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Effect of hyperphenylalaninemia on amino acid metabolism and compartmentation in neonatal rat brain
Brain Research, 67 (1974) 479-488 © ElsevierScientificPublishingCompany,Amsterdam- Printed in The Netherlands
479
EFFECT OF HYPERPHENYLALANINEMIA ON AMINO ACID METABOLISM AND COMPARTMENTATION IN NEONATAL RAT BRAIN
ELLIS L. N O R D Y K E AND M A R Y K. R O A C H
Biochemistry Section, Texas Research Institute of Mental Sciences, Texas Medical Center, Houston, Tex. 77025 (U.S.A.) (Accepted August 23rd, 1973)
SUMMARY
Hyperphenylalaninemia was induced in neonatal rats by p-chlorophenylalanine and phenylalanine treatment up to 21 days of age. Brain and body weights of the hyperphenylalaninemic rats were significantly reduced when compared to controls. Amino acid compartmentation, demonstrated by the ratio of specific radioactivity of glatamine to glutamate after the administration of L-[U-14C]leucine, developed more slowly in the hyperphenylalaninemic group. This result is similar to that seen with thyroidectomized neonatal rats and suggests an inhibition to neuronal process growth. Though the brain levels of amino acids derived through the TCA cycle increased during development, glutamate and glutamine concentrations were significantly less in the hyperphenylalaninemic group than the controls. Body weight and amino acid compartmentation returned to normal 3 weeks after termination of drug treatment whereas brain weight and glutamate and glutamine concentrations did not return to normal.
INTRODUCTION
The relationship between the hyperphenylalaninemic state and the mental retardation often observed in human beings with untreated phenylketonuria (PKU) remains unclear at this time. Investigations have indicated that hyperphenylalaninemia might interfere with metabolic processes that are crucial to the normal development of the nervous system. Disturbances in myelination~, in synthesis of cerebral serotonin and catecholamines7, in brain protein synthesis4 and in brain glycolysis 21 have been hypothesized as responsible for the phenylketonuric lesion. Though these hypotheses are suggestive, the mechanism of the development of mental retardation in PKU is still unknown 1°.
480
E. L. NORDYKE AND M. K. ROACH
Another biochemical process that is important to the maturing nervous system and is indicative of the dynamic properties of metabolism is cerebral amino acid metabolism. This paper examines the effect of hyperphenylalaninemia on the development of amino acid metabolism and compartmentation6 in neonatal brain. The development of this maturational parameter reflects the progression of the brain from a situation of relative biochemical homogeneity to the heterogeneity of maturityis. Further, though its structural and functional significance is still under investigation, the development of amino acid compartmentation has been associated with the growth of neuronal processes s and with neuronal-glial interactionsis. MATERIALS AND METHODS
p-Chlorophenylalanine (p-CPA) supplemented with phenylalanine was prepared by dissolving L-phenylalanine (Sigma Chemical Co., St. Louis, Mo.) in 0.2 ~ agar to make a 1 ~ (w/v) solution and then suspending oL-p-chlorophenylalanine (Sigma) to make a 1 ~ (w/v) suspension. Prior to use, suspensions were ground in a tissue homogenizer for ease in handling. Hyperphenylalaninemia has been developed in rats by subcutaneous injections ofp-CPA and phenylalanine3. Delivery of these two compounds by oral intubation also results in increased plasma levels of phenylalanine (E.L. Nordyke, unpublished observations) but avoids the skin damage caused by daily injections. Timed pregnant Sprague-Dawley rats in the second trimester (Simonsen, Gilroy, Calif.) were caged individually. One day after birth the pups were distributed randomly among the mothers so that litter size remained constant. Two treatment groups were generated: (a) pups intubated once a day from birth with the 0.2~ agar vehicle (control), and (b) pups intubated once a day from birth with 100 mgp-CPA plus 100 mg phenylalanine/kg body weight (experimental). Treatment was terminated after 21 days and one group of experimental rats were maintained on regular laboratory chow for an additional 21 days. At 1, 9, 15 and 21 days after birth pups from each group were intubated as usual; 42-day-old pups were not intubated. Two hours after intubation, pups were injected subcutaneously with 0.2/~Ci/g of L-[U-14C]leucine (specific activity 310 mCi/ mmole; New England Nuclear, Boston, Mass.). Twenty minutes after injection, the pups were killed by immersion in liquid nitrogen. The brains were removed quickly, weighed without thawing and homogenized in 10 ml of ice-cold 3 ~ sulfosalicylic acid. The brains of two animals were pooled at day 1. The homogenates were centrifuged for 20 min at 10,000 × g and 4 °C. The sulfosalicylic extracts of brain were analyzed for glutamate, glutamine, y-aminobutyrate (GABA), aspartate and leucine content and for distribution of the radioactive label in these compounds 2°. Results were analyzed statistically by Student's t-test or by analysis of variance for a 2 × 4 factorial experiment with drug treatment and age being the factors analyzed 22. RESULTS
Rats from the experimental group weighed significantly less than rats from the
481
HYPERPHENYLALANINEMIA AND RAT BRAIN DEVELOPMENT TABLE I EFFECT OF HYPERPHENYLALANINEMIAON RAT BODY AND BRAIN WEIGHT Each value is the mean 4- S.D. Numbers of animals are given in parentheses.
