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The Neurotoxicity o- Valine Deficiency in Rats1

PATRICK K. CUSICK,2 KATHLEEN M. KOEHLER,3'4


BARBARA FERRIER ANDBETTY E. HASKELL 6
Department of Food Science, University of Illinois,
Urbana, Illinois 61801
ABSTRACT When valine, an essential amino acid, was withdrawn
from the diet of weanling rats, the animals rapidly developed a unique
pattern of neurological symptoms characterized by head retraction, stag
gering and aimless circling. At necropsy degenerative changes were most
prominent in the neurons of the red nuclei, brain stem structures which
modulate motor function. To explore the pathogenesis of the neurotoxicity
associated with valine deficiency, we fed rats purified diets deficient in
valine alone or in valine plus other branched chain and neutral amino
acids, and we examined brain tissues by light microscopy. Motor disfunc
tion and red nuclei damage occurred only in rats fed diets lacking valine
alone and not in rats fed diets lacking all three branched chain amino
acids. These results suggest that the neurotoxicity of valine deficiency
results from amino acid imbalance rather than from lack of dietary valine
per se. J. Nutr. 108: 1200-1206, 1978.
INDEXING KEY WORDS valine !amino acid deficiency !amino
acid imbalance !branched chain amino acids
Valine and isoleucine are unique among
the essential amino acids in that deficiency
produces neurological signs. The signs are
species specific. In rats, valine deficiency
(1), but not isoleucine deficiency (2), pro
duces staggering, head retraction and aim
less circling. In human infants, isoleucine
deficiency (3), but not valine deficiency
(4), results in tremors of the arms and
legs and episodes of twitching. Neurologi
cal signs associated with valine deficiency
or isoleucine deficiency disappear when
the missing amino acid is restored to the
diet (1, 3).
To our knowledge, the cause of the cen
tral nervous system damage in isoleucine
deficiency has not been explored. How
ever, Scott (5) has identified sites of brain
damage in valine-deprived rats. He ob
served severe cytoplasmic vacuolization
and chromatolysis in the neurons of the
red nuclei and, to a lesser degree, of the
motor facial and deep cerebellar nuclei.
Chromatolysis as seen via the light
microscope implies the dissolution of Nissl
substance and suggests a change in the
major protein synthesizing organelle of the
cell!specifically, the detachment of ribo-
somes from the endoplasmic reticulum and
the loss of characteristic parallel stacks of
membranes. Such essential changes might
be expected to occur in deficiency of any
amino acid, since withdrawal of an essen
tial amino acid from the diet promptly
depresses protein synthesis (6). One won
ders, however, why valine deficiency dif
fers from a deficiency of other essential
amino acids in that it apparently results in
damage primarily to the red nuclei. Is
Received for publication January 4, 1978.
1Supported by grants from The Nutrition Founda
tion (No. 499) and from the National Institutes of
Health (AM-19303).
2Department of Pathology, College of Veterinary
Medicine, Michigan State University, East Lansing,
Michigan 4X824.
3Based in part on data In Ph.D. thesis submitted
to the University of Illinois, Urbana, Illinois, In May,
' Prsentaddress : Dept. of Nutritional Sciences,
University of Wisconsin, Madison, Wisconsin 53700.
To whom correspondence should be addressed.
1200

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VALINE DEFICIENCY IN RATS
1201
there a unique but presently unrecognized
role for valine in the maintenance of these
brain cells?
This paper describes a nutritional ap
proach to the problem of the neurotoxicity
of valine deficiency. The results suggest
that the neurological disorders characteris
tic of valine-deficient rats are due to amino
acid imbalance rather than lack of dietary
valine per se.
MATERIALS AND METHODS
Weanling, male Sprague-Dawley rats
were housed individually in wire-bottomed
cages in an air-conditioned laboratory
maintained at 23 to 24and fed either a
complete purified diet or a diet lacking
one or more of the essential amino acids.
The complete purified diet had the fol
lowing composition (g/100 g diet): Dex
trin, 75.23; amino acids, 12.26; salts, 4.00;
vitamin mix, 1.50; corn oil, 4.00; cellulose,
2.00; and sodium bicarbonate, 1.00.
