Shuji MATSVEDA and Yoshiaki NIIYAMA1

J. Nutr. Sci. Vitaminol., 28, 557-573, 1982

The Effects of Excess Amino Acids on Maintenance

of Pregnancy and Fetal Growth in Rats

Shuji MATSVEDA and Yoshiaki NIIYAMA1

Department of Nutrition, School of Medicine, University of Tokushima,

Kuramoto-cho, Tokushima, Tokushima 770, Japan

(Received June 17, 1982)

Summary The effects of diets, each with an excess of one essential

amino acid, on the maintenance of pregnancy and fetal growth were

investigated in rats. Rats were fed on 6% casein diet containing 5%, thre

onine, methionine, valine, isoleucine, leucine, tyrosine, phenylalanine,

tryptophan, or lysine from day 1 to day 14 or 21 of pregnancy. Excess

methionine and leucine diets resulted in complete and 80% loss of fetuses,

respectively. This fetal wastage was prevented by daily injection of 0.5ƒÊg

of estrone and 4mg of progesterone. Judging from the total food

consumptions and body weight gains during pregnancy, methionine had

the most severe effects, followed in order by leucine, tryptophan, valine,

lysine, isoleucine, threonine, phenylalanine, and tyrosine. The weights of

fetuses in the excess amino acid groups were significantly lower than those

in the respective pair-fed controls. Excess aromatic amino acids caused

growth retardation of fetal brain, although the levels of free tyrosine and

phenylalanine in fetal brain were not high. The concentrations of free

methionine and threonine were markedly elevated in the maternal plasma

when these amino acids were fed in excess, but those of other amino acids

were not increased appreciably by excess amounts in the diet. Changes in

the maternal plasma levels of individual amino acids other than those in

excess in the diet were small. On the contrary, the levels of not only the

excess amino acids but also of other amino acids in fetal brains were

appreciably elevated by these diets. These findings suggest that the blood

brain barrier is immature and that the synthesis of proteins in fetal brain is

impaired by excess amino acids in the mothers. The importance of

experiments on diets with excess of single amino acids in pregnant animals

is discussed in connection with studies on inborn errors of amino acid

metabolism.

Key Words amino acid excess, pregnancy, fetal brain growth, inborn

error of metabolism

1 松枝秀二,新山喜昭

557

558 S. MATSUEDA and Y . NIIYAMA

Although addition of an appropriate amount of the first limiting amino acid to diet containing poor-quality proteins is beneficial, addition of excess of the amino acid has adverse effects. Thus it is important to know the toxic levels of individual essential amino acids for the determination of appropriate supplementary levels. Furthermore, experimental hyperaminoacidopathies observed when individual amino acids are fed in excess may give some information on inborn errors of amino acid metabolism in which the presence of a single amino acid or its metabolites in excess in the plasma is associated with mental retardation (1-3) . From these two standpoints, the effects of high levels of single amino acids in the diet have been investigated extensively in animals, and particularly in growing rats (4-6). But the relative toxicities of single amino acids in pregnant rats have not been reported (711). Thus, in the present study we examined the effects of dietary excess of single essential amino acids on maintenance of pregnancy and reproductive performance, including fetal growth.

MATERIALS AND METHODS

Virgin Sprague-Dawley rats, weighing about 160g, were obtained com

mercially, and given 20% casein diet until they weighed about 190g. They were then

mated with males of the same strain. Day 1 of pregnancy was determined by daily

examination of vaginal smears. Pregnant rats were fed ad libitum on 6% casein diets

each with an excess of one of the following amino acids: methionine, lysine,

threonine, leucine, isoleucine, valine, tryptophan, phenylalanine, or tyrosine from

day 1 of pregnancy to the day of autopsy. Amino acid-excess diets were prepared by

adding 5% of an L-amino acid at the expense of ƒ¿-starch in the 6% casein diet. Four

groups of control rats (groups A to D) received 6% casein diet ad libitum and in

amounts of 12g, 9g, and 5g per day, respectively. The latter three groups were used

as pair-fed controls: group B for groups given excess tyrosine, threonine, phenyl

alanine, isoleucine, and lysine, group C for those given excess valine and tryptophan

and group D for those given excess leucine and methionine.

