Biochemical and Neuropsychological Effects of Elevated Plasma Phenylalaninein Patients with Treated PhenylketonuriaA Model for the Study of Phenylalanine and Brain Function in Man
Wilma Krause, Margaret Halminski, Linda McDonald, Philip Dembure, Rino Salvo, David Freides, and Louis ElsasDivision of Medical Genetics, Departments of Pediatrics and Psychology, Emory University, Atlanta, Georgia 30322
Abstract
Phenylketonuria provides a human model for the study of theeffect of phenylalanine on brain function. Although irreversiblemental retardation is preventable through newborn diagnosisand dietary phenylalanine restriction, controversy exists re-garding the effects of increased concentrations of phenylalaninein older patients. Wehave studied ten older, treated, phenyl-ketonuric patients using a triple-blind, multiple trials, cross-over design. Each patient was tested at the end of each ofthree 1-wk periods of high or low phenylalanine intakes. Testsincluded a repeatable battery of neuropsychological tests, anal-ysis of plasma amino acids, and measurement of urine aminoacids, phenyl organic acids, dopamine, and serotonin. In all 10patients, plasma phenylalanine rose (9004,000 AM). In 9 of10 patients there was an inverse relationship between plasmaphenylalanine and urine dopamine excretion. When bloodphenylalanine was elevated, these patients had prolonged per-formance times on neuropsychological tests of higher but notlower integrative function. Urinary serotonin fell during phe-nylalanine loading in six patients. The concentration of phen-ylacids in the urine was not proportional to the plasmaphenylalanine at concentrations below 1.5 mM. In one patient,neither performance time nor dopamine excretion varied asblood phenylalanine rose or fell. We interpret these data asfollows: blood phenylalanine above 13 mMimpairs performanceon neuropsychological tests of higher integrative function, thiseffect is reversible, and one mechanism may involve impairedbiogenic amine synthesis.
Introduction
Nearly a half-century ago Folling (1) attributed a syndrome ofmental retardation and aberrant behavior to an inheritedmetabolic error. Since then, phenylketonuria (PKU)' has beenthe prototype for investigations of the effect of phenylalanineon central nervous system function in man. It is clear that if
This work was presented in part at the Combined Plenary SessionAmerican Society for Clinical Investigation/Society for Pediatric Re-search, Washington, DC, 1983.
Address reprint requests to Dr. Krause.Received for publication 17 January 1984 and in revised form 10
July 1984.
1. Abbreviations used in this paper: PKU, phenylketonuria; WISC,Wechsler Intelligence Scale; WRAT, Wide Range Achievement Tests.
plasma phenylalanine is normalized before age 3 wk throughdietary restriction of phenylalanine, irreversible mental retar-dation is prevented (2). The mechanisms for producing thispermanent structural damage remain unclear, but severalhypotheses have developed. Decreased or abnormal myelinformation and/or impaired oligodendroglial migration duringthe first 6 mo of postpartum brain development are the mostprobable mechanisms (3-7).
Controversy persists regarding possible effects of elevatedphenylalanine on brain function when development is nearlycomplete in older, treated patients with PKU. Whether or notelevated concentrations of phenylalanine disturb central nervoussystem function in these patients is unknown. Since -1 in16,000 Caucasian newborns (Georgia statistics) (8) is affectedwith PKU, and effective newborn screening has preventedpermanent brain damage since 1970 in the newborn screenee,an answer to the question of whether high plasma phenylalanineaffects mental function becomes more urgent for this accu-mulating population.
Silverman and Guthrie (unpublished observations) ap-proached the question by administering one loading dose ofphenylalanine to control subjects, heterozygotes, and homo-zygous affected patients with PKU and compared errors inresponse time among the three groups. Their results suggesteda difference among the three groups which related directly tothe concentrations of plasma phenylalanine achieved.
