Effect of experimental hyperphenylalaninemia on biogenic amine synthesis at later stages of brain development

BIOCHEMICAL

MEDICINE

29, 307-3 17 (1983)

Effect of Experimental Hyperphenylalaninemia on Biogenic Amine Synthesis at Later Stages of Brain Development E. H. TAYLOR,’ F. A. HOMMES,’ ANDD. E. STEWART Departments

of Cell

and

Molecular

Biology, and Augusta. Geor,gia

Anatomy, 30912

Medical

College

of Georgia.

Received June 9. 1982

The neurological abnormalities associated with phenylketonuria (PKU) can be prevented with a phenylalanine restricted diet, provided this diet is instituted at a sufficiently young age (1). If this dietary regime is not begun during the period of rapid development or “growth spurt” which occurs near birth, the neurological damage is irreversible. The question of when to terminate dietary treatment is much more difficult and not easily answered. Current opinion is to relax or terminate the dietary treatment at 6-8 years of age, when the brain is thought to be sufficiently mature to tolerate higher levels of phenylalanine. There is much debate as to the effects of dietary termination. There is no agreement on the consequences of the increased intake of phenylalanine at this age on behavior and intellectual development. Some investigators find no difference in intellectual development after dietary termination (2-5) while others demonstrate a drop in IQ (6-9). This research was undertaken to study the effects of increased phenylalanine levels in brain on catecholamine and serotonin synthesis in a suitable animal-model system, at a time in brain development which is equivalent to a PKU patient being removed from a low-phenylalanine diet. MATERIALS

AND METHODS

Animals and Diets Female Harlan Wistar rats, (WI) Br, were obtained from Harlan SpragueDawley, Indianapolis, Indiana. At the age of 25 days, the animals received ’ Present address: Department of Laboratory Medicine, Carolina, 171 Ashley Avenue, Charleston, SC. 29425. ’ To whom requests for reprints should be addressed.

Medical University of South

307 0006-2944/83 $3.00 Copyright Q 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.

308

-I‘Al'L,OK. HCIMMES. AND STEWAKI

one of the following diets: (a) normal laboratory chow (control group): (b) normal laboratory chow supplemented with 0.4% cu-methylphenylalanine (aMP group), Research Organics, Cleveland. Ohio; and (c) normal laboratory chow supplemented with 0.4% aMP and 5% phenylalanine (HyPhe group). All diets were prepared by Ralston Purina Company, Richmond, Indiana. Determinations of’ Catecholamines and Serototlin Water used in these determinations was HPLC grade (Fisher Scientific Springfield, N.J.). Catecholamine standards and alumina were obtained from Bioanalytical Systems (West Lafayette, Ind.). All other chemicals were purchased from Fisher Scientific. A standard stock solution containing 0.5 mgiml in 0. I N perchloric acid (PCA) of norepinephrine, dopa, epinephrine, and dopamine was prepared and then diluted to give five concentrations of 500 @ml through 12.5 rig/ml. The internal standard, dihydroxybenzylamine (DHBA), was prepared at a concentration of 5 mg/liter and IO ~1 of this solution was added to a mixture of 0.5 ml of each dilute standard, 25 mg of alumina, and 0.3 ml of 2.5 M Tris. This solution was vortexed for 2 min and the pH adjusted to 8.6-8.7. After centrifugation in a microfiltration centrifuge (BAS, West Lafayette, Ind.), the pellet was washed twice with 2 ml HzO. The catecholamines were eluted with 0.5 ml of 0.1 N PCA by vortexing 4.5 min and centrifuging. Supernatants were then transferred to a microfilter tube and centrifuged in the BAS microcentrifuge. Twenty microliters of this supernatant was used to inject into the HPLC apparatus. Animals were sacrificed after IO days on the diet by means of decapitation. Each whole brain was dissected and placed into liquid nitrogen. The brain was later weighed and homogenized in 5 ml of 0. I N PCA and centrifuged at 27,OOOg for I5 min in a Beckman 52-21 centrifuge with a J-20 rotor. An aliquot of 0.5 ml was taken from the supernatant and processed just as the standard solutions in the above procedure. The mobile phase for the HPLC consisted of 0.15 M monochloracetic acid. I mM Na,EDTA and 25 ml/liter sodium octylsulfate at pH = 3.0. This solution was filtered through a 0.2-p cellulose nitrate membrane (Fisher Scientific) and degassed under a vacuum with magnetic stirring. Chromatography was done on a Beckman Model 332 gradient liquid chromatograph with a flow rate of 2 ml/min and a pressure of 4000 psi with a Beckman ODS ultrasphere reversed phase column. The detector was an LC-4A amperometric detector (BAS) with a potential of + 0.70 V. Detections were done at 0.5. I, or 2 nA with an offset of approximately 30. The chromatographic tracings were done on a strip chart recorder at a speed of 5 mm/min. Unknown peak heights were read off of the standard curve (nA x peak height vs ng) and converted into units of

