Child Development, November/December 2001, Volume 72, Number 6, Pages

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1 Child Development, November/December 2001, Volume 72, Number 6, Pages Associations between Phenylalanine-to-Tyrosine Ratios and Performance on Tests of Neuropsychological Function in Adolescents Treated Early and Continuously for Phenylketonuria Monica Luciana, Jill Sullivan, and Charles A. Nelson Phenylketonuria (PKU) is a genetic disorder characterized by hyperphenylalaninemia. Treatment involves dietary phenylalanine restriction to prevent mental retardation. Because phenylalanine is involved in tyrosine synthesis and tyrosine is a catecholamine precursor, low tyrosine may lead to brain dopamine deficiencies. Because dopamine is involved in the modulation of prefrontally orchestrated executive functions, deficiencies may lead to executive impairments. Despite treatment, impairments in executive cognitive functions have been reported in young children with PKU. Outcome beyond middle childhood has not been extensively investigated. In this study, PKU-affected adolescents (N 18) with normal-range IQ scores completed neuropsychological tests, and their performance was compared with unaffected peers (N 16) and chronically ill controls (N 17). Results demonstrated that the overall performance of the PKU group did not differ from that of the other two groups, but that performance of the PKU proband was associated with phenylalanine and tyrosine levels, and most strongly with phenylalanine-to-tyrosine ratios at several points in development. These findings provide a preliminary test of the dopamine hypothesis of PKU as it applies to adolescents and young adults. INTRODUCTION Phenylketonuria (PKU) is an inborn disorder of metabolism affecting approximately 1 in 10,000 to 20,000 live births (Benson & Fensom, 1985). PKU is caused by a limited ability to metabolize the amino acid phenylalanine due to the absence or inactivity of the enzyme phenylalanine hydroxylase (PAH). Left untreated, affected individuals experience a rise in serum phenylalanine and subsequent mental retardation. The biochemical mechanisms that explain why high phenylalanine levels result in mental retardation have not been definitively established. However, under normal circumstances, phenylalanine, through the action of PAH, is converted to tyrosine, the precursor enzyme for the production of dopamine and norepinephrine in the central nervous system (McKean & Peterson, 1970). Treated individuals are typically maintained on a diet in which phenylalanine is restricted, but not necessarily entirely eliminated (due to the body s need for protein), and tyrosine is supplemented. Nonetheless, a state of brain tyrosine (and dopamine) depletion is still possible due to (1) the shared competitive mechanism through which both phenylalanine and tyrosine cross the blood brain barrier, and (2) the differential sensitivity of regionally specific brain neurons to modest decreases in tyrosine availability (for a thorough review, see Diamond, Prevor, Callender, & Druin, 1997). Thus, even with treatment, phenylalanine levels in PKU may be elevated in the context of low tyrosine levels. It has been suggested (Diamond et al., 1997) that because of this biochemical state, characterized by moderately high phenylalanine levels in the context of low tyrosine levels, one of the best indicators of dopamine availability in PKU is the phenylalanine-to-tyrosine ratio (Phe:Tyr). This ratio is not typically examined in studies of PKU. Dopamine neurons in the prefrontal cortex and retina, unlike those in other regions, are particularly sensitive to decreased tyrosine availability (Bradberry, Karasic, Deutch, & Roth, 1989; Diamond & Herzberg, 1996; Tam, Elsworth, Bradberry, & Roth, 1990). Reduced levels of the precursor can theoretically occur either when PKU is left untreated, because phenylalanine cannot be converted to tyrosine through PAH activity, or when PKU is treated, if the ratio of phenylalanine to supplemented dietary tyrosine is in the moderate to high range. Under either condition, disruptions in prefrontally mediated behaviors are possible, although specific prefrontal dysfunction is most likely when phenylalanine levels are moderately, but not excessively, elevated. Diffuse damage, extending beyond prefrontal cortex, is more likely when phe by the Society for Research in Child Development, Inc. All rights reserved /2001/

2 1638 Child Development nylalanine levels are excessively high, as in untreated PKU (Diamond et al., 1997). Definitive evidence of central nervous system dopamine deficiency in PKU has not been demonstrated, although there is limited evidence of reduced tyrosine concentrations in the central nervous systems of phenylketonurics (McKean & Petersen, 1970). Additionally, aberrant levels of dopamine precursors, metabolites, and reduced norepinephrine in the plasma, urine, and cerebrospinal fluid of individuals who have discontinued dietary treatment have been reported (Güttler & Lou, 1986). An inverse relationship between plasma phenylalanine and urinary dopamine excretion also has been found (Krause et al., 1985). In laboratory animals, when dopamine is depleted prenatally through tyrosine depletion or neurochemical lesions, animals exhibit decreased responding to dopamine-dependent rewarding stimuli, as well as impaired acquisition of operant responding (Moy, 1995; Palmour, Ervin, Baker, & Young, 1998; Yokogoshi & Nomura, 1991). Supersensitivity to dopaminergic agents has also been observed (Owasoyo, Neri, & Lamberth, 1992). Moreover, when tyrosine or other dopamine agonists are administered prenatally to pregnant females, their offspring show lifetime alterations in response to dopaminergic agents (Nasello & Ramirez, 1978; Santana, Martin, & Rodrigues, 1994). Acute tyrosine depletions in healthy adult humans have been associated with increases in arousal, depression, and anxiety (McCann et al., 1995), as well as impaired cognitive function (McCann et al., 1992) and decreased responses to the rewarding effects of alcohol (Rammsayer & Vogel, 1995). Alternatively, under stressful conditions, tyrosine administration improves learning and working memory performance in animals (Ahlers, Salander, Shurtleff, & Thomas, 1992; Shukitt-Hale, Stillman, & Lieberman, 1996; Shurtleff, Thomas, Ahlers, & Schrot, 1993) and in humans (Deijen & Orlebeke, 1994; Shurtleff, Thomas, & Schrot, 1994). Although experimental studies of tyrosine depletion have yet to result in an animal analogue of human mental retardation, it is well established that dopamine modulates cognitive functions, particularly those mediated by the prefrontal cortex. Specifically, dopamine in the dorsolateral prefrontal cortex facilitates