In 1980, Waisbren et al. (9) reviewed the available literatureon psychological assessment of children after termination ofphenylalanine-restricted diets. Results were mixed, some show-ing a drop in IQ and other achievement test scores and othersshowing no change. Numbers of patients, study design, andassessment tools varied greatly among the reports. The PKUCollaborative Study began a prospective study in 1967 (10).Results of achievement tests (Stanford Binet, Wechsler Intel-ligence Scale (WISC), Wide Range Achievement Tests [WRAT])on 81 children, 38 of whomhad continued the diet beyond 6yr of age and 43 of whom had discontinued at 6 yr of age,were reported in 1982. Results at 8 yr of age showed slightlylower achievement in reading and spelling in the discontinuers.No significant difference in IQ between the groups was observedafter this 2-yr interval (11). Brunner et al. (12) in a recentstudy (1983) reported a negative correlation between perfor-mance on neuropsychological tests and serum phenylalanineconcentration on the day of testing in a group of early treatedpatients age 6-13 yr. Neither of these studies used the patientas his/her own control. Interindividual variation, differencesin phenylalanine concentrations achieved and in techniquesused by collaborating centers have hindered interpretation ofresults.
In in vitro systems, phenylalanine influences the synthesisof two biogenic amines, dopamine and serotonin, which are
40 Krause, Halminski, McDonald, Dembure, Salvo, Freides, and Elsas
critical compounds in neurotransmission (13). Both tyrosine-3-hydroxylase (E.C.C. 1.14.16.2) and tryptophan-5-hydroxylase(E.C.C. 1.14.16.4) are rate-limiting enzymes in the synthesis ofdopamine and serotonin, respectively, and are competitivelyinhibited by phenylalanine at millimolar concentrations (14,15). Another potential inhibitory effect of phenylalanine onbiogenic amine synthesis is through impaired uptake of tyrosineand tryptophan across the blood-brain barrier. Phenylalanine,tyrosine, and tryptophan share the same transport system andcompete for a common transport function at physiologicconcentrations (16). Since transport of amino acids across theblood-brain barrier is the rate limiting step in the movementof amino acids from plasma to brain, and since their plasmaconcentration is near saturation of their transporter proteins,increased concentrations of plasma phenylalanine could limitthe transport of tyrosine and tryptophan and thus their avail-ability to the brain cell membrane for neuropeptide synthesisor conversion to biogenic amines (16-19).
The current study compares specific neuropsychologicaltests with changes in plasma phenylalanine and biogenic amineproduction in young adults and older children with PKU.Although the dopamine excreted in the urine is a reflection ofmultiple sources of dopamine synthesis (20), we chose tomeasure urine dopamine, since it reflects 24-h production ofthe amine, not an acute level, and because urine collection isa noninvasive method of obtaining biologic fluids. Assessmentis made of competitive inhibition by phenylalanine of tyrosineand tryptophan transport by kidney tubule. Weuse a triple-blinded, crossover, clinical protocol to circumvent the influenceof individual variation in this disorder (21, 22).
Methods
Study design. 10 patients with PKU, aged 6-24 yr, were admitted ona 21-d protocol to the Emory University Clinical Research Facility.Informed consent was obtained from adult patients or from the parentsof patients <21 yr of age. Each patient served as his or her owncontrol. Each patient was admitted on one of two double crossoverprotocols and five were studied in each protocol group. Either thepatient entered on a low dietary phenylalanine which was increasedthe second week and decreased the third week (low-high-low) or in thereverse pattern (high-low-high). Patients equilibrated for 7 d after eachchange in dietary phenylalanine. Past plasma concentrations of phe-nylalanine on known intake and genotyping of parents were used todetermine the amount of phenylalanine added to patient formulationfor restriction and loading (23, 24). Patients whose entering concentra-tion of plasma phenylalanine was high either because of poor controlor because of diet discontinuation for several years were on the high-low-high protocol. Five other patients who had been in consistentlygood dietary control entered the study on the low-high-low protocol.
The study diet was based on Phenylfree or Lofenalac as a phenyl-alanine-free amino acid source. A specified amount of tasteless L-phenylalanine was added to the formula during the loading phases.The study was triple-blinded: neither the patients nor their parentscould taste the difference in formula and were unaware of theirexperimental condition; the psychologist administering the neuropsy-chologic tests was uninformed of the patients' blood phenylalanineconcentration; and the laboratory personnel performing amino acid,organic acid, and amine analyses did not know the condition underwhich samples were obtained.
