Comparative diagnostic value of phenyla-lanine challenge and phenylalanine hydroxylase activity in phenylketonuria

Serum phenylalanine (phe) concentrations during and following phe challenges and liver phenylalanine hydroxylase (PH) activity were compared in 13 phenylketonuric (PKU) patients. These patients were separated into two groups: eight patients with no detectable PH activity (PH°) and five patients with residual PH activity (PH^-) ranging from 9 to 24% of the activity obtained in 10 non-PKU subjects. The rise in serum phe concentration during 3 days of oral loading did not differentiate the two groups. However, the difference in serum phe concentration of the PH° and PH^- groups reached statistical significance at 24 h postloading (p<0.01). We concluded that combined results from multiple measurements during the oral challenge, namely serum phe concentration after termination of loading, serum phe clearance rate, post-loading phe tolerance index and urinary metabolite excretion, make a better indicator for predicting residual PH activity for the majority of PKU subjects than peak phe concentrations during phe challenge.     

Differential effects of low-phenylalanine protein sources on brain neurotransmitters and behavior in C57Bl/6-Pahenu2 mice

Phenylketonuria (PKU) is an inborn error of metabolism caused by a deficiency of the enzyme phenylalanine hydroxylase, which metabolizes phenylalanine (phe) to tyrosine. A low-phe diet plus amino acid (AA) formula is necessary to prevent cognitive impairment; glycomacropeptide (GMP) contains minimal phe and provides a palatable alternative to the AA formula. Our objective was to assess neurotransmitter concentrations in the brain and the behavioral phenotype of PKU mice (Pah^enu2 on the C57Bl/6 background) and how this is affected by low-phe protein sources. Wild type (WT) and PKU mice, both male and female, were fed high-phe casein, low-phe AA, or low-phe GMP diets between 3 and 18 weeks of age. Behavioral phenotype was assessed using the open field and marble burying tests, and brain neurotransmitter concentrations were measured using HPLC with electrochemical detection system. Data were analyzed by 3-way ANOVA with genotype, sex, and diet as the main treatment effects. Brain mass and the concentrations of catecholamines and serotonin were reduced in PKU mice compared to WT mice; the low-phe AA and GMP diets improved these parameters in PKU mice. Relative brain mass was increased in female PKU mice fed the GMP diet compared to the AA diet. PKU mice exhibited hyperactivity and impaired vertical exploration compared to their WT littermates during the open field test. Regardless of genotype or diet, female mice demonstrated increased vertical activity time and increased total ambulatory and horizontal activity counts compared with male mice. PKU mice fed the high-phe casein diet buried significantly fewer marbles than WT control mice fed casein; this was normalized in PKU mice fed the low-phe AA and GMP diets. In summary, C57Bl/6-Pah^enu2 mice showed an impaired behavioral phenotype and reduced brain neurotransmitter concentrations that were improved by the low-phe AA or GMP diets. These data support lifelong adherence to a low-phe diet for PKU.     

Phenylketonuria: variable phenotypic outcomes of the R261Q mutation and maternal PKU in the offspring of a healthy homozygote.

Phenylketonuria (PKU) and benign hyperphenylalaninaemia (HPA) result from a variety of mutations in the gene for the hepatic enzyme phenylalanine hydroxylase. PKU has been found in the Israeli population in two variants, classical and atypical. The two are clinically indistinguishable and require treatment with low phenylalanine diet to prevent mental retardation, but show differences in serum phenylalanine levels and in tolerance to this amino acid. Maternal PKU is a syndrome of congenital anomalies and mental retardation that appears in offspring of PKU mothers as a result of fetal exposure to the high phenylalanine level in the maternal blood. We studied a family in which two children with severe, classical PKU and their unaffected brother showed mild signs of maternal PKU. Their mother had no clinical signs of PKU, but the phenylalanine concentration in her serum reached a level that usually characterises PKU patients. This woman represents a rare phenotype, benign atypical PKU. Such 'hidden' PKU in women may lead to maternal PKU in the offspring, similar to overt PKU. Special attention should therefore be paid to women having children with any of the clinical hallmarks of maternal PKU, and to children born to women known to have benign HPA. The mother was also found to be homozygous for a missense mutation at the phenylalanine hydroxylase locus, R261Q, which does not abolish enzymatic activity completely. In two other families, homozygosity for this mutation resulted in atypical PKU in four children. This observation suggests that mutations that do not completely destroy phenylalanine hydroxylase activity may exhibit variable phenotypic expression which is unpredictable. Compound heterozygosity for R261Q and other mutations led in other patients either to classical PKU or to mild benign HPA.     

