Effects of maternal hyperphenylalaninemia on fetal brain development: A biochemical study

-AL

NJ3JR0LDoY

79.641-654

(1983)

Effects of Maternal Hyperphenylalaninemia on Fetal Brain Development: A Biochemical Study DAVIDA.SPJXROANDMANGC.YU~ Departmenr of Anatomy,NewJerseyMedical School,Universityof Medicineand Dentistryof NewJersey,Newark,NewJersey07103 Received March25, 1982;revisionreceived September 15,1982 We examined the effects of maternal hypetphenylalaninemia on body and brain growth, and the biochemical maturation of the fetal and neonatal rat brain. Elevated concentrations of plasma phenylalanine were induced in pregnant rats under two experimental conditions from the 14th through the. Zlst days of gestation. In the first treatment pregnant rats were injected subcutaneously with u-methylphenylalanine (to inhibit maternal liver phenylalanine hydroxylase) at a dosage of 30 mg/lOO g body weight plus phcnylalanine supplementation (to increase maternal and fetal plasma phenylalanine) at a dosage of 60 mg/ 100 g body weight two times daily. In the second treatment, pregnant dams were injected with phenylalanine only at a dosage of 65 mg/lOOg body weight three times daily. Treatment with a-methylphenylalanine/ phenylalanine (mPhe/Phe) resulted in a 76% inhibition in the activity of maternal phenylalanine hydroxybse and a 25-fold increase in the mean daily concentration of phenylalanine in the maternal and fetal plasma. Phenylalanine treatment alone resulted in a 1%fold increase in plasma phenylalanine in the maternal and fetal animals. Significant reductions in body and brain weights in the fetal and neonatal rats were found in both treatment groups. Biochemi~ determinations indicated that the total DNA, RNA, and protein contents of the cerebra were reduced, with the mductions being greater in the mPhe/Phe- than the phenylalanine-treated rats. However, the retardation in body and brain growth of both treatment groups did not appear to be permanent because substantial recovery was noted in the rats alter postnatal day 7. These results suggest that exposure of the fetus to high plasma concentrations of phenylalanine caused a delay in the biochemical matumtion of the fetal rat brain. Abbreviations: Phe, mPhe-phenylalanine, a-methylphenyhtlanine; PKU-phenylketonuria; PCPA-p-chlorophenylalanine; E, P-embryonic, postnatal days; PheH-phenylalanine hydroxylase. ’ This work was supported by General Biomedical Rematch Support Fund from New Jersey Medical School and by National Institutes of Health grant HDl2089. Dr. Spero’s present address is Department of Anatomy, Rutgers Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854. 641 00144886/83/030641-14SO3.00/0 com?ight 0 1983 by Aadcmi Plw, Inc. All rights of mpoduction in any form rtaerwd

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INTRODUCTION Phenylketonuria (PKU) is an inherited metabolic disease of man characterized by a reduced activity in phenylalanine hydroxylase (PheH), an hepatic enzyme necessary for the obligatory conversion of phenylalanine (Phe) to tyrosine (14). The defect in PheH reduces the amount of Phe converted to tyrosine and leads to an elevated amount of Phe in the blood and tissues (20). PKU was one of the first metabolic diseases in which severe mental retardation and seizures could be linked to an enzymatic deficiency (18). The successful treatment of newborn individuals with PKU by dietary restriction of Phe during early childhood has led to an increase in the number of PKU women of child-bearing age in the population. Alter discontinuing the dietary treatment, pregnant PKU women frequently bear offspring who exhibit mental deficiency, retarded growth of the body and brain, and other malformations (22,27,38). These defects, occurring in offspring with normal amino acid metabolism, are believed to be due to the cumulative effects of an imbalance in the amino acid pools in the fetus as a result of elevated rates of transport of Phe across the placenta (17). Although the causes of these brain deficits await elucidation, high concentrations of Phe have been shown to restrict, by competitive inhibition, the transport of large neutral amino acids into the brain (33) and across the placenta ( 17), and cause disaggregation of brain polyribosomes (8, 38) and decreased synthesis of brain proteins (1, 1%. In the past, the experimental approach to produce a phenylketonuric state in animal models has involved the use of para-chlorophenylalanine (PCPA), an inhibitor of PheH, plus Phe supplementation to produce a sustained high concentration of Phe in the plasma (3, 9, 24, 34). Recently, however, PCPA was shown to be toxic to cells in culture (19) and to produce side effects not associated with the phenylketonuric state in animals (21). Another PheH inhibitor, a-methylphenylalanine (mPhe) has been found to be more specific and less toxic to neonatal rats than PCPA (16). Thus, treatment with mPhe plus Phe supplementation is being used for studying the effects of postnatal hyperphenylalanemia on synaptogenesis (32), myelin deposition (1 l), and behavioral alteration (26). In the present study, we report on the effects of maternal PKU, induced by mPhe/Phe, or Phe alone, on the body and brain growth of fetal and neonatal rats, and the biochemical maturation of the brain by determining the total DNA, RNA, and protein contents. MATERIAL Dated pregnant Sprague-Dawley source. High plasma concentrations