Age (days)
1 (8) 9 (8) 15 (8) 21(8) 42 (8)
Body weight (g)
Brain weight (g)
Control
Experimental
Control
Experimental
6.8 4- 1.3 18.4 4- 2.0 33.9 4- 5.3 56.2±10.2 180.3 4- 5.3
7.6 416.6 422.0 430.14175.5 4-
0.33 4- 0.06 0.90 4- 0.04 1.26 4- 0.13 1.434-0.07 1.83 4- 0.05
0.36 4-4-0.03 0.76 4- 0.07* 1.09 4- 0.07* 1.204-0.11" 1.64 4- 0.09*
0.6 2.2 4.5* 7.0* 11.1
* P < 0.01.
control group at 15 and 21 days (Table I). Brain weights of hyperphenylalaninemic animals, though affected less than body weight, were reduced significantly to about 85 ~ of control brain weights at 9, 15 and 21 days. The effect of hyperphenylalaninemia on the development of brain amino acid metabolism and compartmentation was investigated with L-[U-14C]leucine as the source of radioactive label 18. After injection of [14C]leucine, significantly less radioactivity was recovered in the acid soluble fraction of the experimental group as compared to the control group at 9, 15, and 21 days (Table II). There was also a consistent decrease in the percentage of radioactivity remaining as leucine in the experimental group. Hyperphenylalaninemia also had an effect on the distribution of the radioactive label among the amino acids derived from tricarboxylic acid (TCA) cycle intermediates. The specific activities of glutamate, GABA and aspartate at 9, 15 and 21 days were increased in the experimental group (Table III). The specific activity of glutamine showed no difference between the two groups. Compartmentation of amino acids in brain is demonstrated by a ratio of the specific activity of glutamine relative to glutamate that is greater than one after the administration of [14C]leucinelS. This measure of compartmentation increased with age in both groups but was significantly lower in the experimental group compared to the controls (Table IV). The specific activities of GABA and of aspartate relative to glutamate showed an increase with age but no difference between the two groups. The concentration of amino acids derived through the TCA cycle increased with age in both groups (Table V). Glutamate and glutamine levels were lower in the experimental group than in the controls at 9, 15 and 21 days. GABA and aspartate levels were similar in the two groups. Drug treatment was terminated at 21 days, and the animals were weaned gradually and were maintained on regular laboratory chow for an additional 21 days. During this period of time the experimental group recovered the body weight deficit observed during the first 3 weeks of life but not the brain weight deficit (Table I). At
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E . L . NORDYKE AND M. K. ROACH
T A B L E VI SPECIFIC RADIOACTIVITYOF AMINO ACIDS RELATIVE TO THAT OF GLUTAMATE IN BRAINS OF REHABILITATED AND NORMAL RATS Values are m e a n s 4- S.D. G l u t a m a t e = 1. F o u r c o n t r o l a n d 4 e xpe ri me nt a l 42-day-old rats were used. Differences were n o t significant (P > 0.05) by S t u d e n t ' s t-test.
