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Induction of experimental phenylketonuria-like conditions in chick embryo. Effect on amino acid concentration in brain, liver and plasma
Neurochem. Int. Vol. 6, No. 4, pp. 485-489, 1984
0197-0186/84 $3.00 + 0.00 © 1984 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
INDUCTION OF EXPERIMENTAL PHENYLKETONURIA-LIKE CONDITIONS IN CHICK EMBRYO. EFFECT ON AMINO ACID CONCENTRATION IN BRAIN, LIVER AND PLASMA C. MARCO, M. J. ALEJANDRE,M. F. ZAFRA, J. L. SEGOVIA and E. GARCIA-PEREGR1N* Department of Biochemistry, University of Granada, Granada, Spain (Received 31 March 1983; accepted 4 November 1983)
Abstract--Experimental hyperphenylalaninemia has been induced in chick embryos between 11-20 days of incubation by daily injection of ~t-methylphenylalanine and phenylalanine. Brain and liver weight decreased after 8 days of treatment. An increase of nearly 14-fold in the brain phenylalanine/tyrosine ratio was observed after 9 days of treatment. Similar results were obtained in liver, although the increase found in this case was smaller than in brain. Chronic hyperphenylalaninemia induced a clear rise in the levels of plasma and liver valine, leucine and isoleucine, while in brain these levels did not change significantly. Plasma and brain glycine content was also enhanced by this treatment. Brain tyrosine concentration was clearly decreased in these conditions, in contrast to the enhancement reported after this and other treatments in various animal species. Thus, the higher value of the brain phenylalanine/tyrosine ratio obtained by ct-methylphenylalanine plus phenylalanine administration was due to both an increase in the phenylalanine and a decrease in the tyrosine levels, conditions that have been also found in human phenylketonurics. Therefore, the treatment here reported was an excellent method for imitating the conditions of phenylketonuria during the period of rapid myelination in the chick, one of the most dramatic in nervous system development.
One of the most common disorders of amino acid metabolism associated with mental retardation is phenylketonuria (PKU). The primary defect in this disease, an absence of phenylalanine hydroxylase (PHEH) activity (Knox, 1972), produced an elevation in plasma and tissue phenylalanine (PHE). In humans, this basic defect is not evident until shortly after birth when the brain is undergoing its most rapid rate of biochemical differentiation and is most vulnerable to the biochemical insult of PKU. To determine the mechanism leading to abnormal brain development many researchers have induced hyperphenylalaninemic conditions in animals by a variety of methods. Until recently, hyperphenylalaninemia had been produced by injections of PHE alone (Clarke and Lowden, 1969; Johnson and Shah, 1973), but in these conditions tyrosine (TYR) levels are grossly increased, unlike in the clinical picture. Injections of PHE and a PHEH inhibitor, pchlorophenylalanine (DelValle and Greengard, 1976; Shah and Johnson, 1978), permit the maintenance of *To whom correspondence should be sent.
a high P H E / T Y R ratio in the plasma, but the action of the inhibitor is not specific to the PHEH and toxic effects have been reported (DelValle et al., 1978). Greengard et al. (1976) have reported that another analog of PHE, ~t-methylphenylalanine (MPA), inhibited PHEH in newborn rats without other toxic effects on animal growth. More recently Brass et al. (1982) have described a regimen of MPA plus PHE that induces severe and prolonged hyperphenylalaninemia in pregnant rats and their fetuses but does not interfere with the preor postnatal survival of the rats. Several studies have demonstrated a similarity between cerebral myelin abnormalities occurring in phenylketonuric individuals and those occurring in rats with experimentally induced hyperphenylalaninemia. These studies have confirmed that there is a reduced amount of recoverable myelin but this myelin is of normal composition (Shah et al, 1972). The time of birth in relation to the period of maximum rate of myelination is different in different species. Thus, the brains of rats and mice are very immature at birth, the animals are physically quite 485
486
C, MARCt) el al.
helpless, and the periods o f m a x i m u m cellular proliferation and myelination are still to occur. In contrast, chick myelination occurs during embryonic develo p m e n t and chicks are born with a well-developed C.N.S. On the other hand, myelination is more rapid in chick than in other species. With this in mind, we have used M P A plus P H E to produce a chronic hyperphenylalaninemia in chick embryo at the time in which myelination and rapid brain growth occur, so that this new animal model should facilitate the study o f the effects o f hyperphenylalaninemia during the period of rapid myelination, one o f the most dramatic in nervous system development. In the present paper, the ability of M P A to maintain low levels o f TYR and to prolong the hyperphenylalaninemic conditions was examined. In addition, this paper describes the amino acid composition of brain, liver and plasma o f control and hyperphenylalaninemic chick embryos after 8 days of treatment. Previous studies have established the influence o f hyperphenylalaninemia induced by the same treatment on the lipid composition o f brain myelin in 19-day old chick embryos (Alejandre et al., 1984).