Experimental evidence that phenylalanine is strongly associated to oxidative stress in adolescents and adults with phenylketonuria

Few studies have looked at optimal or acceptable serum phenylalanine levels in later life in patients with phenylketonuria (PKU). This study examined the oxidative stress status of adolescents and adults with PKU. Forty PKU patients aged over fifteen years were enrolled, and were compared with thirty age-matched controls. Oxidative stress markers, anti-oxidant enzyme activities in erythrocytes, and blood anti-oxidant levels were examined. Nitric oxide (NO) production was also examined as a measure of oxidative stress. Plasma thiobarbituric acid reactive species and serum malondialdehyde-modified LDL levels were significantly higher in PKU patients than control subjects, and correlated significantly with serum phenylalanine level (P<0.01). Plasma total anti-oxidant reactivity levels were significantly lower in the patient group, and correlated negatively with phenylalanine level (P<0.001). Erythrocyte superoxide dismutase and catalase activities were higher and correlated significantly with phenylalanine level (P<0.01). Glutathione peroxidase activity was lower and correlated negatively with phenylalanine level (P<0.001). The oxidative stress score calculated from these six parameters was significantly higher in patients with serum phenylalanine of 700-800 μmol/l. Plasma anti-oxidant substances, beta-carotene, and coenzyme Q(10) were also lower (P<0.001), although the decreases did not correlate significantly with the phenylalanine level. Serum nitrite/nitrate levels, as stable NO products, were higher together with low serum asymmetric dimethylarginine, as an endogenous NO inhibitor. Oxidative stress status is closely linked with serum phenylalanine levels. Phenylalanine level in should be maintained PKU below 700-800 μmol/l even in adult patients.     

初诊苯丙酮尿症患者微量元素测定

目的探讨初诊PKU患者血清内微量元素铁、铜、锌的变化及与高苯丙氨酸的关系。方法采用分光光度法测定27例PKU患儿及42例正常儿童血清内的铁、铜、锌含量，用HPLC法测定血清内的苯丙氨酸浓度并对两者进行分析比较。结果PKU患儿血清铁、锌明显低于正常时照，血清铜无显著性差异。血清锌的水平与患者体内苯丙氨酸浓度有关。结论PKU患儿体内存在微量元素的缺乏，有必要在治疗时适当的补充微量元素以达到更好的治疗效果。     

Influence of mode and duration of phenylalanine administration on biochemical parameters in rats of various ages

Blood and tissue levels of phenylalanine and tyrosine progressively increase during he first two weeks of feeding a phenylalanine-rich diet to weanling rats. During the same time, liver phenylalanine hydroxylase activity and brain serotonin progressively decline. Hepatic tryptophan hydroxylase activity drops quickly to a constant, low level. With the exception of tryptophan hydroxylase, all of these parameters return towards normal upon continuous treatment. The magnitude of change varies with the age of the animal at initiation of dietary treatment and arises because of Differences in effective dosage and metabolism as the animals mature. A single phenylalanine injection produces a transient elevation in tissue phenylalanine levels without appreciably affecting brain serotonin. Repeated phenylalanine injections produce more marked alterations in tissue phenylalanine and tyrosine levels, lower activity of tryptophan hydroxylase, and increase serum adrenocorticoids; phenylalanine hydroxylase activity and brain serotonin levels are only marginally affected. These changes seriously complicate interpretations of time-consuming behavioral studies in experimental PKU.     

