Biopterin derivatives in normal and phenylketonuric patients after oral loads of L-phenylalanine,L-tyrosine,and L-tryptophan.

Plasma biopterin derivatives studied in 10 normal and 21 phenylketonuric children showed a significantly high concentration in the latter group. Biopterin derivatives correlated with plasma phenylalanine concentration, but in normal adults given an oral phenylalanine load the rate of increase with phenylalanine differed from that in phenylketonuric patients. A patient with hyperphenylalaninaemia, not due to phenylketonuria, had an abnormal biopterin derivatives response to phenylalanine distinct from that of patients with classical phenylketonuria. This may be a useful investigation to differentiate some variants of phenylketonuria.     

Reduced Na(+), K(+)-ATPase activity in erythrocyte membranes from patients with phenylketonuria.

Na(+), K(+)-ATPase activity was determined in erythrocyte membranes from 12 phenylketonuric patients of both sexes, aged 8.8 +/- 5.0 y, with plasma phenylalanine levels of 0.64 +/- 0.31 mM. The in vitro effects of phenylalanine and alanine on the enzyme activity in erythrocyte membranes from healthy individuals were also investigated. We observed that Na(+), K(+)-ATPase activity was decreased by 31% in erythrocytes from phenylketonuric patients compared with normal age-matched individuals (p < 0.01). We also observed a significant negative correlation between erythrocyte Na(+), K(+)-ATPase activity and plasma phenylalanine levels (r = -0.65; p < 0.05). All PKU patients with plasma phenylalanine levels higher than 0.3 mM had erythrocyte Na(+), K(+)-ATPase activity below the normal range. Phenylalanine inhibited in vitro erythrocyte Na(+), K(+)-ATPase activity by 22 to 34%, whereas alanine had no effect on this activity. However, when combined with phenylalanine, alanine prevented Na(+) K(+)-ATPase inhibition. Considering that reduction of Na(+), K(+)-ATPase activity occurs in various neurodegenerative disorders leading to neuronal loss, our previous observations showing a significant reduction of Na(+), K(+)-ATPase activity in brain cortex of rats subjected to experimental phenylketonuria and the present results, it is proposed that determination of Na(+), K(+)-ATPase activity in erythrocytes may be a useful peripheral marker for the neurotoxic effect of phenylalanine in phenylketonuria.     

Determination of phenylalanine hydroxylase activity in patients with phenylketonuria and hyperphenylalaninemia.

The phenylalanine hydroxylase assay was modified by using biopterin, lysolecithin, and dithioerythritol. Liver tissue was obtained by percutaneous needle biopsies in patients with phenylketonuria (PKU) and hyperphenylalaninemia. The use of the naturally occurring cofactor biopterin is essential to measure low enzyme activities. Thirteen of 14 assay specimens in which no activity was detectable correlated with the clinical picture of classic PKU. Twelve assay specimens showed a residual activity up to 6% of normal. This group comprises patients with classic PKU and with so-called hyperphenylalaninemia. Four specimens ranged between 8.7 and 34.5% of the normal values. Patients in this group have developed normally so far without dietary treatment. It seems that patients with residual activity tolerate more phenylalanine in the diet than patients with no detectable activity. One infant with biochemical symptoms of classic PKU was found to have a normal phenylalanine hydroxylase activity.     

Neurological aspects of biopterin metabolism.

Plasma total biopterin concentration was measured by bioassay in 59 infants with hyperphenylalaninaemia and in 50 children with developmental regression and or movement disorder with normal plasma phenylalanine concentrations. In infants with raised phenylalanine concentrations plasma biopterin concentrations were significantly raised in proportion to the phenylalanine values. Five patients had plasma biopterin concentrations at the extremes of the range, and of these two had defective biopterin metabolism. One with low plasma biopterin concentration apparently had a partial defect of biopterin synthesis but died before investigations were complete. One with high plasma biopterin concentration, even when phenylalanine concentrations had fallen to the normal range, had dihydropteridine reductase deficiency. In this patient concentrations of homovanillic acid and 5-hydroxyindolacetic acid in the cerebrospinal fluid (CSF) were severely reduced. In children without hyperphenylalaninaemia plasma biopterin concentrations were normal. Twenty two patients were subjected to lumbar puncture, of whom six with developmental regression without movement disorder had normal CSF biopterin concentrations, and 11 with movement disorder other than torsion dystonia had significantly lower CSF biopterin concentrations. Five patients with torsion dystonia had normal biopterin concentrations.     