EXPERIMENTAL PROCEDURES
Induction o/' experimental hyperphenvlalaninemia
White Leghorn chickens eggs were incubated at 37.8_+0.2 C and 601~, relative humidity. On day 10 of incubation a small hole was open over the air space in the shell and sealed with paraffin. Eggs were divided into two groups and treatments were initiated at the l lth day of incubation. Experimental eggs were injected daily into the air space with 0.05ml of L-phenylalanine (173#mol/ml) at 8 and 20 h and once with 0.05ml of ~-methyl-DL-phenylalanine (190 #mol/ml) at 14 h. Control eggs were injected three times a day at 8, 14 and 20 h with 0.05 ml of 0.9°11NaC1 solution. Blood and tissue collection
Embryos were removed from their shells 12 h after the last injection. Blood was collected from a cardiac puncture, run into heparinized tubes and centrifuged at 2000g for 5 min. Plasma was deproteinized by addition of 1 vol of 0.6 M trichloroacetic acid and after 20 rain the mixture was centri-
fuged at 4000g. Liver and brain were quickly excised and weighed. Hepatic PHEH was measured by the method of McGee et al. (1972). Tissue samples to be used for amino acid analyses were immediately homogenized in 3 vol of ice-cold distilled water and centrifuged at 15000g. Trichloroacetic acid (0.6 M) was added to the supernatants to a final concentration of 0.3 M. Samples were thoroughly mixed, allowed to stand at room temperature for 20 min and centrifuged at 4000g. Ana@~e,~
The tissue and plasma samples were evaporated to dryness and resuspended in salt free buffer containing 10ktm norleucine as an internal standard. Analyses of tissue and plasma amino acids were made with a Chromaspek-J-180 (Rank Hilger) automatic amino acid analyzer and a 3390 A Integrator (Hewlet Packard) computing system calibrated with amino acid standards. RESULTS Experimental hyperphenylalaninemia was induced in chick embryos between the l lth and 20th day o f incubation by daily injections o f ~-methylphenylalanine and phenylalanine into the air space. In preliminary experiments, we tested the capacity of p-chlorophenylalanine in addition to P H E for producing PKU-like conditions in chick embryo, but this treatment resulted in a very high mortality (as much as 80'I~i). For this reason, further studies were limited to the alternative animal model utilizing MPA. As can be seen in Table 1, M P A plus PHE treatment produced a decrease in the brain weight after 8 days of treatment. However, treatments for a short time did not produce any alteration in the brain size. Likewise, liver weight o f hyperphenylalaninemic animals was reduced in 19-day chick embryos (Table 1). In order to check the effectiveness o f an M P A plus PHE treatment for producing and maintaining experimental P K U conditions, we have determined the P H E / T Y R ratio in both brain and liver of chick embryo during the last steps o f development. As shown in Fig. 1A the P H E / T Y R ratio was higher in hyperphenylalaninemic brain embryos than in controls already 3 days after beginning the treatment and increased afterwards. Thus, an increase o f nearly 14-fold in the brain P H E / T Y R ratio was found after
Table 1. Effect of MPA plus PHE administration on brain and liver weight of chick embryo Brain weight (g) Liver weight (gJ Age (days) Control Experimental Conlrol Experimenlal 12 (10) 0.250 + 0.004 0.248 + (I.006 0.072 + 0.005 0.068 _+0.008 16 (10) 0.485 + 0.01 I 0.467 2 0.01 I 0.261 + 0.0 [2 0.254 ± 0.011 19 (30t 0.748 _+0.010 0.677 ± 0.1)14" (I.447 +_0.(/26 0.377 _+0.022t Eggs were injected daily with MPA plus PHE from the l lth day of incubalion as described in Experimental Procedures. Each value representsmean ± SEM. Number of embryos are given in parentheses.*P < 0.001: +P < 0.05.
Hyperphenylalaninemia in chick embryo (a)
487
(b)
10--
~ 4
~
11
14
16
18
20
11
14
16
18
20
Age (doys) Fig. 1. Effect of daily administration of ~t-methylphenylalanine plus phenylalanine on P H E / T Y R ratio in chick embryo brain (A) and liver (B). (O), M P A plus P H E treated embryos; (O), control embryos. Results are expressed as means _+ SEM of three determinations with pools of 4 chick embryos.
