Key Points

• Increasing blood Phe is clearly associated with decreased IQ, with a probability of having an IQ less than 85 exceeding the probability for the general population (approximately 15 percent) at a Phe level of over 400 μmol/L. This finding supports the typical target goal for Phe level in individuals with PKU (120 to 360 μmol/L).
• The probability of having an IQ of <85 does not continue to increase considerably above a blood Phe level of 2000 μmol/L.
• Historical measurements of Phe (taken more than 1 year prior to IQ testing) show a stronger correlation with the probability of having a lower IQ than do concurrent measurements. Even at the highest blood Phe measurement, observed effects differ by when the measurements are taken, both relative to IQ measurement and according to whether measurements were taken during the critical period (<6 years old).
• The best measure of blood Phe for assessing the potential impact on IQ is likely to be a historical measure of dietary control that is taken at least one year prior to the IQ test.
• The probability of low IQ (<85) increases faster with higher blood Phe measurement when historical measurements were taken during the critical period and associated with later IQ, although historical measurements taken after the critical period are also associated with risk of low IQ. Hence, control of blood Phe levels during the critical period is particularly important, but the need for dietary control continues beyond early childhood.
• The relatively modest increase in the probability of low IQ with blood Phe measurement in measurements taken concurrently during the critical period may suggest that effects are unlikely to be observed in this period, either because the IQ test is not stable for young children (under 5 years of age), or because the adverse effects take time to manifest. From a clinical perspective, this provides a basis for being cautious in interpreting measures of cognitive outcomes as they relate to blood Phe in early childhood.

Overview of the Literature

Seventeen unique studies (reported in 21 publications) met our criteria and addressed the relationship between blood Phe levels and IQ (Table 3).^60-79 Age ranges and IQ levels varied widely across studies. Ten studies were conducted in Europe,^{62-66, 69, 70, 73-75} six in the United States,^{60, 71, 72, 78, 80, 81} and one in Iran.⁶¹ We rated one study^{67, 68} as good quality and five studies as fair quality.^{63, 65, 71, 74, 79, 81} The remaining studies^{60-62, 64, 66, 69, 70, 72, 73, 75-78} were rated as poor quality and typically did not document recruitment processes adequately, did not include all eligible participants in analyses, and/or did not assess confounding variables using valid and reliable measures.

Table 3

Overview of studies addressing Phe levels and IQ.

Overall, the number of participants in the studies was low, ranging from 10 to 57. The studies included a total of 432 individuals with PKU. Of the studies that reported on disease classification, 10 included only participants with classic PKU, and the remainder did not provide the classification or included individuals with less severe PKU. Results are therefore most clearly applicable to individuals with classic PKU.

Participant ages ranged from 2 to 34 years. A majority of studies included primarily participants under age 25 at intake,^{60-63, 65, 67, 70, 71, 74, 75, 78, 81} with five studies including only participants under age 15 at intake.^{60, 63, 71, 75, 78} Dietary control varied among the studies, with five studies reporting that all participants were adhering to a restricted diet,^{60, 61, 63, 72, 78} seven reporting a mix of dietary control (some participants on and some off a restricted diet),^{64-70, 81} and three reporting that participants had discontinued a restricted diet.^{62, 71, 73} Dietary status was not clearly reported in the remaining studies.^74-77Table 4 outlines characteristics of study participants.

Table 4

Characteristics of participants in studies addressing Phe levels and IQ.

IQ scores ranged from 44 to 148 across studies. Five studies reported concurrent measures of Phe levels (blood Phe measurement within 6 weeks of IQ measurement),^{61, 62, 64, 67, 70} eight studies reported historical Phe measurements (blood Phe measurements taken more than 12 months before IQ measurement),^{63, 65, 66, 71, 73-75, 78} and four reported both historical and concurrent measurements.^{60, 69, 72, 81} Phe measurements were also taken in the critical period (blood Phe measurement before age 6) in seven studies (Table 5).^{60, 63, 66, 71, 72, 78, 81} The one study that included very young children used developmental quotient as the outcome measurement for the young children.⁶⁰

Table 5

Summary of results of studies addressing Phe levels and IQ.

The degree to which Phe was noted to be correlated with IQ varied across the studies, with some noting a significant negative correlation and others finding little to no relationship. At the individual study level, this variation in outcomes did not appear to be related to the population or when or how the measures were taken. The observed variation is possibly due, however, to the small size of the studies, a consideration that is mitigated by the meta-analysis, below.