AND

METHODS

rats were obtained from a commercial of Phe were induced during the period

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of embryonic days 14 through 21 under two experimental conditions. The first consisted of injecting pregnant dams SUbCUtaUeOUSly with DL-a-I&hylphenylalanine at a dosage of 30 mg/lOO g body weight plus c(-)-pheny&nine at a dosage of 60 mg/ 100 g body weight twice daily at 12-h intervals. The second treatment consisted of injecting pregnant dams subcutaneously with Phe at a dosage of 65 mg/lOO g body weight three times daily at 8-h intervals. The purpose of this latter treatment was to control for the nonspecific effects of mPhe by administering Phe in sufficient dosages to maintain plasma phenylalanine concentrations comparable to those in the mPhe/Phetreated group. For control, another group of pregnant dams was injected with the vehicle only, 1% carboxymethylcellulose, and pair-fed during the treatment period. Blood samples were obtained from the tail veins of pregnant dams on alternate days beginning on the 15th day of gestation. To collect blood from fetal rats, cesarean sections were carried out on randomly selected pregnant dams and fetal blood samples were obtained from decapitated fetuses by collecting the blood in heparinized tubes. Plasma Phe concentrations were determined according to the method of McCaman and Robins (29) and plasma tyrosine was assayed according to the method of Wong et al. (40). The activity of maternal liver PheH was assayed according to the method of McGee et al. (30). Fetal and neonatal rats were killed by decapitation under light ether anesthesia at 4’C on embryonic day (E) 19 and E2 1, and postnatal day (P) 1, P3, P5, P7, and P12. The heads were bissected sag&ally and the cerebra, consisting of the cortical gray matter and underlying white substance, were separated from the subccrtical structures by cutting along the ventricular surfaces. The tissues were homogenized in a chloroform-methanol solution (2: 1, v/v) and the lipids were extracted according to the method of Folch et al. (13). The residue obtained from the lipid extraction was resuspended in cold distilled water, and DNA and RNA were extracted by the combined methods of Schneider (36) and Schmidt and Thannhauser (35). DNA was assayed using the diphenylamine reaction of Burton (6) and RNA by the method of Lin and Schjeide (23). Deoxyadenosine and adenosine-5’-phosphate were used as standards for DNA and RNA, respectively. The method of Lowry et al. (25) was used for protein determinations using bovine serum albumin as a standard. Analysis of variance was used to detect a statistical difference among the treated and control.groups. Comparisons between individual groups were made using Scheffe’s test of multiple comparisons. RESULTS The mean daily concentration of Phe in the maternal plasma was increased approximately 25-times in the mPhe/Phe-treated group and 15-times in the