9 days of MPA plus PHE administration. Similar r e s u l t s were o b s e r v e d in liver (Fig. 1 B), a l t h o u g h t h e i n c r e a s e in t h e P H E / T Y R r a t i o w a s s m a l l e r t h a n t h a t o b s e r v e d in b r a i n . C h r o n i c a d m i n i s t r a t i o n o f M P A p l u s P H E dist o r t e d t h e c o n c e n t r a t i o n s o f m a n y a m i n o a c i d s in p l a s m a , b r a i n a n d liver o f 19-day c h i c k e m b r y o s ( T a b l e 2). I n p l a s m a , t h e a m o u n t o f P H E i n c r e a s e d a b o u t 12-fold while in liver a n d b r a i n this i n c r e a s e w a s a b o u t 9-fold. C h r o n i c 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 c e d a l t e r a t i o n s in t h e c o n c e n t r a t i o n s o f t h e b r a n c h e d - c h a i n a l i p h a t i c a m i n o a c i d s valine, isol e u c i n e a n d leucine. In b o t h p l a s m a a n d liver, levels o f t h e s e a m i n o a c i d s clearly i n c r e a s e d while in b r a i n t h e s e levels d i d n o t c h a n g e s i g n i f i c a n t l y ( T a b l e 2). O n the other hand, plasma and brain glycine content
clearly i n c r e a s e d by M P A p l u s P H E t r e a t m e n t , while in liver n o s i g n i f i c a n t v a r i a t i o n s in t h e level o f this a m i n o acid were o b s e r v e d . Since t h e i n j e c t i o n o f M P A d i d n o t c o m p l e t e l y a b o l i s h t h e P H E H activity a f t e r 8 d a y s o f t r e a t m e n t , a d m i n i s t r a t i o n o f a n e x c e s s o f P H E w a s e x p e c t e d to rise t h e T Y R c o n c e n t r a t i o n in p l a s m a a n d tissues. H o w e v e r , n o s i g n i f i c a n t i n c r e a s e w a s f o u n d in e i t h e r p l a s m a or liver T Y R levels o f 19-day old c h i c k e m b r y o s , while in b r a i n this level w a s l o w e r t h a n in c o n t r o l a n i m a l s ( T a b l e 2). DISCUSSION The present study demonstrates that administ r a t i o n o f M P A p l u s P H E b e t w e e n 11 a n d 20 d a y s o f
Table 2. Amino acid content in chronic hyperphenylalaninemic chick embryos Liver (/zmol/g) Plasma (/~mol/ml) Brain (#mol/g) Aspartic acid Threonine Serine Glutamic acid Glutamine Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine fl-Alanine GABA Lysine
Control 0.763 ± 0.040 0.551 ± 0.048 1.05 | ± 0.113 2.578 ± 0.188 1.210 ± 0.018 0.575 ± 0.120 0.636 ± 0.062 0.303 ± 0.028 0.162 ± 0.012 0.258 ± 0.022 0.339 ± 0.036 0.129 ± 0.016 0.444 ± 0.063 . 0.308 ± 0.057
Experimental 0.761 ± 0.180 0.791 ± 0.157 1.411 ± 0.308 3.645 ± 0.770 1.024 ± 0.315 0.877 ± 0.150 0.670 ± 0.091 0.448 ± 0.048++ 0.218 ± 0.022§ 0.358 ± 0.051§ 0.448 ± 0.068 1.195 ± 0.179" 0.546 ± 0.146 . . 0.403 ± 0.057
Control 0.049 ± 0.006 0.231 ± 0.031 0.299 ± 0.030 0.119 ± 0.032 0.302 ± 0.040 0.167 ± 0.018 0.200 ___0.021 0.206 ± 0.016 0.086 ± 0.009 0.081 ± 0.008 0.105 ± 0.006 0.094 ± 0.001 0.008 ± 0.001 . 0.068 ± 0.031
Experimental 0.073 ± 0.012 0.258 ± 0.029 0.367 ± 0.029 0.175 ± 0.041 0.340 ± 0.075 0.326 ± 0.032* 0.296 ± 0.038 0.376 ± 0.038f 0.181 ± 0.018" 0.146 ± 0.012" 0.127 ± 0.026 1.129 ± 0.091" 0.007 ± 0.001 0.108 + 0.027
Control 0.914 ± 0.084 0.366 ± 0.037 0.941 ± 0.093 5.724 ± 0.564 4.712 ± 0.821 1.073 ± 0.071 0.837 + 0.098 0.190 ± 0.010 0.108 ± 0.015 0.108 ± 0.015 0.150 ± 0.029 0.093 ± 0.010 -1.440±0.116 0.235 ± 0.034
Experimental 1.104 ± 0.065 0.453 ± 0.039 1.043 ± 0.062 6.324 ± 0.441 3.996 ± 0.786 1.615 ± 0.062~ 0.952 ± 0.080 0.185 ± 0.026 0.096 ± 0.029 0.097 ± 0.020 0.094 ± 0.006:~ 0.799 ± 0.039* 1.682±0.115 0.203 ± 0.039
Chronic treatments with MPA plus PHE were given from age 11 to 19 days as described in Experimental Procedures. Each value represents mean +_SEM of six experiments. *P < 0.001; fP < 0.01; ~:P < 0.02; §P < 0.05.