Meta-analysis

We developed two meta-analytic models. The first represents the relationship of blood Phe and IQ when Phe was measured “historically” (more than 12 months before IQ measurement). In the second model, Phe and IQ were measured concurrently (within 6 weeks of IQ measurement). The key model parameters for the relationship between blood Phe and IQ from both models are presented in Table 6. The baseline Phe effect denotes the slope (correlation) of the linear relationship of Phe (either historical or concurrent) and IQ, when both are measured at or after 6 years of age; the critical period effect, then, is the additive effect of using Phe measurements that were taken in the critical period (prior to 6 years of age). The magnitude of association is strongest for the historical measurement of blood Phe versus that seen when Phe and IQ are measured concurrently.

Table 6

Estimates of key parameters by model.

The implications of this relationship for a range of blood Phe levels measured at different points in life are described in Table 7. These probabilities can be used to estimate the chances of an individual's IQ being less than 85, based on blood Phe level, when Phe was measured, and the proximity of the Phe measurement to the IQ measurement. For example, Column 2 provides probabilities of the results of an IQ test showing an IQ less than 85 at different Phe levels when 1) the Phe is measured at least one year prior to the IQ, but 2) when the individual is 6 years old or greater. Note that these probabilities do not have associated levels of uncertainty, such as confidence intervals, because they were derived by integrating over the posterior distribution of the predicted IQ.

Table 7

Summary of probability (IQ<85) for various combinations of predictor variables.

Conversely, Column 3 provides the probabilities for individuals for whom 1) the Phe is measured at least one year prior to IQ, but 2) in the critical period (prior to 6 years of age). As expected, increasing blood Phe in all cases is associated with increasing probability of a low IQ. However, our ability to see the relationship between Phe and IQ is attenuated by when both measurements are obtained. This suggests that although a relationship between high Phe and low IQ clearly exists, the effects may not be observed during early childhood, but become apparent later in life.

The columns in Table 7 provide probabilities for low IQ for four groups of individuals whose Phe levels have been measured at distinct time periods.

• Group 1 represents probabilities for low IQ for individuals who are tested for IQ whose reported blood Phe was measured more than one year prior to their IQ test and at or after age 6.
• Group 2 represents probabilities for low IQ for individuals who are tested for IQ whose reported blood Phe was measured more than one year prior to their IQ test and before age 6.
• Group 3 represents probabilities for low IQ for individuals who are tested for IQ testing whose blood Phe and IQ measurements are occurring within 6 weeks of one another and at or after age 6.
• Group 4 represents probabilities for low IQ for individuals who are tested for IQ testing whose blood Phe and IQ measurements are occurring within 6 weeks of one another and before age 6.

Across all groups, blood Phe of 200 μmol/L is associated with a low probability of about 0.10 (10 percent) of having an IQ less than 85. As Phe increases to 400, probability of low IQ increases considerably to 0.187 (19 percent) only in Group 2 in which Phe has been measured more than one year prior to IQ and before age 6.

The association of a high blood Phe level of 1,200 μmol/L with low IQ is most clearly captured when Phe and IQ measurements take place at least one year apart. The probability of having a low IQ is 0.642 (64 percent) for those individuals who had a Phe level of 1200 before age 6 (critical period) and at least one year before IQ testing. If the Phe level was measured at or after age 6 as opposed to in the critical period, the probability of a low IQ is 0.430 (43 percent).

A less dramatic effect is observed when the blood Phe and IQ measurements are taken concurrently. In individuals whose Phe and IQ are measured concurrently and at or after age 6, if the blood Phe is 1,200 μmol/L, then there is a 0.192 (19 percent) probability of low IQ, compared with a probability of 0.163 (16 percent) when both measurements are taken concurrently in the critical period. This finding may represent tighter dietary control among individuals with frequent and concurrent measurement, or it may suggest that long-term effects of Phe on IQ cannot be seen when the two are measured too closely.

At the highest blood Phe level (3000 μmol/L), the probability of low IQ is substantially different across groups. When both are measured prior to age 6, the probability of low IQ is only 30 percent. This may be because the effects on IQ are not yet observable or because IQ measurements in this young age group are not stable. This also may reflect the fact children are more likely to be in compliance with diet when their diet is substantially controlled by the adults in their lives. Thus, of greater clinical importance is the historical effect of Phe on IQ over the longer term, as observed in Groups 1 and 2, in which earlier blood Phe measures of 3000 μmol/L are associated with approximately 80 and 90 percent probability, respectively, of low IQ measured later. However, even in Group 3, in which both measures are taken concurrently and after the critical period (i.e., in older children, adolescents and adults), very high Phe continues to be associated with low IQ, suggesting a continued effect into adulthood.