Age (days) 42 Glutamine
Control 2.360 4- 0.126 Experimental 2.472± 0.136 GABA
Control 0.868 4- 0.080 Experimental 0.7304- 0.037 Aspartate Control Experimental
0.614 4- 0.077 0.504 4- 0.081
this time no significant differences were observed in the specific activities o f glutamine, GABA and aspartate relative to glutamate (Table VI). However, the pool sizes of glutamate and glutamine remained diminished in the hyperphenylalaninemic animals compared to the control group (Table V). DISCUSSION
p-Chlorophenylalanine is an irreversible in vivo inhibitor of phenylalanine hydroxylase14. However, p-CPA treatment must be supplemented with phenylalanine to increase plasma phenylalanine levels significantly15. Andersen and Guroff showed that hyperphenylalaninemia experimentally induced by subcutaneous injections of p-CPA and phenylalanine resulted in reduced body and brain weights 3. This paper substantiates their results, but extends thereto show that 21 days after termination of drug treatment the body weight deficit i,s recovered whereas the brain weight deficit is not. The results presented here show that the hyperphenylalaninemic state disturbs the normal development of amino acid metabolism. The significant decrease of radioactivity in the acid soluble fraction of the experimental group after injection of [14C]leucine suggests some limitation on the entry of the radioactive label into brain. This observation can be explained by experiments showing that high levels of phenylalanine inhibit the uptake of some amino acids by brain 17. Once the label enters the brain, slightly more leucine is utilized in the experimental brains as indicated by a decrease in the percentage of radioactivity as leucine. That amino acid compartmentation is inhibited in the brains of hyperphenylalaninemic rats is evidenced by the decreased ratio of specific radioactivity of glutamine to glutamate observed after the administration of [14C]leucine. In rats this ratio is
HYPERPHENYLALANINEMIA AND RAT BRAIN DEVELOPMENT
487
less than one at birth but increases rapidly from 9 to 21 days, becoming greater than unity 18. This ratio is thought to reflect the expansion of a glutamate pool that is not available for glutamine synthesis5. There is evidence that the expansion of this pool is associated with the increasing utilization of glucose carbon in the developing brain and the rapid growth of neuronal processes s. Thus an inhibition in the development of compartmentation suggests an interference in the growth of neuronal processes. The inhibition in development of compartmentation in hyperphenylalaninemia is similar to that seen in thyroidectomized rats la. The interpretation of the effects of hypothyroidism on compartmentation as an interference in neuronal process growth is supported by histologicaP1 and electron microscopic data 9. Similar studies are needed for hyperphenylalaninemic animals. Amino acid concentrations, though not as sensitive a marker for development as compartmentation, increase during maturation. In the brains of hyperphenylalaninemic rats, the concentration of glutamate and glutamine is found to be consistently less than controls. In hypothyroid rats, the brain levels of glutamate and aspartate are slightly decreased whereas the level of glutamine is unaffectedla. The reason for the differences between the two animal models is unknown. After drug treatment was terminated at 21 days, 3 weeks of additional time were sufficient for rats to recover normal body weights, but not brain weights. Biochemical measurements made on the brains of these animals indicated that the deficit in the specific radioactivity of glutamine relative to glutamate observed in the experimental animals was recoverable when compared to controls whereas glutamate and glutamine pool sizes were not. Since amino acid compartmentation is thought to be associated with neuronal process growth s, its recovery is not totally unexpected. Even though most process growth occurs by the age of 21 days in the rat, some growth continues after this time 1,1z. The failure of the glutamate and glutamine concentrations to recover shows the effect of amino acid concentration is not transient with age as it is in hypothyroid rats 19. The reduction in body and brain weights perhaps implicates undernutrition as a contributing factor in the hyperphenylalaninemia model. However, recovery of body weight is not observed in rehabilitated undernourished animals~6. Furher, rehabilitated undernourished animals are less active than controls 13 whereas rehabilitated hyperphenylalaninemic rats are more active3. These observations suggest that the biochemical disturbances measured in the experimental group are due to the hyperphenylalaninemic state and not to undernutrition from reduced food intake as a result of drug toxicity. The studies reported here indicate that hyperphenylalaninemia during the developmental period has some injurious effects on the biochemical maturation of rat brain. The inhibition in growth of neuronal processes, reflected by inhibited compartmentation, is reversible and is seen more accurately as a delay in growth. The irreversible changes observed, reduced brain weight and the reduced pool sizes of glutamate and glutamine, indicate that some biochemical parameters of central nervous system maturation in the rat may be altered permanently by hyperphenylalaninemia. Whether the abnormalities observed in hyperphenylalaninemic rats can be extrapolated to
488
E. L. NORDYKE AND M. K. ROACH
h u m a n P K U subjects is speculative, but these defects indicate that i m p a i r e d brain g r o w t h and m a t u r a t i o n does result f r o m h y p e r p h e n y l a l a n i n e m i a i n d u ced during the d e v e l o p m e n t a l period.