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TABLE 1 The Effect of Methylphenylalanine/Phenylalanine (mPhe/Phe) and Phenylalanine (Phe) Treatments on the Activity of Maternal Liver Phenylalanine Hydroxylase and the Average Concentration of Phe and Tyrosine in Maternal and Fetal Plasma

Treatment mPhe/Phe Phe

Percentage inhibition of maternal phenylalanine hydroxylase 76 (4)b -

Maternal phenylalanine nmol/mP

Fetal phenylalanine nmol/ml

Maternal tyrosine nmol/ml

Fetal tyrosine nmol/ml

1839 + 177**

3106 of:298**

186 + 42**

173 t- 48

(16) 1020 + 184*’

(16) Control

-

87+

13

(16)

(‘3) 1939 k 259+*

(12) 165 + 30

03)

(16) 672 k 143**

(16) 99 f

24

(8)

(8) 545 +- 146

(12) 146 A 25

03)

’ The mean daily concentrations of Phe and tyrosine (*SE) were obtained by averaging at 2, 4, 8, and 12 h postinjection in the mPhe/Phe and control groups and at 2, 4, 6, and 8 h postinjection in the Phe-treated group. b Number of animals in each group. ** P < 0.01.

Phe-treated group compared with controls (Table 1). Similarly, the mean daily concentration of Phe in the fetal plasma was increased approximately 24-times in the mPhe/Phe group and 15-times in the Phe-treated group. The experimental regimen with mPhe/Phe reduced maternal liver PheH activity by 76% compared with controls. The slight rise in the concentration of tyrosine in the plasma of the mPhe/Phe-treated group was due to the presence of residual hydroxylase activity. In contrast, treatment with Phe alone resulted in approximately a fivefold increase in the average daily concentration of tyrosine in the maternal and fetal plasma (Table 1). The mean body and wet brain weights are shown in Table 2 and the ratio of the average weights from the mPhe/Phe- and Phe-treated animals to those of the controls are shown in Fig. 1. The growth of the body of fetal and neonatal rats treated with mPhe/Phe or Phe were reduced compared with controls. In the mPhe/Phe group, these reductions were statistically significant at El9 (P < O.Ol), Pl (P < 0.05), and at P5 (P < 0.05). In the Phetreated group, these reductions were significant at El9 (P < 0.05) and Pl (P < 0.05). The total wet brain weights from the mPhe/Phe-treated groups and from the Phe group showed significant reductions (Table 2 and Fig. 1). The cerebrum, in particular, showed the greatest reduction during the late gesta-

Control mPhe/Phe Phe

GXltrol mPhe/Phe Phe

Control mPhe/Phe Phe

Cotltrol mPhe/Phe Phe

Control mPhe/Phe Phe

El9

Pl

P5

F7

P12

35.33 f 1.17 29.55 f 1.63 34.18 f 2.66

15.47 f 0.57 13.94 f 0.59 15.15 f 0.64

12.23 f 0.32 10.56 f 0.46+ 11.08 f 0.46

5.88 i 0.12 5.01 f 0.07* 5.14 f 0.11.

3.45 f 0.11 2.83 + 0.14** 2.92 f 0.12*

1.56 f 0.04 1.42 f 0.03 1.51 f 0.04

Body weiaht 0 3 3’ 4 5 7* 6*

29Ok a 332 f 10 292 + 12

1022 f 28 983 f 24 1020 f 27

601 f 13 586 f 16 612 f 18

339 f 13 292 + 18 332* 17

369 f 12 397 f 12 370 f 11

569 i 18 5061t24 556 f 23

164+ 4 128 f 7** 135 + 6*

134* 105 f 112k

-c -

Cerebrum wet wt (w)

285 f 9 237 f 12* 253 + 11

374 * 7 386 + 11 382 f 10

554 f 12 541 k 18 534 f 15

526 f 14 520 2 18 526 f 16

Brain wt (mg) Body wt (10 Id

383 f 10 402 f 16 398 f 11

484 f 16 4091t 16 431 * 17

228 f 7 186 f 14* 194 f 13

187~~ 6 136 + a*+ 149 f 7**

84f 72 f 77f

Total brain wet w bv3)