In summary, the observed influence of varying blood Phe on the probability of having an IQ <85 depends strongly on when Phe is measured relative to when IQ is tested, and whether or not the Phe measurement takes place in the critical period (before 6 years of age) (Figure 3).

Figure 3

Probability of IQ <85 at varying blood Phe levels and Phe measurement times. IQ = intelligence quotient; Phe = phenylalanine; Pr = probability

Note that in Figure 3 the two lines depicting historical measures of blood Phe (top two lines) both demonstrate increasing probability of low IQ at higher blood Phe levels, regardless of whether IQ was measured during childhood (top line) or beyond (second line). The effect in early childhood is consistently stronger. Nonetheless, effects of Phe on IQ continue beyond early childhood. Therefore, the best measure of Phe for assessing the potential impact on IQ is likely to be a historical measure of dietary control that is taken at least one year prior to the IQ test. The probability of having an IQ <85 does not continue to increase considerably above a blood Phe level of 2,000 μmol/L.

The two lower lines in the figure describe the observed relationship of blood Phe and IQ when they are measured concurrently. The lack of strong association in measurements taken concurrently during the critical period may suggest that effects are unlikely to be observed in this period, either because the IQ test is not stable for young children (under 5 years of age), or because the adverse effects take time to manifest. From a clinical perspective, this provides a basis for being cautious in interpreting measures of cognitive outcomes as they relate to Phe in early childhood.

Key Points

• Too few studies of common outcomes are available to synthesize the relationship of specific Phe levels and executive function measures.
• Among individual studies, data are inconsistent in terms of the direction and degree of association between specific Phe levels and measures of planning ability, inhibitory control and attention.

Overview of the Literature

Nineteen unique studies, reported in 26 papers,^{67, 68, 70, 72, 75-78, 82-99} provided data on blood Phe levels and on measures of executive function. We summarize data from these studies in Appendix H. Of the 19 studies providing Phe and executive function data, only three tests of executive function appeared in at least three studies, suggesting that we could potentially provide some synthesis on these nine studies (Table 8). The nine studies presented here include three using the Tower of London test to assess planning ability,^{61, 78, 84, 85} three studies using a Flanker test for inhibitory control,^{86-88, 90, 97} and three studies using the Color Word Interference Test as a measure of inhibitory control and attention.^{75-77, 91-93} After reviewing these as possible candidates for meta-analysis, clinical and statistical experts determined that a meta-analysis would not be appropriate for any component of executive function.

Table 8

Summary of studies addressing measures of executive function and Phe levels.

Overall, while blood Phe levels correlate with various assessments of executive function in some papers, the degree to which they are correlated, and the correlation on individual measures, are not conclusive. For example, in the three studies of planning skills, one study found no correlation between higher blood Phe and improved planning skills,⁶¹ one found a significant negative correlation,⁷⁸and one did not measure the association.^{84, 85} Two out of three studies of blood Phe and inhibitory control found no association. In none of these studies can a specific Phe threshold as a target be identified to answer the Key Question. Further, these studies cannot be meaningfully aggregated since the measures of executive function relevant for individuals with PKU have not yet been established (see Future Research section also).

Key Points

• Data predominantly from one longitudinal study provide evidence for poor cognitive outcomes in the offspring of women who have high blood Phe during pregnancy.
• Several analyses of the data, including separate analyses for U.S. and German data, suggest that the time it takes for women to achieve dietary control is particularly influential on offspring outcomes, with relatively better outcomes associated with achieving control by 10 weeks postconception, but all studies recommending control as early as possible. Children of mothers with well-controlled PKU prior to pregnancy had the best outcomes.
• One complex analysis using structural equation modeling and splines was able to demonstrate that a threshold of 360 μmol/L of blood Phe is appropriate to prevent poor cognitive outcomes in offspring, and that a linear relationship exists after that threshold.

Overview of the Literature

We identified 20 papers from three unique study populations that provided some data on maternal blood Phe and cognitive outcomes in infants or children.^{19, 21, 24, 31-33, 92, 93, 100-111} Most of the papers in this literature come from the international Maternal PKU (MPKU) Collaborative Study, which prospectively followed women with PKU who were pregnant or planning pregnancy and their offspring from 1984 to 2002 and provides the most complete data currently available on women with PKU and their offspring. The data reported were not suitable for meta-analysis; however, we summarize key findings below and present tables outlining cognitive outcome data in Appendix I.

