Deprivation of Amino Acids and Prenatal Brain

Development in Rats1

Downloaded from https://academic.oup.com/jn/article-abstract/104/8/1002/4776549 by Rudolph Matas Medical Library user on 20 January 2019
S. ZAMENHOF, S. M. HALL," L. GRAUEL, E. VAN MARTHENS
ANDM. J. DONAHUE
Department of Microbiology & Immunology, Department of Bio
logical Chemistry, Mental Retardation Center, and Brain Research
Institute, UCLA School of Medicine, Los Angeles, California 90024

ABSTRACT A protein-free diet (AA) containing a complete chemically defined

mixture of L-amino acids or this mixture deprived of one of the essential amino acids,
tryptophan, lysine or methionine, respectively, was fed to pregnant rats. The feeding
period was 0 to 21 or 10 to 21 days of pregnancy. At birth the following newborn
parameters were measured: body weight, cerebral weight, cerebral DNA (cell number)
and cerebral protein, as well as placental weight, placental DNA and placental protein.
As compared with normal (pelleted) stock diet, feeding AA produced small decreases
that were significant for body weight and some cerebral parameters, but no significant
decreases for placental parameters; thus, it remains uncertain whether our present
knowledge of nutritional factors for optimal fetal development is sufficient to devise a
faultless synthetic diet for pregnancy. Omission of tryptophan, lysine or methionine,
respectively, from AA, resulted in offspring significantly inferior to offspring of dams fed
AA diet in all cerebral parameters; the deficiencies were essentially similar to those
produced in our previous study by total protein deprivation. Thus, omission of single
essential amino acids during pregnancy may be at least as harmful as total absence of
dietary protein. J. Nutr. 104: 1002-1007, 1974.
INDEXING KEY WORDS amino acids, deprivation of •synthetic diet •brain
development, prenatal •fetal development

In the preceding papers of this series we rived; these rats have now been bred in our
have demonstrated that, in rats, maternal laboratory for 34 generations. Virgin fe-
protein restriction before and during the males 3 months old and weighing 200 to
entire pregnancy ( 1-3 ) or during a part 260 g were mated; the presence of a
thereof (4 ) resulted in a significant de- vaginal plug was considered day 0 of preg-
crease or the following neonatal param- nancy.
eters: body weight, cerebral weight, cere- All animals were fed a 20.5% protein
bral DNA (total cell number), cerebral standard pelleted diet (stock)3 with the
protein and placental weight (4). exception of the experimental testing
In the present work we have investigated periods. The second half of gestation ( days
the effects, especially on brain develop- 10 to 21 ) was chosen as the testing period
ment, of total deprivation of each of the for the chemically defined amino acid diet
three essential amino acids separately, omitting tryptophan (AA —Trp) and that
tryptophan, lysine and methionine, during
the entire pregnancy or during the second Receivedfor publicationDecember5, 1973.
halfi 11
of nreffnancv
Preën'mcy- This
m!> studv
!>luuy was
WdS nre-
pie the1Presented In part
goclety for at the Third
Neuroscience, Annual 7-10,
November Meeting of
1973.
ceded by the development or a protein-tree This study was supported by grants HD-05394,
rlipt
diet, «-rmrainincr
containing aq rnmnlptp
compiete rrifmioallv
cnemicaiiy ripae- HD-05615, HD-00345
pubnc Health Service.and HD-04612 from the Ü.S.
fined mixture of L-amino acids, minerals „A^ÎA*«0^0^*6*
îrî,In.ee^
SSPIî?t4™1?,1"^frant
, / A »\ • ij i_ J HD-00345 to Mental Retardation Center. U.C.L.A.
and Vitamins (AA) that would be ade- »The stock pelleted diet (SSOO kcal/1000 g), Wayne
«iiato fr>r nnrmal fptal rlpwplnrwnpnt Mousebreeder Blox, was purchased from Allied Mills.
quate tor normal tetai development. Chicago, 111. This diet has the following L-amlno acid
composition (manufacturer's data; g/kg) : arginine-
EXPERIMENTAL HC1 14.4; histldine-HCl 6.2; Isoleucine 13.8; leucine
18.5 ; lystne-HCl 16.4 ; methionine 4.6 ; phenylalanine
AlViinrv
Albino rat«
rats iisprl
used in trip
me nrpvinin:
previous M—4Ì
(L-*) 11.7; threonine
Rhlt¿mÃ-c 10.3;glycine
acld 46.2; tryptophan 3.1; 5.8;
9.9; serine valinetyroslne
13.5;
and present work are Sprague-Dawley de- 8.3; cystine 4.0.
1002
 AMINO ACIDS AND BRAIN DEVELOPMENT 1003
TABLE 1
The composition of the AA diet