Two experiments were carried out (Experiments 1 and 2). In Experiment 1, the

effect of amino acid-excess diets on maintenance of pregnancy was examined . For

this purpose pregnant animals receiving the experimental diets were killed on day 14

of pregnancy and the products of conception (uterus, amniotic fluid, placenta, and

fetuses) were weighed, because nutritionally induced fetal resorption begins on

about day 10 of pregnancy (12). In Experiment 2, the effects of excess of single

amino acids on the nitrogen balance of the dams, the free amino acid concentrations

in maternal plasma and fetal brain and fetal growth were examined. All pregnant

animals, except those in groups B, C, and D, were given basal or experimental diet ad

libitum during the 21-day period of pregnancy. Fetal resorption caused by reduced

food intake was prevented by injecting 0.5ƒÊg of estrone and 4mg of progesterone

daily.

The urine and feces were collected at 3-day intervals throughout pregnancy to

J. Nutr. Sci. Vitaminol.

EFFECTS OF EXCESS AMINO ACIDS IN PREGNANCY 559

determine nitrogen balance. Food intake and body weight were measured daily. On

day 22 of pregnancy, food was withheld for 12hr, the animals were anesthetized,

blood samples were withdrawn for determination of free amino acids and the

animals were then killed. The products of conception were removed and weighed.

The brains of fetuses were extirpated and stored in a deep freezer for determination

of nucleic acids, protein and free amino acids. The carcasses were dried to constant

weight at 105•Ž and then finely ground to determine lipid and nitrogen contents.

RNA and DNA in fetal brain were measured spectrophotometrically (13).

Protein was determined by the method of Lowry et al. on the 0.3 N KOH

hydrolysate obtained during nucleic acid determination (14). A semi-micro Kjeldahl

method was used for determination of nitrogen in the carcass, urine and feces. The

lipid content of the dried carcass was determined gravimetrically after extraction

with ethyl ether in a Goldfisch apparatus. Free amino acids in the maternal plasma

and fetal brain were determined using an automatic amino acid analyzer (IRICA,

Model A-3300).

RESULTS

Maintenance of pregnancyPregnancy was maintained in 100% of the rats on diets with an excess of amino

acids other than isoleucine, tryptophan, leucine, and methionine. Complete and 80% losses of fetuses were observed in rats receiving excess methionine and leucine, respectively (Table 1), however, daily injections of estrone and progesterone into these animals resulted in growth of conceptuses, as shown in Experiment 2.

Food intake and change in body weight

Table 2 shows the total food consumptions and changes in body weight of the

Table 1. Effects of single essential amino acids on maintenance of pregnancy in rats fed on a 6% casein diet.

Vol. 28, No. 5, 1982


Table 2. Food intake and change in body weight .

a Rats of control groups A, B, C, and D were given 6% casein diet ad libitum, 12g/day,

9g/day, and 5g/day, respectively. Control B for rats fed on diets with excess tyrosine,

phenylalanine, threonine, isoleucine, and lysine. Control C for rats fed on diets with

excess valine and tryptophan. Control D for rats fed on diets with excess leucine and

methionine. b Values represent mean•}SD, c, d Significantly different from control group

A at levels of 5% and 1%, respectively. e Significantly different from control group C at a

level of 5%. f Significantly different from control group D at a level of 1% .

respective groups of animals during gestation (Experiment 2). Control rats fed ad libitum (group A) consumed a total of 328g of 6% casein diet . All groups given excess amino acids ate less than group A, their intakes ranging from 276g for tyrosine-excess diet to 91g for methionine-excess diet. During pregnancy , daily food intakes initially increased gradually, but near term they decreased appreciably in all

pregnant rats. According to their total food consumptions, animals receiving excess amino acid diets were divided into three groups and groups B, C, and D were

prepared as the respective pair-fed controls (see MATERIALS AND METHODS). Control groups A, B, C, and D of rats gained averages of 68g, 44g, 26g, and