REFERENCES 1 AGHAJANIAN,G. K., AND BLOOM,F. E., The formation of synaptic junctions in developing rat brain: a quantitative electron microscopic study, Brain Research, 6 (1967) 716-727. 2 ALVORD,E. C., STEVENSON,L. D., VOGEL,F. S., AND ENGLE,R. L., Neuropathological findings in phenyl-pyruvic oligophrenia (phenylketonuria), J. Neuropath. exp. NeuroL, 9 (1950) 298-310. 3 ANDERSEN,A. E., ANDGUROFE,G., Enduring behavioral changes in rats with experimental phenylketonuria, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 863-867. 4 APPLE, S. H., Inhibition of brain protein synthesis: an approach to the biochemical basis of neurological dysfunction in the aminoacidurias, Trans. N.Y. Acad. Sci., 29 (1966) 63-70. 5 BERL, S., Compartmentation of glutamic acid metabolism in developing cerebral cortex, J. biol. Chem., 240 (1965) 2047-2054. 6 BERL,S., AND CLARKE,D. D., Compartmentation of amino acid metabolism. In A. LAJTHA(Ed.), Handbook ofNeurochemistry, Vol. 2, Plenum, New York, 1969, pp. 447-472. 7 BOYLEN,J. B., AND QUASTEL,J. H., Effects of L-phenylalanine and sodium phenylpyruvate on the formation of adrenaline from L-tyrosine in adrenal medulla in vitro, Biochem. J., 80 (1961) 644-648. 8 COCKS,J. A., BAL.~ZS,R., JOHNSON,A. L., AND EAYRS,J. T., Effect of thyroid hormone on the biochemical maturation of rat brain: conversion of glucose-carbon into amino acids, J. Neurochem., 17 (1970) 1275-1285. 9 CRAGG,B. G., Synapses and membranous bodies in experimental hypothyroidism, Brain Research, 18 (1970) 297-307. 10 D'ELIA, J. A., Phenylketonuria: a review of the mechanism of mental retardation, Georgetown med. Bull., 24 (1971) 13-18. 11 EAYRS,J. T., The cerebral cortex of normal and hypothyroid rats, Acta anat. (Basel), 25 (1955) 160-183. 12 EAYRS,J. T., AND GOODHEAD,B., Postnatal development of the cerebral cortex in the rat, J. Anat. (Lond.), 93 (1959) 385--402. 13 FRANKOVA,S., ANDBARNES,R. H., Influence of malnutrition in early life on exploratory behavior of rats, J. Nutr., 96 (1968) 477-484. 14 GUROEF, G., Irreversible in vivo inhibition of rat liver phenylalanine hydroxylase by p-chlorophenylalanine, Arch. Biochem. Biophys., 134 (1969) 610-611. 15 LIPTON, M. A., GORDON, R., GUROFF, G., AND UDENFRIEND, S., p-Chlorophenylalanine induced chemical manifestations of phenylketonuria in rats, Science, 156 (1967) 248-250. 16 MCCANCE,R. A., AND WIDDOWSON,E. M., Nutrition and growth, Proc. roy. Soc. B, 156 (1962) 326-337. 17 NEAME,K. G., Phenylalanine as inhibitor of transport of amino acids in brain, Nature (Lond.), 192 (1961) 173-174. 18 PATEL,A. J., AND BAL~ZS, R., Manifestation of metabolic compartmentation during the maturation of rat brain, J. Neurochem., 17 (1970) 955-971. 19 PATEL,A. J., AND BAL~.ZS,R., Effect of thyroid hormone on metabolic compartmentation in the developing rat brain, Biochem. J., 121 (1971) 469-481. 20 ROACH, M. K., AND REESE, W. N., Effect of ethanol on glucose and amino acid metabolism in brain, Biochem. Pharmacol., 20 (1971) 2805-2812. 21 WEBER, G., Inhibition of human brain pyruvate kinase and hexokinase by phenylalanine and phenylpyruvate: possible relevance to phenylketonuric brain damage, Proc. nat. Acad. Sci. (Wash.), 63 (1969) 1365-1369. 22 WINER, B. J., Statistical Principles in Experimental Design, 2nd ed., McGraw-Hill, New York, 1971, pp. 431-452.