Plasma biopterin levels and tetrahydrobiopterin responsiveness

Tetrahydrobiopterin (BH4) responsive hyperphenylalaninemia (HPA) with a mutant phenylalanine hydroxylase (PAH) gene was found during neonatal screening for PKU. This study determined blood BH4 and phenylalanine in two patients with hyperphenylalaninemia following oral load with BH4 10 mg/kg. Our patients underwent neonatal screening for PKU, had normal biopterin metabolism and their PAH mutations were determined. Peak plasma biopterin levels in Case 1, which were reached at between 2 and 4h after loading, were 612, 297, and 178 nmol/L at age 30 days, 55 days, and 19 months, respectively, and the maximum phenylalanine decreasing rates, which were found at 24h, were 54, 16, and 4%, respectively. In Case 2, peak plasma biopterin levels were 747 and 327 nmol/L at age 20 and 55 days, respectively, and the maximum phenylalanine decreasing rates were 39 and 32%, respectively. In the BH4 loading test, the peaks of BH4 in both patients lowered ( approximately 50%), on the same dose schedule of BH4, as patients got older.     

Behavioral deficit in phenylketonuric rats: Role of aromatic acid metabolites of phenylalanine

To investigate the relationship between the biochemical and behavioral deficits in phenylketonuria (PKU), we treated rats from Postnatal Days 2 through 21 with p-chlorophenylalanine plus L-phenylalanine to simulate PKU or with one of the following aromatic acid metabolites of phenylalanine: phenylacetate, phenylpyruvate, phenyllactate, and mandelate. Behavioral tests were begun at about 9 weeks of age. Differences between experimental and control animals were found only in the water maze and in the open field. In the former, PKU rats required significantly more trials to reach criterion than controls. None of the single metabolite-treated groups exhibited a similar learning deficit. In the open field, PKU and mandelatetreated rats were hypoactive compared with controls, whereas phenylacetate- and phenylpyruvate-treated rats were hyperactive. These results demonstrate a lack of congruence between morphological and behavioral effects of treatment, suggesting that performance deficits in PKU rats may be due to interactive effects of 2 or more metabolites.     

Mutation analysis of the phenylalanine hydroxylase gene and its clinical implications in two Japanese patients with non-phenylketonuria hyperphenylalaninemia

We describe a mutation analysis for the phenylalanine hydroxylase gene and the clinical outcome of two Japanese patients with non-phenylketonuria (PKU) hyperphenylalaninemia (serum phenylalanine level below 600 μmol/l under a free diet) detected by a mass-screening program. Single strand conformation polynorphism analysis and direct sequencing of their genomic DNAs revealed that non-PKU hyperphenylalaninemia resulted from compound heterozygosity for a mutation causing classical PKU and a mutation with a milder effect on phenylalanine hydroxylase activity. The mutations were R241C and R243Q in exon 7, and R413P in exon 12. The mutation genotypes of the two patients were R241C/R243Q and R241C/R413P. It has been demonstrated that homozygosity for the R243Q or R413P mutation is associated with a severe phenotype of PKU and low in vitro expression activity. In contrast, the R241C mutation has much less effect on phenylalanine hydroxylase activity. The metabolic consequence of each variant allele was confirmed by a phenylalanine loading test in the patients and their parents. The patients achieved normal results in all intellectual and neurologic tests. Brain magnetic resonance imaging revealed no abnormalities. The dietary restriction was continued until 10 years of age in order to maintain the serum phenylalanine level below 400 μmol/l. The genetic analysis to distinguish non-PKU hyperphenylalaninemia from classical PKU helps to determine the principles of dietary management in the early infantile period. Received: June 2, 1998 / Accepted: July 17, 1998     

Allelic phenotype values: a model for genotype-based phenotype prediction in phenylketonuria

PurposeThe nature of phenylalanine hydroxylase (PAH) variants determines residual enzyme activity, which modifies the clinical phenotype in phenylketonuria (PKU). We exploited the statistical power of a large genotype database to determine the relationship between genotype and phenotype in PKU.MethodsA total of 9336 PKU patients with 2589 different genotypes, carrying 588 variants, were investigated using an allelic phenotype value (APV) algorithm.ResultsWe identified 251 0-variants encoding inactive PAH, and assigned APVs (0 = classic PKU; 5 = mild PKU; 10 = mild hyperphenylalaninaemia) to 88 variants in PAH-functional hemizygous patients. The genotypic phenotype values (GPVs) were set equal to the higher-APV allele, which was assumed to be dominant over the lower-APV allele and to determine the metabolic phenotype. GPVs for 8872 patients resulted in cut-off ranges of 0.0–2.7 for classic PKU, 2.8–6.6 for mild PKU and 6.7–10.0 for mild hyperphenylalaninaemia. Genotype-based phenotype prediction was 99.2% for classic PKU, 46.2% for mild PKU and 89.5% for mild hyperphenylalaninaemia. The relationships between known pretreatment blood phenylalanine levels and GPVs (n = 4217), as well as tetrahydrobiopterin responsiveness and GPVs (n = 3488), were significant (both P < 0.001).ConclusionsAPV and GPV are powerful tools to investigate genotype–phenotype associations, and can be used for genetic counselling of PKU families.     