components1Amino
Dietary diet4.0kcal/kg mixtureVit. of
mixture4.50.25
vit.

Downloaded from https://academic.oup.com/jn/article-abstract/104/8/1002/4776549 by Rudolph Matas Medical Library user on 20 January 2019
acid mixture* 789.6 A ester concentrate
Corn oil 50.0 8.9 445.0 (200,000 lU/g)
Lard 50.0 9.0 450.0 Vit. DS concentrate
Vitamin diet fort, mix.f 30.0 105.04.0
3.5 (400,000 lU/g)
Salt mixft 50.0 Alpha tocopherol 5.0
Agar 30.0 Ascorbic acid 45.0
Dextrin 388.4 1,553.6 Inositol 5.0
Sucrose 194.2 4.0 776.8 Choline chloride 75.0
Sodium acetateg/kg197.4
10.01,000.0*Amino 2.5 25.54,145.5(all Menadione 2.25
p-Aminobenzoic acid 5.0
Niacin 4.5
mixtureArginine acid
Riboflavin 1.0
L)11.7 Pyridoxine hydrochloride 1.0
Thiamin hydrochloride 1.0
•
HC1 Calcium pantothenate 3.0
Histidine-HCl 5.3 Biotin 0.02
Isoleucine 12.4 Folie acid 0.09
Leucine 18.3 B-12ttSalt
Vit. 0.00135g/kg
Lysine -HC1 14.2
Methionine 4.7
Phenylalanine 10.1 (13)]Ammonium
mix [Rogers and Harper of
mix0.030
salt
Threonine 8.5
Tryptophan 2.5
Valine 12.0 molybdate-4H»O
Glutamic acid 46.2 Calcium carbonate 292.900
Glycine 9.9 Calcium phosphate -2Hi¡0 4.300
Serine 5.8 Cupric sulfate 1.560
Tyrosine 7.4 Ferric citrate -6H2O 6.230
Cys teine 3.4 Magnesium sulfate'THjO 99.800
Aspartic acid 13.4 Manganese sulfate -H^O 1.210
Proline 2.1 Potassium iodide 0.005
Asparagine 6.0 Potassium phosphate 343.100
Alaninekcal/g 3.5197.4fVitamin Sodium chloride 250.600
Sodium selenite -SH^O 0.020
Zinc chlorideg/kg 0.200
1L-amino acids, vitamin diet fortification mixture, salt mixture, dextrin, and sucrose were purchased from Nutritional
Biochemicals Corp., Cleveland, Ohio. Corn oil and agar were purchased from General Biochemicals, Chagrin Falls, Ohio.
Lard was Farmer John brand (Clougherty Packing Co., Los Angeles, Calif.). Sadium acetate was purchased from J. T.
Baker Chemical Co., Phillipsburg, N. J. The diet was prepared for feeding by mixing with an equal weight of water.