Biochemical tests. Blood and urine samples were obtained on allpatients at the beginning of the first week as a baseline and at the newequilibria achieved at the end of each 7-d interval.
Plasma and urine amino acids were analyzed by ion exchangechromatography on the Beckman model 119 CL using lithium buffers(Beckman Instruments Inc., Palo Alto, CA). Because tryptophan issomewhat labile in extraction from blood, recovery of "spiked" standardsfrom whole blood and urine was quantitated to determine losses. Inthe physiological ranges measured from 50 to 200 AlM, recovery was82-90o efficient. Data are presented normalized to an internal standardwithout correction for these specific losses which are in the range forthe internal standard, S-2-aminoethyl-L-cysteine.
Dopamine assays were performed using the single isotope radioen-zymatic assay developed by Peuler and Johnson (25). This methodused catechol-o-methyl transferase from rat liver to transfer a radioactivemethyl group from S-adenosyl methionine to catecholamine, formingmethyl catecholamine derivatives which were then characterized byradiochromatographic analysis. The assay was sensitive in urine to 120pg/ml for dopamine.
Serotonin was determined by a radioimmunoassay developed byPeskar and Spector (26) using rabbit antibody prepared by couplingserotonin to bovine serum albumin. The antibody bound 50% of3H-serotonin in the absence of free serotonin. Less than I ng of freeserotonin was detected by standard displacement methods.
Urine organic acids were analyzed by gas chromatography on aHP 5992 gas chromatograph/mass spectroscope and quantitated on aHP 5790 gas chromatograph. Organic acids were extracted with ethylacetate and ether and derivatized with trimethylsilane and bis-tri-methylsilyl)trifluoracetamide (27). The level of sensitivity for phenylacidsin urine was -''5 Mg. Specific recovery of phenylacetic, phenyllactic,and phenylpyruvic acids were 68, 91, and 58%, respectively. Allcalculations are corrected for these losses by parallel external andinternal standards used during extraction, derivatization, and quanti-tation.
Renal clearances were calculated for phenylalanine, tyrosine, andtryptophan, from timed 24-h urine collections and mid-point plasmacollections. Both specimens were quantitated for concentrations ofamino acids and creatinine. The glomerular filtration rate (GFR) wascalculated from the creatinine clearance, as were the rates of a specificamino acid filtration, excretion, and reabsorption using the followingformulation (28): FA = GFR X PAA, EA = U.A X V, TAA = FAA- EA, where U was urinary amino acid concentration in mg/ml, V(urine volume) in ml/min, and PAA the plasma amino acid concentrationin mg/ml. The FA (filtered aminoacid), EA (excreted aminoacid), andTAA (reabsorbed aminoacid) were expressed in mg/min. Percent reab-sorption was calculated as TAA/FAA X 100.
Neuropsychological tests. Measurements of general intelligence andachievement were based on the Wechsler Intelligence Scales (TheWechsler Adult Intelligence Scale for adults and the WISC for children)and the WRAT. To determine the influence of phenylalanine concen-trations on neuropsychological performance, a repeatable battery oftests was developed and administered as a baseline on admission tothe study and at the end of each l-wk treatment period. A confoundingvariable inherent in tests given multiple times is subject learning. Twoprocedures were incorporated in the study design to reduce the artifactsdue to learning. For one group of tests (type 1, Table I), the subjectwas allowed to practice the task until the asymptote of the learningcurve was reached. Any changes in performance after becomingmaximally competent with the task then reflected experimental manip-ulation of the patient. This procedure would not eliminate learningartifacts from a second group of tests (type 2, Table I). Because of thislimitation, equivalent forms of this latter group of tests were developedto be given at the end of each of the experimental conditions. Table Ilists the test names and the neuropsychological variables they measured.