488
C. MARco et al.
egg incubation produces hyperphenylalaninemic conditions in a period of chick embryo development that corresponds with rapid brain growth and myelination. Little is known about PKU damage in avian species, although in rats made hyperphenylalaninemic by administration of PHE (Clarke and Lowden, 1969), PHE plus p-chlorophenylalanine (Nordyke and Roach, 1974), or PHE plus MPA (Johnson and Shah, 1980; Lane and Neuhoff, 1980), a microcephaly was observed. However, it is not known whether the reduced brain size in human PKU (Paine, 1957) comes about because of a direct interference of PHE with the synthesis of brain structural components or indirectly by either limiting the availability of amino acids needed for protein synthesis or by causing protein degradation. Hyperphenylalaninemia has profound effects on individual transport mechanisms of different amino acids. Thus, high concentrations of PHE in plasma inhibited brain uptake of methionine and leucine (Agrawal et al., 1970), leucine (Mitoma and Levalley, 1973) or in general the uptake of branched-chain aliphatic amino acids (Vahvelainen and Oja, 1975) probably as a result of the inhibition by PHE of a common transport system of these amino acids. This competition could account for the failure of increased valine, leucine and isoleucine levels in brain from plasma in contrast to the increase observed in liver. On the other hand, the high level of brain glycine found after MPA plus PHE administration can be due to the accumulation from plasma, since PHE did not interfere with glycine uptake into the brain (Agrawal et al., 1970). A similar rise in cerebral glycine content has been observed in hyperphenylalaninemic rats by long exposure to PHE (Lane et al., 1980; Dienel, 1981). Likewise, Isaacs and Greengard (1980) have reported the elevation of glycine content in developing rat brain by MPA plus PHE treatment. These authors proposed that, apparently, chronic hyperphenylalaninemia interferes with normal regulation of intracerebral glycine metabolism during a critical period of early postnatal development of rat. One of the major objectives in introducing MPA to our animal model was to produce hyperphenylalaninemia without concomitantly producing hypertyrosinemic condition. Our results show that the high increase in the brain P H E / T Y R ratio obtained after 8-9 days of MPA plus PHE administration was due to both an increase in the PHE and a decrease in the TYR levels. It is important to remark that the experimental embryos presented nearly a 40~o decrease in brain tyrosine concen-
tration, similar to the 50'~o decrease found in human phenylketonurics (McKean and Peterson, 1970; McKean, 1972), whereas in immature rats an increase in brain and/or plasma tyrosine concentration was obtained by this (Dienel, 1981: Lo and Longenecker, 1981) and other treatments (Lane and Neuhoff, 1980). Therefore, the treatment here reported proved to be an excellent method for imitating conditions of PKU in avian species during myelination, one of the most dramatic period in nervous system development. Further studies will be necessary to elucidate the nature and causes of brain damage in this inborn error of amino acid metabolism. Acknowledgements This work was supported in part by a grant from the -Comisi6n Asesora de Investigaci6n Cientifica y T~cnica" (4113-79). We thank Dr D. GonzalezPacanowska for help in correction of the manuscript. REFERENCES
Agrawal H. C., Bone A. H. and Davison A. N. (1970) Effect of phenylalanine on protein synthesis in the developing rat brain. Biochem. J. 117, 325 331. Alejandre M. J., Marco C., Ramirez H., Segovia J. L. and Garcia-Peregrin E. (1984) Lipid composition of brain myelin from normal and hyperphenylalaninemic chick embryos. Comp. Biochem. Physiol. In press. Brass C. A., Isaacs C. E., McChesney R. and Greengard O. (1982) The effects of hyperphenylalaninemia on fetal development: a new animal model of maternal phenylketonuria. Pediatr. Res. 16, 388-394. Clarke J. T. R. and Lowden J. A. (1969) Hyperphenylalaninemia: effect on the developing rat brain. Can. J. Biochem. 47, 291-295. DelValle J. A., Dienel G. and Greengard O. (1978) Comparison of ~ -methylphenylalanine and pchlorophenylalanine as inducers of chronic hyperphenylalaninemia in developing rats. Bioehem. J. 170, 449459. DelValle J. A. and Greengard O. (1976)The regulation of phenylalanine hydroxylase in rat tissues in vivo. Biochem. J. 154, 613-618. Dienel G. A. (1981) Chronic hyperphenylalaninemia produces cerebral hypergtycinemia in immature rats. J. Neurochem. 36, 34-43. Greengard O., Yoss M. S. and DelValle J. A. (t976) z~-Methylphenylalanine, a new inducer of chronic hyperphenylalaninemia in suckling rats. Science 192, 1007 1009. Isaacs C. E. and Greengard O. (1980) The effect of hyperphenylalaninemia on glycine metabolism in developing rat brain. Biochem. J. 192, 441448. Johnson R. C. and Shah S. N. (1973) Effect of hyperphenylalaninemia on fatty acid composition of lipids of rat brain myelin. J. Neurochem. 21, 1225-1240. Johnson R. C. and Shah S. N. (1980) Effects of -methylphenylalanine plus phenylalanine treatment during development on myelin in rat brain. Neurochem. Res. 5, 709-718. Knox W. E. (1972) Phenylketonuria. In: The Metabolic Basis' o f Inherited Dis'eases (Stanbury J. B., Wyngaarden
Hyperphenylalaninemia in chick embryo J. B. and Frederickson D. S., eds), 3rd edn, pp. 265-295. McGraw-Hill, New York. Lane J. D. and Neuhoff V. (1980) Phenylketonuria. Clinical and experimental considerations revealed by the use of animal models. Naturwissenschaften 67, 227-233. Lane J. D., Sch6ne B., Langenbeck U. and Neuhoff V. (1980) Characterization of experimental phenylketonuria. Augmentation of hyperphenylalaninemia with ~-methylphenylalanine and p-chlorophenylalanine. Biochim. biophys. Acta 627, 144-156. Lo G. S. and Longenecker J. B. (1981) Induction of an experimental phenylketonuria-like condition in infant rats during the first 2 weeks after birth. Am. J. clin. Nutr. 34, 490-497. McGee M. M., Greengard O. and Knox W. E. (1972) The quantitative determination of phenylalanine in rat tissues: Its developmental formation in liver. Biochem. J. 127, 669-674. McKean C. M. (1972) The effects of high phenylalanine concentrations on serotonin and catecholamine metabolism in the human brain. Brain Res. 47, 469-476. McKean C. M. and Peterson N. A. (1970) Glutamine in the phenylketonuric central nervous system. New Engl. J. Med. 283, 1364--1367.