479
EFFECT OF HYPERPHENYLALANINEMIA ON AMINO ACID METABOLISM AND COMPARTMENTATION IN NEONATAL RAT BRAIN
ELLIS L. N O R D Y K E AND M A R Y K. R O A C H
Biochemistry Section, Texas Research Institute of Mental Sciences, Texas Medical Center, Houston, Tex. 77025 (U.S.A.) (Accepted August 23rd, 1973)
SUMMARY
Hyperphenylalaninemia was induced in neonatal rats by p-chlorophenylalanine and phenylalanine treatment up to 21 days of age. Brain and body weights of the hyperphenylalaninemic rats were significantly reduced when compared to controls. Amino acid compartmentation, demonstrated by the ratio of specific radioactivity of glatamine to glutamate after the administration of L-[U-14C]leucine, developed more slowly in the hyperphenylalaninemic group. This result is similar to that seen with thyroidectomized neonatal rats and suggests an inhibition to neuronal process growth. Though the brain levels of amino acids derived through the TCA cycle increased during development, glutamate and glutamine concentrations were significantly less in the hyperphenylalaninemic group than the controls. Body weight and amino acid compartmentation returned to normal 3 weeks after termination of drug treatment whereas brain weight and glutamate and glutamine concentrations did not return to normal.
INTRODUCTION
The relationship between the hyperphenylalaninemic state and the mental retardation often observed in human beings with untreated phenylketonuria (PKU) remains unclear at this time. Investigations have indicated that hyperphenylalaninemia might interfere with metabolic processes that are crucial to the normal development of the nervous system. Disturbances in myelination~, in synthesis of cerebral serotonin and catecholamines7, in brain protein synthesis4 and in brain glycolysis 21 have been hypothesized as responsible for the phenylketonuric lesion. Though these hypotheses are suggestive, the mechanism of the development of mental retardation in PKU is still unknown 1°.
480
E. L. NORDYKE AND M. K. ROACH
Another biochemical process that is important to the maturing nervous system and is indicative of the dynamic properties of metabolism is cerebral amino acid metabolism. This paper examines the effect of hyperphenylalaninemia on the development of amino acid metabolism and compartmentation6 in neonatal brain. The development of this maturational parameter reflects the progression of the brain from a situation of relative biochemical homogeneity to the heterogeneity of maturityis. Further, though its structural and functional significance is still under investigation, the development of amino acid compartmentation has been associated with the growth of neuronal processes s and with neuronal-glial interactionsis. MATERIALS AND METHODS
p-Chlorophenylalanine (p-CPA) supplemented with phenylalanine was prepared by dissolving L-phenylalanine (Sigma Chemical Co., St. Louis, Mo.) in 0.2 ~ agar to make a 1 ~ (w/v) solution and then suspending oL-p-chlorophenylalanine (Sigma) to make a 1 ~ (w/v) suspension. Prior to use, suspensions were ground in a tissue homogenizer for ease in handling. Hyperphenylalaninemia has been developed in rats by subcutaneous injections ofp-CPA and phenylalanine3. Delivery of these two compounds by oral intubation also results in increased plasma levels of phenylalanine (E.L. Nordyke, unpublished observations) but avoids the skin damage caused by daily injections. Timed pregnant Sprague-Dawley rats in the second trimester (Simonsen, Gilroy, Calif.) were caged individually. One day after birth the pups were distributed randomly among the mothers so that litter size remained constant. Two treatment groups were generated: (a) pups intubated once a day from birth with the 0.2~ agar vehicle (control), and (b) pups intubated once a day from birth with 100 mgp-CPA plus 100 mg phenylalanine/kg body weight (experimental). Treatment was terminated after 21 days and one group of experimental rats were maintained on regular laboratory chow for an additional 21 days. At 1, 9, 15 and 21 days after birth pups from each group were intubated as usual; 42-day-old pups were not intubated. Two hours after intubation, pups were injected subcutaneously with 0.2/~Ci/g of L-[U-14C]leucine (specific activity 310 mCi/ mmole; New England Nuclear, Boston, Mass.). Twenty minutes after injection, the pups were killed by immersion in liquid nitrogen. The brains were removed quickly, weighed without thawing and homogenized in 10 ml of ice-cold 3 ~ sulfosalicylic acid. The brains of two animals were pooled at day 1. The homogenates were centrifuged for 20 min at 10,000 × g and 4 °C. The sulfosalicylic extracts of brain were analyzed for glutamate, glutamine, y-aminobutyrate (GABA), aspartate and leucine content and for distribution of the radioactive label in these compounds 2°. Results were analyzed statistically by Student's t-test or by analysis of variance for a 2 × 4 factorial experiment with drug treatment and age being the factors analyzed 22. RESULTS
Rats from the experimental group weighed significantly less than rats from the
481
HYPERPHENYLALANINEMIA AND RAT BRAIN DEVELOPMENT TABLE I EFFECT OF HYPERPHENYLALANINEMIAON RAT BODY AND BRAIN WEIGHT Each value is the mean 4- S.D. Numbers of animals are given in parentheses.