Interpretation of data. Data are arrayed for all subjects in tabularform (Tables II, III, IV, and V) to emphasize intraindividual differencesbecause of the wide interindividual variability in age, sex, intellectualcompetence, and phenylalanine requirements. From these tables, in-dividual differences in dopamine and choice reaction time and thedirection of change between the two dietary conditions are calculated
Phenylketonuria: a Model for Study of Phenylalanine and Brain Function 41
Table L Neuropsychological Tests Usedand the Variables Measured by Them
RAN 2 6+ Verbal visual integrationTrails A 2 6+ Visual-motor coordinationGrooved pegboard 1 5+ Visual-spatial-tactile
coordinationVisual-motor speed
Halstead finger tapping 1 5+ Fine motor skill
* 1, A test which can be repeated over several testing sessions. 2, A test whichcannot be repeated and must be presented as equivalent forms over several test-ing sessions.t +, The age listed in years plus greater ages.
and plotted against changes in plasma phenylalanine during the sameintervals in Figs. 2 and 3.
Results
Patient profiles and study design. Age, sex, IQ, and achievementscores for all patients are listed in Fig. 1. Each patient wasgiven a symbol which was used in subsequent graphs. IQscores below 85 in A.S. and K.K. were explained by their latediagnoses and treatment at 5 and 18 mo of age, respectively.K.K. is the older brother of T.K. Both D.A. and W.J. werediagnosed and treated before 3 wk of age and both had IQ
Full Verbal/ Wide Range Achievement TestSymbol Initial Age Sex Scale Performance
scores which were consistent with parental scores (D.A.'sparents' scores were 103 and 83; W.J.'s were 88 and 70).
Biochemical results. Plasma and urine amino acid andurine organic acid concentrations are presented for all patientsin Table II during each of the three experimental conditions.The level of dietary phenylalanine was calculated from actualintake in the Clinical Research Facility, Emory University.The time interval of 7 d required for stabilizing the plasmaphenylalanine concentration on a constant diet was determinedby sampling one subject daily. A new plateau of blood phe-nylalanine concentration was achieved on the sixth to seventhday after each diet change. The plasma phenylalanine reflectedthe diet changes, and the relationship between intake andplasma concentration demonstrated interindividual variationbetween ingested phenylalanine ranging from 36 to 130 mg/kg per d and plasma phenylalanine concentration rangingfrom 800 to 4,400 MAM.
Urinary phenyl acids are not detected in the urine ofnormal subjects. Four of the five patients who were on thehigh-low-high protocol and had not recently been on restrictedphenylalanine intake were excreting large amounts of phenyl-pyruvate and phenyllactate at the end of the first week of highphenylalanine intake. Excretion of both fell dramatically after1 wk of restricted phenylalanine intake. However, excretionreached the original high levels at the end of the third week(high dietary phenylalanine). In the low-high-low group whohad been on continuous dietary control before entry into thisstudy, excretion of organic acids never reached the high levelsof the other group despite comparable plasma phenylalaninelevels. In general, <50 mg of phenylacids per gram creatininewere excreted until the plasma phenylalanine rose above 1,5001AM. Those with the highest plasma phenylalanine did notconsistently excrete the greatest amount of derived organicacids.
The results of dopamine and serotonin excretion are arrayedin Table III. Results were normalized to creatinine excretion.Interindividual variation in dopamine excretion was great. Ingeneral, the patients who were on the low-high-low protocoland had been on consistent dietary management before thestudy achieved higher levels of dopamine excretion than didthose patients in the high-low-high group who were not wellcontrolled immediately before the study. This kind of separationwas not seen for serotonin excretion. Changes in dopamineexcretion varied inversely with changes in plasma phenylalaninein 9 of 10 patients. The inverse relationship of changes inplasma phenylalanine concentrations and urinary dopamineexcretion are graphed in Fig. 2. Solid symbols representpatients on the high-low-high protocol. Open symbols representpatients on low-high-low protocols. Results from all patientscluster in quadrants I and III regardless of the protocol (high-low-high or low-high-low) where quadrants I and III circum-scribe an inverse relationship between plasma phenylalanineand urinary dopamine concentrations. Symbols in quadrant Ishow an increase in urine dopamine with decrease in plasmaphenylalanine; those in quadrant III show a decrease in urinedopamine with an increase in plasma phenylalanine. Serotoninexcretion did not vary directly with changes in phenylalanine.
