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Amino acid maintenance requirements of

young women and of adult rats fed different

dietary amino acid patterns1
Hans Fisher,4 Ph.D., Paul Griminger,4 Ph.D., and Miriam K. Brush,4 Ph.D.

ABSTRACT Adult rats were fed isonitrogenous diets (5 % protein equivalent) providing
indispensable amino acids either in the whole-egg protein pattern or in the adult rat maintenance
pattern of Smith and Johnson (S & J, Brit. J. Nutr. 21 : 17, 1967). In two separate experiments
the threonine and the methionine (total sulfur amino acid) requirements were assessed. Similar
responses were obtained with rats on the threonine experiment independent of the pattern of
indispensable amino acids fed. In the methionine experiment the rats fed the S & J pattern

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showed a lower need for this amino acid than the rats fed the egg pattern . When the latter group
was switched to the S & J pattern, they too required less methionine . In the experiment with
college students (female) fed 7 g of nitrogen/subject per day, the lysine requirement on the
whole-egg pattern was compared to the requirement on a pattern equal to twice the previously
published requirements for young women. No evidence was obtained over the relatively short
experimental period that the lysine requirement differed substantially with change in the pattern
of indispensable amino acids. Am. I. Clin. Nun. 30: 932-938, 1977.

Despite several studies concerned with (7), who used a lower level of total dietary
the maintenance requirement for indispen- nitrogen and a different starting pattern of
sable amino acids of adult rats (1-3), uncer- indispensable amino acids.
tainty remains as to a reliable set of values Young et al (8) have also drawn
. atten-
that might be used in formulating adequate tion to reported differences among re-
purified diets for this species. The Subcom- searchers in requirements for indispensable
mittee on Laboratory Animal Nutrition of amino acids. They concluded that the cx-
the National Research Council (4), selected tent to which the pattern and absolute in-
the results of Benditt et al. (1) for its re- take of the various essential amino acids
quirement recommendations This is sur-
. may be modified without affecting the mini-
prising, since single animals only were used mum needs for a specific essential amino
to study any one level of a given amino acid, acid is unknown . Studies designed to estab-
and the amino acids used were with few
, lish the minimum requirement for an essen-
exceptions, composed of racemic mixtures tial amino acid given various patterns
of the indispensable amino acids Of course,
. and levels of the essential amino acids are
no statistical evaluation of the results could required to resolve this uncertainty(8)
be performed. Based on parenteral feeding While it is possible that pattern and levels
studies with adult rats, Sitren and Fisher (5) of amino acids may, indeed, be responsible
have recently suggested that the Benditt et
From the Departments of Nutrition and Home
al. values, as given by the National Re-
Economics, Rutgers University, New Brunswick, New
search Council, may be excessively high for Jersey 08903
at least four indispensable amino acids. In 2 Paper of the Journal Series. New Jersey Agricul-
studies with adult human subjects, there is tural Experiment Station.
also need to reconcile divergent published 3 This study was part of Regional Research Project
NE-73, Nutritional Improvement in the Northeast Re-
amino acid requirement values. Rose Lev- ,
gion, and was supported in part by regional research
erton and others (6) have generally reported funds.
much higher requirements than Fisher et al. 4 Professor.

932 The American Journal ofClinicoi Nutrition 30: JUNE 1977, pp. 932-938. Printed in U.S.A.

for the reported differences in requirement order to more nearly equalize the nitrogen intake of the
two groups of animals, the concentration
of the S & J
for specific amino acids of adult animals and amino acid mixture was increased by 25 % (Table 1,
man, the possibility cannot be overlooked, column 2 of amino acid mixtures); this had the desired
as Hegsted recently emphasized, that effect of increasing food intake to the level of the
much of the data obtained by bal- animals on the amino acid mixture simulating the egg
pattern (Table 1).
ance studies must be artifactual for one rea-
The composition of the basal diet together with that
son or another (9). Hegsted proceeded to of the amino acid mixtures is given in Table 1 . In
point out that in his view there is no satisfac- experiment 1 L-threonine was the variable dietary con-
tory means to avoid some or most of the stituent and in experiment 2 L-methionine was the
difficulties inherent in nitrogen balance variable and served as the only source of sulfur amino
acids (cystine was omitted). Isonitrogenous additions
studies, and he warned that until new meth-
or substitutions for the variable amino acid were made
ods are available to measure nutritional re- with glycine.
quirements, the results obtained with bal- The study with college women followed the same
ance techniques must be viewed with cau- protocol as described in detail in earlier papers on the
tion and skepticism. reassessment of amino acid requirements of young
women receiving low nitrogen diets (7, 10, 1 1). As in
The present studies were carried out in an the rat studies, either of two diets were given that
attempt to elucidate the relationship, if any, provided the same amount of nitrogen per day (7 g).
of dietary amino acid pattern on the require-