Age (days)
1 (8) 9 (8) 15 (8) 21(8) 42 (8)
Body weight (g)
Brain weight (g)
Control
Experimental
Control
Experimental
6.8 4- 1.3 18.4 4- 2.0 33.9 4- 5.3 56.2±10.2 180.3 4- 5.3
7.6 416.6 422.0 430.14175.5 4-
0.33 4- 0.06 0.90 4- 0.04 1.26 4- 0.13 1.434-0.07 1.83 4- 0.05
0.36 4-4-0.03 0.76 4- 0.07* 1.09 4- 0.07* 1.204-0.11" 1.64 4- 0.09*
0.6 2.2 4.5* 7.0* 11.1
* P < 0.01.
control group at 15 and 21 days (Table I). Brain weights of hyperphenylalaninemic animals, though affected less than body weight, were reduced significantly to about 85 ~ of control brain weights at 9, 15 and 21 days. The effect of hyperphenylalaninemia on the development of brain amino acid metabolism and compartmentation was investigated with L-[U-14C]leucine as the source of radioactive label 18. After injection of [14C]leucine, significantly less radioactivity was recovered in the acid soluble fraction of the experimental group as compared to the control group at 9, 15, and 21 days (Table II). There was also a consistent decrease in the percentage of radioactivity remaining as leucine in the experimental group. Hyperphenylalaninemia also had an effect on the distribution of the radioactive label among the amino acids derived from tricarboxylic acid (TCA) cycle intermediates. The specific activities of glutamate, GABA and aspartate at 9, 15 and 21 days were increased in the experimental group (Table III). The specific activity of glutamine showed no difference between the two groups. Compartmentation of amino acids in brain is demonstrated by a ratio of the specific activity of glutamine relative to glutamate that is greater than one after the administration of [14C]leucinelS. This measure of compartmentation increased with age in both groups but was significantly lower in the experimental group compared to the controls (Table IV). The specific activities of GABA and of aspartate relative to glutamate showed an increase with age but no difference between the two groups. The concentration of amino acids derived through the TCA cycle increased with age in both groups (Table V). Glutamate and glutamine levels were lower in the experimental group than in the controls at 9, 15 and 21 days. GABA and aspartate levels were similar in the two groups. Drug treatment was terminated at 21 days, and the animals were weaned gradually and were maintained on regular laboratory chow for an additional 21 days. During this period of time the experimental group recovered the body weight deficit observed during the first 3 weeks of life but not the brain weight deficit (Table I). At
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T A B L E VI SPECIFIC RADIOACTIVITYOF AMINO ACIDS RELATIVE TO THAT OF GLUTAMATE IN BRAINS OF REHABILITATED AND NORMAL RATS Values are m e a n s 4- S.D. G l u t a m a t e = 1. F o u r c o n t r o l a n d 4 e xpe ri me nt a l 42-day-old rats were used. Differences were n o t significant (P > 0.05) by S t u d e n t ' s t-test.