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nanomoles per gram wet weight of brain. Samples from the three diet groups were then compared using analysis of variance (ANOVA). The determination of serotonin and SHIAA was also done by the method of HPLC. Standards of serotonin and SHIAA were prepared in similar concentrations, as those of the catecholamine determination. The standards were diluted to give six concentrations of 500 rig/ml through 5 rig/ml. The internal standard, N-methylserotonin, was prepared at a concentration of 10 pg/ml. To 500 ~1 of each of the dilute standards were added 10 ~1 of internal standard and 20 ~1 of this solution was injected into the HPLC. Rats were sacrified under the same conditions as the catecholamine determination, except only brainstem was dissected. Each brainstem was homogenized in 2 ml of 0.1 N PCA in a polytron tissue homogenizer (Brinkman Instruments, Westbury N.Y.). Samples were centrifuged as described in the catecholamine assay, and 500 ~1 of the supernatant was added to 10 ~1 of internal standard. Twenty microliters was then used to inject into the HPLC. Chromatographic conditions were identical to the catecholamine assay with the exception of the mobile phase which consisted of 0.1 M chloroacetic acid, 1 mM EDTA. 9% methanol, and pH = 3.0. Standard curves were plotted and unknowns determined as described in the catecholamine assay. Determination

of Amino Acids

Solid sulfosalicylic acid (30 mg/ml) was added to plasma samples and spun at 3000~ for 10 min in a refrigerated centrifuge. The supernatant (0.4 ml) was diluted to 2 ml with lithium citrate buffer (0.21 N, pH = 2.8) and amino acid analysis performed on a JEOL Amino Acid Analyzer. (JEOL, U.S.A., Cranford, N.J.). Brain samples were prepared for amino acid analysis by placing whole brains into liquid nitrogen and later homogenizing in 5 ml of 0.1 N perchloric acid and centrifuging at 27,000g for 15 min. An aliquot of this supernatant was taken for tryptophan determinations by the revised method of Denkla and Dewey (28). The remainder of the supernatant was used for Phe and Tyr determinations under the same conditions described for plasma samples. RESULTS

The dietary treatment of normal chow plus 5% Phe + 0.4% aMP (HyPhe) provides a very successful model to approximate the plasma phenylalanine (Phe) values of the human PKU patient (Table 1). A group treated with the phenylalanine hydroxylase inhibitor, aMP, added to the diet, showed a threefold increase in plasma Phe without a significant increase in plasma tyrosine (Table 1). One drawback to any chemically induced experimental hyperphenylalaninemia is the plasma tyrosine values which were also elevated, and this condition is not analogous to the human PKU patient. Plasma tyrosine was elevated in the HyPhe group and this demonstrates the inability of 0.4% (uMP to completely block the

310

TAYLOR.