Studies of membrane transport. To explore the possibilitythat increased concentrations of phenylalanine might compet-itively inhibit tyrosine or tryptophan uptake by the onlyplasma membrane transport function available for study in
42 Krause, Halminski, McDonald, Dembure, Salvo, Freides, and Elsas
Figure 1. Identification of patients by age, sex, IQ, and achievement.The Wechsler Adult Intelligence Scale and the WISC-revised wereused to determine IQ. Scores given for the WRATare standardscores.
1111
Table II. Effects of Dietary Manipulation of Phenylalanine (PHE) on Concentrations of PHE,Tyrosine (TYR), and Tryptophan (TRP) in Plasma and Urine and on Excretion of Three Organic Acids
np, not processed; nd, none detected. Data are single measurements. The space between patients A.S. and D.A. separates the patients on thehigh-low-high protocol above from those on the low-high-low protocol below.
children, we quantitated their renal tubular transport. Renaltubular reabsorption data were obtained on eight patientsunder these conditions of phenylalanine loading and arepresented in Table IV. Phenylalanine did not inhibit tyrosinereabsorption by renal tubular epithelium at the levels of filteredphenylalanine reached in these patients. At the highest rate offiltered phenylalanine (45 mg/min/M2 in patient A.S.), weobserved no less than 99% reabsorption of tyrosine. Maximumrenal uptake of tryptophan was also seen at these filtered loadsof phenylalanine. These findings differ from earlier resultsreported by Lines and Waisman (29), who reported a gener-
alized aminoaciduria in PKU patients and suggested thepossibility of competitive inhibition of reabsorption by highfiltered loads of phenylalanine. However, their data were notadjusted for surface area. Our data for renal tubular transportprovide negative evidence for a significant effect of phenylala-nine on tyrosine uptake in the proximal renal tubule at thesame time that dopamine excretion is reduced. Whether ornot the lack of effect of increased phenylalanine reabsorptionof amino acids in the proximal renal tubule is an appropriatereflection of transport across the blood-brain barrier is notknown. Evaluation of blood-brain barrier transport using in-
Phenylketonuria: a Modelfor Study of Phenylalanine and Brain Function 43
Table III. Effects of Dietary Manipulation of Phenylalanine(PHE) on the Excretion of Dopamine and Serotonin
The space between patients A.S. and D.A. separates the patients on the high-low-high protocol above from those on the low-high-low protocol below.
vasive techniques is not ethical in healthy children. Nuclearimaging techniques may be useful in the future.
Neuropsychological tests. Part of the purpose of this studywas to determine the kinds of tests most suitable for determiningpossible changes in performance in treated PKU childrenchallenged with phenylalanine. We found that many of thestandard tests were too difficult to be applicable across the agegroup we were assessing and data could not be obtained onall 10 subjects. More complete analysis of these issues will bepresented in a separate paper. Results are presented here forthose tests on which data were obtained for all subjects. Datawere obtained from all 10 subjects on the Choice ReactionTime when figures were used for matching. They were alsocomplete on the Pegboard Test, the Tapping Test, and onTrails "A". Table V summarizes the results of the ChoiceReaction Time and the Grooved Pegboard Test. The latter isa test of visual-spatial-tactile coordination and motor speed,whereas the Computerized Choice Reaction Time is a test ofvisual-perceptual discrimination, and by comparison is a test
of higher integrative function. The Grooved Pegboard Testresults are typical of results of the tests of lower integrativefunction, i.e., no significant differences were seen betweenconditions. In three other tests of lower integrative functionof which the Grooved Pegboard is representative, <3 of 10showed changes consistent with changes in plasma phenylala-nine. Those results are not reported here. In the ChoiceReaction Time Test, 7 out of 10 subjects showed changesconcomitant with changes in plasma phenylalanine, i.e., reac-tion time was prolonged with increased plasma phenylalanine.M.F., who did not demonstrate typical changes in his ChoiceReaction Time, also did not have a decrease in urinarydopamine when plasma phenylalanine concentrations were
