A. Niederwieser

GTP cyclohydrolase I deficiency, a new enzyme defect causing hyperphenylalaninemia with neopterin, biopterin, dopamine, and serotonin deficiencies and muscular hypotonia

A 4-year-old patient is described with hyperphenylalaninemia, severe retardation in development, severe muscular hypotonia of the trunk and hypertonia of the extremities, convulsions, and frequent episodes of hyperthermia without infections. Urinary excretion of neopterin, biopterin, pterin, isoxanthopterin, dopamine, and serotonin was very low, although the relative proportions of pterins were normal. In lumbar cerebrospinal fluid, homovanillic acid, 5-hydroxy-indoleacetic acid, neopterin and biopterin were low. Oral administration ofL-erythro tetrahydrobiopterin normalized the elevated serum phenylalanine within 4 h, serum tyrosine was increased briefly and serum alanine and glutamic acid for a longer time. Urinary dopamine and serotonin excretion were also increased. Administration of an equivalent dose ofd-erythro tetrahydroneopterin was ineffective and demonstrated that this compound is not a cofactor in vivo and cannot be transformed into an active cofactor.GTP cyclohydrolase I activity was not detectable in liver biopsies from the patient. The presence of an endogenous inhibitor in the patients liver was excluded. This is the first case of a new variant of hyperphenylalaninemia in which the formation of dihydroneopterin triphosphate and its pterin metabolites in liver is markedly diminished. Normal activities of xanthine oxidase and sulfite oxidase were apparent since uric acid levels were normal and no increase in hypoxanthine, xanthine, andS-sulfocysteine concentrations could be observed in urine. It is concluded that the molybdenum cofactor of these enzymes may not be derived from dihydroneopterin triphosphate in man. Also, since no gross abnormalities in the patients immune system could be found, it seems unlikely that dihydroneopterin triphosphate metabolites, such as neopterin, participate actively in immunological processes, as postulated by others. See Note added in proof.

Atypical phenylketonuria due to tetrahydrobiopterin deficiency. Diagnosis and treatment with tetrahydrobiopterin, dihydrobiopterin and sepiapterin.

Abstract The effect of administration of several pterins on serum phenylalanine concentration (Phe) and urinary pterin excretion was investigated in two patients with atypical phenylketonuria (PKU) due to L -erythro-7,8-dihydrobiopterin (BH2) deficiency, one patient with atypical PKU caused by dihydropteridine reductase (DHPR, EC 1.6.99.7) deficiency, 5 patients with classical PKU (defective phenylalanine-4-hydroxylase, EC 1.14.16.1) and two adult controls. A drastic and comparable decrease of serum Phe, lasting for about 48 h, was observed in the 2 patients with BH2 deficiency after oral administration of L -erythro-5,6,7,8-tetrahydrobiopterin (BH4), BH2 (both 8μmol/kg body weight) and L -sepiapterin (2.8 and 4.2 μmol/kg in the 2 patients, respectively). BH4 and partially even BH2 decreased serum Phe also in the patient with DHPR deficiency. BH4, intravenously, had no effect on serum Phe of the classical PKU patients and of the controls. d -erythro-5,6,7,8-Tetrahydroneopterin (NeH4) had no effect on serum Phe in one patient with BH2-deficiency. The patients with BH2 deficiency excreted large amounts of neopterin, some dihydroneopterin and dihydroxanthopterin (XH2), but no trace of biopterins, indicating a BH2 synthetase deficiency in both patients. The patient with DHPR deficiency excreted remarkable amounts of BH2 and XH2 and traces of biopterin and neopterin. After BH4 administration, excessive amounts of BH2 and XH2 were excreted. Untreated PKU patients excreted more pterins than did patients under PKU diet or normal controls; after BH4 administration, PKU patients and controls excreted large amounts of BH4, BH2, biopterin. The results demonstrate that BH4, BH2, L -sepiapterin and NeH4, in admixture with ascorbic acid (10 mg/kg body weight) can be absorbed easily upon oral administration. The PKU diet of patients with BH4-deficiency can be replaced by oral BH4 supplementation. The two patients with BH2 synthetase deficiency need about 4μmol BH4 kg−1 d−1 (1.25 mg BH4 · 2 HC1 kg-1 d−1), and the patient with DHPR deficiency needs about 8 μmol BH4 kg-1 d-1. Substitution of neurotransmitter precursors (dopa, 5-hydroxytryptophan and carbidopa) cannot be discontinued. Screening of all newborn hyperphenylalaninemics for BH4 deficiency is suggested by a single oral administration of BH4 · 2 HCl, 8 μmol kg−1, 1 h before a meal, and measurement of serum Phe before and 4 or 6 h after loading.