URINARY PHENYLALANINE EXCRETION IN HYPERPHENYL-ALANINEMIC CHILDREN

24-hour phenylalanine loading tests were done in 5 children with persistent hyperphenyl-alaninemia off diet. The urinary excretion of phenylalanine were compared to the excretion after loading of phenylketonuric children on diet. Serum phenylalanine of children with persistent hyperphenylalaninemia returned to preloading levels (285 μmol/l) within 24 hours. These children, however, excreted phenylalanine during phenylalanine loading in lesser quantities than patients with phenylketonuria on diet. Serum phenylalanine of phenylketonuric children on diet attained preloading levels (300 μmol/l) approximately 14 days after loading. Thus, the present data do not support the hypothesis that hyperphenylalaninemic patients are phenylketonurics protected by increased urinary excretion of phenylalanine.     

Nutrition, physical growth, and bone density in treated phenylketonuria

Dietary treatment of phenylketonuria is well established to be safe and to prevent developmental and mental impairment in patients with low or absent phenylalanine hydroxylase activity. The use of semi-synthetic diets necessitates careful and longitudinal control not only of physical and intellectual development, which are both near normal in well treated patients, but also of potential diet inherent insufficiencies of essential nutrients. Concern has been raised by some reports on growth retardation in young patients on strict diets and on decreased bone density in older phenylketonuric children. The clinical significance of these findings is not known. Conclusion Changes have been found, although inconsistently, in connection with selenium, zinc, iron, retinol and polyunsaturated fatty acid status in dietetically treated patients with phenylketonuria. Both the mechanism and significance of these changes is doubtful at present.     

Repeated adverse fetal outcome in pregnancy complicated by uncontrolled maternal phenylketonuria

Phenylketonuria in pregnancy carries with it an increased risk of spontaneous abortion and development of a fetus that is affected by the maternal phenylketonuria syndrome. This syndrome is characterized by low birthweight, congenital heart disease, microcephaly, childhood growth failure, and cognitive impairment. It is the result of the hyperphenylalaninemia that accompanies the phenylketonuric state, and may therefore be avoided by maintaining maternal serum phenylalanine levels within the normal range. Phenylalanine is an essential amino acid and may be controlled by dietary manipulation. Presented here is a case history of a woman with phenylketonuria who was unable to satisfactorily control her serum phenylalanine levels in each of her three pregnancies. All three children were adversely affected by the fetopathy of the maternal phenylketonuria syndrome, each with evidence of growth failure and impaired neurodevelopment. This patient illustrates the difficulties that may be encountered when providing obstetric care to the woman with phenylketonuria who is not able or not willing to restrict her dietary intake of phenylalanine. The discussion includes consideration of management strategies, including dietary therapy and legal intervention.     

Phenylalanine-fetopathia in twins of an undiagnosed phenylketonuric mother (author's transl)

A case report of microcephalic twins born to a hithertoo undedected phenylketonuric mother is given. The female twin suffered from phenylketonuria, too. The clinical findings included microcephaly, growth retardation, retarded bone age and an unusual facies. The psychomotor development was retarded in both twins, but more so in the phenylketonuric twin despite appropriate low phenylalanine diet.     

Early breastfeeding is linked to higher intelligence quotient scores in dietary treated phenylketonuric children

Strict control of phenylalanine intake is the main dietary intervention for phenylketonuric children. Whether other dietary-related factors improve the clinical outcome for treated phenylketonuric children in neurodevelopmental terms, however, remains unexplored. We retrospectively compared the intelligence quotient (IQ) score of 26 school-age phenylketonuric children who were either breastfed or formula fed for 20-40 days prior to dietary intervention. Children who had been breastfed as infants scored significantly better (IQ advantage of 14.0 points, p = 0.01) than children who had been formula fed. A 12.9 point advantage persisted also after adjusting for social and maternal education status ( p = 0.02). In this sample of early treated term infants with phenylketonuria there was no association between 1Q scores and the age at treatment onset and plasma phenylalanine levels during treatment. We conclude that breastfeeding in the prediagnostic stage may help treated infants and children with phenylketonuria to improve neurodevelopmental performance.     

Malignant hyperphenylalaninemia--clinical features, biochemical findings, and experience with administration of biopterins

Four cases of malignant hyperphenylalaninemia (MHPA) are described. Pretreatment serum phenylalanine levels were 1.5, 3.0, 2.4, and 0.9 mmoles/l. Dihydropteridine reductase (DHPR) deficiency was proven in one patient by assays on cultured fibroblastic cells and was presumed in her sibling and in another deceased patient whose parents' fibroblastic cells show approximately 50% of normal enzyme activity. DHPR and phenylalanine hydroxylase deficiency were excluded by assays on liver obtained at autopsy in the 4th patient. Parenteral administration of tetrahydrobiopterin (BH4) corrected the hyperphenylalaninemia and increased the levels of catecholamines and 5-hydroxy-indoles in the one patient studied in life, but BH4 did not reach the cerebrospinal fluid. A 3-wk course of BH4 therapy had no clinical effect. Oral biopterin was absorbed and excreted in the urine, but did not alter the serum phenylalanine level. The frequency of MHPA in Australia was estimated as 7 in 258 patients with phenylketonuria.     