3 3 2

6 7 6 322 f 10 303 f 10 315 + 11

18Ok 169& 174f

167 + 5 1422 6 148zt 4

53* 49* 49k

-

-

1 3 4 4

2

was

99~~6 93 f4 92 + 7

32 f 30-+2 30 f 50 f 45 f 50 f

-

Cerebellum wet w-t (me)

‘The mean values and the standard error nre shown. The number of animals in each age group was 10. The sianifrcance of dikence calculated using analysis of variance and Sch&e’s test of multiple comparisons. b E-embryonic day, P-postnntal day. c Insuakient tissue for weight determinntions. l P < 0.05. *+ P < 0.01.

Control mPhe/Phe Phe

El7

Treatment

Effect of cu-Methylphenylnlanine/Phenylalanine (mPhe/Phe) and Phenylalanine (Phe) on Growth of the Body and Brain of Fetal and Neonatal Animal@

TABLE 2

% VI

P

s

tJ

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FI9

PI

PS

PI

FIG. 1. Effect of cy-methylphenylalanine/phenylalanine (mP/P, diagonal) and phenylalanine (P, solid) treatments on the growth of the body, brain, and cerebrum expressed as a percentage of the control value. The numbers above the ba,rs represent the percent of the control weights. *P -C 0.05, **P -C 0.01. E, P-embryonic, postnatal days.

tional and early postnatal periods. In contrast, the growth of the subcortical structures and the cerebellum were not signihcantly a&ted by mPhe/Phe and Phe treatments. In spite of the retarded body and brain growth, no significant differences between the brain:body weight ratios of the mPhe/Phe, Phe, and control groups could be detected. The effects of mPhe/Phe and Phe treatments on the total DNA, RNA, and protein contents of the cerebrum are shown in Table 3 and the ratio of

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TABLE 3 Ell’ect of a-MetbylphenyManine/phenylalanine (mPhe/Phe) and Phenylalanine (We) Treatments on the Total DNA, RNA, and protein Contents of the Q&rum of Fetal and Neonatal Aniw Treatment

DNA ocg) per-m

RNA ha) per cerebrum

Protein (ms) per cerebrum

El9

Control mPhe/Phe Phe

283 f 10 208 f 7* 220 f 8”

246 f 6 183 + 4” 189 + 4**

4.1 f 0.3 2.9 f 0.20, 3.1 f 0.2*,

15.2 + 0.4 13.5 k 0.3 14.0 * 0.3

Pl

Control mPhe/Phe Phe

406+ 9 307 f SC* 323 + 7**

447 f 14 345 f 6** 371 + 8’

9.3 + 0.3 6.7 + 0.4** 7.1 f 0.04”

22.3 f 0.3 19.9 + 0.4 20.9 2 0.4

PS

Control mPhe/Phe Phe

589 f 15 483 f 8” 521& 7

731 f 22 628 f 7+ 705 k 8

18.9 + 0.6 16.1 f 0.8’ 17.8 + 0.9

30.7 f 1.0 31.2 + 1.1 31.9 + 0.7

P7

Control mPhe/Phe Phe

691 + 14 625 + 13 671 + 12

884 f: 20 824 f 15 868 f 16

3.1.2 f 1.4 27.3 + 0.6 31.4 f 0.7

43.8 f 1.4 43.6 + 1.6 42.8 & 1.5

P12

Control mPhe/Phe Phe

816 * 1s 783 + 10 807 f 14

1457 + 24 1444+31 1437 + 19

75.9 f 4.3 78.6 f 8.3 75.6 + 2.3

78.6 zk 4.8 84.1 k 3.6 76.9 f 6.4

Protein/DNA

’ Mean values and standard errors are shown. The number of animals in each group was 10. ’ E-embryonic day, P-postnatal day. * P < 0.05. ** P < 0.01.

