; basal + arginine; basal-targinine + histidine; basal + lysine (excess); basal + lysine + arginine; and basal +
lysine + histidine.
In the final investigation of the specificity of the lysine-arginine interaction
(experiment 6), further metabolic support for the growth observations were sought
by measurement of nitrogen-retention in addition to plasma amino acid status.
The basal diet was prepared deficient primarily in threonine (0*55 per cent of the
diet) and marginally lacking in arginine (0-85 per cent of the diet). The following
sequence of treatments was imposed: basal diet alone; basal + arginine; basal4threonine; basal + arginine + threonine; basal + lysine (excess); basal + lysine +
arginine; basal + lysine 4- threonine; and basal + lysine + arginine 4- threonine.
RESULTS

Experiments 1-2: Occurrence of the lysine-arginine interaction. The results of experi-

ment 1 are shown in Table 3. The basal diet was clearly not limiting in arginine
TABLE 3

Experiment 1: Effect ofsupplementation ofarginine and ofexcess lysine on mean liveweight gain (g/d), and efficiency offood conversion {g gainjgfoodingested) during
the period J-SI d

Values represent mean of 4 replicate groups of 4 chicks each. Basal diet

marginally adequate in arginine, fully adequate in all other essential
amino acids
Treatment
Basal diet
Basal+o-2 per cent L-arginine
Basal+0-6 per cent L-lysine
Basal+o-8 per cent L-lysine
S.E.M.
L.S.D. (P<o-O5)

Live-weight
gain

Efficiency of
food conversion

19-2
19-0
18-1
14-8

o-57
o-57
0-56
0-48
o-o 1
0-03

o-39
i-3

since supplementation with this amino acid did not result in enhanced growth or in
an improvement of efficiency of food conversion. Addition of o-6 per cent lysine
resulted in a growth inhibition (which just failed to reach significance 0-05 < P < o-1 o).
Further supplementation with lysine at 0-8 per cent of the diet, was accompanied by
a severe growth depression (P<o-65). The efficiency of food conversion ratios
reflected the live-weight gain observations.
In experiment 2 (Table 4), the basal diet was demonstrated to be inadequate in
arginine, though not significantly so (P>O"O5), when the response was measured
in terms of weight gain and efficiency of food conversion ratios. The response was
not reflected in the nitrogen retention data. Excess lysine arrested growth markedly,
and significantly lowered the efficiency of food conversion and nitrogen retention
values in animals consuming this surplus (P<o-O5). Arginine largely counteracted
this retardation of growth and the ill-effects on efficiency of food conversion and

INTERRELATIONSHIP BETWEEN LYSINE AND ARGININE

305

nitrogen retention. However, this reversal was not complete, indicating that excess
lysine in some manner altered the arginine requirement of the chick.
The plasma amino acid data relevant to experiment 2 are shown in Table 5.
As expected on supplementation of the basal diet with arginine or lysine, there was
an accumulation of these amino acids in plasma. However, the addition of lysine
TABLE 4

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Experiment 2: Efficacy of arginine in alleviating the growth depression by excess lysine. Response
measured in terms of live-weight gain {gjd), efficiency of food conversion (g gainjgfoodingested),
and nitrogen retention (g JV retainedjg JV ingested), during the period j-21 d
Values represent means of 4 replicate groups of 4 chicks each. . Basal diet deficient in
arginine
Treatment

Li
Live-weight
gain

Efficiency of
food conversion

Nitrogen
retention

Basal diet
Basal+0-3 per cent L-arginine
Basal+0-6 per cent L-lysine
Basal + lysine+arginine

17-6
18-7
67
H-9

0-56
o-59
0-36
o-53
0-O2
0-05

'59

S.E.M.
L.S.D.

o-43
t-3

o-59
0-50
0-58
o-oi
0-03

to the diet also caused a lowering of the arginine levels in plasma. Other amino
acids in plasma remained relatively constant except glycine which also showed a
marked depression on addition of excess lysine to the diet; histidine, a basic amino
acid like arginine and lysine, also appeared in considerably diminished quantities
in plasma. An interesting feature of the plasma amino acid data is that although
TABLE 5