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The egg pattern of indispensable amino acids was sup-
ment of specific indispensable amino acids
in adult rats and in college women. TABLE 1
Composition of basal diet and of
Experimental procedures amino acid mixtures

Ingredient Basal diet Amount

Two experiments were carried out with male
Sprague-Dawley rats . In the first rat experiment the
animals weighed approximately 200 g at the start of the Corn starch 38.5
experiment, by which time they had received the exper- Corn dextrin 20.0
imental diets for an 1 1-day adjustment period. In the Corn oil 12.0
second experiment, the same rats were used approxi- Fiber 5.0
mately 6 months after the completion of the first exper- Minerals 4.0
iment . During this time interval the rats had been fed a Vitamins 0.2
commercial stock diet (Purina Rat Chow). In both Choline chloride 0.1
experiments separate groups of rats were fed either of Amino acids 6.6 or 8.2
two patterns of indispensable amino acids: a whole egg Sucrose to 100
pattern based upon the amounts of indispensable Amino acid mixtures
amino acids provided by 5 % whole egg protein, and a Ammo acid
pattern S & 3 pattern Egg pattern
pattern based upon the reported maintenance require- I II
ments of the adult rat as given by Smith and Johnson
- %of diet
Lysine 0.05 0.06 0.345
Because of concern over accuracy in measuring food
Histidinee 0.07 0.09 0.12
nitrogen intake and urinary and fecal nitrogen excre-
Phenylalanine 0.075 0.09 0.29
tion, the rats were given limited access time to their
Tyrosine 0.056 0.07 0.205
food. This procedure was adopted to minimize food
Tryptophan 0.04 0.05 0.08
spillage and to increase the possibility of separating out
Methionine 0.166 0.20 0.165
any food that might have contaminated the collection
Cystine 0.124 0.16 0.115
compartments of the metabolism cages. In experiment
Threonine 0.18 0.22 0.25
1 the rats were given access to their food for 2 hr in the
Leucine 0.10 0.12 0.47
morning. Since the rats seldom ate much food during
the second hour of access, it was found more effective
Isoleucine 0.24 0.30 0.345
Valine 0.15 0.19 0.37
in experiment 2 to provide 1 hr access time in the
Glycine 1.17 1.47 0.852
morning and a second hour in the afternoon.
Glutamic acid 2.31 2.87 1.68
In experiment 1 , the diet containing the Smith and
Aspartic acid 2.09 2.61 1.52
Johnson (S & J) pattern of amino acids was fed as
shown in the first column under amino acid mixtures in a Salt mixture 4164; Draper et al.
Table 1 , with a protein intake equivalent to 5 .0% (N x (19). b Adamson and Fisher (18). C Supplied as
6.25) of diet. The egg pattern was provided at the same the L-isomer. d Supplied as the hydrochlo-
level of N intake . In experiment 2 , where food was ride. e Supplied as the hydrochloride monohy-
provided for two 1-hr periods instead of one 2-hr pe- drate. Supplied 25% more of all amino acids than
riod, the animals on the S & J pattern consumed much the original S & J (3) mixture given in the adjacent
less food than did those receiving the egg pattern. In column.

plied in concentrations equivalent to 31 .25 g of whole

egg protein per day, while the other pattern (herein
referred to as the requirement pattern) represented oq
twice the reported requirement values for women (6)
and had been previously used by Fisher and associates a. +1
(7, 10, 11). The composition ofthese amino acid mix-
turns as consumed by the women is given in Table 5.
The remainder of the diet, consisting of nonprotein .
items only was identical to that previously reported 22

(7, 11).