-20g body weight , respectively, during gestation. Most of the rats given diets with an excess of amino acids other than methionine or leucine also gained weight, the gains ranging from 63g with phenylalanine excess to 34g with tryptophan excess, depending on the food intakes. The weight gains in the experimental groups were almost comparable to those of the respective pair-fed controls. The rats fed methionine and leucine-excess diets lost body weight and the decrease in weight of the former was greater than that of pair-fed control (group D). The maternal body weights in most of the experimental groups at term, estimated by subtracting the


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weight of conception products from the total body weight, were lower than the

initial body weights, indicating that fetal growth occurred at the expense of the

maternal body.

Nitrogen balanceData on nitrogen balance determined every three days during two periods of

pregnancy (days 1-16 and 17-22) are shown in Table 3. Control animals other than group D retained nitrogen throughout the entire period and the increments in nitrogen retention depended on the nitrogen intakes. Although the nitrogen intakes of rats on amino acid-excess diets were considerably larger than those of the respective pair-fed controls, the nitrogen retentions of these groups were comparable to, or less than, those of the controls, indicating that increased degradation and/or excretion of the added amino acids had occurred. Rats receiving diets containing excess leucine or methionine and rats in control group D showed negative nitrogen balances, due mainly to their low food intakes. Values for nitrogen balance in all animals during pregnancy were closely related to weight

Table 4. Effects of single essential amino acids in excess on the maternal body

composition.

a Fat -free carcass mass . b Rats of control groups A, B, C, and D were given 6 % casein

diet ad libitum, 12g/day, 9g/day, and 5g/day, respectively. Control B for rats fed on diets

with excess tyrosine, phenylalanine, threonine, isoleucine and lysine. Control C for rats

fed on diets with excess valine and tryptophan. Control D for rats fed on diets with

excess leucine and methionine. C Values represent mean•}SD. d, e Significantly different

from control A at levels of 5% and 1%, respectively. f, g Significantly different from

control C at levels of 5% and 100, respectively.



gains in the 21-day period.

Gross carcass compositionsTable 4 shows data on the gross carcass compositions on day 22 of pregnancy.

The fat contents in the excess-amino acid and control rats were closely related to total food consumptions during pregnancy, ranging from 28.1g (15.2%) for control

group A to 5.6g (5.5%) for rats on excess methionine diet. Although the percentages of protein and water in the carcasses differed in different groups, the percentages of both components per 100g of lean body mass were relatively constant.

Reproductive performancesThe reproductive performances on day 22 of pregnancy in rats fed control or

excess diets are shown in Table 5. The products of conception were significantly lighter in control group D than in control groups A, B, and C, but those of the latter

Table 5. Reproductive performances on day 22 of pregnancy of rats given diets with

excess of single essential amino acids.

a Products of conception consist of the uterus, placenta, fetus, and amniotic fluid. b Rats

of control groups A, B, C, and D were given 6% casein diet ad libitum, 12g/day, 9g/day,

and 5g/day, respectively. Control B for rats fed on diets with excess tyrosine,

phenylalanine, threonine, isoleucine, and lysine. Control C for rats fed on diets with

excess valine and tryptophan. Control D for rats fed on diets with excess leucine and

methionine. c Values represent mean•}SD. d, e Significantly different from control A at

levels of 5% and 1%, respectively. f, g Significantly different from control B at levels of 5%

and 1%, respectively. h Signiffiicantly different from control C at a level of 5%.

i, j Significantly different from control D at levels of 5% and 1%, respectively.

Val. 28, No. 5, 1982

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three groups were comparable. In all animals given excess amino acids other than

threonine and tryptophan, the weights of conception products were similar to those

of the corresponding pair-fed controls. But in most groups given excess amino

acids, the weights of fetuses were significantly lower than those of the respective

controls. The litter size was almost normal in all rats on excess amino acid diets,

except that of rats on excess threonine diet, in which some fetuses were resorbed.