Age (days) 42 Glutamine
Control 2.360 4- 0.126 Experimental 2.472± 0.136 GABA
Control 0.868 4- 0.080 Experimental 0.7304- 0.037 Aspartate Control Experimental
0.614 4- 0.077 0.504 4- 0.081
this time no significant differences were observed in the specific activities o f glutamine, GABA and aspartate relative to glutamate (Table VI). However, the pool sizes of glutamate and glutamine remained diminished in the hyperphenylalaninemic animals compared to the control group (Table V). DISCUSSION
p-Chlorophenylalanine is an irreversible in vivo inhibitor of phenylalanine hydroxylase14. However, p-CPA treatment must be supplemented with phenylalanine to increase plasma phenylalanine levels significantly15. Andersen and Guroff showed that hyperphenylalaninemia experimentally induced by subcutaneous injections of p-CPA and phenylalanine resulted in reduced body and brain weights 3. This paper substantiates their results, but extends thereto show that 21 days after termination of drug treatment the body weight deficit i,s recovered whereas the brain weight deficit is not. The results presented here show that the hyperphenylalaninemic state disturbs the normal development of amino acid metabolism. The significant decrease of radioactivity in the acid soluble fraction of the experimental group after injection of [14C]leucine suggests some limitation on the entry of the radioactive label into brain. This observation can be explained by experiments showing that high levels of phenylalanine inhibit the uptake of some amino acids by brain 17. Once the label enters the brain, slightly more leucine is utilized in the experimental brains as indicated by a decrease in the percentage of radioactivity as leucine. That amino acid compartmentation is inhibited in the brains of hyperphenylalaninemic rats is evidenced by the decreased ratio of specific radioactivity of glutamine to glutamate observed after the administration of [14C]leucine. In rats this ratio is
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487
less than one at birth but increases rapidly from 9 to 21 days, becoming greater than unity 18. This ratio is thought to reflect the expansion of a glutamate pool that is not available for glutamine synthesis5. There is evidence that the expansion of this pool is associated with the increasing utilization of glucose carbon in the developing brain and the rapid growth of neuronal processes s. Thus an inhibition in the development of compartmentation suggests an interference in the growth of neuronal processes. The inhibition in development of compartmentation in hyperphenylalaninemia is similar to that seen in thyroidectomized rats la. The interpretation of the effects of hypothyroidism on compartmentation as an interference in neuronal process growth is supported by histologicaP1 and electron microscopic data 9. Similar studies are needed for hyperphenylalaninemic animals. Amino acid concentrations, though not as sensitive a marker for development as compartmentation, increase during maturation. In the brains of hyperphenylalaninemic rats, the concentration of glutamate and glutamine is found to be consistently less than controls. In hypothyroid rats, the brain levels of glutamate and aspartate are slightly decreased whereas the level of glutamine is unaffectedla. The reason for the differences between the two animal models is unknown. After drug treatment was terminated at 21 days, 3 weeks of additional time were sufficient for rats to recover normal body weights, but not brain weights. Biochemical measurements made on the brains of these animals indicated that the deficit in the specific radioactivity of glutamine relative to glutamate observed in the experimental animals was recoverable when compared to controls whereas glutamate and glutamine pool sizes were not. Since amino acid compartmentation is thought to be associated with neuronal process growth s, its recovery is not totally unexpected. Even though most process growth occurs by the age of 21 days in the rat, some growth continues after this time 1,1z. The failure of the glutamate and glutamine concentrations to recover shows the effect of amino acid concentration is not transient with age as it is in hypothyroid rats 19. The reduction in body and brain weights perhaps implicates undernutrition as a contributing factor in the hyperphenylalaninemia model. However, recovery of body weight is not observed in rehabilitated undernourished animals~6. Furher, rehabilitated undernourished animals are less active than controls 13 whereas rehabilitated hyperphenylalaninemic rats are more active3. These observations suggest that the biochemical disturbances measured in the experimental group are due to the hyperphenylalaninemic state and not to undernutrition from reduced food intake as a result of drug toxicity. The studies reported here indicate that hyperphenylalaninemia during the developmental period has some injurious effects on the biochemical maturation of rat brain. The inhibition in growth of neuronal processes, reflected by inhibited compartmentation, is reversible and is seen more accurately as a delay in growth. The irreversible changes observed, reduced brain weight and the reduced pool sizes of glutamate and glutamine, indicate that some biochemical parameters of central nervous system maturation in the rat may be altered permanently by hyperphenylalaninemia. Whether the abnormalities observed in hyperphenylalaninemic rats can be extrapolated to
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h u m a n P K U subjects is speculative, but these defects indicate that i m p a i r e d brain g r o w t h and m a t u r a t i o n does result f r o m h y p e r p h e n y l a l a n i n e m i a i n d u ced during the d e v e l o p m e n t a l period.
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