spatial working memory performance (Diamond, 1996; Luciana & Collins, 1997; Sawaguchi & Goldman-Rakic, 1991). Nonhuman primates with depleted prefrontal dopamine exemplify impairments in working memory, and dopamine augmentation under acute conditions improves performance (Brozoski, Brown, Rosvold, & Goldman, 1979). Thus, although mental retardation as a consequence of PKU has been successfully averted through dietary intervention (Williamson, Koch, Azen, & Chang, 1981), it has yet to be determined whether phenylketonurics exemplify dopamine-dependent cognitive deficits. Although language, memory, and basic motor skills appear to be normal, several researchers have reported deficits in reaction times, attention, problem solving, abstract reasoning, and executive abilities in affected groups of all ages (Waisbren, Brown, de Sonneville, & Levy, 1994). Executive functions involve the ability to maintain a problem-solving set that is appropriate for goal acquisition, including formulation, planning, self-monitoring, mental flexibility, and the ability to change strategies in response to new information. Tests of executive functioning typically require information to be held in mind while other pieces of information are simultaneously processed (e.g., multitasking or working memory). Several studies have reported that infants and young children with early treated PKU show deficits in working memory and other high-level reasoning skills, despite the fact that these children have normal-range IQs (Diamond et al., 1997; Pennington, van Doorninck, McCabe, & McCabe, 1985; Spreen, Tupper, & Risser, 1984; Welsh, Pennington, Ozonoff, Rouse, & McCabe, 1990). Outcome in young adults treated since birth has not been reliably ascertained, because findings have been inconsistent in studies of older patients. Mazzocco et al. (1994) studied PKU probands, ages 6 to 13 years, and found no evidence of prefrontal dysfunction, suggesting that the cognitive deficits observed in younger children represent a developmental delay. In contrast, Weglage, Pietsch, Fünders, Koch, and Ullrich (1996) found evidence of dysfunction in PKU subjects aged 8 to 13 years on the Stroop test, a conventionally used executive measure of attention and inhibitory control. Ris and colleagues (Ris, Williams, Hunt, Berry, & Leslie, 1994) compared the neuropsychological performance of adults who had PKU with that of their unaffected siblings and found that the PKU probands were relatively impaired on measures of attention, visual reasoning, and on the Wisconsin Card Sort. Smith and colleagues (Smith, Klim, Mallozzi, & Hanley, 1996) studied the neuropsychological performance of hyperphenylalaninemic adults and found that the hyperphenylalaninemic group performed worse than control subjects on both frontal and temporal/parietal lobe tests. Only performance on the frontal measures, however, was related to concurrently measured phenylalanine levels. Interpretation of these studies is complicated, as has been noted (Stemerdink et al., 1999), because the neuropsychological measures vary widely between

3 Luciana, Sullivan, and Nelson 1639 studies, and several of the available reports (e.g., Ris et al., 1994; Smith et al., 1996) focused on samples of young adult PKU probands who had been early but not continuously treated. Most recently, Stemerdink et al. (1999) assessed neuropsychological function in early and continuously treated PKU probands, ages 8 to 20, relative to an age-matched control group. The cognitive measures utilized involved a test of visual contrast sensitivity, the Wisconsin Card Sort, the Tower of Hanoi planning test, and the Corsi Milner test of recency judgments. Several control measures were also employed to assess nonexecutive functions. These authors reported that their PKU sample was impaired relative to normal controls on a composite index of the prefrontal measures and specifically on the Wisconsin Card Sort and the test of recency judgments. Performance on nonexecutive measures was indistinct between the groups. Moreover, concurrent levels were related to task performance in the predicted direction within the youngest (e.g., age 8 years) and oldest (age 20 years) subjects, although findings were not consistent across the tasks employed. In the intermediate age groups, concurrent phenylalanine levels were related to task performance in a direction opposite to that predicted. That is, high concurrent phenylalanine levels were associated with better task performance. There have been several reports of associations between concurrent phenylalanine levels and composite indices of prefrontal function (Diamond et al., 1997; Ris et al., 1994; Weglage et al., 1996; Welsh et al., 1990), although the relationship between phenylalanine levels and performance on specific tests of executive function has been inconsistent. None of these studies of adolescent and young adult probands has examined the relationship between Phe:Tyr ratios and neuropsychological performance, despite its potential importance for the study of dopamine availability in PKU. This literature raises several questions relevant to the longer term outcome of adolescents and young adults with PKU. Not only does it remain uncertain as to whether early and continuous restriction of phenylalanine prevents cognitive dysfunction, particularly with respect to behaviors mediated by the prefrontal cortex, but evidence for a dopaminergic mechanism underlying the problems that have been reported is lacking. In this study, we compared the neurocognitive performance of adolescents and young adults who had early and continuously treated PKU with that of two comparison groups one consisting of age-matched peer-nominated controls and the other an age-matched group of individuals with chronic illnesses. All individuals completed measures of prefrontally mediated executive function including spatial working memory, set shifting and look-ahead planning. The measures of executive function used in this study are sensitive to alterations in catecholamine neurotransmission (Coull, Middleton, Robbins, & Sahakian, 1995; Dias, Robbins, & Roberts, 1996; Downes et al., 1989; Lange et al., 1992; Morris et al., 1988) and have been used to detect cognitive impairments in developmentally disordered populations (Hughes, Russell, & Robbins, 1994; Luciana, Lindeke, Georgieff, Mills, & Nelson, 1999; Ozonoff, 2001). It was predicted that relative to controls, individuals with PKU would show impairments in executive functions such as spatial working memory and set shifting that are modulated by dopamine activity in the frontal cortex. It was also predicted that individual differences in the cognitive performance of PKU probands on these tests of executive function would be associated with