Methods a. +1
In the rat studies food consumption was measured PS

daily but fecal and urine collections were made only

from Tuesday to Friday or 4 days per week. The ani-
mals were fed each experimental diet for periods of 2 to
3 weeks, mostly the latter. In the human study, the
time on each level of indispensable amino acids is listed E
in the table of results, and urine and fecal collections a. +1 +1 +1
were continuous throughout the entire study. Feces

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were accumulated for each period on each level of the m


amino acid being fed, while urine was analyzed on a

daily basis. Nitrogen determinations on food, urine and
feces and creatinine determinations were carried out by
procedures previously described (12, 13).
a 000
+1 +1 +1
Results 4

In experiment 1 (Table 2) adult rats re-

ceiving two different amino acid patterns E
showed a similar response at each level of U
m tt

+1 +1 +1
dietary threonine , irrespective of the amino a. o 0
acid pattern into which the threonine had C., C.,,

been incorporated . Both groups of rats lost

an insignificant amount of weight during the E
experimental period; however, during the U

initial adjustment period to the purified a. +1 +1 +1

diets and the restricted food access time (2 4 c-c-c-

weeks) animals in both groups lost about 20

g each. Food consumption increased signifi-
cantly on the egg pattern as the level of E
+1 +1 +1
threonine was increased. No such effect was a. ir, n 0
noted for the S &J-fed rats. The nitrogen C., C., N


els of threonine
both diets and in response

to meet
of the rats was similar on
to increasing
The level of 0 1 0% ap-
the requirement

for this
V a. +1 +1 +1
amino acid. Quantitatively, nitrogen reten- 4 N

tion was higher on the highest intake of
threonine (0 .1 0%) for the rats on the egg H

pattern, as compared to those on the S & J Iz V

I v N 0
diet. This was not surprising in view of the I * 000
significantly greater food and, therefore , ni- f-u

trogen and amino acid intake on this diet.

The results of the methionine study (ex-
periment 2) indicated a statistically signifi-
cant break at a level of 0.15% on the egg

pattern; however, the higher level of methi- tern; therefore the concentration
, of all
omne (0 .225 %) afforded significantly bet- amino acids was increased by 25 % This .

ter nitrogen retention (Table 3). Body change successfully brought food consump-
weight changes on the egg pattern were tion and nitrogen retention for the S & J rats
small and non-significant; food consumption into line with those getting the egg pattern
remained relatively constant but was higher (compare lines 1 and 2 Table 4). Although,

in this experiment than in the previous one, the nitrogen retention results with a dietary
undoubtedly due to the changed food access methionine level of 0.07% seem out of line,
time (twice for 1 hr each versus once for 2 a break in the response pattern is clearly
hr). When the rats that had received the egg evident at 0.10 to 0.12% methionine. If the
pattern were switched to the S & J pattern good nitrogen retention with only 0.07%
(Table 3 last 2 lines), nitrogen
, retention on methionine is also considered, then it ap-
the reduced methionine intake (0.10%) was pears that for methiomne the requirement is
as good as that previously observed with the lower on the S & J pattern between one
higher level of 0.1 5 % on the egg pattern. third and one half in comparison to that
As mentioned in the experimental sec- noted on the egg pattern It is interesting
. to
tion, food intake was much lower with the S note that the rats that had previously been
& J pattern (Table 4) than for the egg pat- fed the egg pattern when switched to the S

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Body weight changes, food consumption, and nitrogen retention of adult male rats fed the
egg patterna of indispensible amino acids with varying levels of L-methionine

Body Weight
L,-methionine Food consumption N retention
Start End
% ofdiet g giday mg/day

0.3 278 71b 280 8 11.1 0.5k 30.3 2.61

0.225 282 71 284 7 13.5 0.7k 42.2 3.0
0.15 281 8 280 9 1 1 .9 1 .0 21 .3 401
0.225 277 91 284 9 13.3 0.3k 45.3 2.3
0.225c 281 10 276 9 11.7 0.71.2
0.10C 262 91 264 9 12.1 0.2 30.9 3.9
a Provided in the amounts shown in Table 1. b Mean SE for nine rats; the same animals were consecu-
tively given the indicated methionine levels for periods of 2 to 3 weeks; collections were made daily from Tuesday
through Friday of each week; only values within a column that do not share the same superscript(s) are
significantly different (P < 0.05). C The rats were switched to the S & J (3) pattern increased by 25% (column
2, Table 1).