Detailed Analysis

Effects of BH4 on Blood Phe Levels and Phe Tolerance

Phe levels were reduced by at least 30 percent in up to half of treated participants (32 to 50 percent) at dosages of 5 to 20 mg/kg/day and for up to 22 weeks of observation in comparative studies (Table 11). In the one RCT that compared the effect of placebo on likelihood of a 30 percent reduction in blood Phe, only 9 percent of those on placebo achieved this effect, compared with 44 percent of the treated group after 6 weeks.¹¹³ Data from the uncontrolled open label trial¹¹⁴ following this RCT¹¹³ suggested a sustained response for up to 22 weeks' duration, with 46 percent achieving a 30 percent reduction in blood Phe levels with most participants receiving 10 and 20 mg/kg/day doses compared with 5 mg/kg/day.

Table 11

Summary of effects of BH4 on Phe in comparative studies.

Similarly positive effects were reported at a dosage of 20 mg/kg/day in children on Phe-restricted diets. Reduction in blood Phe levels sampled at week 3 (before supplemental medical foods began) was greater among those receiving BH4.¹¹⁵ In the other nonrandomized clinical trial,¹¹⁶ BH4 (7 to 20 mg/kg/day) was associated with a reduction of blood Phe levels among participants both on and off Phe-restricted diets. Overall, participants' responses to different dosages of BH4 varied, with individualized dose adjustments needed according to target plasma Phe and dietary intake. Dosages of 10 to 20 mg/kg/day were most effective across the studies. Response also varied by different baseline Phe levels, with those with the highest baseline levels having lower response rates. As noted above, some participants from the RCTs and extension study have now been followed for up to three years; almost all participants for whom data were available achieved Phe levels within clinically recommended ranges, although specific Phe levels are not reported.¹¹²

Studies of Phe tolerance (total Phe intake an individual can tolerate without raising blood Phe to an unacceptable level) all reported improvements over time.^{115-118, 120} Data from the one RCT¹¹⁵ measuring this outcome indicate that participants in the treatment group were able to increase the supplementary Phe added in controlled amounts to a patient's usual dietary intake from 0 mg/kg at baseline to 20.9 mg/kg/day, while maintaining blood Phe levels at <360 μmol/L, compared with an increase of 2.9 mg/kg/day in the placebo group. However, response varied substantially within the treatment group, with 33 percent tolerating an increase of between 31 and 50 mg/kg/day in medical food form, but the rest of the participants tolerating lower levels of supplementary Phe. Similarly, total Phe intake (medical food plus diet) in the treatment group doubled from baseline to a mean of 43.8 mg/kg/day, response varied substantially within the treatment group.¹¹⁵ In the one open label trial that assessed changes in tolerance,¹¹⁶ participants on a Phe-restricted diet taking 10 to 20 mg/kg of BH4 per day increased their Phe tolerance by an average of 21 to 41 mg/kg/day. Participants tolerated a wide range of dietary Phe, ranging from increases of 20 to 22 mg/kg/day up to a full non-protein restricted diet. For some individuals, increasing the dose of BH4 to 20 mg/kg/day allowed further liberalization of the diet. Trials did not evaluate the impact of increasing natural protein sources on micronutrient levels, nutritional status, or quality of life.

Three case series^{117, 118, 120} also reported improved Phe tolerance. Among 11 children with mild or moderate PKU, participants reduced or discontinued Phe-free medical foods with 12 months of BH4 treatment. These reductions in special formula and replacement with unrestricted diet did not result in deficiencies of essential nutrients.

Although the mean blood Phe level is an important predictor of IQ, Phe variability may also be an important determinant. In one small retrospective case series (N=37), blood Phe variability as well as blood Phe levels decreased on BH4 20 mg/kg/day.¹¹⁹

Effect of BH4 on Longer Term Effectiveness

After nearly 3 years of following participants in the longer term extension study of BH4, most of the 90 study completers (of 111 enrolled) were reported to have reached clinical targets in Phe levels.¹¹² Only one small prospective case series (N=11) reported on IQ and nutritional outcomes following one year of 5 mg/kg/day BH4 treatment.¹¹⁸ After one year of treatment, 11 participants with mild to moderate PKU discontinued use of a medical food and normalized their diet. IQ scores after 12 months on BH4 were maintained, or developmental quotients were within normal limits. Treatment was not adversely associated with anthropometric or nutritional status indicators, and all participants had normal levels of micronutrients. In another case series with 16 participants,¹²⁰ treatment duration ranged from 24 to 110 months (mean=56 months), 14 individuals responded to BH4 treatment (blood Phe reduction of ≥30 percent in loading test). Among these responders, the mean blood Phe decrease was 54.6 percent, and 13 were able to maintain Phe control while increasing Phe intake or eliminating dietary restrictions. The study noted that psychomotor development was in the normal range among children between 5 and 6 years of age.