The amino acid mixture was that of
Ranhotra and Johnson' (7) Diet C III.
Amino acids were purchased.8
The salt mix was that of Draper et al.
(8). This formulation supplied at 4% of
the diet provides 60% of the National
Research Council requirement for phos
phorus, the assumption being that addi
tional phosphorus will be supplied by di
etary protein (9). For example, diets con
taining 20% casein or soy protein would
provide an additional 140 mg P/100 g diet.
Since crystalline amino acids were sub
stituted for protein in our diet, we added
additional phosphorus ( 100 mg/100 g diet )
as dibasic potassium phosphate.
The vitamin mixture was that described
by Parsons, Shrader and Zeman9 ( 10) with
minor modifications.
The amino acid-free diet was prepared
by substituting an equal weight of dextrin
for the missing amino acid mixture. In
amino acid-deficient diets, the nitrogen
content of the diet was maintained at a
constant level by appropriate increases in
the L-glutamic acid content of the diet and
by appropriate decreases in the dextrin
content.
Unless indicated otherwise, the diet was
supplied as a gel prepared as follows: 40
g agar 10was dissolved in 1 liter hot water.
One kg of the purified diet mixture was
added with vigorous stirring. The slurry
was ladled into plastic trays, cooled over
night at 3and stored frozen.
Fresh food and water were supplied
daily. Diet was supplied ad libitum except
to a group (pair-fed controls) whose food
intake was restricted to that of the amino
acid-deficient rats. Food intake values are
reported for dry weight of diet. Suitable
correction was made for a weight loss
averaging 10% which occurred during ex
posure of the gel diet to air for 24 hours.
Rats were assigned randomly to a com
plete or experimental diet according to the
plan indicated in table 1. Data for weight
and food intake are reported only for sur
vivors. Deaths occurred among the valine-
deprived groups: 1 in experiment 2 and 4
in experiment 3. In anticipation of a higher
death rate among valine-deprived rats,
we assigned additional animals to this
group. Differences in growth were evalu
ated statistically with Student's i-test
(2-tailed).
Tissue samples were collected from rats
anesthetized with pentobarbital sodium
and killed by vascular perfusion. Imme
diately before perfusion, the chest was
opened, the heart exposed and 1 ml of
heparin (1,000 units/ml) and 2.5 ml of
\% sodium nitrite solution were injected
into the left ventricle. The right ventricle
slit to permit escape of blood and
was
perfusion fluid, the descending aorta
clamped with a hemostat and hypertonic
perfusion fluid introduced into the left
ventricle. The perfusion mixture contained:
2.5% reagent grade glutaraldehyde " and
2.0% paraformaldehyde in 0.08 M sodium
" Sprague-Dawley Division of Mogul Corporation,
Madison, Wisconsin.
7 It supplied (mg/100 g diet): L-arginlne-HCl,
500; L-histidine-HCl-HzO, 338; L-lsoleuclne, 550;
L-leuelne, 730; L-lysine-HCl, 1,184; L-methlonine,
539 ; L-phenylalantne, 763 ; L-threonlne, 500 ; L-tryp-
tonhnn, 150; DL-alanlne, 230; i.-anpartic acid, 230;
L-glutumlc acid, 3,290; glycine, 1,394; L-prollne,
230; L-cystlne, 230; DL-serlne, 230; L-tyroslne, 230;
L-asparaglne, 395 ; and L-vallne, 550 mg.
"Ajlnomoto Company, Tokyo, Japan.'
! It supplied per 100 g diet : chollne chloride, 7.5
mg ; inosltol, 37.5 mg ; ascorbic acid, 1.75 mg ; cal
cium pantothenate, 3.75 mg ; nlacln, 2.25 mg;
thiamin-HCl, 2.25 mg ; menadione, 1.87 mg; p-amlno-
benzoic acid, 750 ng; riboflavin, 750 wg ; folie acid.
45 tig; blotin, 19 ne : vitamin B-12, 2 us ; DL-a
tocopheryl acetate, 9 IU, cholecalciferol, 112 ICU ;
retinyl acetate, 1136 IU ; pyrldoxlne-HCl, 2.25 mg.