Impact of lipophilic antioxidants and level of antibodies against oxidized low-density lipoprotein in Polish children with phenylketonuria

The treatment of phenylketonuria (PKU) patients constitutes a phenylalanine (Phe) intake restriction in their diet, which is achieved by adding a special Phe-free amino acid mixture to the diet. It has been reported that this diet could have some micronutrient deficiency. Several authors have also reported an increased oxidative stress or impaired antioxidant status in human and experimental PKU. Our project assessed the concentrations of retinol, alpha-tocopherol, coenzyme Q10, and anti-oxidized low-density lipoprotein (ox-LDL) antibodies in PKU children's plasma. It was found that retinol concentration in PKU children remains within the norm despite a low intake. The lower plasma alpha-tocopherol concentration in PKU children compared with normal children was associated with the lower level of antibodies against ox-LDL. This raises the question whether higher than observed circulatory alpha-tocopherol is indeed beneficial to lower plasma ox-LDL levels. Further studies are needed to explain the genetic factor in PKU patients (e.g., CD36/FAT polymorphism gene). The open clinical question is whether daily supplementation of alpha-tocopherol changes the PKU patients' level of antibodies against ox-LDL.     

Treatment of phenylketonuria during pregnancy

A woman with apparently classic phenylketonuria (PKU) was treated from the sixth week of her pregnancy with a diet restricted in phenylalanine and supplemented with tyrosine. Serum phenylalanine levels were monitored weekly and documented good patient compliance. A female infant was born who was examined at age 8.5 months. Physical examination was notable for a heart murmur suggestive of patent ductus arteriosus. Developmental quotient was normal. It is important to continue to monitor the outcome of pregnancies in women with PKU whose diet is restricted in phenylalanine in an effort to better define risks and to optimize treatment.     

Physical growth of children treated for phenylketonuria.

The growth of 133 children participating in the Collaborative Study of Children Treated for Phenylketonuria (PKU) was compared to growth data from the National Center for Health Statistics (NCHS) to determine whether the growth patterns of the children with PKU were the same as those of unaffected children. Height and weight by age, and weight by height, were analysed for ages 2-10 years. Head circumference by age was analysed for ages 2-7 years. Median height by age of the PKU children was consistently near the 50th percentile of the NCHS growth curves for males and females. However, for both sexes, median weight by height and by age was between the 50th and 75th percentiles for children over 3 years old. Two-sample t-tests showed mean weight was significantly different (p less than 0.05) between the PKU and NCHS groups at most ages for both sexes. Median head circumference for the PKU children tended to be smaller than NCHS standards; however, the maximum difference at any age was less than 0.5 cm. Polynomial growth curves fitted to the PKU and NCHS growth data showed that, on average, the PKU males and females weighed more than their unaffected counterparts, while height and head circumference for both groups were very close. Including median serum phenylalanine (phe) level (mg/dl) in the growth curves suggested that the weight differences between the PKU and NCHS groups are related to degree of diet adherence. Higher phe levels in the PKU group were associated with higher weight levels, more so for girls (p less than 0.001) than for boys (p = 0.08). No relationship was found between phe level and height or head circumference. We conclude that growth in children treated for PKU differs from national standards for weight by age and weight by height, but not for height by age. We speculate that diet adherence may be an important factor in determining which children have a tendency to become overweight.     

Phenylalanine ammonia lyase, enzyme substitution therapy for phenylketonuria, where are we now?