elevated (compare Table V with Table III, week 1 to week 2).Differences in Choice Reaction Time were not as consistentin K.K. and A.S. as in the other subjects. It is pertinent tonote that K.K. and A.S. were not treated effectively early inlife, and were less competent by achievement testing thanmany of the other patients (see Fig. 1). A graphic display ofchanges in phenylalanine and changes in Choice ReactionTime among these 10 patients is shown in Fig. 3. A directrelationship was seen between changes in plasma phenylalanineconcentration and reaction time. When plasma phenylalanineincreased, the choice reaction time increased; that is, perfor-mance worsened. Conversely, when phenylalanine concentra-tions fell, choice reaction times were shorter, which indicatedimproved performance. Solid symbols again represent patientson high-low-high dietary protocol and open symbols those on
low-high-low dietary protocol. Symbols in quadrant II representchanges of increased choice reaction time with changes reflectingincreased plasma phenylalanine concentrations. Symbols inquadrant IV indicate decreased choice reaction time withdecreased plasma phenylalanine (Fig. 3).
By comparing Figs. 2 and 3, one sees the inverse relationshipbetween choice reaction time and dopamine excretion. Asurinary dopamine fell, choice reaction time increased; that is,performance worsened.
C
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Change in Plasma Phe (mM)
Figure 2. The relationship of changes in plasma phenylalanine tochanges in urine dopamine during dietary manipulation of phenylala-nine. Each patient is represented by a symbol that appears twice onthe graph, indicating the difference in dopamine between the first andsecond week and the second and third week plotted against parallelchanges in plasma phenylalanine.
44 Krause, Halminski, McDonald, Dembure, Salvo, Freides, and Elsas
Table IV. Absence of an Effect of Increased Filtered Phenylalanine onRenal Tubular Reabsorption of Tyrosine (Tyr) and Tryptophan (Trp)
Urine Tyrosine TryptophanPatient Wk volume FPhe FTyr TTyr transport FT.p TTrp transport
mI/24 h mg/minIM2 mg/minIM2 mg/minIM2 %Reabsorption mg/minIM2 mg/minIM2 % Reabsorption
* np, not processed. The space between patients A.S. and M.B. separates the patients on the high-low-high protocol above from those on thelow-high-low protocol below. Calculations for renal tubular reabsorption are detailed in the methods section. FPhe, FTyr, FTrp, filtration rate ofphenylalanine, tyrosine, and tryptophan, respectively. TPhe, TTyr, TTrp, tubular reabsorption rate of phenylalanine, tyrosine, and tryptophan,respectively. mg/min/M2, mg of indicated amino acid filtered or reabsorbed per minute normalized to meters squared of body surface area.
Discussion
Although mechanisms are unclear, the negative effect of in-creased blood phenylalanine on the developing human brainduring infancy and early childhood is clear (1-7). Early dietaryrestriction of phenylalanine prevents irreversible brain damagein children detected and treated for phenylalanine hydroxylasedeficiency (2). The studies reported here investigate whetherelevated blood phenylalanine in the older child and youngadult is associated with altered mental function, and if so, bywhat mechanism.
Early studies by Weil-Malherbe (30), Nadler and Hsia (31),and McKean (13) demonstrated decreased levels of catechol-amines in blood, urine, and autopsied brains of untreatedpatients with phenylketonuria. McKean (13) also demonstratedimprovements in visual evoked response in three severelyretarded untreated patients when dietary phenylalanine was
restricted or when catecholamine precursors were administeredwithout restricting phenylalanine in the diet. He postulated
that although concentrations of tyrosine (1.2 X 10-4 M/g ofbrain) in brain of hyperphenylalaninemic patients were wellabove the Km reported for tyrosine hydroxylase in mammalianbrain tissue (5 X i0-s M) (32), phenylalanine itself mightinhibit tyrosine hydroxylase activity directly. This hypothesiswas supported by in vitro observations of Udenfriend (33),who found that phenylalanine was a competitive inhibitor ofrat brain tyrosine hydroxylase with a Ki = 1.7 X l0-s M. Sincethe concentrations of phenylalanine found by McKean (13) inhis autopsy material averaged 8.4 X l0-4 M, such a mechanismof competitive inhibition was possible.