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Mitoma C. and LeValley S. E. (1973) Transport and incorporation of labeled compounds in experimental phenylketonuric rats. Proc. Soc. exp. Biol. Med. 144, 710-713. Nordyke E. L. and Roach M. K. (1974) Effect of hyperphenylalaninemia on amino acid metabolism and compartmentation in neonatal rat brain. Brain Res. 67, 479--488. Paine R. S. (1957) The variability in manifestation of untreated patients with phenylketonuria (phenylpyruvic aciduria). Pediatrics 20, 290-302. Shah S. N. and Johnson R. C. (1978) Effect of postweaning hyperphenylalaninemia on brain development in rats: Myelination, lipid and fatty acid composition of myelin. Exp. Neurol. 61, 370-379. Shah S. N., Peterson N. A. and McKean C. M. (1972) Lipid composition of human cerebral white matter and myelin in phenylketonuria. J. Neurochem. 19, 23692376. Vahvelainen M. L. and Oja S. S. (1975) Kinetic analysis of phenylalanine-induced inhibition in the saturable influx of tyrosine, tryptophan, leucine and histidine into brain cortex slices from adult and 7-day old rats. J. Neurochem. 24, 885-892.
0197-0186/84 $3.00 + 0.00 © 1984 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
INDUCTION OF EXPERIMENTAL PHENYLKETONURIA-LIKE CONDITIONS IN CHICK EMBRYO. EFFECT ON AMINO ACID CONCENTRATION IN BRAIN, LIVER AND PLASMA C. MARCO, M. J. ALEJANDRE,M. F. ZAFRA, J. L. SEGOVIA and E. GARCIA-PEREGR1N* Department of Biochemistry, University of Granada, Granada, Spain (Received 31 March 1983; accepted 4 November 1983)
Abstract--Experimental hyperphenylalaninemia has been induced in chick embryos between 11-20 days of incubation by daily injection of ~t-methylphenylalanine and phenylalanine. Brain and liver weight decreased after 8 days of treatment. An increase of nearly 14-fold in the brain phenylalanine/tyrosine ratio was observed after 9 days of treatment. Similar results were obtained in liver, although the increase found in this case was smaller than in brain. Chronic hyperphenylalaninemia induced a clear rise in the levels of plasma and liver valine, leucine and isoleucine, while in brain these levels did not change significantly. Plasma and brain glycine content was also enhanced by this treatment. Brain tyrosine concentration was clearly decreased in these conditions, in contrast to the enhancement reported after this and other treatments in various animal species. Thus, the higher value of the brain phenylalanine/tyrosine ratio obtained by ct-methylphenylalanine plus phenylalanine administration was due to both an increase in the phenylalanine and a decrease in the tyrosine levels, conditions that have been also found in human phenylketonurics. Therefore, the treatment here reported was an excellent method for imitating the conditions of phenylketonuria during the period of rapid myelination in the chick, one of the most dramatic in nervous system development.
One of the most common disorders of amino acid metabolism associated with mental retardation is phenylketonuria (PKU). The primary defect in this disease, an absence of phenylalanine hydroxylase (PHEH) activity (Knox, 1972), produced an elevation in plasma and tissue phenylalanine (PHE). In humans, this basic defect is not evident until shortly after birth when the brain is undergoing its most rapid rate of biochemical differentiation and is most vulnerable to the biochemical insult of PKU. To determine the mechanism leading to abnormal brain development many researchers have induced hyperphenylalaninemic conditions in animals by a variety of methods. Until recently, hyperphenylalaninemia had been produced by injections of PHE alone (Clarke and Lowden, 1969; Johnson and Shah, 1973), but in these conditions tyrosine (TYR) levels are grossly increased, unlike in the clinical picture. Injections of PHE and a PHEH inhibitor, pchlorophenylalanine (DelValle and Greengard, 1976; Shah and Johnson, 1978), permit the maintenance of *To whom correspondence should be sent.