these values of those of the control group are shown in Fig. 2. The cell number of the cerebrum from the mPhe/Phe-treated group, as estimated in terms of the total DNA content, was significantly reduced at El9 (P < O.Ol), Pl (P < 0.01) and P5 (P < 0.05). Thereafter, the DNA content of the cerebrum from the mPhe/Phe-treated group gradually approached the control value so that by P12, the DNA content from the mPhe/Phe group was 94% of the control value. The total DNA content from the Phetreated group was significantlyreducedatE19(P<0.01)andatP1 (P
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Cerebrum

mP/P P El9

elf/P

P PI

mpfP P P5

mP/P

DNA

P P7

El9

PI

P5

P7

El9

PI

P5

P7

FIG. 2. Effect of a-methyIphenylalanine/phenylalanine (mP/P, diagonal) and phenylalanine (P, solid) treatments on the total DNA, RNA, and protein contents of the cerebrum expressed as a percentage of the control value. The numbers above the bars represent the percent of the control values. *P < 0.05, **P < 0.01. E, P-embryonic, postnatal days.

and 7 1% for DNA, RNA, and protein, respectively, and for the Phe-treated group, these values were 78%, 77%, and 76%, respectively. The mean cell size as estimated in terms of the protein:DNA ratio in the mPhe/Phe group was significantly reduced compared with controls at El9

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TABLE 4 Effect of a-Methylphenylalan&/phenylalanine (mPhe/Phe) and Phenylalank. (Phe) Treatment on the Concentrations of DNA, RNA, and Protein in the Cerebrum of Fetal and Neonatal Allimalse DNA w100

RNA

m g wet

(~~100

mg

Protein wet

cerebrum wt)

Ww

a

Treatment

cenbrum wt)

El9

Control mPhe/Phe Phe

207 f 6 204& 8 213 + 5

184* 174f 170*

5 8 6

30.6 f 0.8 27.6 i 0.8 27.4 i 0.9

Pl

Control mPhe/Phe Phe

248 + 5 250 f 3 239 + 6

273 f 270 f 275 f

8 5 9

56.7 + 1.4 52.3 + 1.5 52.9 i 1.7

PS

Control mPhe/Phe Phe

207 f 204* 206k

9 8 6

256 f 5 279 + 8 265 * 11

66.3 + 2.5 63.9 + 3.0 67.9 i 2.5

P7

Control mPhe/Phe Phe

205 f 8 214 f 10 202 * 5

261* 6 282 f 11 256 f 9

92.0 f 2.3 93.4 + 4.7 91.1 f 3.5

Pi2

Control mPhe/Phe Phe

136* 8 134* 7 145 * 11

242 f 246 f 251f

7 8 5

cerebrum wt)

101.4 f 3.6 97.2 + 2.9 110.1 * 4.9

’ Mean values and standard errors are shown. The number of animals in each treatment group was 10. b E-embryonic day, P-postnatal day.

(P < 0.05). Thereafter, there were no signilicant differences between the protein:DNA ratios of the mPhe/Phe and control groups. The DNA, RNA, and protein concentrations (micrograms per unit wet tissue weight) are shown in Table 4. The DNA concentration @g/100 mg wet cerebrum weight) in the normal developing cerebrum increased between El9 and Pl, followed by a gradual decrease in the DNA concentration. In the mPhe/Phe- and Phe-treated groups, the cerebrum DNA concentrations followed the same pattern; however, the values were slightly higher than those of the control group at several time periods. These differences were not statisticahy significant. The RNA concentration (rcg/lOO mg wet cerebral weight) in the normal cerebrum increased gradually between E 19 and P 12. In the mPhe/Phe- and the Phe-treated groups, the concentrations of RNA followed the same pattern, but were less than the controls at several time periods; however, these differences were not statistically significant.

