Dihydropteridine Reductase Deficiency Research Articles

A specific and sensitive assay for succinylacetone used in the antenatal diagnosis and management of hereditary tyrosinaemia

Tetrahydrobiopterin (BH4) deficiency among newborns with hyperphenylalaninemia must be rapidly diagnosed and distinguished from classical phenylketonuria (PKU) to initiate immediately specific treatment and to prevent irreversible neurological damage. The characteristic pattern of urinary pterins makes it possible to differentiate between PKU and BH4 deficiencies, and to identify different variants of BH4 deficiency. However, collection, storage, and shipment of urine samples for pterin analysis is cumbersome. A method for the measurement of different pterins (neopterin, biopterin, and pterin) in blood collected on filter paper was developed as a potential alternative to the screening for BH4 deficiencies in urine and for the monitoring of BH4 pharmacokinetics. Pterins pattern in blood spots was comparable with those in plasma and urine. We thus established reference values for pterins in blood spots in patients with hyperphenylalaninemia and identified new patients with GTP cyclohydrolase I deficiency, 6-pyruvoyl-tetrahydropterin synthase deficiency, and dihydropteridine reductase deficiency using dried blood spots on filter paper.

Dihydropteridine reductase deficiency in an 18‐year‐old boy

Dihydropteridine reductase deficiency in an 18‐year‐old boy

URINARY SCREENING FOR ABNORMALITIES OF AMINO ACID OR MUCOPOLYSACCHARIDE METABOLISM IN PATIENTS IN A HOSPITAL FOR THE MENTALLY HANDICAPPED IN WESSEX

Urine from 348 patients of a hospital for the mentally handicapped in Wessex was screened for abnormalities of aminoacid and mucopolysaccharide excretion. Persistent aminoacid abnormalities were found in 16 patients: two had phenylketonuria and one a severe variant form of hyperphenylalaninaemia (dihydropteridine reductase deficiency). In at least nine of the other patients, the observed abnormality was probably a secondary phenomenon. Two patients excreted excessive amounts of a glycosaminoglycan identified tentatively as hyaluronic acid. The study also uncovered one undiagnosed diabetic and three patients with urinary tract infection.

Phenylalanine Hydroxylase Activity-Reply

In Reply .—For most metabolic disorders, a diagnosis based on a single symptom would not be acceptable. Hyperphenylalaninemia is a symptom that in a newborn infant suggests either complete or partial PH deficiency (PKU), dihydropteridine reductase deficiency, or deficiency in biopterin synthesis. The last two defects were recognized only after liver biopsy specimens showed near-normal PH activity in patients who were thought to have PKU. Phenylketonuria has been studied intensively for nearly 30 years. There are as many unanswered questions today as there were then. We have no clue as to why some untreated patients with classic PKU attained normal mental development, while the majority were severely mentally retarded. 1 There is still no consensus as to which children with hyperphenylalaninemia need a phenylalanine-restricted diet to ensure normal intellectual development. 2,3 There is no agreement as to whether the phenylalanine-restricted diet can be terminated without adverse effects. 4 Many other

Hyperphenylalaninemia Due to Dihydropteridine Reductase Deficiency: Diagnosis by Enzyme Assays on Dried Blood Spots

Enzymatic diagnosis of hyperphenylalaninemia due to a deficiency of dihydropteridine reductase (DHPR) has previously been made by assay on liver biopsy samples, cultured skin fibroblasts, cultured lymphoid cell lines, or peripheral leukocytes. These procedures have some disadvantages for the purpose of early diagnosis of the disease. A simple method of DHPR assay using erythrocytes or dried blood spots on filter papers is described. The mean DHPR activity erythrocytes of control subjects was 3.20 +/- 0.70 (SD) nmoles/min/mg of hemoglobin, those of two patients were undetectable, and those of obligate heterozygotes for DHPR deficiency were approximately 50% of the mean control value. The assay on erythrocytes required only a 5-microliters volume of whole blood for one test. The DHPR activities in dried blood spots on filter papers from 100 normal newborns were 5.77 +/- 1.16 nmoles/min per 5-mm diameter disc; those from normal older infants, children, and adults were 3.37 +/- 0.72 nmoles/min per disc; and those from two adolescent patients with DHPR deficiency were undetectable. No false-positive results were obtained. The stability of DHPR in dried blood on filter papers was enough to mail samples in an ordinary form to a specialist laboratory. The DHPR assay on erythrocytes of dried blood spots can be easily applied to all newborn infants with hyperphenylalaninemia detected using the Guthrie tests, and will facilitate the quick and confirmative detection of DHPR deficiency among them.

Pterin metabolism in normal subjects and hyperphenylalaninaemic patients.