a high P H E / T Y R ratio in the plasma, but the action of the inhibitor is not specific to the PHEH and toxic effects have been reported (DelValle et al., 1978). Greengard et al. (1976) have reported that another analog of PHE, ~t-methylphenylalanine (MPA), inhibited PHEH in newborn rats without other toxic effects on animal growth. More recently Brass et al. (1982) have described a regimen of MPA plus PHE that induces severe and prolonged hyperphenylalaninemia in pregnant rats and their fetuses but does not interfere with the preor postnatal survival of the rats. Several studies have demonstrated a similarity between cerebral myelin abnormalities occurring in phenylketonuric individuals and those occurring in rats with experimentally induced hyperphenylalaninemia. These studies have confirmed that there is a reduced amount of recoverable myelin but this myelin is of normal composition (Shah et al, 1972). The time of birth in relation to the period of maximum rate of myelination is different in different species. Thus, the brains of rats and mice are very immature at birth, the animals are physically quite 485
486
C, MARCt) el al.
helpless, and the periods o f m a x i m u m cellular proliferation and myelination are still to occur. In contrast, chick myelination occurs during embryonic develo p m e n t and chicks are born with a well-developed C.N.S. On the other hand, myelination is more rapid in chick than in other species. With this in mind, we have used M P A plus P H E to produce a chronic hyperphenylalaninemia in chick embryo at the time in which myelination and rapid brain growth occur, so that this new animal model should facilitate the study o f the effects o f hyperphenylalaninemia during the period of rapid myelination, one o f the most dramatic in nervous system development. In the present paper, the ability of M P A to maintain low levels o f TYR and to prolong the hyperphenylalaninemic conditions was examined. In addition, this paper describes the amino acid composition of brain, liver and plasma o f control and hyperphenylalaninemic chick embryos after 8 days of treatment. Previous studies have established the influence o f hyperphenylalaninemia induced by the same treatment on the lipid composition o f brain myelin in 19-day old chick embryos (Alejandre et al., 1984).
EXPERIMENTAL PROCEDURES
Induction o/' experimental hyperphenvlalaninemia
White Leghorn chickens eggs were incubated at 37.8_+0.2 C and 601~, relative humidity. On day 10 of incubation a small hole was open over the air space in the shell and sealed with paraffin. Eggs were divided into two groups and treatments were initiated at the l lth day of incubation. Experimental eggs were injected daily into the air space with 0.05ml of L-phenylalanine (173#mol/ml) at 8 and 20 h and once with 0.05ml of ~-methyl-DL-phenylalanine (190 #mol/ml) at 14 h. Control eggs were injected three times a day at 8, 14 and 20 h with 0.05 ml of 0.9°11NaC1 solution. Blood and tissue collection
Embryos were removed from their shells 12 h after the last injection. Blood was collected from a cardiac puncture, run into heparinized tubes and centrifuged at 2000g for 5 min. Plasma was deproteinized by addition of 1 vol of 0.6 M trichloroacetic acid and after 20 rain the mixture was centri-
fuged at 4000g. Liver and brain were quickly excised and weighed. Hepatic PHEH was measured by the method of McGee et al. (1972). Tissue samples to be used for amino acid analyses were immediately homogenized in 3 vol of ice-cold distilled water and centrifuged at 15000g. Trichloroacetic acid (0.6 M) was added to the supernatants to a final concentration of 0.3 M. Samples were thoroughly mixed, allowed to stand at room temperature for 20 min and centrifuged at 4000g. Ana@~e,~
The tissue and plasma samples were evaporated to dryness and resuspended in salt free buffer containing 10ktm norleucine as an internal standard. Analyses of tissue and plasma amino acids were made with a Chromaspek-J-180 (Rank Hilger) automatic amino acid analyzer and a 3390 A Integrator (Hewlet Packard) computing system calibrated with amino acid standards. RESULTS Experimental hyperphenylalaninemia was induced in chick embryos between the l lth and 20th day o f incubation by daily injections o f ~-methylphenylalanine and phenylalanine into the air space. In preliminary experiments, we tested the capacity of p-chlorophenylalanine in addition to P H E for producing PKU-like conditions in chick embryo, but this treatment resulted in a very high mortality (as much as 80'I~i). For this reason, further studies were limited to the alternative animal model utilizing MPA. As can be seen in Table 1, M P A plus PHE treatment produced a decrease in the brain weight after 8 days of treatment. However, treatments for a short time did not produce any alteration in the brain size. Likewise, liver weight o f hyperphenylalaninemic animals was reduced in 19-day chick embryos (Table 1). In order to check the effectiveness o f an M P A plus PHE treatment for producing and maintaining experimental P K U conditions, we have determined the P H E / T Y R ratio in both brain and liver of chick embryo during the last steps o f development. As shown in Fig. 1A the P H E / T Y R ratio was higher in hyperphenylalaninemic brain embryos than in controls already 3 days after beginning the treatment and increased afterwards. Thus, an increase o f nearly 14-fold in the brain P H E / T Y R ratio was found after
Table 1. Effect of MPA plus PHE administration on brain and liver weight of chick embryo Brain weight (g) Liver weight (gJ Age (days) Control Experimental Conlrol Experimenlal 12 (10) 0.250 + 0.004 0.248 + (I.006 0.072 + 0.005 0.068 _+0.008 16 (10) 0.485 + 0.01 I 0.467 2 0.01 I 0.261 + 0.0 [2 0.254 ± 0.011 19 (30t 0.748 _+0.010 0.677 ± 0.1)14" (I.447 +_0.(/26 0.377 _+0.022t Eggs were injected daily with MPA plus PHE from the l lth day of incubalion as described in Experimental Procedures. Each value representsmean ± SEM. Number of embryos are given in parentheses.*P < 0.001: +P < 0.05.