the Phe:Tyr ratio measured at various time points during individuals lifetimes. A high ratio score, indicating low tyrosine in the context of high phenylalanine levels, was expected to be associated with relatively poor cognitive performance. METHODS Participants PKU probands were recruited from the patient database at the Fairview-University Medical Center s Department of Pediatrics at the University of Minnesota. All had received treatment at the hospital s PKU Clinic during childhood, had been diagnosed with classic PKU (i.e., based on the clinic s criteria of serum phenylalanine concentrations 20 mg/dl on an unrestricted diet, normal or low tyrosine levels, and increased phenylketones present in urine), and had begun treatment within the first 3 months of life. This clinic serves Minnesota and several surrounding states, as well as southern central Canada. The clinic database was screened for individuals between the ages of 14 and 25 who had measured full-scale IQ scores above 80 on the age-appropriate version of the Wechsler Intelligence Scales. A total of 58 probands were identified as possible study participants, and all were sent letters of invitation. Letters were followed by phone calls from a study representative. Recruitment was complicated by the fact that many families were currently living out of state or in Canada. Of the initial pool of 58 possible participants, 19 could not be contacted, 11 refused to participate, and 28 expressed some interest in the study. Of those who expressed interest, 10 participants initially agreed to participate but did not make

4 1640 Child Development their scheduled appointments even when multiple visits were scheduled. The final number of PKU probands who agreed to participate (N 18) were asked (but not required) to nominate a friend of the same age and gender to co-participate in the study. These peer controls (N 16) were contacted separately and invited to participate in a study of adolescent health. In an attempt to address whether the PKU probands who had agreed to participate in this study were representative of the group as a whole, the IQ scores and phenylalanine levels of the 18 PKU probands who participated in this study were compared with those of 30 affected individuals who were identified as possible participants but could not be contacted or declined to participate. The nonparticipants had phenylalanine, tyrosine, and IQ data available through medical chart review. Although scores remained within the normal range, there was a trend toward lower full-scale IQ scores in the nonparticipating (M 97.2, SD 11.3) versus participating (M 104.8, SD 10.9) PKU sample, F(1, 37) 2.76, p.10. Phenylalanine levels did not differ between the groups, F(1, 37) 1.14, ns. In order to control for the nonspecific effects of chronic illness on behavioral functioning, a group of age-matched controls with lifelong illnesses was also recruited. This comparison group was comprised of patients with cystic fibrosis (n 6) and juvenile onset diabetes (n 4) identified through records of the Pediatric Pulmonary and the Pediatric Endocrinology Clinics at the Fairview-University Medical Center, as well as asthmatic patients (n 7) from the Pulmonology Clinic at Children s Health Care in St. Paul, MN. Of 95 potentially recruitable chronically ill patients, 73% (N 32) of patients with cystic fibrosis, 73% (N 22) of patients with diabetes, and 64% (N 14) of patients with asthma declined to participate. Eleven patients agreed to participate but either cancelled or failed to attend their scheduled visits. The 17 individuals who agreed to participate were also invited to nominate a peer to participate with them. There was no difference between the PKU and chronic illness groups in their willingness to recruit peers, 2 (1, N 37) 2.45, p 12. Participants under the age of 18 years (65%) were significantly more likely to recruit peers that were older participants, 2 (1, N 37) 8.40, p.01. The final study sample consisted of 18 PKU probands, 16 peer controls, and 17 patients with other chronic illnesses. All individuals were assessed on an outpatient basis. Demographic characteristics of the groups are presented in Table 1. As indicated, the three samples were comparable in terms of gender representation, age, F(2, 51) 1.61, ns, ethnicity, dwelling (rural versus urban settings), marital status, and living arrangements. As would be expected, age at initial diagnosis was significantly different between PKU probands (M days, SD days) and chronically ill participants (M 4.74 years, SD 4.13 years). All but one chronically ill participant was currently participating in daily medical and/or dietary treatment; the majority of these participants (N 13) reported that they complied with treatment as prescribed at least 75% of the time, with no significant differences in rates of compliance between the chronically ill subgroups, 2 (2, N 17) 5.42, p.49. Socioeconomically, the groups were similar in terms of their completed years of education, F(2, 51) 1.18, ns, level of education of their parents, and their annual median incomes (their own if living independently; parental if living with parents). The three groups did not differ in terms of history of academic difficulties, F(2, 51) 1.05, ns, the number of individuals who repeated a grade in school, F(2, 51).26, ns, nor in the number of current clinical scale elevations on the Minnesota Multiphasic Personality Inventory Adolescent Version (MMPI-A; Butcher et al., 1992), F(2, 51).64, ns. There was a group difference in the number of individuals who had received past psychiatric treatment, with approximately equal numbers of PKU and chronically ill individuals reporting such histories. No one in the peer control group reported a psychiatric treatment history, F(2, 51) 6.31, p.01. Cognitive Assessment Tests of Executive Function Three tasks were selected to represent executive functions mediated by the prefrontal cortex. These included a test of spatial working memory (Owen, Downes, Sahakian, Polkey, & Robbins, 1990), the Tower of London planning task (Shallice, 1982), and a set-shifting task, analogous to the Wisconsin Card Sort (Downes et al., 1989). All tasks were administered through the use of a touch-screen computer (Fray, Robbins, & Sahakian, 1996). Spatial working memory. This self-ordered searching task measures working memory for spatial stimuli and requires the participant to use mnemonic information to work toward a goal (Owen et al., 1990; Owen, Evans, & Petrides, 1996; Petrides & Milner, 1982). Two variables of interest are computed. The first is a measure of strategy whereby a high score represents poor strategy use. The second consists of mnemonic errors for searches of two, three, four, six, and eight items.