omitting methionine (AA —Met). For
comparison, the complete AA diet was fed
for the same time period. The diet omitting
lysine ( AA —Lys ) was fed during the
entire pregnancy ( days 0 to 21 ). The group
fed the complete AA diet during the entire
pregnancy was used as a comparison for
the AA —Lys group. The groups fed the
complete AA diet either for the entire
pregnancy or for the second hau were com
pared to those fed the stock diet.
The composition of the AA diet is repre
sented in table 1.
The food intakes per 24 hours were mea
sured for all animals, by feeding known
amounts and weighing the remainder (in
cluding any spilled food). These food in
takes for all groups are listed in table 2.
After delivery, the mothers were dis
sected and their uteri examined for résorp
tion sites. The newborns were weighed
and decapitated within 6 hours after de
livery. The brains (cerebral hemispheres,
without cerebellum and olfactory lobes)
were immediately removed and weighed;
they were then frozen and subsequently
used for analysis of DNA and protein con
tent. In order to recover placentas, in some
animals the offspring were removed by
cesarean section approximately 6 hours be
fore delivery. The placentas were weighed
and then frozen for analysis.
1004 ZAMENHOF ET AL.

TABLE 2 (compared to complete AA), had no sig

Food intakes during pregnancy nificant effect on body weights on day 20.
On the other hand, the omission of trypto
Total pregnancy phan or methionine produced significant
intake1/ deficiencies in maternal body weights on

Downloaded from https://academic.oup.com/jn/article-abstract/104/8/1002/4776549 by Rudolph Matas Medical Library user on 20 January 2019
hrStock015.1
24 amino
acids1 day 20. Similar results were obtained for
Diet1StockAAAAAA-
period2days0-2110-210-2110-210-2110-21Food kcal"1046
intakeg57.7±5.752.0maternal liver weights (day 21; not shown
in table 3), except that the deficiencies
produced by omission of tryptopan or
±1.511.8 ±104983methionine were only on the borderline of
significance (0.05 > P > 0.02).
±1.513.0 ±1.953.8±381046 The effects of the various amino acid test
±0.9—12.8
diets on neonatal parameters are repre
±130818sented in table 4. The results indicate that
±1.69.4 ±6.743.5±7.446.9
when the complete AA diet was fed during
TrpAA-LysAA-MetTest ±1.7—14.0 ±3.012.2 ±144981the entire pregnancy or during the last half
of pregnancy, fetal growth was signifi
±1.410.4 ±5.347.6 ±110900cantly inferior to that when the pelleted
stock diet was fed with respect to body
±2.3AAa—14.1
±1.4Total±6.7Total±122 weight and some brain parameters. How
ever, there were no deleterious effects on
'Stock: Normal stock pelleted diet (see Text). AA: Diet placental weight, placenta! DNA or pla-
synthetic with respect to amino acids. AA —Trp : As above,
with tryptophan omitted. aPeriod of pregnancy when diet cental protein content. The decrease in
used ; stock diet at other times. 3 ±SD.
cerebral DNA was not significant for 0 to
21 feeding days but was significant for 10
DNA was determined by a modification to 21 days: however, this may only mean
of the diphenylamine colorimetrie method that with respect to cerebral DNA the
(5, 6); protein was determined by a modi complete AA diet was on the borderline of
fication of the Lowry colorimetrie method adequacy, and may or may not result in
(7). cerebral DNA deficiency.
The single omission of any of the three
RESULTS essential amino acids gave highly signifi
Maternal body weights during preg cant decreases in neonatal body weight
nancy are represented in table 3. The com and all cerebral parameters when com
plete AA diets, fed 10 to 21 days or 0 to 21 pared with their respective full AA diet
days (compared to stock diet), as well as control. Placental weight and DNA were
lysine-déficientAA diet fed 0 to 21 days significantly lower for AA —lysine and
AA —methionine only, but otherwise, there
were no substantial differential effects be
TABLE 3 tween the omission of any of the three
Maternal body weights during pregnancy amino acids tested.
Since 80 to 10090 of the females main
Maternal body weights tained pregnancy, it was not necessary to
pregnancy00219 of administer ovarian steroids. This 20% fail
DietiStockAAAAA ure to maintain pregnancy was within the
normal range and therefore, was not at
tributable to the AA test diets.
±23220 ±24247 ±27275 ±28301
±18242 ±20274 ±13292
±16218±25214
±26239 ±47234 ±41241* DISCUSSION
±32232 ±39259
±25224 ±26226±11232
A-TrpAA—LysAA-MetNo.104138g9Testperiod^days0-2110-210-2110-210-2110-21Day
The purpose of this work is to provide
±15206 ±18236 ±38249*
information on two subjects:
±29
±28150268 ±2720(7310
±22100245 1. The feasibility of devising a synthetic
diet that is equivalent to a natural stock
'-' As in table 2. * The deficiency of body weight com diet supporting fetal development, espe
pared to AA (diet 10-21) statistically significant at P < 0.01
level. cially prenatal brain development.
 AMINO ACIDS AND BRAIN DEVELOPMENT 1005