Phenylketonuria (PKU) is an autosomal recessive genetic disorder in which mutations in the phenylalanine-4-hydroxylase (PAH) gene result in an inactive enzyme (PAH, EC 1.14.16.1). The effect is an inability to metabolize phenylalanine (Phe), translating into elevated levels of Phe in the bloodstream (hyperphenylalaninemia). If therapy is not implemented at birth, mental retardation can occur. PKU patients respond to treatment with a low-phenylalanine diet, but compliance with the diet is difficult, therefore the development of alternative treatments is desirable. Enzyme substitution therapy with a recombinant phenylalanine ammonia lyase (PAL) is currently being explored. This enzyme converts Phe to the harmless metabolites, trans-cinnamic acid and trace ammonia. Taken orally and when non-absorbable and protected, PAL lowers plasma Phe in mutant hyperphenylalaninemic mouse models. Subcutaneous administration of PAL results in more substantial lowering of plasma and significant reduction in brain Phe levels, however the metabolic effect is not sustained following repeated injections due to an immune response. We have chemically modified PAL by pegylation to produce a protected form of PAL that possesses better specific activity, prolonged half-life, and reduced immunogenicity in vivo. Subcutaneous administration of pegylated molecules to PKU mice has the desired metabolic response (prolonged reduction in blood Phe levels) with greatly attenuated immunogenicity.     

Tetrahydrobiopterin,its Mode of Action on Phenylalanine Hydroxylase,and Importance of Genotypes for Pharmacological Therapy of Phenylketonuria

In about 20%–30% of phenylketonuria (PKU) patients (all phenotypes of PAH deficiency), Phe levels may be controlled through phenylalanine hydroxylase cofactor tetrahydrobiopterin therapy. These patients can be diagnosed by an oral tetrahydrobiopterin challenge and are characterized by mutations coding for proteins with substantial residual PAH activity. They can be treated with a commercially available synthetic form of tetrahydrobiopterin, either as a monotherapy or as adjunct to the diet. This review article summarizes molecular and metabolic bases of PKU and the importance of the tetrahydrobiopterin loading test used for PKU patients. On the basis of in vitro residual PAH activity, more than 1,200 genotypes from patients challenged with tetrahydrobiopterin were categorized as predictive for tetrahydrobiopterin responsiveness or non‐responsiveness and correlated with the loading test, phenotype, and residual in vitro PAH activity. The coexpression of two distinct PAH mutant alleles revealed possible dominance effects (positive or negative) by one of the mutations on residual activity as result of interallelic complementation. The treatment of the transfected cells with tetrahydrobiopterin showed an increase in residual PAH activity with several mutations coexpressed.     

A simplified PKU gene carrier detection test using fasting blood

By fluorometric analysis of fasting phenylalanine and tyrosine plasma levels, we could discriminate classic gene PKU carriers from non-carriers with 99% confidence in 67 of 74 adults. Results on the remaining seven subjects were non-discriminating. However, we could not determine their carrier status by other accepted testing parameters either (such as phenylalanine dosing). In contrast to the latter, our method: (1) allows for 90% of the population a relatively accurate but more benign test for carriers of the classical PKU gene (requiring only fluorometry on a single fasting blood specimen); (2) identifies the remaining 10% of the population who require the more cumbersome-noxious testing by phenylalanine dosing.     

Sapropterin therapy increases stability of blood phenylalanine levels in patients with BH4-responsive phenylketonuria (PKU)

It has recently been demonstrated that variability in blood phenylalanine levels is inversely correlated with IQ and is a better predictor of IQ in early and continuously treated patients with phenylketonuria (PKU) than mean blood phenylalanine levels. This suggests that stability of blood phenylalanine should be a therapeutic goal in patients with PKU. The purpose of this study was to determine if treatment with sapropterin in patients with BH4-responsive PKU would increase the stability of blood phenylalanine levels. The records of all patients treated with sapropterin in the PKU Clinic at Children's Memorial Hospital in Chicago were examined retrospectively. Patients were included in the study if they were responsive to sapropterin during a 2- to 4-week challenge (reduction of blood phenylalanine level of at least 25% after 2 weeks of therapy or, in the case of patients with well-controlled blood phenylalanine at the time of testing, increased dietary phenylalanine tolerance by 4 weeks of treatment). A total of 37 subjects were eligible for inclusion (16 male; 21 female); the mean age was 12.6 years (range, 1.5–32.0). The total number of observations (phenylalanine levels) for all subjects was 1391 with a mean of 39 per subject (range, 13–96). Linear mixed modeling was utilized to estimate variances of the blood phenylalanine before (pre) and after (post) starting sapropterin. Likelihood ratio test was performed using SAS 9.1. Means and standard deviations for phenylalanine as estimated by the model were 6.67 mg/dl (4.20) and post 5.16 (3.78). The mean level post-sapropterin was significantly lower (p = .0002). The within-subject variances (mean and SD) of phenylalanine were: pre 6.897 (2.62) and post 4.799 (2.19). These two variances are significantly different with a p = .0017. We conclude that sapropterin therapy results in increased stability of blood phenylalanine levels. This effect is likely to improve cognitive outcome in BH4-responsive patients with PKU.     