Experiment 2: Effect of lysine and arginine supplementation on levels of selected amino acids in plasma (n moles/100 ml)
Values represent single determinations of pooled samples
Plasma amino acid levels (fi moles/100 ml)
Treatment

Arginine

Histidine

IsoLysine Leucine leucine

Basal diet
Basal+0-3 per cent
L-arginine
Basal+0-6 per cent
L-lysine
Basal+lysine+arginine

4-5
18-6

19-0
13-2

28-5
34-4

39-8
44'0

4-2
4-8

12-4
8-3

46-8
69-2

20-4
2i-o

Valine

Glycine

Threonine

11-5
11-3

16-8
14-5

74-3
83-0

128-9
83-7

8-6
8-0

14-1
12-3

46-9
42-9

92-4
86-6

additional arginine alleviated to a large extent the growth-depressing effects of

excess lysine, it did not alter the concentrations of lysine in the plasma, thereby
casting some doubt on the reciprocity of the interaction.
Experiments 3-6: Specificity of the lysine-arginine interaction. T h e results of the initial

experiments suggested that the interaction between lysine and arginine required
further investigation particularly in terms of its specificity. Accordingly the next
phase involved experiments designed to examine alternative target amino acids.
These experiments were based on the premise that in specific interactions, the effective
target amino acid would be sensitive to a surplus of the agent even if the target
were not itself first limiting in the diet.

306

j . P. F. D'MELLO AND D. LEWIS

The live-weight gain and efficiency of food conversion data of experiment 3

are listed in Table 6. The basal diet was deficient primarily in methionine as shown
by the response to methionine alone, but not to arginine alone. The simultaneous
addition of methionine and arginine to the basal diet yielded the best growth
performance (P<O'O5). Excess lysine precipitated a severe growth retardation
(P<o-O5) which was reversed by arginine but not by methionine supplementation.
The concomitant supply of methionine and arginine to the high lysine diet improved
performance even further. This was attributed to the fact that arginine alleviated
the effects of excess lysine while methionine addition merely resolved a normal,
uncomplicated deficiency of the sulphur amino acids.

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TABLE 6

Experiments: Specificity of the lysine-arginine interactioneffect of supplementingfirstand

second limiting amino acids on live'iveight gain (gjd), and efficiency of food conversion
(g gainjgfood ingested), during the period y-si d

Each figure is a mean of 4 replicate groups of 4 chicks each. Basal diet first
limiting in methionine and second limiting in arginine
Treatment
Basal diet
Basal+o-3 per cent L-arginine
Basal+0-3 per cent DL-methionine
Basal+arginine+methionine
Basal+o-6 per cent L-lysine
Basal+lysine+arginine
Basal+lysine+methionine
Basal+lysine+arginine+methionine
S.E.M.
L.S.D. (P<o-O5)

Live-weight
gain

Efficiency of
food conversion

16-3

o-57
o-55

16-0
17-1
18-1

'59

167

0-63
0-48
o-55
0-47
0-58

o-53

O'OI

I2-O

'5*4

i-6

0-04

Experiment 4 was the second of the series of experiments examining the

specificity of the lysine-arginine interaction. The ability of arginine in alleviating
the ill-effects of excess lysine was tested when tryptophan was arranged to be the
first limiting amino acid. The results are listed in Table 7. The significant improvement (P<o-O5) in performance on addition of tryptophan alone and in combination
with arginine confirmed that tryptophan was first limiting. Excess lysine depressed
weight gain and efficiency of food conversion (P < 0-05), which was restored to normal
only by the addition of arginine and not by tryptophan supplementation.
The results of experiment 5 are shown in Tables 8 and 9, and confirm again the
unique specificity of the interrelationship between lysine and arginine. No response
in performance occurred to arginine supplementation alone, but arginine in combination with histidine improved weight gain over the control group, establishing
that arginine was not first limiting. However, the ill-effects of excess lysine were
reversed by increasing the dietary supply of arginine; histidine supplementation
merely aggravated the growth retardation (P<o-O5). The plasma concentration
of lysine and arginine (Table 9) increased in response to their supplementation in the
diet. In addition excess dietary lysine induced a pronounced drop in the concentration of arginine in plasma but exerted no effect on the histidine concentration, for
instance while plasma arginine declined from 14-6 p. moles/100 ml to 7-4 p moles/100
ml of plasma on addition of excess lysine the concentration of histidine remained