f psf s s

Downloaded from https://academic.oup.com/jn/article-abstract/104/8/1002/4776549 by Rudolph Matas Medical Library user on 20 January 2019
-"-
OH

-H

O
co>oö

oo oo^l öo oo^l

0 oofl«
vT ' v
1
'2
a

g
O

3 .1
£2 CO•—
IQ »-O •—
'C

i-«
o
öo' ^¡qo'O
ööv l rt o °t-
öövl H 5__
ööv' rHOOOO
öövl
•w
'S
* ""ft, ^ ^
^ ""

e
-.006? o^i
•8-1
i irf rH O !ffl ^- —: Tt<ÖOO

ft,

%
o s 2
Q / o
03
1006 ZAMENHOF ET AL.
2. The effect of omission of single essen
tial amino acids on fetal development.
With respect to the first subject there is
very little information in the literature.
The possibility of some unknown nutri
tional factors cannot be excluded (8). In
fact, one such factor has been reported re
cently (9) for weanling rats; there are no
corresponding reports for prenatal devel
opment in general, or prenatal brain de
velopment in particular.
Our results (table 4) do not exclude the
need for some unknown growth factors
since our diet AA was inferior to the natu
ral stock diet. Obviously, much more work
will be needed for a definite answer.
Before 1950 many synthetic diets were
unsuccessful because L-amino acids were
contaminated with D isomers which are
known to be inhibitory ( 10 ). Recently sev
eral improved synthetic diets have been
proposed for weanling rats (8-17). In pre
liminary experiments we found that these
diets did not support optimum weanling
growth in our rats, indicating the possi
bility of strain differences. A proper formu
lation of a synthetic diet is important be
cause the levels of dietary amino acids
influence the size of the free amino acid
pool, which, in turn, could be one of the
principal regulators of protein synthesis in
the cell and thus regulate development
(18). With respect to pregnant rats, the
published diets were mainly studied for
the effects on maintenance of pregnancy
(16, 17). Niiyama et al. (16) found that
pregnancy was maintained in 100% of the
animals receiving the complete semipuri-
fied amino acid diet ad libitum. It is of
interest that, when one essential amino acid
( with the exception of lysine ) was omitted,
the proportion of résorptionswas as high
as 100%. When daily subcutaneous injec
tions of steroids were given, pregnancy
was maintained in all animals. These
authors have demonstrated that among
others, omission of any of the three essen
tial amino acids gave a decrease in neo
natal body weight and placental weight,
and that the decrease in body weight was
most pronounced in case of methionine
(16) or tryptophan (17), and least pro
nounced in case of lysine (16, 17). To our
knowledge, the omission of single essential
amino acids from the diet of pregnant rats
was studied only in this work. Brain devel
opment of the offspring was not studied.
The amino acid composition of our AA
diet was based on the stock diet since none
of the other diets tested in our laboratory
Downloaded from https://academic.oup.com/jn/article-abstract/104/8/1002/4776549 by Rudolph Matas Medical Library user on 20 January 2019
gave comparable weanling growth rates.
The amounts of aspartic acid and proline
were the same as those used by Swendseid
(19), and those of alanine and asparagine
were the same as used in the Rogers and
Harper (13) diet. Our AA diet was tested
on weanling rats for 30 days; the growth
curves were comparable to those obtained