Mental development of phenylketonuric children on or off diet after the age of six.

Eleven children with phenylketonuria (PKU) taken off diet after the age of six showed significant decreases in rate of mental development compared with 26 control children children of comparable IQ and with 17 PKU children of comparable IQ who remained on the diet. Changes in rate of mental development were significantly and inversely correlated with plasma phenylalanine levels in treated PKU.     

Optimizing the use of sapropterin (BH4) in the management of phenylketonuria

Phenylketonuria (PKU) is caused by mutations in the phenylalanine hydroxylase (PAH) gene, leading to deficient conversion of phenylalanine (Phe) to tyrosine and accumulation of toxic levels of Phe. A Phe-restricted diet is essential to reduce blood Phe levels and prevent long-term neurological impairment and other adverse sequelae. This diet is commenced within the first few weeks of life and current recommendations favor lifelong diet therapy. The observation of clinically significant reductions in blood Phe levels in a subset of patients with PKU following oral administration of 6R-tetrahydrobiopterin dihydrochloride (BH₄), a cofactor of PAH, raises the prospect of oral pharmacotherapy for PKU. An orally active formulation of BH₄ (sapropterin dihydrochloride; Kuvan^®) is now commercially available. Clinical studies suggest that treatment with sapropterin provides better Phe control and increases dietary Phe tolerance, allowing significant relaxation, or even discontinuation, of dietary Phe restriction. Firstly, patients who may respond to this treatment need to be identified. We propose an initial 48-h loading test, followed by a 1–4-week trial of sapropterin and subsequent adjustment of the sapropterin dosage and dietary Phe intake to optimize blood Phe control. Overall, sapropterin represents a major advance in the management of PKU.     

Response of patients with phenylketonuria in the US to tetrahydrobiopterin

Tetrahydrobiopterin (BH4) responsive forms of phenylketonuria (PKU) have been recognized since 1999. Subsequent studies have shown that patients with PKU, especially those with mild mutations, respond with lower blood phenylalanine (Phe) concentrations following oral administration of 6-R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4). To determine the incidence of BH4 responding PKU patients in the United States and characterize their phenylalanine hydroxylase (PAH) mutations, a study was undertaken at UTMB in Galveston and the Children's Hospital of Los Angeles on 38 patients with PKU. Patients were screened by a single oral dose of BH4, 10 mg/kg and blood Phe and tyrosine were determined at 0, 4, 8, and 24 h. Twenty-two individuals (58%) responded with marked decrease in blood Phe (>30%) at 24h. Some of the patients that responded favourably were clinically described as having Classical PKU. Blood tyrosine concentrations did not change significantly. Twenty subjects with PKU, responsive and non-responsive to BH4, were enrolled in a second study to evaluate blood Phe response to ascending single doses of BH4 with 10, 20, and 40 mg/kg and to evaluate multiple daily doses, for 7 days each, with 10 and 20 mg/kg BH4. The 7-day trial showed a sustained decrease in blood Phe in 14 of 20 patients taking 20 mg/kg BH4 (70%). Of these 14 patients, 10 (71%) responded with a significant decrease in blood Phe following 10 mg/kg BH4 daily. To understand the mechanism of response to BH4, the kinetics and stability of mutant PAH were studied. We found that mutant PAH responds with increase in the residual enzyme activity following BH4 administration. The increase in activity is multi-factorial caused by increased stability, chaperone effect, and correction of the mutant Km. These studies indicate that BH4 can be of help to patients with PKU, including some considered to have Classical PKU. The PKU population in US is heterogeneous and mutations can be varied so mutations need to be characterized and response to BH4 tested. It is more likely that mutations with residual activity should respond to BH4, therefore the clinical definition of "Classical PKU" should be reconciled with the residual activity of PAH mutations.