Body weight changes, food consumption, and nitrogen retention of adult male
rats fed the S & J maintenance pattern of indispensible amino acids
with varying levels of L-methiomne

Body weight
UMethionine Food consumption N retention
Start End

% ofdiet g g/day mg/day

0.Y 277 61b 270 5 8.5 0.2 7.2 1.8

0.3 258 32 259 32 12.4 0.8k 50.2 3#{149}72

0.225 256 32 257 32 13.0 0.82 38.1 5#{149}42

0.15 252 32 255 32 13.4 0.52 38.3 3#{149}72

0.10 256 32 256 32 15.4 0.6 28.4

0.07 249 4 252 42 13.7 0.42 38.3 4#{149}32

0.12 250 42 251 42 13.0 0.3 25.6 2.5

ap diet provided amino acids according to the S & J (3) pattern as given in column 1 , Table 1 ; in all other
diets the amino acid levels were increased by 25% as given in column 2, Table 1 . iS Mean SE for eight rats;
the same animals were consecutively given the indicated methionine levels for periods of 2 to 3 weeks; collections
were made daily from Tuesday through Friday of each week; only values within a column that do not share the
same superscript(s) are significantly different (P < 0.05).

& J pattern (Table 3 last 2 lines),

, re-
sponded similarly to those animals that had a

been continuously fed the S & J diet, thus a.

providing corroboration for the apparently B
lower methionine need with the S & J pat- .B

In the human lysine study (Table 5) no I
with either
in body weight
of the two amino
were detected
acid patterns I. a
used. With the egg pattern a statistically a.
significant break in nitrogen retention oc-
curred at a lysine intake of 50 mg/day: re-
tention decreased significantly in reducing
the intake from the high level provided by E
egg protein (2 140 mg) and increased signifi- a.
cantly when 300 mg of lysine were pro- C

vided . No difference was observed in the
quantitative nitrogen retention response be- C

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0 U

tween lysine intakes of 300 and 2140 mg. C

With the amino acid pattern based on

twice the reported requirements (require- E
ment pattern) nitrogen retention was higher
at each lysine intake level as compared to
the egg pattern response Among
. the three
lysine increments the nitrogen retention fol-
lowed a similar response as previously noted
with the egg pattern. From these, admit-
tedly limited results, it does not appear C

likely that the lysine requirement is very B

different from 50 mg/day with either amino a.


acid pattern, at least as judged from these

short-term studies. .

It is interesting to compare the present Lu

amino acid requirement results,for rats with

those recently reported by Said and Hegsted .
(14). These workers used adult female rats
that were fed an amino acid mixture that U

provided five of the amino acids at levels

eight between the egg and the S & J pattern . I ..

and the other six amino acids at levels some-

what lower than those provided by the S & J a

pattern . The threonine requirement, esti- Lu

mated from body water determination, was

calculated to be 0 .1 2 % of diet compared to r .

the present estimate of 0 1 0 % The methio-

. . 8 .%

nine requirement, similarly estimated, was

also 0 . 1 2 % of diet a value very similar to -

the one observed for the S & J pattern (with

which the pattern used by Said and Hegsted
had considerable similarity).

In contrast to our observations and those large intakes. We have already pointed out
of Said and Hegsted (14), the requirements that the direction of the change in the con-
reported by Smith and Johnson (3) were centration of an indispensable amino acid
considerably higher for both threonine may also influence the response This is par-

(0 .1 8 %) and methionine (0 1 7 % methio- . ticularly relevant in moving from an made-

nine plus 0.12% cystine). Steffee et al. (15) quate to a higher intake . We have earlier
have reported an increased need for amino shown (13) that human subjects in negative
acids in the adult, protein-depleted rat. It is nitrogen balance as a result of ingesting an
possible that the adjustment period and the inadequate level of an indispensable amino
nature of the diet used by Smith and John- acid may retain inordinately large amounts
son (3) could have led to a greater state of of nitrogen during a subsequent period
depletion and thus to an increased require- when the concentration of the limiting
ment in comparison to the present study and amino acid is only slightly increased.
that of Said and Hegsted. It is unfortunate that the present results
The methionine study in rats suggested a dont permit generalization that the amino
difference in requirement resulting from the acid pattern does or does not influence the
change in the pattern of indispensable requirements of all indispensable amino
amino acids. It is not likely that this differ- acids. Clearly, more work is necessary in