INTERRELATIONSHIP BETWEEN LYSINE AND ARGININE

307

TABLE 7

Experiment 4: Specificity of the interaction between lysine and arginineeffect of supplementing first and second limiting amino acids on live-weight gain (gjd), and efficiency
of food conversion (g gainfgfood ingested), during the period 7-21 d
Each figure in the table is a mean of 4 replicate groups of 4 chicks each. Basal
diet first limiting in tryptophan and second limiting in arginine

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Treatment

Live-weight
gain

Efficiency of
food conversion

16-5

0-56
0-57
0-56
o-6i
0-41
o-53
0-41
o-57
0-03
0-08

Basal diet
Basal+o-25 per cent L-arginine
Basal+0-10 per cent DL-tryptophan
Basal+arginine+tryptophan
Basal+o-6 per cent L-lysine
Basal+lysine + arginine
Basal+lysine + tryptophan
Basal+lysine+arginine+tryptophan
S.E.M.
L.S.D. (P<o-O5)

I8-I

20-2

8-3
14-2
8-2

16-o
o-73
2-O

TABLE

Experiment j : Specificity of the interaction between lysine and arginineeffect of supplementing

first and second limiting amino acids on live-weight gain (g]d), and efficiency of food conversion (g gain/g food ingested), during the period J-si d
Values in the table represent a mean of 4 replicate groups of 4 chicks each. Basal
diet first limiting in histidine and second limiting in arginine
Treatment

Live-weight
gain

Efficiency of
food conversion

'9-5
19-8
20-9
13-9
17-8
9-5
0-71

o-59
o-61
o-61
0-46
o-54
0-38
0-02

2*1

O-07

Basal diet
Basal+0-3 per cent L-arginine
Basal+arginine+o-i 5 per cent L-histidine
Basal+ o-6 per cent L-lysine
Basal+lysine+arginine
Basal+lysine+histidine
S.E.M.
L.S.D. (P<o-O5)

TABLE 9

Experiment 5 : Effect of dietary supplementation of arginine, histidine and excess lysine on post-prandial levels of some amino
acids in plasma {JX. moles! 100 ml)
Plasma values represent single determinations of pooled samples
Plasma amino levels (^ moles/100 ml)
Treatment
Basal diet
Basal+arginine
Basal-l-arginine
-(-histidine
Basal+lysine
Basal+lysine
+ arginine
Basal+lysine
+histidine
U

Threonine

Glutamic
acid

Isoleucine

Leucine

155-6
91-0

35-o
35-2

17-6
14-6

21-4
22-2

13-8

150-6

26-8

14-2
18-8

130-0

3 6-8

164-2

3 6-8

Phenylalanine

Lysine

Histidine

Arginine

I2-O

62-4
49-2

19-2
13-2

14-6
33-2

19-4
22-6

n-8
14-8

55-4
146-2

22-0
21-2

30-0

15-8

'9-4

13-4

127-0

15-6

17-0

20-2

24-0

12-4

i33-o

27-8

8-4

7-4

308

j . P. F. D'MELLO AND D. LEWIS

unchanged. The concentration of most other amino acids increased marginally on

addition of excess lysine.
Additional metabolic support for a specific relationship between lysine and
arginine was provided in the final investigation (experiment 6; Tables 10 and n ) .
TABLE IO

Experiment 6: Specificity of the interaction between lysine and arginineeffect of supplementingfirstand

second limiting amino acids on live-weight gain (gjd), efficiency of food conversion (g gainjg food ingested), and nitrogen retention (g N retained)g N ingested), during the period y-si d

Each value in the table is a mean of 4 replicate groups of 4 chicks each. Basal diet calculated to be first limiting in threonine and second limiting in arginine

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Treatment

Live-weight
gain

Efficiency of
food conversion

12-9
12-9
19-5
20-6

0-49
0-48
o-57
o-59

7-8

-37

Basal diet
Basal+0-3 per cent L-arginine
Basal+0-3 per cent DL-threonine
Basal+arginine + threonine
Basal+o-6 per cent L-lysine
Basal+lysine + arginine
Basal+lysine+threonine
Basal+lysine+arginine+threonine
S.E.M.
L.S.D. (P<0-O5)