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The protein concentration (&mg wet tissue weight) in the control cerebrum increased rapidly between El9 and P7; thereafter the increase was moderate. In the mPhe/Phe- and Phe-treated groups, the cerebral protein concentrations showed a similar rise between El9 and P7. The protein concentrations from the mPhe/Phe-treated group were reduced compared with controls during the late gestational and early postnatal periods. In the Phetreated group, these values were less than controls at El9 and Pl . The reductions in the concentrations of protein from the cerebra of the mPhe/Phe groups were not significantly different from control values at any time tested. DISCUSSION This study indicates that treatment with mPhe/Phe resulted in a 24-fold increase in the concentration of Phe in the fetal plasma and the concentration of tyrosine was only slightly elevated. The activity of maternal liver PheH was reduced by 76%. Because the activity of fetal PheH is negligible (30), it was assumed to be unaffected by mPhe treatment. The present regimen of treating pregnant rats with mPhe/Phe brought forth a number of conditions which closely paralleled those found in untreated pregnant PKU women (20, 27, 38). These consisted of a 20- to 30-fold increase in maternal and fetal plasma Phe, a high fetahmatemal plasma Phe ratio, indicating a carriermediated transport of Phe across the placenta, and a reduction in body weights of the offspring at birth. Treatment with Phe alone also produced many of the signs and symptoms associated with maternal PKU, but to a much lesser extent. However, a substantial increase in maternal and fetal plasma tyrosine was observed to follow Phe treatment, an effect not associated with the human condition. The total DNA, RNA, and protein contents of the cerebral cortex of rats treated with mPhe/Phe or Phe showed a significant reduction during the late gestational and early postnatal periods, with the maximum reduction occurring at E19. The cerebral protein contents, in particular, appeared to be more affected than the DNA and RNA contents, indicating that high concentrations of Phe interfered with protein synthesis. It has been shown that elevated Phe in the plasma inhibited the transport of certain amino acids into the brain (7), altered the concentrations of free amino acids in the brain (3 1), interfered with protein synthesis (l), and caused disaggregation of brain polyribosomes (39). Some of these factors may also account for the reduced protein content in the present study. The reduction in total DNA content, which is an index for cell number (41), indicates that treatment with mPhe/ Phe or Phe caused a significant suppression of cell acquisition in the cerebrum during the treatment period. The decrease in cell number appears to be also the result of an increase in cell death, because our studies (37) confirmed

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that there was an increase in the number of pyknotic nuclei in the cortical plate and in the intermediate zone of rats treated with mPhe/Phe. It is not known, however, whether high concentrations of Phe inhibit cell proliferation directly or does so indirectly through a general inhibition of protein synthesis. The cerebrum appeared to be a&cted to a much greater extent than the cerebellum or the brain stem after mPhe/Phe and Phe treatments. This may reflect the selective vulnerability of cerebral metabolism to high concentrations of Phe due to differences in the rate of growth among various brain regions (2, 5). Also, the cerebrum was shown to undergo greater polyribo somal disaggregation than the cerebellum in response to high concentrations of Phe (39). An interesting finding of the present study is that in spite of the deficits in body and brain weights in both mPhe/Phe- and Phe-treated groups, no increase in the ratio of brain weightbody weight was observed. This indicated that there was a lack of brain sparing, an observation similar to that found in the offspring of pregnant rats treated with PCPA during pregnancy (9). Neither mPhe nor hyperphenylalaninemia was lethal to the developing fetus. Maternal rats treated with mPhe/Phe showed no changes in litter size or in the survival of the newborn rats. However, treatment of maternal rats with PCPA has been reported to result in an increase in maternal muricide (3,9). It was suggested that the increase in muricide was caused by a PCPAinduced depletion in maternal serotonin, because it could be prevented by the administration of 5-hydroxytryptophan to the dams prior to delivery (9). Thus, the high rate of maternal muricide resulting from PCPA treatment appeared to be specific to this analogue because these effects were not ob served in mPhe-treated animals. In spite of the cellular reductions (DNA, RNA, and protein) observed in the late embryonic and early postnatal periods, treatment with mPhe/Phe or Phe did not result in permanent deficits after the age of postnatal day 7. The apparent recovery of the cerebrum could be explained by either an increase in the capacity of the cerebmm for cell multiplication and growth a&r the cessation of treatment, or by cellular deficits being obscured by the increase in DNA, RNA, and protein which occurs during the first postnatal week (12,28). Evidence that the brain has increased capacity to undergo cell multiplication after birth following a period of intrauterine insult is well documented (4, 10). However, further studies will be needed to determine whether the recovery was the result of an adaptive increase in the formation of neurons after birth, or was solely a compensory increase in the number of glial cells. The present method of treatment with mPhe/Phe in rats appears to be a suitable model for studying maternal PKU. Our studies indicated that rats exposed to high concentrations of mPhe/Phe or Phe during intrauterine life