Hyperphenylalaninemia due to impaired dihydrobiopterin biosynthesis

A fourteen month-old boy with atypical phenylketonuria was treated with 5-hydroxytryptophan, L-dopa and peripheral aromatic amino acid decarboxylase inhibitor (Ro 4-4602:benserazide). Despite the good control of plasma phenylalanine on a low phenylalanine diet, he had shown no improvement in his development but progressive neurological symptoms, such asiirritability, convulsions and decrease voluntary movement. After beginning neurotransmitter therapy, his irritability disappeared promptly and the other symptoms diminished. He gradually reached his developmental milestones. At two and a half years of age, he had recovered sufficiently to be able to walk freely on treatment with 13 mg/kg/day of 5-hydroxytryptophan, 11 mg/kg/day of L-dopa and 2.7 mg/kg/day of benserazide in combination with slight restriction of phenylalanine intake (100 mg/kg/day).Levels of serotonin and 5-hydroxyindoleacetic acid were low in the patient's CSF. His urinary biopterin (Crithidia factor) excretion was low. An increase in serum biopterin following L-phenylalanine loading was not found. Dihydropteridine reductase activity in his skin fibroblasts was normal. He excreated large amounts of erythro- and threo-neopterins (but only a trace of biopterin) in his urine. After loading with phenylalanine the urinary excretion of neopterins was even more enhanced, but biopterin remained at low levels. These findings indicated that the patient has a dihydrobiopterin synthetase deficiency.     

Biopterin synthesis defects: problems in diagnosis

Hyperphenylalaninemia due to a biopterin synthesis defect was detected in an infant with decreased biopterin and increased neopterin levels in plasma and urine. Tetrahydrobiopterin (BH4) administration normalized plasma phenylalanine levels. CSF biopterin and neurotransmitter metabolite levels were normal and with the infant's normal growth and development suggest that the defect in biopterin synthesis did not affect CNS biopterin metabolism. Comparison of plasma and urine pterin levels from this patient with levels reported in patients who have neurologic complications fails to reveal differences that would distinguish patients at risk for neurologic problems. CSF pterin and neurotransmitter levels may correlate with neurologic function in these patients. CSF pterin and neurotransmitter determinations should be performed prior to initiation of neurotransmitter precursor and BH4 replacement therapies in patients who were determined to have biopterin synthesis defect(s).     

Atypical cases of phenylketonuria

Conclusion Neonatal HPA can be caused by deficiency of PAH or of its cofactor. At present, conventional methods are not able to delineate the molecular basis of the mutations in PKU patients. DNA analysis might in future visualize the different genotypes, but might not solve the problem of therapeutic decision.All infants with HPA should be screened for THB deficiency. Diagnostic tools are now available for the recognition of these variants among hyperphenylalaninemic infants. The most important question-which infants can achieve normal development if treated early-remains tobe answered. Efforts have to be directed toward better characterization of individual residual capacity to synthesize THB and toward and definition of protocols for the follow-up of neurotransmitter replacement therapy.Abbreviations PKU phenylketonuria - HPA hyperphenylalaninemia - PAH phenylalanine hydroxylase - DHPR dihydropteridine reductase - BS biopterin synthetase - THB tetrahydrobiopterin - GTP guanosine triphosphate - GTP-ch GTP-cyclohydrolase - N/B ratio neopterin/biopterin ratio     

Effects of totally synthetic,low phenylalanine diet on adolescent phenylketonuric patients

The long-term responses of 5 adolescent phenylketonuric patients to chemically-defined, synthetic diets with normal and low phenylalanine content were determined.The synthetic preparations were found capable of sustaining good health and rapid growth in this group of profoundly retarded, behaviourally disturbed patients over a 3½-year period without clinical or biochemical evidence of nutritional inadequacy. 4 of these patients who were treated for 6 months on a comparable diet, in which 80% of the phenylalanine was replaced by tyrosine, continued to show weight maintenance and height increases. There was no evidence of poor acceptability of the imbalanced diet, whether the blood phenylalanine concentrations were at phenylketonuric or treatment levels. The phenylalanine intake required to maintain blood phenylalanine concentrations of 3-5 mg/100 ml in these 4 patients was well below normal requirements, and ranged between 6·8 and 20·1 mg/kg per day. Predictably, the phenylalanine requirement varied with individual growth rates.All 4 treated patients had objective signs of improved central nervous system function during the six-month period on the phenylalanine-restricted diet. These electrophysiological and behavioural improvements were manifest after blood phenylalanine concentrations fell below 12 mg/100 ml in 3 cases and below 5 mg/100 ml in the fourth.     