12-8
9-8

Nitrogen

retention
0-55
o-55
0-56
0-58
0-46
o-53
o-49
0-56
0-02
0-05

0-48
0-41

1&7

o-53

0-41
1-a

o-oi
0-04

Typical responses were obtained in terms of weight gain, extent of nitrogen retention
and efficiency of food conversion, confirming that the desired sequence of limitation
of amino acids had been achieved. A significant response (P < 0-05) occurred to
threonine supplementation indicating that it was first limiting in the basal diet.
TABLE I I

Experiment 6: Effect of lysine addition and supplementation with first and second limiting amino acids {threonine and
arginine, respectively) on post-prandial levels of some amino acids in plasma (ft moles) 100 ml)

Values are given for single determinations of pooled samples

Plasma amino acid levels (fi moles/100 ml)
t

Treatment
Basal diet
Basal+arginine
Basal+threonine
Basal+arginine
+ threonine
Basal+lysine
Basal+lysine + arginine
Basal+lysine+threonine
Basal+lysine+arginine
+threonine

IsoAspartic Threacid
onine leucine Leucine
26-6
23-4
8i-6

15-8

94-8
33-6

15-4

5-0

3.8
4-4

40-4
133-6

3-8

8o-o

5-2

6-6
5-2

5-6

Tyrosine

Phenylalanine Lysine

Histidine

Arginine

83-8
9i-4
58-8

22-8
17-4
15-8

16-6
20-0

28-8
7-4
io-8
7.6

22-4
22-0
18-6

27-8
27-6

8o-o

IO-O

11-8

119-8
169-6

108

188

24-8
24-2
23-4
25-0

12-0

ia-8

23-6
19-0
23-8

10-2

115-0

*2-5
19-6
17-8
16-o

90

18-2

21-0

8-6

I2O-O

16-2

I2'O
12-0

n-8

24-6

12-4
12-0
10-0

13-6

6-8

Excess lysine induced the predictably severe inhibition of growth and nitrogen
retention (P<o-O5). The ill-effects were counteracted by arginine but not by
threonine supplementation.
The relevant plasma amino acid data are shown in Table 11. Excess dietary
lysine depressed the concentration of arginine in plasma from 16-6 /x moles/100 ml

INTERRELATIONSHIP BETWEEN LYSINE AND ARGININE

309

to 7*4 /x moles/100 ml plasma, without imposing much alteration on the threonine

concentration. Lysine status remained consistently high in plasma when a surplus
was present in the diet, even when arginine supplementation corrected the growth
retardation.

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DISCUSSION

Since there is essentially no storage of amino acids in the body, it has been
frequently assumed that any surplus ingested and not subsequently utilised in
protein synthesis is disposed of without impairing growth. It is now acknowledged
that in most instances a surplus of an essential amino acid will impose a limitation
upon the efficiency of nutrient utilisation commensurate with the magnitude of the
deviation from a perfect balance. There are also recognised to be certain occasions
when a dietary excess of an amino acid or of a mixture of amino acids will precipitate
an ill-effect that is totally disproportionate to the degree of imbalance. Harper
(1958, 1964) grouped these effects into three categories: imbalances, toxicities and
antagonisms without examining in detail the aetiological basis for this separation.
Lewis (1965) suggested that this classification could not be justified and proposed
that adverse effects of amino acids could best be considered as specific interactions
between pairs of amino acids. The interrelationship between lysine and arginine
was therefore examined in the light of the agent-target hypothesis.
The results of experiments i and 2 demonstrate the occurrence of the lysinearginine interaction in chick nutrition. These findings support the observations of
other authors (O'Dell et ah, 1958; Jones, 1961, 1962 and 1964; Smith and Lewis,
1966). Excess lysine depresses growth severely when the arginine content of the
diet is marginally adequate. A concomitant supply of arginine in the diet precludes
the onset of this phenomenon.
Information regarding the specificity of the interaction between lysine and
arginine is virtually absent due presumably to the assumption that arginine is a
general non-specific detoxifying amino acid and as such is not involved in a specific
interaction (Snetsinger and Scott, 1961). More recently, Boorman and Fisher
(1966), arrived at the conclusion that the lysine-arginine interrelationship was not
unique in spite of some contradictory evidence. These authors showed that lysine
was singularly potent as an agent of interaction when compared with other amino
acids in excess in the diet. They further demonstrated that the ill-effects of excess
lysine were reversed by arginine supplementation in the diet, but the adverse effects
of large quantities of methionine or phenylalanine were not similarly alleviated.
Since massive doses of any amino acid might be expected to be toxic (Almquist,
1952), mere demonstration of a growth depression on addition of a surplus to a diet
need not necessarily constitute a basis for establishing or disputing the existence of
a particular interaction. Evidence of complete reversal of the adverse effects of the
agent by the target amino acid should also be taken into account. It is therefore
only possible to decide upon the specificity of an interaction when the above criteria
have been satisfied. It is conceivable that much of the uncertainty regarding the
specificity of the interaction between lysine and arginine is due to lack of evidence
concerning the significance and function of arginine as an effective target. For this
reason the lysine-arginine pair was examined in detail for possible alternative target
amino acids in the interaction (experiments 3-6).