5 Luciana, Sullivan, and Nelson 1641 Table 1 Patients Demographic Characteristics of Phenylketonuria (PKU) Probands, Peer Controls, and Chronically Ill PKU Probands Peer Controls Chronically Ill Patients Age Gender (male:female) 9:9 8:8 11:6 Ethnicity (% White) 100% 98% 100% Years of education Number with reported history of academic difficulties 35 2 Number who repeated a grade in school Number with history of psychiatric treatment Average number of MMPI clinical scale elevations Dwelling Urban Rural Marital status Single Married Living arrangements With parents or guardian Dormitory Rented apartment Homeowner Employment status Student Student, plus part-time employment Part-time employment Full-time employment Unemployed Mothers education 12 years years a or GED Some college b College degree 34 3 Advanced degree Unknown Fathers education 12 years years a or GED Some college b College degree Advanced degree 0 32 Unknown 4 30 Incomes c Parental $40 $50,000 $40 $50,000 $40 $50,000 Own $20 $30,000 $20 $30,000 $20 $30,000 Note: MMPI Minnesota Multiphasic Personality Inventory; GED Graduate Equivalency Diploma. a Indicates completion of high school degree. b Includes trade school. c Participants reported personal income if financially dependent, otherwise parental income was used. d Two participants refused to provide financial information. Intradimensional/extradimensional set shifting. One of the most popularly used measures for the experimental assessment of frontal lobe dysfunction is the Wisconsin Card Sort Test (Milner, 1964). Deficient performance is typically attributed to the inability to shift response set between conceptual categories. However, the precise nature of the cognitive deficit underlying task failure has yet to be determined, be-

6 1642 Child Development cause the task actually requires several abilities. To dissociate the various abilities underlying set-shifting performance, we utilized a task called the Intradimensional/Extradimensional Set-Shift task (Dias, Robbins, & Roberts, 1996; Downes et al., 1989). This task measures discrimination and reversal learning under conditions that require the subject to shift attention to changing patterns of visual stimuli. Briefly, this task progresses along a series of nine stages of increasing difficulty. The first two stages involve simple discrimination and reversal learning. The next three stages involve extending the discrimination and reversal learning to instances in which a distractor stimulus is present. The sixth stage demands an attentional shift. Termed the intradimensional (ID) shift stage, novel or never-seen exemplars of each of the two prior dimensions are introduced, and the participant generalizes the rule from previous learning in order to achieve correct responses. Subsequently, another extradimensional (ED) attentional shift is required. Novel exemplars of each stimulus dimension are presented, and the subject must shift response set from the previously relevant dimension to the previously irrelevant dimension. This stage is analogous to the between-category shifts required by the Wisconsin Card Sort (Milner, 1964). Variables coded for each participant included the stage reached, the trials to criterion, and number of errors for each completed stage. Tower of London. This task measures planning and behavioral inhibition (Shallice, 1982). The participant views two displays on a computer screen, one of which is the target display and one that comprises a workspace. The participant must move colored balls on the screen to make the workspace display appear like the target. There are a minimum number of moves in which to complete a trial perfectly, and the participant is told at the start of a trial that the trial should be completed in that minimum number of moves. The following variables were computed for each problem set representing four levels of problem difficulty (2-move, 3-move, 4-move, and 5-move problems): average number of moves to complete each set, planning time, and total number of problems perfectly completed in the minimum number of moves. Performance expectations for PKU probands. Relative to comparison groups, we anticipated the performance of PKU probands on measures of executive function to be consistent with frontal lobe impairment. Specifically, the PKU group was expected to demonstrate increased mnemonic errors and higher strategy scores (indicative of poor use of strategy) on the spatial working memory task, an inability to proceed through all nine stages of the ID/ED set-shift task, and fewer perfect solutions in completing Tower of London problems. Tests of Nonexecutive Function Three additional cognitive measures were included to represent nonexecutive functions related to psychomotor speed and accuracy, sequencing ability, and recognition memory. Motor screening task. This test measures psychomotor speed and accuracy. The participant s task is to quickly and accurately touch visual targets that are presented one at a time on the computer screen. Accuracy and response latency are recorded. Spatial span task. This task measures memory for a figural sequence and is a computerized analog of the Corsi block task (Milner, 1971). It yields a measure of the participant s nonverbal memory span, the number of items that can be accurately remembered in a sequence. Pattern/spatial recognition. This delayed-match-tosample task is a measure of recognition memory for visual patterns and spatial locations. First, the participant views geometric patterns presented consecutively on screen. After a brief pause, the participant is presented with two geometric patterns. One of the two designs is from the previously viewed list, while the other is novel. The participant indicates the pattern that has already been seen. Accuracy and response latency are recorded. An additional series of trials comprises a delayed-match-to-location task. The participant attends to the locations of five empty boxes presented at various locations on the screen. Then the participant views two empty boxes, one of which is in a location that was just targeted, and touches the box that represents a previously targeted location. Notably, performance on these recognition memory tasks is highly sensitive to temporal and parietal lobe lesions in adult patients and appears to be unaffected by frontal lobe pathology (B. J. Sahakian, personal communication, April 16, 1998). Participants completed cognitive testing in the course of one 3-hour testing session. In addition, each participant completed the age-appropriate version of the MMPI-2/A (Butcher et al., 1992), the Tennessee Self- Concept Scale (Fitts & Warren, 1996), and a structured clinical interview (the Diagnostic Interview for Children and Adolescents Revised Adolescent Version for DSM-IV: Reich, Leacock, & Shanfeld, 1995; or the Structured Clinical Interview for DSM-IV, Non-Patient Version: First, Spitzer, Gibbon, & Williams, 1994), Cognitive testing took place during either the first or second hour of the assessment. The cognitive tasks were administered in the same order for all participants.