HOMMES. ‘TABLE

AROMATIC

AMINO

ACID

AND STEWAR’I I

CONTENT OF PLASMA HYPERPHENYLALANINEMIA”

AND

BRAIN

Brain’

Plasma” Phe Control 0.4% aMP 5% Phe f 0.4% (YMP

IN EXPERIMENIAI.

Tyr

Phe

52* 5 147 -c 23

90 2 10 108 -r- I4

42t 5 83 zk 26

2217 + 889

423 f 41

1260 + 52

Tyr

Tw

65 k 29 92 5 26 l92-+

4

20 k I I9 k 2 I8 !I 2

” Animals of 35 days on respective diets for the previous IO days. (For explanation of diets see Materials and Methods.) ’ Values expressed as PM: mean ? SD (n = 4). ’ Values expressed as nmole/g: mean 2 SD (n = 4 or 5).

phenylalanine hydroxylase. Intracerebral values of Phe were dramatically increased in the HyPhe group, while the tyrosine values were approximately three times that of the control group and tryptophan was not significantly different between the three diet groups (Table I). Since experimental hyperphenylalaninemia causes increased levels of tyrosine in brain and phenylalanine is known to competitively inhibit tyrosine hydroxylase (IO), it is of interest to investigate the effects of experimental hyperphenylalanemia on catecholamine synthesis and equate these findings with the human condition. The separation of norepinephrine, dopa, epinephrine, and dopamine of control animal’s brain at 35 days is shown in Fig. IA. The epinephrine peak was noticeably absent in brains from 35day-old animals which has been treated with 5% Phe + (uMP from Day 25 (Fig. IB). The question as to the effects of (wMP on catecholamine synthesis must be evaluated before conclusions can be drawn about the effects of experimental hyperphenylalaninemia. (YMP proved to be a very potent inhibitor of tyrosine hydroxylase in viva, since animals treated with a diet supplemented with 0.4% CXMP showed substantially decreased levels of dopa in brain (Table 2). Dopamine P-hydroxylase is also inhibited by aMP as norepinephrine values dropped to half the control values in the 0.4% aMP group, and this drop was not due to depressed levels of dopa, as dopamine values were slightly lower in controls (Table 2). Thus, (wMP appears to inhibit the hydroxylases in catecholamine metabolism in addition to inhibition of phenylalanine hydroxylase. Surprisingly, (YMP also caused a decrease in epinephrine to about 25% of control values. Since the norepinephrine was a little more than 50% of controls in the aMP group, inhibition of phenylethanolamine iV-methyltransferase by (YMP may occur beyond the decreased availability of norepinephrine for epinephrine synthesis.

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PKU

6

30

20

A

Time

10

30

0

6

(minutes)

20

Time

tb

(minutes)

FIG. 1. Separation of catecholamines by HPLC from acid-soluble extract of brain from a 35-day-old rat maintained on: (A) control diet for the previous 10 days and (B) control diet supplemented with 5% Phe + 0.4% cr-methylphenylalanine. Identification of peaks: (1) norepinephrine; (2) dopa; (3) epinephrine; (4) dihydroxybenzylamine (DHBA), internal standard: (5) 5-hydroxyindoleacetic acid: and (6) dopamine.

Even though the tyrosine in brain of the HyPhe group was three times the value in the arMP group, there was no difference in dopa concentrations in brain, suggesting that substantially increased phenylalanine had little effect in further depressing the dopa levels beyond those observed in the cxMP group. The levels of norepinephrine were significantly different among all diet groups, and the increased norepinephrine in the HyPhe TABLE BRAIN CATECHOLAMINE

Control (n = 4Y 0.4% (uMP (n = 5) 5% Phe + 0.4% aMP (n = 5)

2

LEVELS IN EXPERIMENTAL

HYPEIWHENYLALANINEMIA”