Hyperphenylalaninemia in chick embryo (a)
487
(b)
10--
~ 4
~
11
14
16
18
20
11
14
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Age (doys) Fig. 1. Effect of daily administration of ~t-methylphenylalanine plus phenylalanine on P H E / T Y R ratio in chick embryo brain (A) and liver (B). (O), M P A plus P H E treated embryos; (O), control embryos. Results are expressed as means _+ SEM of three determinations with pools of 4 chick embryos.
9 days of MPA plus PHE administration. Similar r e s u l t s were o b s e r v e d in liver (Fig. 1 B), a l t h o u g h t h e i n c r e a s e in t h e P H E / T Y R r a t i o w a s s m a l l e r t h a n t h a t o b s e r v e d in b r a i n . C h r o n i c a d m i n i s t r a t i o n o f M P A p l u s P H E dist o r t e d t h e c o n c e n t r a t i o n s o f m a n y a m i n o a c i d s in p l a s m a , b r a i n a n d liver o f 19-day c h i c k e m b r y o s ( T a b l e 2). I n p l a s m a , t h e a m o u n t o f P H E i n c r e a s e d a b o u t 12-fold while in liver a n d b r a i n this i n c r e a s e w a s a b o u t 9-fold. C h r o n i c 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 c e d a l t e r a t i o n s in t h e c o n c e n t r a t i o n s o f t h e b r a n c h e d - c h a i n a l i p h a t i c a m i n o a c i d s valine, isol e u c i n e a n d leucine. In b o t h p l a s m a a n d liver, levels o f t h e s e a m i n o a c i d s clearly i n c r e a s e d while in b r a i n t h e s e levels d i d n o t c h a n g e s i g n i f i c a n t l y ( T a b l e 2). O n the other hand, plasma and brain glycine content
clearly i n c r e a s e d by M P A p l u s P H E t r e a t m e n t , while in liver n o s i g n i f i c a n t v a r i a t i o n s in t h e level o f this a m i n o acid were o b s e r v e d . Since t h e i n j e c t i o n o f M P A d i d n o t c o m p l e t e l y a b o l i s h t h e P H E H activity a f t e r 8 d a y s o f t r e a t m e n t , a d m i n i s t r a t i o n o f a n e x c e s s o f P H E w a s e x p e c t e d to rise t h e T Y R c o n c e n t r a t i o n in p l a s m a a n d tissues. H o w e v e r , n o s i g n i f i c a n t i n c r e a s e w a s f o u n d in e i t h e r p l a s m a or liver T Y R levels o f 19-day old c h i c k e m b r y o s , while in b r a i n this level w a s l o w e r t h a n in c o n t r o l a n i m a l s ( T a b l e 2). DISCUSSION The present study demonstrates that administ r a t i o n o f M P A p l u s P H E b e t w e e n 11 a n d 20 d a y s o f
Table 2. Amino acid content in chronic hyperphenylalaninemic chick embryos Liver (/zmol/g) Plasma (/~mol/ml) Brain (#mol/g) Aspartic acid Threonine Serine Glutamic acid Glutamine Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine fl-Alanine GABA Lysine
Control 0.763 ± 0.040 0.551 ± 0.048 1.05 | ± 0.113 2.578 ± 0.188 1.210 ± 0.018 0.575 ± 0.120 0.636 ± 0.062 0.303 ± 0.028 0.162 ± 0.012 0.258 ± 0.022 0.339 ± 0.036 0.129 ± 0.016 0.444 ± 0.063 . 0.308 ± 0.057
Experimental 0.761 ± 0.180 0.791 ± 0.157 1.411 ± 0.308 3.645 ± 0.770 1.024 ± 0.315 0.877 ± 0.150 0.670 ± 0.091 0.448 ± 0.048++ 0.218 ± 0.022§ 0.358 ± 0.051§ 0.448 ± 0.068 1.195 ± 0.179" 0.546 ± 0.146 . . 0.403 ± 0.057
Control 0.049 ± 0.006 0.231 ± 0.031 0.299 ± 0.030 0.119 ± 0.032 0.302 ± 0.040 0.167 ± 0.018 0.200 ___0.021 0.206 ± 0.016 0.086 ± 0.009 0.081 ± 0.008 0.105 ± 0.006 0.094 ± 0.001 0.008 ± 0.001 . 0.068 ± 0.031
Experimental 0.073 ± 0.012 0.258 ± 0.029 0.367 ± 0.029 0.175 ± 0.041 0.340 ± 0.075 0.326 ± 0.032* 0.296 ± 0.038 0.376 ± 0.038f 0.181 ± 0.018" 0.146 ± 0.012" 0.127 ± 0.026 1.129 ± 0.091" 0.007 ± 0.001 0.108 + 0.027
Control 0.914 ± 0.084 0.366 ± 0.037 0.941 ± 0.093 5.724 ± 0.564 4.712 ± 0.821 1.073 ± 0.071 0.837 + 0.098 0.190 ± 0.010 0.108 ± 0.015 0.108 ± 0.015 0.150 ± 0.029 0.093 ± 0.010 -1.440±0.116 0.235 ± 0.034