7 Luciana, Sullivan, and Nelson 1643 Chart review of medical records maintained by the Department of Pediatrics and the Biomedical Genetics Laboratory were utilized to obtain IQ and phenylalanine and tyrosine levels for PKU probands. IQ scores for PKU probands were obtained from each participant s most recent neuropsychological assessment (N 3, Wechsler Adult Intelligence Scale Revised; N 15, Wechsler Intelligence Scale for Children Revised, 3rd edition). The average IQ obtained (M 104.8, SD 10.9) was not significantly different from the normal population mean (M 100, SD 15). IQ was not measured in the comparison groups. Additionally, each participant s lifetime history of recorded phenylalanine and tyrosine levels was recorded and used in subsequent data analyses. Over the course of their lifetimes, PKU probands each contributed an average of assessments (M 58.67, SD 36.06, range 1 120) to this analysis. From each assessment, consisting of a phenylalanine and a tyrosine level, the following values were computed for each of the PKU probands: 1. The most recently obtained phenylalanine and tyrosine levels obtained within 6 months prior to the cognitive assessment. All probands had values available. 2. Yearly grand averages of phenylalanine and tyrosine for each year of life; the grand average computed for the first year of life always excluded the first value recorded subsequent to the proband s birth (e.g., the value from which the diagnosis of PKU was made). From these yearly averages, five variables were constructed separately for phenylalanine and tyrosine, representing grand averages for the years (a) 0 to 2 years, (b) 3 to 4 years, (c) 5 to 8 years, (d) 9 to 13 years, and (e) 14 to 15 years. The period encompassing ages 16 to 18 was not included in the analysis because only a small percentage of the PKU sample (n 10 or 55%) was old enough to contribute these values. These age ranges were selected based on developmental research using cognitive tasks identical or similar to the ones used in this study (Diamond et al., 1997; Luciana & Nelson, 1998). 3. For each of these five time periods, the average Phe:Tyr ratio was computed. This method of obtaining phenylalanine and tyrosine levels from medical records has been used in other PKU studies (Diamond et al., 1997). To assess whether the most recently obtained levels were representative of each participant s lifetime pattern of dietary control, correlations between these levels and lifetime averages were computed. These findings are presented in Table 2. As indicated in Table 2, the most Table 2 Intercorrelations among Lifetime and Most Recent Phenylalanine (Phe) and Tyrosine (Tyr) Levels Most Recent Phe Level Most Recent Tyr Level Lifetime Phe Level Most recent Tyr level.502 Lifetime Phe level.840**.600* Lifetime Tyr level **.338 Note: Values represent Pearson correlations. * p.05; ** p.01; p.10. recent and lifetime levels of phenylalanine and tyrosine were significantly intercorrelated. RESULTS Data were analyzed using SPSS for Windows, version 10.0 (SPSS, 1999). Two primary questions were addressed in the data analyses: (1) Do PKU probands differ from the two comparison groups with respect to cognitive functions in general, and, specifically, with respect to executive functions? and (2) In individuals with PKU, what is the relationship between lifetime measures of phenylalanine and tyrosine levels and cognitive function? Group Differences in Cognitive Function Cognitive task variables were compared between groups using one-way analyses of variance (ANOVA). When group differences were found, post hoc comparisons using Tukey s honestly significant difference were evaluated to determine the nature of significant group differences. As indicated in Table 3, the cognitive performance of adolescents with PKU was comparable with that exhibited by the two age-matched comparison groups. There were no statistically significant differences noted between groups on any cognitive task variables. Following the methodology of Welsh et al. (1990), a composite score representing executive function (EF) was derived by converting raw scores on the frontal lobe measures to Z scores, using the total sample mean and standard deviation. After the Z score conversions, the following variables, each of which is the best representative of frontal lobe dysfunction for the particular task, were averaged to obtain composite EF scores: spatial working memory strategy score, number of minimum-move Tower of London solutions, and ID/ED stage reached. Composites were computed so that a Z score above the mean reflected below-average performance. As a discriminant measure, a second composite

8 1644 Child Development Table 3 Cognitive Task Performance among Phenlyketonuria (PKU) Probands, Peer Controls, and Chronically Ill Patients Task Variable PKU Probands Peer Controls Chronically Ill Patients F Ratio p Value Motor error ns Motor latency Memory span ns Spatial working memory Easy-item errors ns Hard-item errors ns Strategy score ns Tower of London planning Minimum move solutions ns Average moves to complete 3-move problems ns 4-move problems ns 5-move problems ns Planning times 3-move problems move problems ns 5-move problems ns ID/ED set shifting Stage reached ns ID errors ID trials ns ED errors ns ED trials ns Reversal errors ns Recognition memory Patterns: % correct ns Spatial: % correct ns Executive function composite ns Nonexecutive function composite ns Note: ID intradimensional; ED extradimensional. score was similarly derived to represent performance on tasks measuring nonexecutive functions (NEF). This measure was derived from the following variables: motor screening accuracy, motor screening latency, spatial memory span, pattern recognition percent correct performance, and spatial recognition percent