These results show that fetal growth was influenced by the quality of the diet as well

as its amount, and that threonine-excess had a special effect.

Growth of fetal brainGrowth of fetal brain is shown in Table 6 in terms of weight, protein and

nucleic acid contents. As shown above (Table 5), fetal growth was impaired by food restriction and excess of single amino acids in the maternal diet. To examine the adverse effects of amino acid excess on growth of fetal brain, we plotted fetal brain weight (Y: mg) against fetal weight (X: g) (Fig. 1). In the four control groups, the brain weight was proportional to the fetal body weight, the relation conforming to the following equation:

Y=14.09X+38.3 (n:48, r=+0.91, p<0.001)

Similar plots for rats on diets with excess of amino acids other than tyrosine and

phenylalanine also fitted this regression line; with excess tyrosine or phenylalanine values fell below this line. This finding indicates that prenatal brain growth is

Fig. 1. Relation between fetal weight and fetal brain weight. •œ, control A (n =14); •£,

control B (n=10); •›, control C (n=10); •¡, control D (n=14). Values for excess

amino acid groups (1-9) are expressed as means•}SD. 1, Tyr (n=7); 2, Phe (n=8);

3, Ile (n=10); 4, Thr (n=12); 5, Lys (n=11); 6, Val (n=10); 7, Trp (n=14); 8, Leu

(n=10); 9, Met (n=10).

Vol. 28, No. 5, 1982


severely and specifically affected by excess of aromatic amino acids.The total amounts of DNA in the brain of fetuses in control groups A and B

were identical, but those in control groups C and D and all groups given excess amino acids were significantly lower. Moreover , values for total DNA in the brain of rats given excess amino acids other than methionine , leucine, and valine were significantly lower than those for the respective controls . Due to decreases in cerebral weight and protein concentration , the total amounts of brain protein in control groups B, C, and D were proportional to the food intakes . The total amounts of brain proteins in rats on excess amino acid diets were considerably lower than those of the respective pair-fed controls , with some exceptions. The RNA/DNA ratio of fetal brain was signicantly lower in control group D than in

groups A, B, and C. However, the ratios in groups given excess amino acids were higher than those in the respective pair-fed controls. These results suggest that maternal food restriction or excess of single amino acids during pregnancy impaired

proliferation and/or protein synthesis in fetal brain cells and that amino acid excess had more affected brain cell proliferation than protein synthesis .

Free amino acid concentrations in maternal plasma and fetal brainDespite differences in food intakes, the patterns and concentrations of free

amino acids in maternal plasma in the four control groups were similar . This was also the case for brain amino acids in their fetuses . Thus, combined data for the four

Table 7. Free amino acid concentrations in fetal brain and plasma of pregnant rats fed on different amounts of 6% casein diet.

Values represent means of 16 rats with SD .



Fig. 2. Relative amino acid concentrations of maternal plasma and fetal brain in rats fed on 6% casein diet containing 5% methionine .

Fig. 3. Relative amino acid concentrations of maternal plasma and fetal brain in rats fed on 6% casein diet containing 5% threonine .

control groups are shown in Table 7. The ratios of amino acid concentrations in fetal brain to those in maternal plasma were about 2 to 3 for lysine, arginine, histidine, methionine and branched chain amino acids (BCAAs) , and about 5 to 8 for serine, threonine, glycine, alanine, and aromatic amino acids.

Figures 2 to 10 show the relative changes in amino acid concentrations in maternal plasma and fetal brain in rats given amino acid-excess diets ad libitum during pregnancy. In general, the concentrations of free amino acids increased in both the plasma and brain and the alterations were far greater in brain than in

plasma.

Vol. 28, No. 5, 1982

568 S, MATSUEDA and Y. NIIYAMA

Fig. 4. Relative amino acid concentrations of maternal plasma and fetal brain in rats fed on 6% casein diet containing 5% isoleucine.

Fig. 5. Relative amino acid concentrations of maternal plasma and fetal brain in rats fed on 6% casein diet containing 5% leucine.