10Bacto Agar, Dlfco, Detroit, Michigan.
" Eastman Kodak, Rochester, New York.

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1202 CUSICK, KOEHLER, FERRIER AND HASKELL
TABLE 1
Effect of diet deficient in one or more of the branched
chain amino acids on growth and food intake
in weanling rats1
DietExpt.
1CompleteStock
dietVal-freeExpt.
2CompleteVal-freePair-fedExpt.
3*CompleteVal-freePair-fedvai-,
ile-,leu-freeExpt.
4<CompleteVal-,
ile-,leu-freeVal-,
ile-,leu-,
try-freeVal-,
ile-.leu-,
try-,phe-,
tyr-freeAmino
acid-freelie-freeLeu-freeTry-freeInitial
wtg63.00
1.44W63.4
1.40(5)59.8
1.42(5)56.2
2.35(5)55.2
1.27(12)49.6
3.07(5)62.5
3.52(6)58.2
0.81(10)63.5
1.14(6)69.5
0.72(9)64.6
3.25(6)68.1
3.19(6)66.5
1.78(6)64.7
2.85(6)62.0
2.29(6)71.0
2.61(7)65.7
5.44(7)77.3
2.39(6)Weight
gaing/dav5.06
0.197.
16 0.34-0.65
0.064.31
0.17-0.61
0.030.20
0.1 16.40
0.13-0.44
0.020.23
0.11-0.49
0.045.63
0.20-0.32
0.01-0.25
0.11-0.24
0.03'-0.55
0.03'-0.88
0.05'-0.32
0.12-0.46
0.07'Food
intakeg/day14.81
0.554.36
0.1314.03
0.644.85
0.044.84
0.1917.901.084.29
0.094.37
2. 145.170.10
1Values shown are the mean BE. Data are reported only for
survivors. The number surviving is shown in parentheses.
Duration of experiment: 25 days. Deaths occurred only in the
valine deprived groups, 1 in Expt. 2 and 4 in Expt. 3. ' Diet
mixed with an equal weight of water and fed as slurry.
1Wayne Lab-Blox, Allied Mills, Incorporated, Peor-a,Illinois.
4Diet supplied as a gel. ' Pair-fed to valine deficients.
' Weight loss not significantly different (P > 0.05) from that in
valine-deprived rats (expt. 3). ' Weight loss significantly
different as compared to that in rats fed vai-, ile-, leu-free diet
(Expt. 4); for rats fed the vai-, ile-, leu-, try-, phe-, tyr-free
diet and for rats fed the try-free diet, P < 0.01 ; for rats fed the
amino acid-free diet and for rats fed the ile-free diet, P < 0.001.
phosphate buffer, pH 7.4. The final os-
molarity of the solution was 1,080 to 1,100
milliosmoles per liter. It was stored at 4
no longer than 72 hours before use and
was filtered by gentle suction early on the
day it was to be used. After the rat had
been perfused for 30 minutes, the brain
was removed and stored at 4in the same
solution. Brain tissue was sliced, processed
and embedded in paraffin by standard
methods. The brain tissues in paraffin
blocks were sectioned at 6 /A, deparaffin-
ized, stained with hematoxylin-eosin, and
examined via light microscopy.
RESULTS
When weanling rats were fed a purified
diet lacking valine, food intake dropped
sharply and the animals lost weight stead
ily. After 25 days of valine-depletion, rats
typically weighed about 10 g less than at
weaning. Growth and food intake data are
shown in table 1.
Clear signs of central nervous system
damage were apparent after rats had
eaten the valine-free diet for 18 to 20 days.
A characteristic early sign was inability of
the rat to cling to the wire mesh side of
the cage without falling. By 23 to 25 days,
severe motor incoordination was apparent.
The rats walked with a staggering gait
and frequently fell. They appeared dis
oriented and circled aimlessly. Head move
ments characteristic of normal exploratory
behavior became grossly exaggerated in
that extreme retraction of the head oc
curred. The rats appeared unusually sensi
tive to touch. They failed to groom them
selves. Their paws and nose became caked
with diet. Death was common in rats fed
a valine-free diet more than 25 days. In
the terminal stages, rats huddled in the
corner of the cage and moved only when
gently prodded. These signs were not ap
parent in pair-fed controls or in the con
trols fed a valine-supplemented diet ad
libitum.