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310

j . p. F. D'MELLO AND D. LEWIS

The results demonstrate conclusively that the interaction between lysine and
arginine cannot be shown not to be specific. In experiment 3, for example,
methionine was tested as the alternative target amino acid to arginine. It is clear
that the basal diet was primarily deficient in methionine and only marginally lacking
in arginine. However, the addition of excess lysine reversed the sequence of limitation in that the accruing growth depression was reversed only by arginine and not
by methionine therapy. The growth data of experiments 4-6 confirm further that
the disproportionate effects of excess dietary lysine are alleviated by only arginine
and not by the alternative amino acids tested, although these were first limiting in
the original diets. Tryptophan, histidine and threonine were each tested as alternative targets in these experiments due to allegations that these amino acids could
interact with lysine (Winje, Harper, Benton, Boldt and Elvehjem, 1954; Henderson,
Koeppe and Zimmerman, 1953; Rosenberg, Culik and Eckert, 1959 respectively).
In addition, histidine is a basic amino acid and may share a common transport
mechanism with lysine and arginine during absorption from the intestine and during
renal reabsorption (Boorman, 1969). The possibility that histidine could replace
arginine as the target amino acid was therefore considered. However, the growth
observations in the present study demonstrate adequately that arginine alone is the
target for lysine action. Nesheim (1968) illustrated this specificity further in studies
employing two strains of chicks which differed substantially in their requirements
for arginine. Chicks1 with a high requirement were less able to tolerate dietary
excesses of lysine than were chicks with a low requirement for arginine. In
addition, excess dietary lysine enhanced significantly the excretory output of
arginine.
It is evident from the observations of Jones, Petersburg and Burnett (1967)
that the mechanism whereby the ill-effects of excess dietary lysine are brought
about is via an alteration of the metabolic fate of arginine. The present
results support these observations. Surplus lysine precipitates a deficiency of
arginine in spite of the impending lack in the basal diets of adequate supply of
methionine, tryptophan, histidine or threonine. This accounts for the partial
reversal of the ill-effects of excess lysine by arginine supplementation in some
of the present experiments. Complete reversal would certainly be attained at
higher supplementary doses of arginine only (see D'Mello, Hewitt and Lewis,
1967).
The plasma amino acid data in experiments 5 and 6 support the relevant growth
observations. Excess lysine in the diet induces a specific drop in the plasma concentration of arginine while exerting no alteration on the metabolism of the alternative target amino acids tested (histidine in experiment 5; threonine in experiment 6).
On the basis of evidence from the present study it is reasonable to conclude that
in chick nutrition, lysine and arginine are inextricably engaged in an unique
interaction.
ACKNOWLEDGEMENTS

One of us (J. P. F. D'M.) received a research studentship from the Ministry of

Agriculture, Fisheries and Food. The technical assistance of Miss C. Mott and
Mrs J. Hall is gratefully acknowledged.

INTERRELATIONSHIP BETWEEN LYSINE AND ARGININE

3II

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