Fig. 6. Relative amino acid concentrations of maternal plasma and fetal brain in rats fed on 6% casein diet containing 5% valine.

J. Nutr. Sci. Vitaminole


Fig. 7. Relative amino acid concentrations of maternal plasma and fetal brain in rats fed on 6% casein diet containing 5% lysine.

Fig. 8. Relative amino acid concentrations of maternal plasma and fetal brain in rats fed on 6% casein diet containing 5% tyrosine.

Fig. 9. Relative amino acid concentrations of maternal plasma and fetal brain in rats fed on 6% casein diet containing 5% phenylalanine.

Fig. 10. Relative amino acid concentrations of maternal plasma and fetal brain in rats fed on 6% casein diet containing 5% tryptophan.

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570 S. MATSUEDA and Y. NIIYAMA

In rats receiving methionine-excess diet, the methionine concentration increased about 20-fold in the maternal plasma and 22-fold in fetal brain. In addition, moderate increases in aspartic acid, BCAAs, threonine and tyrosine in the plasma, and isoleucine, arginine and histidine in the brain were observed.

In rats given threonine-excess diet, the threonine concentration in the plasma and brain increased about 13-fold and 16-fold, respectively. This diet induced appreciable increases in cerebral amino acids other than phenylalanine, aspartic acid, and glutamic acid.

In rats given excess BCAAs, the plasma concentrations of the excess amino acids were comparable to those of controls, but the concentrations of brain amino acids were abnormal and most brain amino acids, particularly arginine, leucine, isoleucine, methionine, and histidine increased 5-8 times. Moreover, the changes in

patterns of brain amino acids were similar with excess of the respective BCAAs and excess threonine.

Ingestion of diet containing excess lysine or tyrosine did not result in appreciable alterations in plasma or brain amino acid concentrations.

No changes in maternal plasma or fetal brain amino acid concentrations were observed in rats given phenylalanine-excess diet, except that the histidine concentration in fetal brain increased about 8-fold (15).

Excess tryptophan diet resulted in no appreciable changes in free amino acids in the plasma but about 2 to 3-fold increases in acidic and aromatic amino acids in the brain.

DISCUSSION

The toxicities of dietary excesses of individual essential amino acids have been studied extensively in growing animals (4-6). Results have shown that excess amino acids all produce growth depression to varying degrees, depending on their degrees of excess and on the dietary protein level. However, only the effects of excess methionine, lysine, leucine, and aromatic amino acids in pregnant rats have so far been examined (7-11, 15). Judging from the effects on food consumption and body weight gain during pregnancy observed in the present work, methionine had the most severe effects, followed in decreasing order by leucine, tryptophan, valine, lysine, isoleucine, threonine, phenylalanine and tyrosine. The present findings that aromatic amino acids had the least effects on growth are quite different from those obtained in growing animals. Muramatsu et al. (6), for example, reported that 10% casein diet containing 5% tyrosine, phenylalanine, or methionine caused the most severe growth inhibition in growing rats. The reasons for this difference in the toxicities of phenylalanine and tyrosine in pregnant rats from those in growing rats are unknown, but could be due to differences in dietary needs for these amino acids or in their catabolic rates.

The fact that a dietary excess of one amino acid results in increase in its plasma level has been generally recognized. Peng et al. reported that the plasma levels of



excess amino acids were elevated 3hr after the force-feeding of young rats with liquid diets containing excess amino acids (16). In the present study, however, the maternal plasma concentrations of the amino acids present in excess in the diet were not markedly elevated, except in groups given excess methionine and threonine . This may in part be attributable to the decreased food intakes during the last 2 to 3 days of pregnancy. This problem requires further study.