On light microscopic examination, brains
of valine-deprived rats had cellular dam
age apparently confined to the neurons of
the red nuclei. Neuronal cell bodies were
swollen with large round eccentrically
placed nuclei. Neuronal cytoplasm was
pale, devoid of Nissl clumps, and contained

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VALINE DEFICIENCY IN RATS
1203
prominent empty spaces, particularly ad
jacent to cell borders (fig. 1A). White
matter changes were not noted. However,
specific staining techniques to detect early
myelin degeneration were not used. Exami
nation of tissues from liver, kidney, heart,
and skeletal muscle were unremarkable.
Pair-fed controls and valine-supplemented
controls fed ad libitum had no apparent
significant changes in brain or other organs.
Light micrographs of red nuclei from con
trols fed ad libitum ( fig. IB ) and pair-fed
controls (fig. 1C) are shown.
When rats were fed a diet lacking all
three branched-chain amino acids (valine,
isoleucine and leucine), the characteristic
neurological signs of valine deficiency did
not appear. Although such a diet resulted
in growth depression as severe as that pro
duced by valine deficiency (see table 1,
expt. 3), it produced no signs of motor in
coordination. When the red nuclei of rats
deprived 25 days of all three branched-
chain amino acids were examined by light
microscopy, these neurons were indistin
guishable from those of controls. A light
micrograph of red nuclei from rats fed a
diet deficient in valine, isoleucine and leu-
cine is shown in figure ID.
No neurologic signs characteristic of
valine deficiency were observed when rats
were fed diets lacking (1) leucine alone,
(2) isoleucine alone, (3) tryptophan alone,
(4) valine, isoleucine, leucine, and trypto
phan, (5) valine, isoleucine, leucine, tryp
tophan, phenylalanine, and tyrosine, and
(6) all amino acids. All of these diets pro
duced severe growth retardation (table 1,
expt. 4) but none produced changes in the
red nuclei detectable by histological exami
nation. Of these experimental diets, only
the tryptophan-free diet produced an indi
cation of mild nerve disfunction. Trypto
phan deficient rats appeared hyperirri-
table but did not display the motor in
coordination and other neurologic signs
characteristic of valine deficiency.
DISCUSSION
The first report of the neurological dis
orders characteristic of valine deficiency
was that of Rose and Eppstein (1) who
noted "peculiar symptoms unlike any we
have encountered in other types of amino
acid deficiencies" including severe lack of
coordination, staggering gait, head retrac
tion and a rotary motion "resembling that
of a dog chasing his tail." These observa
tions have been confirmed (5, 11).
Scott (5) traced the apparent cause to
neuronal deterioration and myelin degen
eration in nerve pathways related to co
ordination and orientation. In young rats
depleted of valine for 22 days, severe dam
age occurred in the cell bodies of the red
nuclei, and the myelin of the vestibular
nerve showed clear signs of deterioration.
Ih adult rats which tolerated 70 days of
depletion, myelin damage was even more
extensive in the facial and vestibular
nerves and was apparent, to a lesser de
gree, in the medial longitudinal fasciculus.
Scott reported that these changes were
specific for valine deficiency and were not
observed in rats depleted of phenylalanine,
threonine, histidine, tryptophan and isoleu
cine. An obvious extension of Scott's work
was to determine whether the neurotox-
icity of valine deficiency is due to lack of
valine per se or due to amino acid imbal
ance. Specifically, it seemed important to
determine whether the signs of valine
neurotoxicity could be observed not only
in rats fed a diet lacking valine alone but
also in rats fed a diet lacking all three
branched-chain amino acids.
Our experiments demonstrate that the
neurological symptoms attributed to valine
deficiency can be reproduced with rats fed
an improved purified diet which permits a
satisfactory rate of growth in controls
(4.3 0.50 to 6.4 0.13 (see table 1) as
compared to 3.0 g/day in Scott's experi
ments) (5).