Dopa

Dopamine

Norepinephrine

Epinephrine

0.239 k 0.024

3.39 -r- 0.51

2.00 k 0.23**

0.087 ” 0.054

0.086 2 0.069*

2.94 ” 0.35

1.08 k 0.14**

0.021 f 0.004*

0.067 2 0.055*

3.78 2 0.68

1.51 2 0.29**

Not detected

a Animals of 35 days on respective diets for the previous 10 days. (For explanation of diets see Materials and Methods.) ’ Number of animals. ’ Values expressed as nmolelg: mean f SD. * P SC0.05 with respect to controls (ANOVA). ** P CC0.05 with respect to all groups (ANOVA).

312

TAYLOR.HOMMES.AND

I

STEWART

b

2 (

A

Time (minutes)

I3 Time (minutes)

FIG. 2. Separation of 5-hydroxytryptamine (5HT) and 5-hydroxyindoleacetic acid (SHIAA) by HPLC from acid-soluble extract of brain from a 35-day-old rat maintained on: (A) control diet for the previous 10 days and (B) control diet supplemented with 5% Phe + 0.4%~methylphenylalanine. Identificationofpeaks: SHT-Shydroxytryptamine(serotonin); IS-internal standard (N-methylserotonin); and SHIAA-5-hydroxyindoleacetic acid.

group versus the CXMP group may be related to increases in dopamine observed in the HyPhe group (Table 2). Hyperphenylalaninemia depresses the epinephrine levels in brain more than that observed in the aMP group (Table 2), as epinephrine could not be detected in the HyPhe group (Fig. IS). In PKU patients, decreased plasma values of serotonin (SHT) and 5hydroxyindoleacetic acid (SHIAA) occur (32). Therefore, one can ask how experimental hyperphenylalaninemia and (-uMP affect serotonin metabolism. The separation of 5HT and SHIAA from normal rat brainstem is shown in Fig. 2A and it is apparent that a substantial reduction of 5HT and SHIAA occurs in the hyperphenylalaninemic condition (Fig. 2B). The (YMP group did not vary from the control group in concentrations of 5HT or SHIAA (Table 3). Although no difference in intracerebral values of tryptophan in the HyPhe group versus controls was observed, a substantial reduction (>50%) of both 5HT and SHIAA was demonstrated in the HyPhe group which was independent of the presence of (YMP. This inhibition was indeed due to the presence of excess phenylalanine (Table 3). DISCUSSION Necropsied brains of untreated PKU patients contain 3040% of normal levels of serotonin, dopamine, and norepinephrine with a 40-50% decrease

BIOGENIC

SEROTONIN

AND

AMINES

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PKU

TABLE 3 SHIAA LEVELS FROM BRAINSTEMS IN EXPERIMENTAL HYPERPHENYLALANINEMIA”

Serotonin 2.30 k 0.27 Controls (n = 5)b 0.4% (rMP (n = 5) 2.26 rf- 0.47 5% phe + 0.4% cvMP (n = 4) 1.07 t 0.29* ____ .~~~__ ” Animals of 35 days on respective diets for the previous IO days. (For diets see Materials and Methods.) ’ Number of animals. ’ Values expressed as nmoleig: mean k SD. * P < 0.001 with respect to controls (ANOVA).