Atypical phenylketonuria with defective biopterin metabolism. Monotherapy with tetrahydrobiopterin or sepiapterin, screening und study of biosynthesis in man

Administration of a single dose of tetrahydrobiopterin dihydrochloride, 10–20 mg/kg orally, to a patient with dihydrobiopterin deficiency led to disappearance of clinical symptoms for 4 days, normalization of urinary phenylalanine and serotonin and decrease of elevated neopterin for 2–3 days. A dose-dependent stimulation of serotonin production was observed. A similar effect was note with even lower doses of L-sepiapterin. The patient is now under monotherapy with tetrahydrobiopterin·2 HCl, 2.5 mg/kg daily. Other patients with this disease may not respond as well.Results of screening for tetrahydrobiopterin deficiency in 228 cases with hyperphenylalaninemia, including 140 newborns, are reported.There is evidence that biopterin biosynthesis in human kidney and liver proceeds via a dioxo compound and L-sepiapterin.

Atypical phenylketonuria caused by 7,8-dihydrobiopterin synthetase deficiency

A patient with atypical phenylketonuria and normal liver dihydropteridine reductase and phenylalanine-4-hydroxylase activities excreted neopterin but not biopterin or dihydrobiopterin in urine. The oral administration of L-sepiapterin (1 mg/kg body weight) lowered serum-henylalanine from 17.1 to 1.1 mg/dl within 6 h. Comparable responses were observed after oral administration of L-erythro-7, 8-dihydrobiopterin or L-erythro-5, 6, 7, 8-tetrahydrobiopterin (each given in a dose of 2.5 mg/kg body weight). The results indicate a 7, 8-dihydrobiopterin synthetase deficiency in the patient.

High-performance liquid chromatography with column switching for the analysis of biogenic amine metabolites and pterins☆

An automatic high-performance liquid chromatographic system with facilities for column switching is described which involves minimal pre-treatment of biological samples, separates complex mixtures of compounds in a short period of time and uses fluorimetric or amperometric detection. The system has been applied to the analysis of oxidized pterins in urine and reduced pterins in cerebrospinal fluid and rat brain fractions (R- and S-enantiomers of tetrahydrobiopterin resolved). The system can also be used for the analysis of most of the dopamine and serotonin metabolites in cerebrospinal fluid and brain fractions from norepinephrine to serotonin.

Serotonin and dopamine synthesis in phenylketonuria.

Two regulation systems of the serotonin and dopamine biosynthesis in patients with classical and atypical PKU were investigated. In classical PKU, the serotonin and dopamine biosynthesis is inhibited by high L-phenylalanine in blood and tissues. The dopamine formation in vivo was inhibited by phenylalanine blood concentrations higher than 25 mg/dl: the serotonin formation was inhibited even at a phenylalanine blood concentration of only 8 mg/dl. In two patients with dihydrobiopterin synthetase deficiency, the dopamine, and even more pronounced the serotonin, excretions are considerably reduced. The dopamine excretion was reduced to about 50% and the serotonin excretion to only 10% compared to controls. Under BH4 therapy (16 mg daily), the dopamine values increased about twice, serotonin threefold and the phenylalanine blood concentration normalized to 1-1.5 mg/dl. On loading a patient with BH2 synthetase deficiency with 50 mg/kg deuterated tryptophan-d5 and 150 mg/kg deuterated tyrosine d2 (phenylalanine blood concentration of 16 mg/dl), deuterated dopamine d1 and serotonin d4 could only be formed in detectable amounts after BH4 administration. During BH4 therapy the amount of dopamine d1 and serotonin d4 formed was lower than but comparable to normal controls.