The nature of the cellular changes ob
served in the red nuclei in valine deficiency
suggests that valine may be the rate-limit
ing amino acid for protein synthesis in
these cells. Withdrawal of valine from the
diet resulted in a deterioration of Nissl
substance!that is, in a disruption of the
chief protein synthesizing machinery of
the cell. Similar cellular changes have been
observed in liver of rats fed a tryptophan-
free diet (12). Removal of tryptophan
from the diet caused an abrupt decrease
in the rate of protein synthesis. The de
pression of protein synthesis is manifested

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1204 CUSICK, KOEHLER, FERRIER AND HASKELL
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B
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VALINE DEFICIENCY IN RATS
1205
r
'
Fig. 1 Neurons of the red nuclei from rats fed the following diets for 25 days: (A)
valine-free; (B) control, ad libitum; (C) control, pair-fed to valine deficients; (D) iso-
leucine-, leucine-, and valine-free. Cell swelling, chromatolysis and nuclear displacement are
apparent in neurons from valine-deprived rats but not in the neurons of controls or of rats
deprived of all three branched chain amino acids. Photographed at a magnification of 450X.
Stain: hematoxylin-eosin.
at the cellular level as a disaggregation of
polysomes. The cellular response of liver
was characteristic of tryptophan deficiency
and not of a deficiency of other essential
amino acids, apparently because trypto
phan is the rate-limiting amino acid for
protein synthesis in this organ (12). That
branched-chain amino acids limit the rate
of protein synthesis in muscle has been
demonstrated ( 13). Our data are consistent
with the possibility that valine may limit
the rate of protein synthesis in the red
nuclei!and perhaps, judging from Scott's
work, in other parts of brain.
We observed that neurological signs did
not occur and the red nuclei appeared
normal when rats were fed diets lacking
all three branched-chain amino acids.
These data strongly suggest that the nerve
tissue damage characteristic of valine de
ficiency actually is a consequence of amino
acid imbalance. In other words, it ap
parently was not simply a lack of valine
which caused the nerve damage but the
presence in the valine-free diet of isoleu-
cine and leucine.
Branched-chain amino acids compete
with one another and with other neutral
amino acids for transport into brain (14).
The essential amino acid supply for pro
tein synthesis comes both from diet and
from tissue breakdown (15). In protein
deficiency, amino acids from muscle (and
presumably from other extracerebral tis
sues) are salvaged to maintain brain pro
tein ( 16). In an animal fed a diet lacking
all three branched-chain amino acids, one
might anticipate that a small but balanced
supply of isoleucine, leucine and valine
from breakdown of extracerebral tissues
would compete for entry into brain. In the
rat fed a diet deficient in valine alone, the
relatively high concentrations of leucine
and isoleucine in the diet might inhibit the
transport into brain of valine from tissue
breakdown. Thus, because of its unique
transport system which limits entry of
amino acids, brain might suffer more from
valine deficiency than other organs. Suit
able experiments to test this hypothesis
are in progress.
LITERATURE CITED
1. Rose, W. C. & Eppstein, S. H. (1939) The
dietary indispensability of valine. J. Biol.
Chem. 127, 677-684.

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120G CUSICK, KOEHLER, FERRIER AND HASKELL
2. Womack, M. & Rose, W. C. (1936) The
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to growth. J. Biol. Chem. 116, 381-391.
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Roitman, E. & Holt, L. E., Jr. (1964) The
essential amino acid requirements of infants.
IX Isoleucine. Am. J. Clin. Nutr. 15, 313-
321
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F., Boyer, A. & Westall, R. G. (1959) The
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8. Draper, H. H., Bergan, J. G., Chi!, M.,
Csallany, A. S. & Boaro, A. V. (1964) A
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395-400.
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with rats. Ann. Rev. Biochem. 40, 549-572.
10. Parsons, P. L., Shrader, R. E. & Zeman, F. J.
(1976) Adrenal function in young of pro
tein-deprived pregnant rats. J. Nutr. 106,
392-404.