SHIAA 2.73 -t 0.48 2.14 t 0.73 0.87 + 0.23* explanation of

in their amino acid precursors tryptophan and tyrosine; reduced levels of biogenic amines were reversed following restriction of phenylalanine intake (11). The catecholamines are seemingly unaffected in rat animal models as treatment with 5% phenylalanine in weanling rats did not alter the brain levels of dopamine or norepinephrine at a dose that caused depletion of serotonin (12). Norepinephrine was significantly altered with a diet nine times the basal diet of Phe intake; however, dopamine was increased (13). The effects of phenylalanine on biogenic amines at 10 and 30 days in an cYMP-model system was limited to decreased levels of serotonin and SHIAA with the levels of dopamine and norepinephrine being unaffected (14). The differences noted in the catecholamine determinations from whole brains do not preclude regional variations in certain areas of brain, since McKean (11) observed severely decreased levels of dopamine in the dopamine-rich area of the caudate nucleas with no change evident in brainstem. Although all three experimental groups were fed ad libitum, there was a significant decrease in body weight observed in the 5% Phe + 0.4% (YMP group when compared to controls. No differences in body weight were observed in the group fed a normal diet supplemented with only 0.4% (YMP, thus eliminating the possibility of growth failure or less-rapid maturation in the presence of 0.4% (YMP in the diet. The decreased body weight and food intake observed in the HyPhe group raises the possibility of lack of substrate for catechol synthesis (29) and serotonin synthesis (23). which has been observed by Wurtman and co-workers. This possibility of lack of substrate can be eliminated since tyrosine values were elevated in the experimental PKU condition due to the inability of aMP to completely block phenylalanine hydroxylase (Table 3). Similarly no significant difference was observed in brain tryptophan concentrations between the three diet groups.

314

I‘AYLOK.

~0~~~s.

AND STEWART

Udenfriend rt crl. t IO) demonstrated inhibition of tyrosine hydroxylase by a-methylphenylalanine in vitro and Torchiana rt ul. (IS) demonstrated inhibition in IYvo. The 0.4% aMP group also showed substantially reduced levels of dopa in brain suggesting inhibition of tyrosine hydroxylase as well as phenylalanine hydroxylase. These reductions in dopa were observed even in the presence of increased tyrosine in the Phe + (uMP group. possibly due to increased competition by the excess Phe. Phenylatanine is present in the HyPhe group at 30 times the value of the control group: Phe is 40 times less potent than cvMP as an inhibitor of tyrosine hydroxylase (IO). The presence of a less-potent inhibitor (Phe) at 30 times the control value could act to depress the dopa values even in the presence of elevated tyrosine, so that no difference was noted in the (YMP group versus the HyPhe group. Although dopa was decreased in the cvMP group, dopamine did not vary between the three diet groups due to the inability of (wMP to inhibit dopa decarboxylase, a very ubiquitous enzyme, while exerting an inhibition on dopamine p-hydroxylase thus preserving no net change in the flux of dopamine. These observations of no dopamine changes were also observed in another Phe -k cuMP-model system (14). A 50% decrease in norepinephrine was observed in the (wMP group which would indicate (YMP also has an inhibitory effect on dopamine ,0hydroxylase. It is interesting that the addition of excess Phe increased the norepinephrine concentration between the CUMP group and the control group and this effect may account for the fact that no differences were observed by Lane ef rrl. (14) with controls and Phe + aMP treatment. CXMP also had a profound influence on epinephrine production. possibly by interference with phenylethanolamine f~-methyltransferase by Phe and crMP since the presence of excess Phe decreased the levels of epinephrine beyond those observed with cvMP alone. This evidence of decreased synthesis of the catecholamines in the presence of 5%’ Phe + 0.4% aMP should be extrapolated to the human condition with great care. because the decreased levels of dopa. norepinephrine, and epinephrine were also observed in the 0.4% crMP-treated group, thus attributing these deficits to the presence of the enzyme inhibitor and not to an increased Phe. The effects of decreased serotonin and SHIAA levels in brains of PKU patients (I I) have been reproduced in brains of weanling rats (age 17 days) which were fed a high phenylalanine diet ( 13,16,17). A decrease in serotonin was seen in rats treated from birth in a ~-chlorophenylalanine(PCPA) model system; however. this effect was shown to be due to the presence of PCPA alone, i.e.. in the absence of excess phenylaianine (18-20) because of the inhibition of tryptophan hydroxylase by PCPA (21). This side effect was eliminated by using crMP as an inhibitor of phenylalanine hydroxylase. A decrease in 5HT was observed in weanling rats but not in neonatal rats following treatment from birth in an (YMP-