correct performance. The composite was scored such that a high score represented poor performance. EF and NEF composite scores were compared among the three groups in a one-way ANOVA yielding no significant main effect for group for the EF nor for the NEF composite. Because this pattern of findings was inconsistent with what has been observed in younger samples of PKU probands, we considered whether performance in the comparison groups accurately represented normal cognitive performance. Akin to the notion of assortative mating, it may be that individuals with PKU select friends who are similar in having some level of cognitive deficit. As indicated in Table 1 (items 4 8), the three groups did not differ in terms of average educational level, whether school services had ever been received in the past, or in the average number of clinical scale elevations obtained on the MMPI. A more thorough analysis of the emotional functioning of these groups has indicated that the three groups were generally healthy with respect to current psychopathology (Sullivan, in press). However, each group consisted of some individuals who experienced difficulties in one or more of these areas, as indicated in Table 1. Accordingly, to further address the representativeness of the two comparison samples (peers and chronically ill), their cognitive performance was compared with that of a third group (n 19) of age-matched normal controls whose performance on the same battery of tests was measured for a separate study. This sample was recruited from the undergraduate population of students at the University of Minnesota and from a private area high school. Their data have been reported elsewhere (Luciana & Nelson, 1998). No one in this additional sample had a history of school difficulty, special

9 Luciana, Sullivan, and Nelson 1645 education, chronic illness, or psychological disorder. There were no significant differences in cognitive performance on any task variable among the peernominated controls, chronically ill adolescents, and this third normative sample of young adults. Thus, we have no reason to conclude that our comparison groups were not representative of individuals in this age range. Associations between Biochemical Variables and Cognitive Outcome Associations between phenylalanine levels, tyrosine levels, the Phe:Tyr ratio and measures of executive and nonexecutive function were examined. Several tables are included to describe these data: Table 4 presents correlations between biochemical variables at various points in development and the PKU probands ages and IQs, Table 5 presents correlations between measures of executive function and biochemical variables, and Table 6 presents correlations between measures of nonexecutive function and biochemical variables. Phenylalanine and Cognitive Function Of the 18 PKU probands studied, 77% (14/18) indicated by self-report that they were currently complying with dietary restrictions. However, the range of most recently obtained phenylalanine levels varied in this group from 5.3 to 28.8 mg/dl (M 16.3, SD 6.8). Since adequate control of phenylalanine levels is represented by a value of less than 10 to 15 mg/dl (Diamond et al., 1997), many of these participants were either not maintaining the diet adequately or were not maximally benefiting from phenylalanine restriction. The correlation between the most recently obtained phenylalanine level and IQ was in the predicted direction, with high phenylalanine levels associated with lower full-scale IQ scores. Additionally, older participants tended to exhibit higher phenylalanine levels (see Table 4). As indicated in Tables 5 and 6, there were significant associations between phenylalanine levels obtained at various points in development and several cognitive task variables. Executive function. With respect to spatial working memory performance, phenylalanine levels at 0 to 2 years of age were related to the total number of mnemonic errors, but in a manner opposite to that predicted. High phenylalanine levels were associated with lower numbers of errors. However, poor use of strategy was associated with high phenylalanine levels at ages 3 to 5 years, 9 to 13 years, 14 to 15 years, and in the past 6 months. In terms of set-shifting performance, high phenylalanine levels at ages 3 to 4, 5 to 8, and 9 to 13 years were associated with a decreased stage reached on the task. Tower of London performance, as assessed by a high number of items solved in the minimum number of moves, was associated with higher phenylalanine levels at 0 to 2 years, a finding similar to what was found for spatial working memory errors and counter to prediction. High phenylalanine levels at 9 to 13 years of age were associated with poor overall performance as represented by the EF composite. Nonexecutive function. Increased motor latency was marginally associated with higher phenylalanine levels at ages 9 to 13 and 14 to 15. Low memory spans were associated with high phenylalanine levels at ages 9 to 13, 14 to 15, and in the past 6 months. High phenylalanine levels at ages 9 to 13 also related to cur- Table 4 Associations among Age, IQ, and Biochemical Indices Age When Blood Levels of Phe and Tyr Were Recorded 0 2 Years 3 4 Years 5 8 Years 9 13 Years Years Past 6 Months Age Phe/age *.58**.64*.61* Tyr/age **.25.44*.31 Ratio/age **.74**.75**.74** IQ Phe/IQ.49*.47*.71**.75**.56**.69* Tyr/IQ Ratio/IQ.39.57*.52*.54*.48*.39 Note: Age and IQ correlations are Pearson correlations. Bold-faced values indicate correlations that conform to the expectations that high phenylalanine (Phe), low tyrosine (Tyr), and/or a high Phe:Tyr ratio will be associated with lower IQs and increased age. Note that values recorded during ages 0 2 exclude those used to diagnose phenylketonuria. * p.05; ** p.01; p.10.