Guanosine triphosphate cyclohydrolase I assay in human and rat liver using high-performance liquid chromatography of neopterin phosphates and guanine nucleotides

D-erythro-7,8-Dihydroneopterin triphosphate (NH2TP) formed from guanosine triphosphate (GTP) by GTP cyclohydrolase I (EC 3.5.4.16) in the presence of EDTA was oxidized to neopterin triphosphate (NTP) by iodine, separated from substrate and other compounds by ion-paired reverse-phase HPLC, and quantitated by fluorometric detection at 365/446 nm. Excess GTP at the end of reaction was controlled by simultaneous detection of guanine nucleotides at 254 nm. The method required only 15 mg of liver tissue for the measurement of GTP cyclohydrolase I and is suitable for activity measurement in liver biopsies. The detection limit was 4 pmol of NTP at a signal to noise ratio of 10:1. The activity of GTP-cyclohydrolase I in homogenates of human liver (n = 10) was 45 pmol NH2TP (range 32-60) formed per milligram protein per hour at 37 degrees C. Liver homogenates from Wistar rats (n = 8) formed 47 pmol NH2TP (range 35-61) per milligram protein per hour.

Increase of GTP cyclohydrolase I activity in mononuclear blood cells by stimulation: detection of heterozygotes of GTP cyclohydrolase I deficiency

An assay is described for GTP cyclohydrolase I activity in human mononuclear cells isolated from 20 ml of heparinized blood. The activity of this enzyme was low in unstimulated cells and increased 5-10 times after stimulation by phytohemagglutinin (formation of 0.8-1.3 pmol dihydroneopterin triphosphate/min per mg protein at 37 degrees C, n = 15) or mixed lymphocyte culture. No activity was detected in phytohemagglutinin-stimulated mononuclear cells of a patient with proven GTP cyclohydrolase I deficiency in liver; the samples from the father and mother of the patient showed 30 and 46%, respectively, of the mean of 15 healthy controls. In unstimulated cells, neopterin was the main component of the total intracellular pterins (after oxidation). After stimulation, dihydroneopterin triphosphate, measured as neopterin triphosphate by high performance liquid chromatography, was increased 10-30 times; neopterin and pterin were increased only 2- to 6-fold. Since the immunoreactive cells from this patient were unable to produce pterins and all immunological tests on the patient were normal, it is concluded that neither dihydroneopterin triphosphate, nor one of its metabolites are of primary importance for an immune reaction. The assay described can be used for the detection of heterozygotes of GTP cyclohydrolase I deficiency.

Atypical phenylketonuria with “dihydrobiopterin synthetase” deficiency: Absence of phosphate-eliminating enzyme activity demonstrated in liver

An assay for the phosphate-eliminating enzyme (PEE) activity in liver was developed which required only 5–10 mg tissue. PEE catalyses the elimination of inorganic triphosphate from dihydroneopterin triphosphate, which is the second and irreversible step in the biosynthesis of tetrahydrobiopterin (BH4). In the presence of substrate, magnesium, NADPH, and a sepiapterin reductase fraction from human liver, PEE catalysed the formation of BH4 which was measured by HPLC and electrochemical detection. In adult human liver, a PEE activity of 1.02±0.134 μU/mg protein (mean ±1 SD; n=5) was observed. In liver needle biopsy material from five patients with defective biopterin biosynthesis, no PEE activity was found (less than 2% and 6% of the control values, respectively). The presence of an endogenous inhibitor was excluded. In a patient who died without definite diagnosis and in a patient with β-thalassaemia liver PEE activity was increased. Sepiapterin reductase activity was present in all cases. Results indicate that in “dihydrobiopterin synthetase” deficiency, the most frequent of the rare BH4-deficient variants of hyperphenylalaninaemia, the molecular defect consists in a defect of PEE.

Tetrahydrobiopterin: Efficacy in endogenous depression and Parkinson's disease

One patient with agitated, 2 patients with inhibited endogenous depression, and 2 patients with Parkinsons disease were treated with tetrahydrobiopterin (BH4) in single oral doses of 0.9–1 g. Part of the clinical symptoms disappeared in the 2 patients with inhibited endogenous depression after 4–5 hours and the symptoms reappeared approximately 10 hours after the loading. In these two patients urinary free dopamine and serotonin increased from low to high normal values parallel to the clinical improvement. Both patients with Parkinsons disease lost their hypokinesia and rigidity and part of the tremor 4 hours after BH4 for a period of approximately 5 hours. These observations support the theories that depression may be related to a relative lack of certain biogenic amine neurotransmitters and that Parkinsons disease is primarily related to the loss of dopamine neurons. Furthermore, the potential causal role of altered BH4 metabolism in certain cases of depression and Parkinsons disease should be considered. The administration of BH4 may also be of diagnostic importance in diseases related to altered biogenic amine metabolism.