with the stock diet. In contrast, when a
commercially available synthetic amino
acid diet4 was fed to weanling rats, a de
creased somatic growth was observed.
Our AA diets deficient in one amino acid
did not require steroid supplementation
for maintenance of a pregnancy. In addi
tion, the body weights of the offspring of
rats fed our complete AA diet were sub
stantially higher than those of Niiyama et
al. at comparable times. These differences
are probably due to a different ratio of
amino acids present in the diet; however,
the difference in rat strain also cannot be
excluded.
Contrary to Niiyama et al. we found
lysine omission to be as harmful to neonatal
development as the omission of tryptophan
or methionine. In addition, as far as placen
tal parameters are concerned, the omission
of lysine was more harmful than the omis
sion of tryptophan or methionine. On the
other hand, our data in general do confirm
the original finding (16) that omission of
these single essential amino acids results
in lower neonatal body weights, and, ex
cept for tryptophan, also lower placental
weights. In addition, we found lower neo
natal cerebral weight, cerebral DNA (cell
number ) and cerebral protein. The placen
tal weights and their DNA and protein
content were decreased in all diets omit
ting any of the essential amino acids tested,
when compared with their respective com
plete AA diet, but the decreases were sig
nificant generally only in the case of omis
sion of lysine and methionine.
Of some interest is the fact that the omis
sion of tryptophan was not the most harm
ful. This essential amino acid was believed
«General Blochemicals, Chagrin Falls, Ohio, Diet
no. 176140.
 AMINO ACIDS AND BRAIN DEVELOPMENT
to have a regulatory function since its de
ficiency in vivo causes disaggregation of
polysomes (20, 21 ). However, further work
has demonstrated that the polysome pro
files in the liver of the rat can also be in
fluenced by other amino acids provided
that a sufficiently severe amino acid de
ficiency can be created (22).
The omission of single essential amino
acids produced deficiencies essentially sim
ilar to those produced by total protein
deprivation in the comparable period of
pregnancy (4). Thus, the absence of single
dietary essential amino acids, even during
half of pregnancy, may be as harmful as
total absence of dietary protein during this
period, at least as far as prenatal brain de
velopment is concerned. Obviously, more
study is required before this view may be
fully accepted. However, such a study ap
pears to be of importance in view of well
known single or double essential amino
acid deficiencies in many foods of plant
origin.
ACKNOWLEDGMENTS
The authors wish to thank Mrs. Juanita
Garcia and Mrs. Facie Miles, U.C.L.A. for
their excellent technical assistance, and
Dr. Sanford A. Miller, Massachusetts Insti
tute of Technology, for his advice in the
course of these experiments.
LITERATURE CITED
1. Zamenhof, S., van Marthens, E. & Margolis,
F. L. (1968) DNA (cell number) and pro
tein in neonatal brain: alteration by maternal
dietary protein restriction. Science 160, 322-
323.
2. Zamenhof, S., van Marthens, E. & Grauel, L.
(1971) DNA (cell number) in neonatal
brain: Second generation (F2) alteration by
maternal dietary protein restriction. Science
172, 850-851.
3. Zamenhof, S., van Marthens, E. & Grauel, L.