10 1646 Child Development Table 5 Associations among Executive Function (EF) Task Performance, Phenylalanine (Phe), and Tyrosine (Tyr) Age When Blood Phe and Tyr Levels Were Recorded 0 2 Years 3 4 Years 5 8 Years 9 13 Years Years Past 6 Months EF composite Phe/EF * Tyr/EF.76** Ratio/EF.67* *.01 Spatial working memory Strategy/Phe *.71** Strategy/Tyr * Strategy/ratio.89** Errors/Phe Errors/Tyr.81** * Errors/ratio Set shifting Stage/Phe *.57*.03### Stage/Tyr ### Stage/ratio **.67**.40 ### Tower of London Perfect solutions/phe.54* Perfect solutions/tyr.92**.51* * Perfect solutions/ratio.58* * Note: Values are partial correlation coefficients, covarying for age and IQ. Bold-faced values indicate correlations that conform to the study s predictions that cognitive function will be impaired by high Phe, low Tyr, and/or a high Phe:Tyr ratio. * p.05; ** p.01; p.10; ### coefficient cannot be determined because of lack of variance. rent spatial recognition memory performance. Otherwise, correlations between phenylalanine levels and measures of nonexecutive function were nonsignificant. Tyrosine and Cognitive Outcome The range of most recently obtained tyrosine levels varied from.50 to 1.7 mg/dl (M 1.05, SD.36). The reference range for normal tyrosine levels in individuals above the age of 12 years is from.6 to 2.4 mg/dl according to the University of Minnesota s Department of Biomedical Genetics, where the assays were conducted. Tyrosine levels were correlated with all cognitive task variables, partialing out the effects of both IQ and age on performance. These findings are represented in Tables 5 and 6. Executive function. Spatial working memory impairment, as measured by a high number of mnemonic errors and poor use of strategy, was associated with low tyrosine levels at ages 0 to 2 years. However, counter to prediction, higher tyrosine levels were associated with poor performance on this task at ages 5 to 8 (increased mnemonic errors) and 9 to 13 (increased errors and high strategy scores). Stage reached on the set-shifting task was unrelated to tyrosine levels. A high number of Tower of London problems solved in the minimum number of moves was associated with higher tyrosine levels at ages 0 to 2 years and in the past 6 months. Counter to prediction, high tyrosine levels at ages 3 to 4 and 5 to 8 were associated with poorer Tower of London performance. The EF composite was most related to tyrosine levels at ages 0 to 2 years, with low tyrosine levels associated with poorer current performance. Nonexecutive function. In terms of nonexecutive performance, longer motor response latencies were associated with high tyrosine levels at ages 0 to 2 years. A relatively higher spatial memory span was related to high tyrosine levels at ages 0 to 2 years. Finally, better pattern recognition memory was associated with higher tyrosine levels in the most recent period. The Phe:Tyr Ratio and Cognitive Outcome As indicated in Table 2, phenylalanine and tyrosine levels measured within the past 6 months were inversely correlated to a moderate degree, r(17).50. To address Diamond and colleagues (1997) hypothesis that the ratio between phenylalanine and tyrosine leads to dopamine deficiency in the prefrontal cortex and associated cognitive dysfunction, ratio scores were correlated with all tasks, covarying once again for IQ

11 Luciana, Sullivan, and Nelson 1647 Table 6 Associations among Nonexecutive Function (NEF) Task Performance, Phenylalanine (Phe), and Tyrosine (Tyr) Age When Phe and Tyr Levels Obtained 0 2 Years 3 4 Years 5 8 Years 9 13 Years Years Past 6 Months NEF composite Phe/NEF Tyr/NEF Ratio/NEF.64* Motor screening Accuracy/Phe Accuracy/Tyr Accuracy/ratio Latency/Phe Latency/Tyr.59* Latency/ratio **.64 Memory span Span/Phe **.93** Span/Tyr Span/ratio ** ### Recognition memory Patterns/Phe Patterns/Tyr ** Patterns/ratio *.32 Locations/Phe Locations/Tyr ** Locations/ratio Note: Values are partial correlation coefficients, covarying for age and IQ. Bold-faced values indicate correlations that conform to the study s predictions that cognitive function will be impaired by high Phe, low Tyr, and/or a high Phe:Tyr ratio. * p.05; ** p.01; p.10; ### coefficient cannot be determined because of lack of variance. and age. The findings, represented in Tables 5 and 6, suggest that the Phe:Tyr ratio is moderately to strongly associated with several aspects of cognitive function. Executive function. Increased errors on the spatial working memory task were associated with increased Phe:Tyr ratios at ages 0 to 2 and 14 to 15. Poor use of strategy was also associated with a higher ratio score at these ages, as well as within the past 6 months. A decreased stage reached on the ID/ED set-shifting task was associated with higher Phe:Tyr ratios at ages 3 to 4, 5 to 8, 9 to 13, and 14 to 15. Similarly, a decrease in the number of Tower of London problems solved in the minimum number of moves was related to increased ratio scores at ages 0 to 2, 14 to 15, and in the past 6 months. As expected from this pattern of findings within individual tasks, a high current EF composite score was associated with increased ratio scores at ages 0 to 2, 5 to 8, 9 to 13, and 14 to 15 years. Nonexecutive function. With respect to nonexecutive functions, increased motor response latencies were associated with higher ratio scores at ages 14 to 15 and in the past 6 months. Higher ratio scores at ages 14 to 15 were also associated with poor pattern recognition memory performance. In the age range from 0 to 2 years, high ratio scores were associated with better current NEF performance. However, high ratio scores at ages 9 to 13 and 14 to 15 were associated with poorer NEF performance, as indicated by the composite index. Implications of high versus low ratio scores. To better understand the implications of high Phe:Tyr ratio scores for current levels of cognitive function, the median ratio score (represented by a value of 14.6) at ages 14 to 15 years was used to group PKU probands into those with high versus low scores. This age range was selected because all PKU probands had values available for this time period and because it was significantly associated with performance on a variety of tasks. The performance of these two groups was compared once again with that of the control samples. Findings indicated that PKU probands with high ratio scores performed significantly worse than the control sample in terms of the NEF composite, F(2, 43) 3.62, p.05, and marginally worse in terms of the EF composite, F(2, 43) 2.49, p.10. With respect to specific tasks, the high-ratio PKU sample performed worse than controls in terms of the maximum stage reached on the set-shifting task, F(2, 43) 3.29, p