Our findings that the concentrations of methionine and threonine in the plasma of rats given excess methionine and threonine, respectively, were very high agree with previous findings (16, 17) and suggest that the rates of disappearance of these amino acids from the amino acid pool by catabolism and/or excretion are low in

growing and pregnant animals. Changes in the plasma levels of amino acids other than those given in excess were not marked except in rats given excess methionine. In rats given excess methionine the normal balance of the free amino acid pool was considerably disturbed (Fig. 2), in contrast to the findings of Daniel and Waisman (3). This shows that excess methionine has various effects, as Benevenga

pointed out (18).The fetal brain concentration of the amino acid given in excess was highest in

the case of methionine, followed in order by threonine, leucine, isoleucine, and valine. In rats given excess lysine, tyrosine, or phenylalanine, the brain concentrations of these respective amino acids were comparable to those of the controls. Changes in the amino acid patterns in the brain were also slight in the latter rats. Peng et al. reported that when excess amino acids were given in the diet , the concentrations of most other amino acids in the brain decreased (16). High circulating levels of phenylalanine (2) or methionine (3) caused depletion of most free amino acids in the brain. On the contrary, in our study the concentrations of most amino acids were elevated in the brain of fetuses from dams given excess methionine, threonine, or BCAAs. Our findings may indicate immaturity of the blood-brain barrier and decreased protein synthesis in the brain of fetuses of rats

given excess amino acids. In addition, the changes in the amino acid patterns in the brain of fetuses of rats given excess threonine and BCAAs (Figs. 3-6) were similar, suggesting a relation between the metabolisms of these amino acids.

As shown in Table 5, the fetuses in most of the excess amino acid groups weighed significantly less than those of the respective pair-fed controls, indicating that excess of single essential amino acids in the maternal diet during pregnancy affects intrauterine fetal growth. Excesses of aromatic amino acids and threonine had specific and significant influences on fetal growth. Thus, although the mothers ate a relatively large quantity of these diets with excess amino acids, fetal growth , including fetal brain growth, was markedly impaired . The remarkably high threonine concentration in the plasma of rats given threonine-excess diet and in the brain of their fetuses may contribute in part to fetal growth retardation . However, this was not the case in rats on excess tyrosine or phenylalanine diet, in which the concentrations of these respective amino acids in the plasma and brain were almost normal. The exact mechanism of fetal brain damage in these animals , or the relation

Vol. 28, No. 5, 1982

572 S. MATSUEDA and Y. NIIYAMA

of excess amino acids to brain damage, is unknown and must be examined further by the procedures used in some previous investigations (19-22).

Finally the significance of experiments on feeding diets with excess of single essential amino acids must be considered in connection with studies on inborn errors of amino acid metabolism. In these anomalies, increased concentrations of the amino acids concerned or their metabolites are commonly found in body fluids in association with mental retardation. Consequently, diets with an excess of single amino acids have been used for determining the etiology of mental disorders in hyperaminoacidemic animals. Experiments on postweaning or young adult animals are meaningless because brain growth has already been completed in these animals. However, studies on the influences of hyperaminoacidemia in pregnant animals on neuronal development in their fetuses should be useful. Recently, Lenke and Levy (23) reported that untreated maternal phenylketonuria may result in non

phenylketonuric offspring with mental retardation, microcephaly, congenital heart disease, and low birth weight. They recommended that the best way of preventing these adverse effects on the offspring is dietary therapy before conception. Our results showed only the relative toxicities of single amino acids given in excess on fetal brain growth. The details of the mechanisms by which neuronal development is retarded and protein and nucleic acid syntheses in the brain are impaired by excess amino acids must be further investigated.

REFERENCES

1) Nyhan, W. L. (1981): Inheritable abnormalities of amino acid metabolism, in Amino Acid Metabolism and Sulphur Metabolism, ed. by Neuberger, A., Elsevier, New York, pp. 305-439.

2) McKean, C. M., Boggs, D. E., and Peterson, N. A. (1968): The influence of high phenylalanine and tyrosine on the concentrations of essential amino acids in brain. J.