Experimental 1.104 ± 0.065 0.453 ± 0.039 1.043 ± 0.062 6.324 ± 0.441 3.996 ± 0.786 1.615 ± 0.062~ 0.952 ± 0.080 0.185 ± 0.026 0.096 ± 0.029 0.097 ± 0.020 0.094 ± 0.006:~ 0.799 ± 0.039* 1.682±0.115 0.203 ± 0.039
Chronic treatments with MPA plus PHE were given from age 11 to 19 days as described in Experimental Procedures. Each value represents mean +_SEM of six experiments. *P < 0.001; fP < 0.01; ~:P < 0.02; §P < 0.05.
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C. MARco et al.
egg incubation produces hyperphenylalaninemic conditions in a period of chick embryo development that corresponds with rapid brain growth and myelination. Little is known about PKU damage in avian species, although in rats made hyperphenylalaninemic by administration of PHE (Clarke and Lowden, 1969), PHE plus p-chlorophenylalanine (Nordyke and Roach, 1974), or PHE plus MPA (Johnson and Shah, 1980; Lane and Neuhoff, 1980), a microcephaly was observed. However, it is not known whether the reduced brain size in human PKU (Paine, 1957) comes about because of a direct interference of PHE with the synthesis of brain structural components or indirectly by either limiting the availability of amino acids needed for protein synthesis or by causing protein degradation. Hyperphenylalaninemia has profound effects on individual transport mechanisms of different amino acids. Thus, high concentrations of PHE in plasma inhibited brain uptake of methionine and leucine (Agrawal et al., 1970), leucine (Mitoma and Levalley, 1973) or in general the uptake of branched-chain aliphatic amino acids (Vahvelainen and Oja, 1975) probably as a result of the inhibition by PHE of a common transport system of these amino acids. This competition could account for the failure of increased valine, leucine and isoleucine levels in brain from plasma in contrast to the increase observed in liver. On the other hand, the high level of brain glycine found after MPA plus PHE administration can be due to the accumulation from plasma, since PHE did not interfere with glycine uptake into the brain (Agrawal et al., 1970). A similar rise in cerebral glycine content has been observed in hyperphenylalaninemic rats by long exposure to PHE (Lane et al., 1980; Dienel, 1981). Likewise, Isaacs and Greengard (1980) have reported the elevation of glycine content in developing rat brain by MPA plus PHE treatment. These authors proposed that, apparently, chronic hyperphenylalaninemia interferes with normal regulation of intracerebral glycine metabolism during a critical period of early postnatal development of rat. One of the major objectives in introducing MPA to our animal model was to produce hyperphenylalaninemia without concomitantly producing hypertyrosinemic condition. Our results show that the high increase in the brain P H E / T Y R ratio obtained after 8-9 days of MPA plus PHE administration was due to both an increase in the PHE and a decrease in the TYR levels. It is important to remark that the experimental embryos presented nearly a 40~o decrease in brain tyrosine concen-
tration, similar to the 50'~o decrease found in human phenylketonurics (McKean and Peterson, 1970; McKean, 1972), whereas in immature rats an increase in brain and/or plasma tyrosine concentration was obtained by this (Dienel, 1981: Lo and Longenecker, 1981) and other treatments (Lane and Neuhoff, 1980). Therefore, the treatment here reported proved to be an excellent method for imitating conditions of PKU in avian species during myelination, one of the most dramatic period in nervous system development. Further studies will be necessary to elucidate the nature and causes of brain damage in this inborn error of amino acid metabolism. Acknowledgements This work was supported in part by a grant from the -Comisi6n Asesora de Investigaci6n Cientifica y T~cnica" (4113-79). We thank Dr D. GonzalezPacanowska for help in correction of the manuscript. REFERENCES
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