12 1648 Child Development.05. All other specific task variables yielded nonsignificant differences between the high-ratio PKU, lowratio PKU, and control samples. A large number of correlations have been presented in these analyses, and control for multiple correlations has not been stringently applied. In Tables 5 and 6, there are a total of 194 correlations reported. We utilized one-tailed tests of significance and have highlighted p values less than.10. Hence, we would expect 10% of these correlations to be significant by chance. In Table 5, which represents 87 correlations between measures of executive function and three biochemical indices (phenylalanine, tyrosine, and the Phe:Tyr ratio), 31 correlations (or 36%) were significant in the predicted direction, a number that exceeds chance expectancies. Moreover, 48% of the correlations that were significant in the expected direction represent associations that involved the Phe:Tyr ratio. Table 6 presents 107 correlations between measures of nonexecutive function and the same biochemical indices. Here, 16 of 107 correlations (15%) were significant in what might loosely be described as the predicted direction (e.g., poor cognitive performance associated with high phenylalanine and low tyrosine levels). Of these, 38% involved the ratio score. This was, admittedly, a crude analysis. Biochemical levels obtained at one age are highly associated with those obtained at past and subsequent ages, and the ratio score is related to each of the two indices that comprised it. Moreover, the cognitive task variables, particularly those derived from the same task, are significantly intercorrelated. A larger study sample would permit the use of factor-analytic techniques to reduce the data into smaller components, but data reduction techniques were not appropriate with this sample. Thus, because of the number of comparisons reported, we stress that these findings await replication, although the observed pattern of effects was clearly in the predicted direction. DISCUSSION Most studies of cognitive function in early and continuously treated PKU have included participants who are infants or preschoolers (Diamond et al., 1997). Long-term cognitive outcome is uncertain, particularly in adolescents and young adults who have achieved normal IQ scores. The results of the current study suggest that with continuous dietary treatment, individuals with PKU who have achieved normal IQ scores do not differ substantially from their peers or from an age-matched group of individuals with other chronic illnesses in terms of cognitive performance. The cognitive measures used in this study are particularly sensitive to frontostriatal dysfunction, as evidenced by both clinical and functional neuroimaging studies (Baker et al., 1996; Fray et al., 1996; Morris, Ahmed, Syed, & Toone, 1993; Owen, Doyon, Petrides, & Evans, 1996; Owen, Evans, & Petrides, 1996; Owen et al., 1992; Owen, Morris, Sahakian, Polkey, & Robbins, 1996; Owen, Roberts, Pokley, Sahakian, & Robbins, 1991; Veale, Sahakian, Owen, & Marks, 1996). Although relatively low scores on tasks that index frontal lobe function have been reported in younger samples of PKU-affected individuals, it cannot be generally concluded on the basis of the current data that adolescents and young adults with PKU will necessarily demonstrate a deficit level of performance on measures of executive function. Despite what appears to be a relatively positive outcome for the PKU sample as a whole, dietary control of phenylalanine and tyrosine levels nevertheless appears to contribute to individual differences in cognitive function. Diamond and colleagues (Diamond et al., 1997) have discussed the importance of the Phe:Tyr ratio as an index of the level of tyrosine that will be available to the brain for dopamine synthesis. A high ratio indicates that phenylalanine levels are in a range that would theoretically impede the transport of available tyrosine across the blood brain barrier, resulting in a decreased availability of tyrosine for dopamine synthesis. The findings of this study support the hypothesis that in individuals with PKU who may have decreased tyrosine availability, frontal lobe functions that are dopamine dependent may be compromised. High Phe:Tyr ratio scores in the past 6 months, at ages 14 to 15 and at ages 0 to 2 years were associated with relatively poor current spatial working memory performance (both in terms of number of errors and use of strategy) and a relatively poor ability to solve Tower of London problems with maximal efficiency. Relatively poor set-shifting performance was associated with high ratio scores in all age ranges between 3 and 15 years of age. Moreover, below-average scores on a composite index of EF performance were associated with high Phe:Tyr ratios at ages 0 to 2, 5 to 8, 9 to 13, and 14 to 15 years. However, counter to prediction, high ratio scores, particularly at ages 9 to 13 and 14 to 15, were also associated with relatively poor nonexecutive function performance. Indeed, individuals with high ratio scores at ages 14 to 15 performed significantly worse than control participants in terms of the NEF composite. If these findings can be replicated, then the specificity of dopamine s role in mediating performance on tests of executive function, particularly in the context of what is hypothesized to be a lifetime dopamine deficit, should be re-evaluated. In addition, to better examine the significance of