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model system (14). Parallel increases in serotonin and its rate-limiting enzyme tryptophan hydroxylase have been described with adult values attained at 4 weeks postnatally (30.31). Since animals were placed on the respective diets at 25 days of age, the possibility of retarded maturation of enzymes involved in the synthesis of serotonin can be eliminated and the alterations in serotonin and SHIAA due to elevated Phe and not to the presence of the inhibitor, aMP. Treating adult rats (100 days old) for 10 days with 5% Phe did not alter brain serotonin levels appreciably from those of controls (22). Phenylalanine can decrease the tryptophan content (I 1) in brain by its competition across the blood-brain barrier, and this depletion of substrate is offered as a possible explanation for the decrease in brain serotonin (23). The mechanism of reduction of serotonin in the Phe + aMP group may be due to the inhibition of tryptophan hydroxylase by phenylalanine (24,25) rather than a lack of substrate as intracerebral tryptophan values did not vary between the three diet groups (Table I). This does not preclude regional differences which may occur in brainstem, since tryptophan values were measured in whole brains and serotonin and SHIAA measured in brainstems. The K, values of 32 PM for tryptophan and 287 PM for phenylalanine for tryptophan hydroxylase indicate that phenylalanine can act as a competing substrate for tryptophan hydroxylase (25). The Phe concentration in brain of 1.26 pmole/g or approximately 1260 FM was sufficiently above the K, of 287 PM to result in inhibition of tryptophan hydroxylase, by phenylalanine. This hypothesis confirms results of Yuwiler et al. (12) in which tryptophan hydroxylase activity was shown to be 75% decreased in animals fed a 5% Phe diet. The possibility of increased degradation of 5HT has been ruled out since monoamine oxidase activity was unaffected (22). A similar decrease in 5HT was observed in weanling rats from birth with a Phe + (uMP model (14), and the control group of aMP in this study verifies the results being due to excess Phe and not due to the presence of the inhibitor. The decrease in serotonin observed in this study are relevant to the PKU patient, since CXMP had no effect on serotonin synthesis and the results of decreased serotonin can be attributed to phenylalanine alone. Tryptophan supplementation (26) to increase serotonin synthesis and also supplementation with the branched-chain amino acids (27) is being applied successfully in PKU patients. These new approaches together with dietary phenylalanine restriction may provide an effective means of treatment to overcome the neurochemical abnormalities observed in this study. The nature of the mental retardation remains unresolved: however, the evidence presented here may contribute to the elucidation of some underlying neurochemical abnormalities associated with PKU at later stages

316

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of brain development and offer evidence for dietary supplementation tryptophan and tyrosine to increase neurotransmitter synthesis.

of

SUMMARY The effects of experimental hyperphenylalaninemia on catecholamine and serotonin synthesis in brain at a later stage of brain development were investigated. A group of 35day-old rats treated with normal chow supplemented with 5% Phe + 0.47 c a-methylphenylalanine, (YMP. for the previous 10 days showed decreases in dopa. norepinephrine, and epinephrine versus controls. A group treated with a normal diet supplemented with 0.4% (-uMP showed similar decreases and these differences could be attributed to the presence of the phenylalanine hydroxylase and tyrosine hydroxylase inhibitor, aMP, rather than the hyperphenylalaninemia condition. No differences in dopamine were observed. Serotonin and Shydroxyindoleacetic acid (SHIAA) were decreased 50% in the HyPhe condition and were unaffected in the presence of CXMP alone. indicating that the decreases in serotonin and SHIAA were due to the increases in phenylalanine rather than the presence of the inhibitor. These abnormalities in serotonin metabolism at later stages of brain development may be relevant to early discontinuation of dietary therapy in the PKU patient and implies a role in tryptophan supplementation to increase intracerebral serotonin values. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. IO.

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