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displayed a consistent reduction in the same developmental parameters ob served in the human condition (22, 27, 38), and that the difference in the severity of the deficits between these two treatments seem to be related to the concentrations of plasma Phe and not to any side effects of the inhibitor. However, the long-term effects of these treatments are more difficult to assess. This is due to the fact that the human fetal brain growth spurt occupies a much greater proportion of the fetal period than the rat brain growth spurt (10). Thus, the human fetus may be subjected to a more prolonged state of hyperphenylalaninemia during the growth period, and the resulting impairment in brain growth may be more profound and render subsequent recovery more difficult. REFERENCES 1. AGRAWAL, H. D., A. H. BONE, AND A. N. DAVISON. 1970. Effect of phenylaIanine on protein synthesis in the developing rat. Eiochem. J. 117: 325-33 1. 2. ALTMAN, J. 1969. Autoradiographic and histological studies of postnatal neurogenesis. III. Dating the time of production and onset of differentiation of cerebellar microneurons in rats. J. Camp. Neural. 1361 269-294. 3. ANDERSEN, A. E. 1976. Maternal hyperphenylalaninemia: an experimental model in rats. Dev. Psychobiol. 9: 157-166. 4. BARNES, D., AND J. ALTMAN. 1973. Effects of two levels of gestational-lactational undernutrition on the preweaning growth of the rat cerebellum. Exp. Neural. 38: 420-428. 5. BERRY, M., AND A. W. ROGERS. 1965. The migration of neuroblasts in the developing cerebral cortex. J. Anat. (London) 9: 691-709. 6. BURTON, K. 1956. A study of the conditions and mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. B&hem. J. 62: 3 15-323. 7. CARVER, M. J. 1965. Influence of phenylalanine administration of the free amino acids of brain and liver in the rat. J. Neurochem. 12: 45-50. 8. COPENHAVER, J. H., J. P. VACANTI, AND M. J. CARVER. 1973. Experimental maternal hyperphenylalaninemia: disaggregation of fetal brain polyribosomes. J. Neurochem. 21: 273-280. 9.

COPENHAVER,J. H., M. J. CARVER, AND R. L. SCHALOCK. 1974. Experimental maternal hyperphenylalaninemia: biochemical effectsand offspring development. Dev. Psychobiol. 7: 175-184.

10. DOBBING, J., AND J. SANDS. 1971. Vulnerability of developing brain. IX. The effect of nutritional growth retardation on the timing of the brain growth spurt. Biol. Neonate. 19: 363-378.

11. FIGELWICZ, D. A., AND M. J. DRUSE. 1980. Experimental hyperphenylalaninemia: effect on central nervous system myelin subfractions. Exp. Neural. 67: 315-329. 12. FISCH,I., AND M. WINICK. 1969. Effect of malnutrition on regional growth of the developing brain. Exper. Neural. 25: 534-540. 13. FOLCH, J., M. LEES, AND G. H. SLOANE-STANLEY. 1957. A simple method for isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: 497-509. 14. FRIEDMAN, P. A., S. KAUFMAN, AND E. S. KANG. 1972. Nature of the molecular defect in phenylketonuria and hyperphenylalaninemia Nature (London) 240: 157-l 59.

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