Neurochem., 15, 235-241.3) Daniel, R. G., and Waisman, H. A. (1969): The influence of excess methionine or the

free amino acids of brain and liver of the weanling rat. J. Neurochem.,16, 787-795.4) Sauberlich, H. E. (1961): Studies on the toxicity and antagonism of amino acids for

weanling rats. J. Nutr., 75, 61-72.5) Daniel, R. G., and Waisman, H. A. (1968): The effects of excess amino acids on the

growth of the young rat. Growth, 32, 255-265.6) Muramatsu, K., Odagiri, H., Morishita, S., and Takeuchi, H. (1971): Effect of excess

levels of individual amino acids on growth of rats fed casein diets. J. Nutr.,101, 1117-1126.

7) Cohlan, S. Q., and Stone, S. M. (1961): Effects of dietary and intraperitoneal excess of L-lysine and L-leucine on rat pregnancy and offspring. J. Nutr., 74, 93-95.

8) Boggs, D. E., and Waisman, H. A. (1964): Influence of excess dietary phenylalanine on

pregnant rats and their fetuses. Proc. Soc. Exp. Biol. Med., 115, 407-410.9) Vian, A. T., Leathem, J. H., and Wannemacher, R. W., Jr. (1969): Pregnancy and

excess dietary methionine in rats. Fed. Proc., 28, 638.10) Knipfel, J. E., and Elliot, J. I. (1974): Gestational dietary methionine level as related to



growth and 35S-distribution in maternal and fetal rats. Nutr. Rep. Int., 10, 103-112.11) Kerr, G. R., Chamove, A. S., Harry, M. A., Harlow, H. F., and Waisman, H. A. (1968):

"Fetal" PKU: The effect of maternal hyperphenylalaninemia during pregnancy in the

rhesus monkey (Macaca mulatta). Pediatrics, 42, 27-36.12) Niiyama, Y., Sakai, K., Kishi, K., and Inoue, G. (1972): Effect of ovarian steroids

during early and mid pregnancy on uterine protein metabolism in malnourished rats. Jpn. J. Physiol., 22, 505-516.

13) Wannemacher, R. W., Jr., Banks, W. L., Jr., and Wunner, W. H. (1965): Use of a single tissue extract to determine cellular protein and nucleic acid concentrations and rate of

amino acid incorporation. Anal. Biochem., 11, 320-326.14) Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951): Protein

measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275.15) Carver, M. J., Copenhaver, J. H., and Serpan, R. A. (1965): Free amino acids in foetal

rat brain, Influence of L-phenylalanine. J. Neurochem., 12, 857-861.16) Peng, Y., Gubin, J., Harper, A. E., Vavich, M. G., and Kemmerer, A. R. (1973): Food

intake regulation: amino acid toxicity and changes in rat brain and plasma amino acids. J. Nutr., 103, 608-617.

17) Yamashita, K., and Ashida, K. (1971): Effect of excessive levels of lysine and threonine on the metabolism of these amino acids in rats. J. Nutr., 101, 1607-1614.

18) Benevenga, N. J. (1974): Toxicities of methionine and other amino acids. J. Agric. Food Chem., 22, 2-9.

19) Appel, S. H. (1967): Inhibition of brain protein synthesis: An approach to the biochemical basis of neurological dysfunction in the amino-acidurias. Trans. N. Y. Acad. Sci., 29, 63-70.

20) Rudolph, N., and Betheil, J. J. (1970): Protein synthesis in liver and brain microsomes isolated from rats fed a high phenylalanine diet. J. Nutr., 100, 21-29.

21) Battistin, L., Grynbaum, A., and Lajtha, A. (1971): The uptake of various amino acids by the mouse brain in vivo. Brain Res., 29, 85-99.

22) Korpi, E. R. (1981): Aromatic amino acid incorporation into proteins in rat brain slices: influence of other amino acids affecting transport. Acta Physiol. Scand.,112, 353-358.

23) Lenke, R. R., and Levy, H. L. (1982): Maternal phenylketonuria-Results of dietary therapy. Am. J. Obstet. Gynecol.,142, 548-553.

Vol. 28, No. 5, 1982

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Shuji MATSVEDA and Yoshiaki NIIYAMA1