The selenium status of children with phenylketonuria: results of selenium supplementation

The selenium status of children with phenylketonuria on a synthetic low phenylalanine diet was assessed. Correlation between blood selenium and red cell glutathione peroxidase was unsatisfactory (r = 0.65) due to the poor discrimination of red cell glutathione peroxidase with a low selenium diet. No symptoms of deficiency were observed. Supplementation with 50 micrograms per week of selenium as brewers yeast tablets over a period of 6 months significantly increased the blood selenium of the phenylketonuric children. Plasma Vitamin E levels were within normal limits. The supplementation effectively doubled their selenium intake to 15-17 micrograms per day, which is probably sufficient for this group with an adequate Vitamin E status, though considerably lower than the recommended minimum intake of 50 micrograms per day.     

Free amino acids in the saliva of children with phenylketonuria

Free amino acids were determined quantitatively in saliva of 23 children with phenylketonuria (male = 11, female = 12) (ages 6-17 years). Saliva was deproteinised by adding an equal volume of 5% sulphosalicylic acid and the amino acids were separated by ion exchange column chromatography. Results: The comparison of the amino acid concentrations in saliva of phenylketonuric children with healthy children (male = 34, female = 31) (ages 6-13 years) showed, that vast majority of amino acids - taurine, serine, glutamine, glycine, alanine, citrulline, a-aminobutyric acid, valine, isoleucine, leucine, tyrosine, phenylalanine, ornithine, lysine, delta-aminovaleric acid - were excreted significantly lower in saliva of phenylketonuric children.     

Differential effects of phenylalanine on Rac1, Cdc42, and RhoA expression and activity in cultured cortical neurons

Phenylketonuria (PKU) is characterized by a high concentration of phenylalanine, which can lead to mental retardation. One of the characteristic pathologic changes in untreated phenylketonuria patients is a reduction in the number of axons, dendrites, and synapses in the brain. This is thought to be due to the toxic effects of phenylalanine and/or its metabolites, however, the underlying mechanism remains unclear. In this study, we observed that phenylalanine reduced the number of dendrites and dendritic spines in cultured neurons. We further demonstrated that phenylalanine down-regulated Rac1, Cdc42, and RhoA mRNA and protein expression. Pull-down assays indicated that phenylalanine caused a decrease in Rac1/Cdc42 activity but increased RhoA activity. Expression of a dominant negative RhoA or treatment with a Rho-associated kinase specific inhibitor, Y-27632, partly inhibited the phenylalanine-induced decrease in dendrite numbers. In conclusion, we have demonstrated that phenylalanine affects the expression and activity of Rac1, Cdc42, and RhoA. Furthermore, RhoA signaling is involved in the inhibitory effect of phenylalanine on dendritic branching. These results may provide an important insight into the molecular mechanism underlying phenylalanine-induced abnormalities of dendrites, specifically in phenylketonuria neuronal injury.     

PHENYLKETONURIA: REDUCTION OF SERUM LEVELS OF PHENYLALANINE FOLLOWING ORAL ADMINISTRATION OF B-2 THIENYLALANINE

8 mentally retarded but otherwise healthy phenylketonuric individuals were given two oral loads of phenylalanine (100 mgm/Kg). One of these loads was accompanied by β-2-thienylalanine (20 mgm/Kg). Blood levels of phenylalanine in the 6 hours after the two phenylalanine loads were compared. β-2-thienylalanine reduced the levels of phenylalanine in every case. This suggests that if the phenylketonuric child were to take an appropriate amount of β-thienylalanine with meals, he might be able to eat more phenylalanine, and this would allow a considerably more liberal diet. As diet has been shown to be a dominant factor in the emotional climate of families of phenylketonuric children, this stress would be considerably reduced. Thus, the administration of β-thienylalanine might lead to a more normal social development of the patient.     

Correlation between plasma and urine phenylalanine concentrations

In this pilot study, we show that plasma phenylalanine concentration can be predicted from urine concentration if the age of the patient is taken into consideration. This observation could open the way to a new monitoring of phenylketonuric patients in which painful frequent blood sampling, mandatory to adapt the low phenylalanine diet, could be mostly replaced by urinalysis. Compliance to treatment would be improved and hence also the ultimate mental development. Since this study was based on a small number of patients, validation of the model in a large multicentric survey is needed before it can be recommended.     

Maternal phenylketonuria: successful outcome in four pregnancies treated prior to conception

The management of four pregnancies in two phenylketonuric women is described. A successful outcome in these pregnancies is ascribed to the initiation of treatment prior to conception and the maintenance of tight control with serum phenylalanine between 100 and 400 mol/l throughout the gestation period. A trial of diet is desirable before a decision is made about pregnancy and before contraception is ceased. Close contact must be maintained with female phenylketonurics throughout their reproductive life to ensure that this process is followed.Abbreviations PKU phenylketonuria - Phe phenylalanine - Tyr tyrosine