Clinical Trials

The first RCT¹¹³ evaluating the efficacy of BH4 was carried out in 16 centers in North America and 14 centers in Europe. Between 2005 and 2006, 89 participants with PKU were randomized to receive either 10 mg/kg of BH4 (N=42) or placebo (N=47) once daily for 6 weeks. Eligible participants were responsive to BH4 in a previous phase I screening study, had a blood Phe of 450 μmol/L or more, were 8 years of age and older, and had relaxed or abandoned a strict low phenylalanine diet. The primary outcome was the change in blood Phe from baseline to week 6. Participants' mean age was 21.5 years in the treatment group and 19.5 years in the placebo group. Adherence to treatment was high, with 82 percent of participants taking all doses correctly during the 6 week period.

After 6 weeks of treatment, participants in the BH4 group had a significant decrease in mean blood Phe levels of -235.9 ± 257 μmol/L from baseline (843 μmol/L) compared with a 2.9 ± 239.5 μmol/L increase in mean Phe levels from baseline (888 μmol/L) in the control group (p<0.0001). The mean blood Phe decreased in the BH4 group at 1 week and remained at that lower level until the 6 week end point, when the mean Phe level was 607 μmol/L. The estimated difference between treatment and placebo groups in the mean change in blood Phe at 6 weeks compared with baseline was -245 (p<0.0002). A significantly higher proportion of participants receiving BH4 (44 percent) had a 30 percent or greater reduction in blood Phe levels compared with controls (9 percent).

The proportion of individuals in the BH4 group who had blood Phe levels under 600 μmol/L increased significantly from 17 percent at baseline to 54 percent at 6 weeks compared with controls (baseline: 19 percent, week 6: 23 percent). Almost all participants (16 of 17) for whom genotyping was performed had at least one mutation known to be associated with residual enzymatic activity. Responsiveness was not consistently linked to specific mutations. Despite enrolling only those participants who had at least a 30 percent reduction in blood Phe while taking BH4 in a one week loading test, not all participants were responsive to BH4 in the trial.

A 22 week uncontrolled open label trial¹¹⁴ followed this RCT.¹¹³ This was conducted in three parts: the first period was a 6 week forced dose titration phase in which all participants received doses of 5, 20, and 10 mg/kg/day of BH4 consecutively for 2 weeks each. This phase was followed by a dose analysis phase in which all participants received 10 mg/kg/day for 4 weeks followed by a 12 week fixed dose phase in which participants received doses of 5, 10, or 20 mg/kg/day based on their plasma Phe concentrations during the dose titration at weeks 2 and 6. All participants enrolled in the previous RCT were eligible if they had taken at least 80 percent of their scheduled does in the trial and were willing to continue their current diet during the study. The primary endpoint was mean plasma Phe levels at week 22 and mean changes from week 0. Plasma Phe levels at weeks 2, 4, and 6 were used to estimate the effects of dose on plasma Phe levels.

Of 87 participants who completed the previous RCT,¹¹³ 80 were enrolled in the extension trial,¹¹⁴ of whom 39 had previously received BH4 and 41 placebo. Participants' mean age was 20.4 years. Overall, 60 percent reported taking all doses correctly, 18 percent reported missing at least one dose and no incorrect doses, 9 percent took at least one dose incorrectly but did not miss any doses, and 14 percent took at least one dose incorrectly and missed at least one dose.

During the dose titration phase, individuals receiving 10 or 20 mg/kg/day had significantly greater mean reductions in blood Phe at week 6 compared with week 0 than those receiving 5 mg/kg/day. Additionally, those receiving 20 mg/kg/day had significantly greater reductions from week 6 to week 22 compared with week 0 than those receiving 10 mg/kg/day. By the end of the dose analysis phase with 10 weeks at 10 mg/kg/day, 46 percent of participants had a decrease in plasma Phe of at least 30 percent compared with week 0. During the fixed dose phase, most participants (92 percent) received either 10 (46 percent) or 20 mg/kg/day (46 percent). By week 22, plasma Phe was reduced by 190.5 μmol/L compared with week 0. The mean Phe level at 22 weeks for those on 5, 10, and 20 mg/kg/day was 438 μmol/L, 450 μmol/L, and 896 μmol/L, respectively.