Phenylalanine may impair production of two psychoactiveamines, namely dopamine and serotonin. Curtius et al. (34)described both decreased serotonin and dopamine synthesis inpatients with high plasma phenylalanine concentrations causedby both phenylalanine hydroxylase deficiency and disorders inthe tetrahydrobiopterin pathway. He also postulated competitiveinhibition of both tyrosine and tryptophan hydroxylase byhigh phenylalanine at 1,500 and 600 gM concentrations,
Phenylketonuria: a Modelfor Study of Phenylalanine and Brain Function 45
Table V. Effects of Dietary Manipulation of Phenylalanine (PHE)on Choice Reaction Time and Grooved Pegboard Assembly
Pegboardi (s/IO pegs)
Plasma ChoicePatient Wk PHE reaction time Right Left
B.R.
W.J.
;AM
1 1,2552 2523 797
1 1,7902 1973 1,303
1 2,3172 1,4263 3,296
1 2,0582 7353 2,647
K.K.
T.K.
A.S. 1 4,4052 4413 3,900
D.A. 1 6682 3,2603 1,632
M.B. 1 3042 1,5493 634
M.F. 1 1992 1,4023 329
M.K. 1 7932 2,4603 577
T.W. 1 3522 2,2903 536
M/s
811601654
1,9401,4071,594
1,085967901
811621767
669678769
1,6582,1131,518
1,2151,4851,221
998980791
9101,062
744
1,2861,2971,193
The space between patients A.S. and D.A. separates tithe high-low-high protocol above from those on the kprotocol below. The choice reaction time represents atrials. The pegboard results represent a mean of four
respectively (34). Katz et al. (35) demonstraconversion of 20 MMphenylalanine to dihydrox)without the release of free tyrosine in rat braintosomal preparations. Phenylalanine was onlysubstrate for this enzyme as tyrosine. Howeverthat phenylalanine could be a substrate for tyrosiin the presence of saturating concentrations of toterin, and could be a competitive inhibitor as M
Our results in vivo in treated PKU patienthe hypothesis that high phenylalanine inhilsynthesis, since 24-h urine dopamine excrete
57 7357 6851 66
30 10050 3350 50
34 3135 3229 31
19 1919 2118 18
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--200
--300
--400
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--600
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II
*
III
RR.E D.A. 0W.J. * M.B. AK.K. * M.K. LT.K. A T.W. OA.S. * M.F. *
Change in Plasma Phe (mM)
Figure 3. The relationship of changes in plasma phenylalanine tochanges in choice reaction time during dietary manipulation of phe-nylalanine. Each patient is represented by a symbol which appearstwice on the graph, indicating the difference in mean choice reactiontime between the first and second week and the second and thirdweek plotted against the parallel change in plasma phenylalanine.
26 29 plasma phenylalanine concentrations were maintained at an26 35 elevated concentration for days by dietary manipulation. A25 28 consistent relationship was not found between plasma phenyl-
alanine and serotonin excretion in our study. Serotonin is52 31 stored in many tissues (36), and this inconsistency may be56 29 related to the high "background noise" of excretion of stored36 31 serotonin during a 24-h period.
36 36 The results from our experiments do support the hypothesis33 48 that brain function is altered by phenylalanine at the equilib-40 39 rium concentrations achieved in the study. The battery of
neuropsychological tests showed differences in a performance25 25 test which required higher integrative function rather than fine20 20 motor coordination. This was consistent over the whole group,25 25 regardless of the age or competence of the patients. Although
20 24 many test batteries have been used in other surveys, the
21 32 computerized reaction time has not been reported (9, 11, 12).