(1972) DNA (cell number) and protein in
rat brain. Nutr. Metabol. 14, 262-270.
4. Zamenhof, S., van Marthens, E. & Grauel, L.
( 1971 ) DNA ( cell number ) and protein in
neonatal rat brain: Alteration by timing of
maternal dietary protein restriction. J. Nutr.
101, 1265-1269.
5. Zamenhof, S., Bursztyn, H., Rich, K. & Zamen
hof, P. J. (1964) The determination of de-
oxyribonucleic acid and of cell number in
brain. J. Neurochem. 11, 505-509.
6. Zamenhof, S., Grauel, L., van Marthens, E. &
Stillinger, R. A. (1972) Quantitative deter
1007
mination of DNA in preserved brains and
brain sections. J. Neurochem. 19, 61-68.
7. Lowry, O. M., Rosebrough, N. J., Farr, A. L.
& Randall, R. J. (1951) Protein measure
ment with the Folin phenol reagent. J. Biol.
Chem. 193, 265-275.
Downloaded from https://academic.oup.com/jn/article-abstract/104/8/1002/4776549 by Rudolph Matas Medical Library user on 20 January 2019
8. Salmon, W. C. (1964) Relative potency of
diets containing amino acids and proteins for
promoting growth of rats. J. Nutr. 82, 76-82.
9. Schwarz, K. (1970) An agent promoting
growth of rats fed amino acid diets. J. Nutr.
100, 1487-1499.
10. Maruyama, K., Sunde, M. L. & Harper, A. E.
( 1972 ) Effect of D-alanine and o-aspartic
acid on the chick. J. Nutr. 102, 1441-1451.
11. Greenstein, J. P., Otey, M. C., Birnbaum,
S. M. & Winitz, M. ( 1960) Quantitative nu
tritional studies with water-soluble, chemi
cally defined diets. X. Formulation of a nu
tritionally complete liquid diet. J. Nat. Cancer
Inst. 24, 211-219.
12. Clark, A. J., Peng, Y. & Swendseid, M. E.
(1966) Effect of different essential amino
acid deficiencies on amino acid pools in rats.
J. Nutr. 90, 228-234.
13. Rogers, Q. R. & Harper, A. E. (1965) Amino
acid diets and maximal growth in the rat. J.
Nutr. 87, 267-274.
14. Ranhotra, G. S. & Johnson, B. C. (1965)
Effect of feeding different amino acid diets on
growth rate and nitrogen retention of wean
ling rats. Proc. Soc. Exp. Biol. Med. 118,
1197-1201.
15. Greenstein, J. P. & Winitz, M. (1971)
Chemistry of the Amino Acids, vol. 1, J.
Wiley & Sons, Inc., New York, pp. 397-402.
16. Niiyama, Y., Kishi, K. & Inone, G. (1970)
Effect of ovarian steroids on maintenance of
pregnancy in rats fed diets devoid of one
essential amino acid. J. Nutr. 100, 1461-1469.
17. Niiyama, Y., Kishi, K. & Inone, G. (1973)
Effects of diets devoid of one essential amino
acid on pregnancy in rats maintained by
ovarian steroids. J. Nutr. 103, 207-212.
18. Miller, S. A. (1970) Nutrition in the neo
natal development of protein metabolism. Fed
eration Proc. 29, 1497-1502.
19. Swendseid, M. E., Villalobos, J. & Friedlich,
B. ( 1963 ) Ratios of essential-to-nonessen-
tial amino acids in plasma from rats fed dif
ferent kinds and amounts of proteins and
amino acids. J. Nutr. SO, 99-102.
20. Fleck, A., Shepherd, J. & Munro, H. N.
(1965) Protein synthesis in rat liver: Influ
ence of amino acids in diet on microsomes
and polysomes. Science 150, 628-629.
21. Sidransky, H., Bongiorno, M., Sarma, D. S. R.
& Verney, E. ( 1967 ) The influence of tryp-
tophan on hepatic polyribosomes and protein
synthesis in fasted mice. Biochem. Biophys.
Res. Commun. 27, 242-248.
22. Pronczuk, A. W., Rogers, Q. R. & Munro,
H. N. (1970) Liver polysome patterns of
rat fed amino acid imbalanced diets. J. Nutr.
100, 1249-1258.