Mean plasma Phe decreased from 844 μmol/L at baseline to 645 μmol/L at week 10 and was maintained at a mean of 652 μmol/L at week 22. At week 22, 46 percent of participants had achieved a 30 percent reduction in plasma Phe concentration compared with week 0. The corresponding reductions for those receiving 5, 10, and 20 mg/kg/day were 50 percent, 49 percent, and 42 percent respectively.

Another RCT¹¹⁵ of fair quality was carried out in the United States, Germany, Spain, and Poland between 2005 and 2006 and enrolled children with PKU between 4 to 12 years of age who were on a Phe-restricted diet, had maintained blood Phe control (blood Phe level <480 μmol/L) and had an estimated Phe tolerance of ≤1000 mg/d. The objective was to determine the safety and efficacy of BH4 at 20 mg/kg/day for 10 weeks in increasing Phe tolerance while maintaining blood Phe control. Investigators randomized BH4 responders in a 3:1 ratio to receive either 20 mg/kg of BH4 or placebo once daily for 10 weeks. Participants maintained a stable, Phe-restricted diet, monitored by food diaries. Starting at the third week, a medical food was added or removed every 2 weeks based on Phe levels. Children with blood Phe level of ≥1200 μmol/L in 2 consecutive weeks were withdrawn from study drug treatment and received dietary counseling.

The primary endpoint was daily Phe tolerance at week 10 compared with week 0. Phe tolerance was defined as the cumulative increase or decrease in medical food at the last visit for which blood Phe level was ≤360 μmol/L. Secondary endpoints were the difference in blood Phe levels in the BH4 group between week 0 (before dosing) and week 3 (before Phe supplementation), and the comparison of Phe tolerance between treatment and placebo groups at week 10. Thirty-three children were randomized to 20 mg/kg/day of BH4 for 10 weeks, and 12 children received a placebo. Baseline characteristics, including blood Phe levels, were similar between groups.

After 10 weeks of treatment, the total mean ± SD of medical food tolerated by participants on BH4 increased significantly from 0 mg/kg/day at baseline to 20.9 ± 15.4 mg/kg/day. In contrast, the placebo group tolerated only an increase of 2.9 mg/kg/day of medical food. The adjusted mean difference between the groups in Phe tolerance was 17.7 ± 4.5 mg/kg/day(p<0.001). Total Phe intake (dietary Phe intake plus total medical food) also increased significantly from baseline in the BH4 group, approximately doubling to 43.8 mg/kg/day at 10 weeks. The placebo group had a slight increase in total Phe intake from 16.3 mg/kg/day at baseline to 23.5 ± 12.6 mg/kg/day at 10 weeks.

The BH4 group tolerated a range of medical food supplementation over the 10 weeks: 36 percent tolerated an increase of 10 mg/kg/day or less, 30 percent tolerated an 11 to 30 mg/kg/day increase and 33 percent tolerated an increase of 31 to 50 mg/kg/day. No one in the placebo group tolerated an increase of more than 10 mg/kg/day, and 58 percent could not tolerate any medical food supplement. Mean blood Phe levels decreased significantly in the BH4 group between baseline and the beginning of supplementation in week 3 (decrease of 148.5 ±_134.2 μmol/L). Some participants in the BH4 group had transient low blood Phe levels (<26 μmol/L) corrected with increased medical food supplementation.

More recently,¹¹⁶ an uncontrolled open label trial of good quality conducted at one U.S. clinic from 2008 to 2009, included participants with classic or variant PKU with any Phe level or diet. Eligible subjects received 7 days of open label BH4 at 10 mg/kg/day with plasma Phe measurement on day 8 and weekly during a dietary modification period. The study defined response as a 30 percent reduction in plasma blood Phe or reduction to treatment range of <360 μmol/L after day 7. Investigators increased the dosage to 20 mg/kg/day for nonresponders and rechecked Phe levels after 8 days. Individuals who were still nonresponders continued on 20 mg/kg/day until day 30. Responders who were on a Phe-restricted diet underwent gradual liberalization of their diet to the maximum tolerated natural protein intake while still maintaining plasma levels in the range of 120 to 360 μmol/L.