19 23 We are currently attempting to determine whether patientcompetency, age, attention, or other factors influence the
22 34 neuropsychological response to increased plasma phenylalanine,32 33 and deriving tests to maximize changes in accordance with24 36 patient competency.
Our data support a mechanism for prolonged performancehe patients on through an inhibition by phenylalanine of biogenic amineow-high-low synthesis. In our study when dopamine excretion fell, bloodmean of 20 phenylalanine rose and performance times were prolonged.
trials. Data from two patients deserve special attention: the patientwith the lowest IQ (K.K.) who was not diagnosed until 18 moof age demonstrated expected biochemical changes in urinary
ited the direct dopamine excretion when phenylalanine concentrations wereyphenylalanine increased, but test scores on the Computerized Choice Reactionstriatal synap- Time were unchanged. It is not surprising, in view of his
1/10 as good a overall low performance and achievement, that reaction timer, he suggested improved over the 3-wk period independent of the plasmane hydroxylase phenylalanine, which suggested a gradual learning effect ratheretrahydrobiop- than relationship to biochemical status. It is also likely thatvell. intellectual competency of a patient will control the amountits conform to of change produced by altered phenylalanine. The lower thebits dopamine individual's competency, the less change might be expected.ion fell when The one major outlier (M.F.) did not show consistent trends
46 Krause, Halminski, McDonald, Dembure, Salvo, Freides, and Elsas
I
in neuropsychological tests or in catecholamine excretion.Despite attaining a concentration of 1,402 AM plasma phe-nylalanine, he excreted barely measurable amounts of derivedorganic acids. Wecan speculate that he has other "protective"functions. Possible mechanisms include impaired transport ofphenylalanine across the blood brain barrier or an increasedrate of phenylalanine incorporation into new protein synthesis.He could also have some "protective" variation in tyrosinehydroxylase which prevents inhibition by phenylalanine. Heemphasizes the individuality of patients with phenylketonuriaand the "sensitivity" of brain function to phenylalanine loading.
The impairment in choice reaction time and decrease indopamine excretion seen with increased plasma phenylalaninewere reversible within the week periods studied. We arecurrently investigating a variety of repeatable neuropsycholog-ical and electrophysiological tests with which to assess perfor-mance in patients with varying competency, age, and achieve-ment scores.
These data support the hypothesis that high concentrationsof phenylalanine reversibly affect neuropsychological perfor-mance, probably through reduction in L-dihydroxyphenylal-anine and dopamine production. The mechanisms may bethrough increased intracellular phenylalanine and competitiveinhibition of brain tyrosine-3-hydroxylase. Whether intracellularconcentrations of brain tyrosine are diminished is unknown.Although the concentrations of blood phenylalanine attainedin our studies did not inhibit renal tubular reabsorption oftyrosine, it should be noted that the transport Km of phenyl-alanine, tyrosine, and tryptophan in brain and kidney differ.Additionally, the blood-brain barrier is saturated at normalplasma concentrations, whereas the renal tubular epitheliumis not (16-19). Since nearly 80% of all brain dopamine isfound in the corpus striatum, decreases in dopamine synthesiscould affect neuropsychological functions that involve boththe nigrostriatal and corticostriatal pathways (37). This couldexplain the deterioration in response of our patients to a timedtest, the Computerized Choice Reaction Time Test, whichrequired integration of stimuli and a motor response. Wehaverecently observed a change in the mean power frequency ofelectrical impulses detected by EEG in a different group ofpatients with phenylketonuria who were studied under similarclinical research protocols (38). This type of electrophysiologicalapproach could assist in anatomical localization of changes inbrain function.
Acknowledgments
The authors wish to express their appreciation to Dr. Bahjat Faraj andMr. Vernon Camp of the Department of Radiology for performingthe catecholamine assays. Wealso want to thank Mr. Robert Mapoufor modifying the computerized choice reaction time tests.
References
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48 Krause, Halminski, McDonald, Dembure, Salvo, Freides, and Elsas