11. Ferrare, A., Roizin, L. & Givner, I. (1947)
Ocular changes in rats on diets deficient in
amino acids. II. Corneal dystrophy due to
valine deficiency. Arch. Ophth. 38, 342-352.
12. Munro, H. N. (1968) Role of amino acid
supply in regulating ribosome function. Fed.
Proc. 27, 1231-1237.
13. Fulks, R. M., Li, J. B. & Goldberg, A. L.
( 1975 ) Effect of insulin, glucose, and amino
acids on protein turnover in rat diaphragm.
J. Biol. Chem. 250, 290-298.
14. Oldendorf, W. H. & Szabo, J. (1976)
Amino acid assignment to one of three blood-
brain barrier amino acid carriers. Am. J.
Physiol. 230, 94-98.
15. Wannemacher, R. W., Jr. (1967) Protein
synthesis in various tissues from animals at
different stages of protein-calorie malnutrition.
Seventh Int. Cong. Nutr. 5, 205-210.
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Brain response to protein undemutrition:
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Errata
Cusick, P. K., Koehler, K. M., Ferner, B. & Haskeil, B. E. (1978) The
neurotoxicity of valine deficiency in rats. J. Nutr. JOS, 1200-1206. The vita
min mixture supplied (per 100 g diet) 75 mg choline chloride (not 7.5) and
7.5 mg ascorbic acid (not 1.75).
Tagari, H. & Bergman, E. N. (1978) Intestinal disappearance and portal
blood appearance of amino acids in sheep. J. Nutr. 108, 790-803. The fol
lowing section of text was deleted from tibe manuscript.
Some of the missing amino acids may
have been utilized by intestinal bacteria to
produce diaminopimdic acid or other N
compounds (11, 29, 30) and may have
never left the intestinal lumen.
Quantitative correlations of gut disap
pearance and portal appearance of amino
acids. While the above metabolic pathways
may account for significant amounts of
missing amino acids, the great SE one finds
in this type of work (5, 7) must be taken
into consideration. One of the main aims of
this study was to find a possible correla
tion between gut disappearance of amino
acids and some easily sampled and deter
mined blood criterion. Indeed, when gut
disappearance was correlated with portal
appearance, it was found that when smaller
amounts of amino acids disappear from the
gut, the amounts of the same amino acid
appearing in the portal blood usually were
even smaller. The same was true when the
amounts of amino acids passing through
the pylorus were correlated with portal
appearance.
Also of importance is that the arterial
concentrations of several essential amino
acids were significantly correlated with the
quantities of amino acids passing through
the pylorus and also to the quantities dis
appearing from the intestine (table 9).
One major exception, however, was lysine
and, in this case, part of the missing lysine
might have been metabolized to NML
where concentrations in arterial circulating
blood were greater in sheep fed the H.P.
TABLE 9
Significance of correlations between amounts of amino
acids (g) passing through the pylorus (Py) or dis
appearing between pylorus and ileum (Py-I)
with amounts of amino acids circulating in ar
terial blood (A) in sheep fed high protein
(H.P.) or medium protein (Af.P.) diets1
Correlation coeffi
cients between2Amino
acidThreonineJ
Cystine
ValineIsoleucineLeucinePhenylalanineLysineHistidineMethionineNon-essential
amino acidsPy
and Ar
= 0.76N.
S.3
r = 0.88"r
= 0.79"r
= 0.96r
= 0.72"N.S.N.S.N.S.N.S.Py-Iand
Ar
= 0.84N.S.
r = 0.79N.
S.r
= 0.88"N.S.N.S.N.S.N.S.N.S.
'H.P. diet: 5 replicates; M.P. diet: 4 replicates,
for each amino acid. 2Determined on 9 pairs of
results for each amino acid. ' N.S. : Not signifi
cant; P < 0.05, *P < 0.01.
diet. This may explain the lack of correla
tion between the amounts of intestinal dis
appearance and arterial circulating lysine.
The glutathione pathway for cystine was
previously discussed and this may be one
of the reasons why arterial cystine concen
tration differences were not found. It must
be considered that dietary differences for
arterial plasma amino acid concentrations
will tend to be masked by the regulatory
effect of the liver. In spite of this, however,
2076

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