Of the 36 participants (mean age 23.4) who began treatment with BH4, 29 (74 percent) completed the study. Of these 29 individuals, 59 percent were on some form of protein restricted diets and had a mean baseline blood Phe of 587 μmol/L. Forty-one percent were not following protein restricted diets and had a mean baseline blood Phe level of 1372 μmol/L. Overall, 62 percent were determined to be responders, with variable doses required for response; 14 participants required a dose of 7 to15 mg/kg/day, and four participants required a dose of 15 to 20 mg/kg/day. Four (29 percent) of the classic PKU participants (defined as off diet plasma Phe of >1200 μmol/L) were responders, and 100 percent of the variant PKU participants (>400 and <1200 μmol/L) were responders.

Of the 12 participants who were not on a Phe-restricted diet, 33 percent were responders with a significantly decreased mean blood Phe of 554 μmol/L compared with baseline Phe level (1049 μmol/L). Of the 17 participants who were on a Phe-restricted diet, 82 percent were responders with significantly reduced mean blood Phe level of 226 μmol/L compared with baseline (485 μmol/L). Among individuals who were responders and on a Phe-restricted diet, the average Phe tolerance increased from 21 to 41 mg/kg/day. However, responders' Phe tolerance varied widely from an increase of 20 to 22 mg/kg/day to a non-protein restricted diet in two participants. Of the 11 who were nonresponders, three were on a Phe-restricted diet with a mean blood Phe level at the end of the trial of 978 μmol/L (baseline mean=1363 μmol/L). Eight of the 11 nonresponders were on an unrestricted diet with an end of trial mean blood Phe level of 1465 μmol/L (baseline mean=1524 μmol/L). Overall, nonresponders had an end trial blood Phe of 1333 and a baseline of 1422 μmol/L compared with responders who had significant decrease in blood Phe from a baseline of 1049 μmol/L to an end of trial level of 554 μmol/L.

Prospective Cohort Study and Case Series

One poor quality prospective cohort study¹²¹ assessed variability in blood Phe. Participants included nine children who were responsive to a 20 mg/kg BH4 loading test and 25 who were nonresponsive. Among those who were BH4 responsive, two were treated with BH4 alone and the rest also needed dietary modifications. From 2002 to 2010, there were 1384 blood samples available from BH4 responders and 4415 samples available from non-responders. Overall, there appeared to be no significant difference in mean and median blood Phe levels between the groups; however, above blood Phe levels of 600 μmol/L, confidence intervals around the mean were wider among BH4 nonresponsive participants. The authors equate these differences with variability in response.

Four poor-quality case series^117-120 evaluated dosages of BH4 ranging from 5 to 26 mg/kg/day for duration of 6 months to up to 9 years among BH4-responsive participants. All reported positive outcomes in terms of reduction in blood Phe and increased Phe tolerance. As reported above, one case series¹¹⁸ also examined longer term functional outcomes, including IQ and developmental quotient after one year of treatment, reporting no adverse effects as participants' Phe tolerance increased and the diet was liberalized. Nutritional status was unchanged with the exception of increases in selenium. In another case series,¹¹⁷ 12 participants were studied for up to 7 years on a dosage of 10 mg/kg twice a day. In this group, ranging in age from 2 to 16 years old, all participants eventually stopped medical food supplementation and relaxed dietary restrictions.

Another longer-term case series¹²⁰ assessed Phe levels and increase in Phe tolerance (presented as the number of times Phe intake increased from baseline level for those on dietary restriction) in 16 individuals receiving BH4 for between 24 months to 9 years (mean=56 months). Of the 16 patients, 15 (94 percent) patients were initial responders. The mean blood Phe level in responders was 321 ± 236 μmol/L, and the mean decrease in blood Phe was 54.6 percent (range 28.4 to 85.6 percent). Two patients, ages 10 and 13 years at the start of treatment, were non-responders and had high fluctuations in blood Phe levels. Seven patients had stable Phe control (defined as that recommended by the 2000 NIH consensus development panel), without any dietary restriction. Of the remaining seven patients who were on dietary restrictions, six increased their Phe intake from a baseline of 200 to 300 mg/day to 800 to 1000 mg/day. Psychomotor development, (measured using the Hamburg Wechsler Intelligence test-HAWIK III) among children 5 to 6 years of age was reported to be within normal range; however, results were not presented. Finally, one case series¹¹⁹ provided data on Phe variability by measuring blood Phe at least six times before and after treatment initiation. Individual variability in Phe levels was lessened after treatment.