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ence can be explained on the basis of the 20 relation to the issue raised, particularly to-
g differential in body weight between the wards the evolution of better requirement
groups of animals. A similar weight differ- criteria than nitrogen retention alone The

ence also prevailed during the threeonine use of plasma amino acid changes and of
study where no difference in requirement C402 monitoring after C4 amino acid ad-
for this amino acid was noted. ministration in conjunction with graded 1ev-
The lysine requirement for women has els of the amino acid under study (8 18) ,

been reported to be much higher (500 mg/ may deserve more consideration in this re-
day (16, 17)) than the 50 mg/day observed spect.
in our earlier studies (10) and again in the
present experiment. Perhaps this difference The authors express their appreciation to Mrs.
Olga Donis, Mrs. Anna Sekowski, Miss Elaine Grimm-
is explainable by an effective conservation
ger and Miss Eileen Liddy for excellent technical assist-
of lysine when the dietary supply is low or ance.
inadequate and further
, compounded by the
short experimental diet periods. However,
the experimental procedures of earlier
workers did not differ much in these regards 1 . BENDrrT, E. P., R. L. WOOLRIDGE, C. H. STEFFEE
from the present study. Alternatively, since AND L. E. FRAZIER. Studies in amino acid utiliza-
the amino acid pattern did not influence the tion. IV. The minimum requirements of the indis-
pensable amino acids for maintenance of the adult
requirement for lysine over the short-term well-nourished male albino rat. J. Nutr. 40: 335,
period, it is possible that the lysine provided 1950.
from cereal sources (55 to 1 00 %) in the 2. NASSET, E. S. Comparison of amino acid mixtures
earlier study of Clark et al (1 7) might have .
and egg protein as sources of dietary nitrogen. J.
Nutr. 61: 555, 1957.
been as completely available as the crystal-
3. SMim, E. B., AND B. C. JOHNSON. Studies of
line lysine used exclusively in our studies amino acid requirements of adult rats. Brit. J.
(10). Nutr. 21: 17, 1967.
Other sources of variation may include 4. National Research Council-National Academy of
difficulties with the nitrogen balance tech- Sciences. Nutrient Requirements of Laboratory
Animals, no. 10, NAS, Washington, D.C.: 1972.
niques, to which Hegsted (9) has called at-
5. SrranN, H. S., H. FISHER AND R. ALl. Compari-
tention . It is also possible that the size of the son of two amino acid solutions for total parenteral
increment in the concentration of the varia- nutrition of normal and traumatized rats. J. Nutr.
ble amino acid may be an influencing factor. 105: 1318, 1975.
Some workers have moved from large in- 6. ROSE, W. C. The amino acid requirements of adult
man. Nutr. Abstr. Rev. 27: 631, 1957.
takes of a given indispensable amino acid to 7. FISHER, H., M. K. BRUSH AND P. GRIMINGER.
very low intakes and back again to relatively Reassessment of amino acid requirements of

young women on low nitrogen diets. IH. Isoleu- ture. J. Nutr. 87: 306, 1965.
cine, threonine, phenylalanine, and summation. 14. Sm,A.K.,41tnD.M.HsosmD.Responseof
Am. J. Chin. Nutr. 27: 130, 1974. adult rats to low dietary levels of essential amino
8. YOUNG, V. R., K. Toimsiami, I. Ozi.p, F. acids. J. Nutr. 100: 1363, 1970.
LxsmNri AND N. S. Scitmisisw. Plasma 15. Smmn, C. H., R. W. WISSLER, E. M. HuM-
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10. Fisnn&, H. , M. K. BRUSH, AND P. GazMiriaaR. 16. JONES, E. M., C. A. BAUMANN AND M. S. REY-
Reassessment of amino acid requirements of NOLDS. Nitrogen balances of women maintained
young women on low nitrogen diets. I. Lysine and on various levels of lysine. J. Nutr. 60: 549, 1956.
tryptophan. Am. J. Chin. Nutr. 22: 1190, 1969. 17. Cix, H. E., E. T. MERTZ, E. H. KWONG, J. M.
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Reassessment of amino acid requirements of ments of men and women. J. Nutr. 62: 71 , 1957.
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1216, 1971. 106: 717, 1976.
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WESSELS, C. D. BERDANIER, P. GRIMINGER AND Csuiy AND A. V. Boiuto. A further study of
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high nitrogenlow tryptophan diets. J. Nutr. 81: reproduction. J. Nutr. 84: 395, 1964.
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