Summary of Effects

One RCT¹²⁴enrolling 16 participants reported on measures of cognition, including executive functioning. The study reported that LNAAs supplementation had a positive effect on executive functioning, specifically improving verbal generativity, cognitive flexibility, and self-monitoring. Despite improvements in some aspects of executive functioning with LNAAs supplementation, studies reported considerable individual variation. In all three studies, blood Phe decreased after one week of treatment, but remained above clinically acceptable levels. The one trial that measured correlation between blood and brain Phe found no association.¹²⁴ Overall, participants who were using a Phe-free formula did not experience a decrease in blood Phe, although those not adhering to diet or not using Phe-free formula did. This finding suggests that LNAAs may be helpful in lowering blood Phe in participants unable to adhere to medical treatment, but current research suggests a lack of clinical impact.^{16, 124, 125}Table 14 summarizes key outcomes of comparative studies.

Table 14

Comparative studies of LNAAs for the treatment of PKU.

Clinical Trials

One fair quality, double blind, randomized, crossover study¹²⁴ was carried out in one center in Australia. Sixteen participants with early treated, classic PKU (plasma Phe >1000 μmol/L) were enrolled. The objective was to evaluate the relationship between LNAAs supplementation and cognitive and affective outcomes under four different therapeutic combinations with PKU amino acid products. Participants followed their usual Phe-restricted diet and PKU Phe-free medical food. All subjects completed four phases, each lasting 14 days with a 4 week washout period between phases. Phase 1 consisted of taking their usual medical food, usual Phe-restricted diet and LNAAs at 250 mg/kg/day in three equal daily doses.

During Phase 2, participants maintained their usual medical food, usual Phe-restricted diet and placebo. In Phase 3, participants did not take their usual medical food, maintained their usual Phe-restricted diet and received LNAAs. In Phase 4, participants did not take their usual medical food, maintained their usual Phe-restricted diet and placebo. For the phases without medical food, advice was provided on energy supplements needed to replace energy intake usually obtained from a medical food. Blood Phe levels from the previous year were used to determine baseline Phe. Of the 16 participants, nine were determined to have good control (median Phe level 450 to 750 μmol/L), six participants had marginal control (median Phe level 750 to 1000 μmol/L), and two participants had poor control (median Phe level >1000 μmol/L).

Dietary analysis demonstrated that both total protein and LNAAs intake were highest in phase 1 followed by phase 2, phase 3, and lowest in phase 4, consistent with the phased study. There was no significant difference in brain Phe between the 4 phases (range 176 to 365 μmol/L). Brain Phe levels were determined by magnetic resonance spectroscopy. No participant was determined to have excellent control (median blood Phe level<450 μmol/L), and no difference in Phe reduction was observed with or without LNAAs as long as participants were on the Phe-free medical food. However, in the absence of medical food, the LNAAs arm was associated with greater reductions in Phe than placebo. However, plasma Phe levels remained high for all participants, including those taking the LNAAs formulation (958 for those not on Phe-free formula).

The second RCT¹²⁵ was a crossover trial of poor quality that was carried out in 6 centers located in the United States, Italy, Denmark, Ukraine, Russia, and Brazil. Blood Phe levels dropped significantly on LNAAs from a mean level of 932.9 μmol/L at baseline to 568.4 μmol/L at one week, an average decline of 365.5 ±_233.2 (39 percent). Seven participants who adhered to PKU formula had a significant reduction in blood Phe from a baseline mean of 531.6 μmol/L to 281.5 μmol/L at one week, an average decline of 250.1 ± 173.7 (47 percent). The average decline of Phe on placebo from a baseline mean of 932.9 μmol/L to 882.66 μmol/L at one week (5.4 percent) was not significant.

Uncontrolled Open-Label Trial

The third trial¹⁶ was an uncontrolled open-label trial of poor quality that included 11 participants. Participants were not on a Phe-restricted diet and Phe intake was over 500 mg/day. Of 11 participants enrolled, eight received 0.5 mg/kg/day of LNAAs and three received 1.0 g/kg/day. Blood Phe levels decreased significantly from baseline after one week of LNAAs, an average decline of 601 μmol/L ± 370. In the eight participants taking 0.5 g/kg/day, Phe levels decreased significantly from the baseline mean of 957.4 μmol/L to 458.4 μmol/L, a decline of 52 percent. Among the three participants who took 1 g/kg/day of LNAAs, the mean blood Phe level decreased from the baseline level of 1230 μmol/L to 549.0 μmol/L, a decline of 55 percent.