The application of high performance liquid chromatography to the estimation of urinary pterins is illustrated by results from normal subjects and from patients with phenylketonuria, dihydropteridine reductase deficiency and biopterin synthetase deficiency. In normal subjects following a phenylalanine load the is a temporary increase in pterin elimination, the pattern being different to that seen in chronic hyperphenylalaninaemia.

Diagnosis of Dihydropteridine Reductase Deficiency by Erythrocyte Enzyme Assay

Patients with hyperphenylalaninemia caused by dihydropteridine reductase (DHPR) deficiency, who show progressive cerebral degeneration despite adequate dietary treatment, may respond to neurotransmitter therapy if the diagnosis is made early.1 The diagnosis of this disorder is therefore urgent. A number of methods that had been shown to be of value in the diagnosis of the disease were discussed previously.2-4 A newer screening method for DHPR mutants that was based on measurement of the reduction state of biopterin in urine by high performance liquid chromatography was described by Milstien et al.5 Furthermore, recently leukocyte enzyme assays6,7 have been used in the diagnosis of DHPR deficiency.

Heterogeneity of the molecular defect in human dihydropteridine reductase deficiency.

Radioimmunoassay, immunoprecipitation, affinity chromatography and two-dimensional gel electrophoresis were used to test cultured cells from three families with dihydropteridine reductase deficiency for a catalytically incompetent product of the mutant gene. No mutant enzyme was detected in one dihydropteridine reductase-deficient homozygote or in her parents. A second homozygote and both her parents had easily detectable concentrations of inactive mutant enzyme. In a third family one parent fitted into each of these categories.

Neurotransmitter defects and treatment of disorders of hyperphenylalaninemia

The disordered biosynthesis of dopamine, norepinephrine, and serotonin in brain in untreated PKU is corrected by dietary restriction of phenylalanine. Low levels of biogenic amine metabolites were measured in cerebrospinal fluid from three patients with classical PKU; metabolite concentrations increased after dietary treatment. In a variant of hyperphenylalaninemia caused by deficiency of dihydropteridine reductase, there is defective metabolism of biogenic amines despite dietary restriction of phenylalanine. Two siblings with DHPR deficiency had low amine metabolite values in CSF; in one patient the metabolic defect was corrected by administration of hydroxylated amino acid precursors. Defective biosynthesis of biogenic amines in brain in disorders associated with hyperphenylalaninemia and evaluation of specific dietary treatments can be determined by analysis of neurotransmitter metabolites in CSF.

Neopterin and biopterin levels in patients with atypical forms of phenylketonuria.

The pattern of unconjugated pterins in liver tissue and in urine from patients with atypical forms of phenylketonuria with hyperphenylalaninemia (HPA) has been investigated with a high performance liquid chromatographic technique. Two patients with defects in the biosynthesis of biopterin have been shown to have higher than normal levels of neopterin and lower than normal levels of biopterin. In contrast, a patient with HPA due to a deficiency of dihydropteridine reductase has the reverse urinary pattern, i.e., high biopterin, low neopterin. These results indicate that the ratio of neopterin to biopterin in urine can be of value in discriminating between HPA due to a deficiency of phenylalanine hydroxylase (classic PKU), HPA due to dihydropteridine reductase deficiency, and HPA due to a block in the biosynthesis of biopterin.

Dihydropteridine reductase deficiency: diagnosis by leukocyte enzyme assay

An assay procedure for dihydropteridine reductase in peripheral leukocytes is described. The assay utilizes the tetrahydropterin-dependent reduction of ferri-cytochrome C in the presence of NADH and requires a smaller number of cells than assays described for cultured skin fibroblasts. Dihydropteridine reductase activity was not detectable in the peripheral leukocytes nor in the cultured skin fibroblasts from two adolescent patients with malignant hyperphenylalaninemia. The parents of the patients showed approximately 50% of normal dihydropteridine reductase activity in their peripheral leukocytes. Immunochemical experiments using antibodies against bovine liver dihydropteridine reductase suggest that normal leukocytes and skin fibroblasts contain dihydropteridine reductase which is immunologically similar to that of human liver. The present studies indicate that the determination of dihydropteridine reductase activity in peripheral leukocytes can be used to diagnose hyper-phenylalaninemia due to a defect in dihydropteridine reductase.

Dihydropteridine reductase variation in man and the characid fish "Cheirodon axelrodi": evidence for a dimeric enzyme structure.

Genetic evidence for a dimeric structure of dihydropteridine reductase in man and in the fish species "Cheirodon axelrodi" and "Salmo irideus" is presented. A single locus in man and two loci in the fishes examined encode this enzyme. Zymograms revealed two alleles for the locus in man and two alleles for each locus in the fish "Cheirodon axelrodi". The liver homogenate of a patient with dihydropteridine reductase deficiency showed no detectable activity in the gel, while his parents showed the normal electrophoretic phenotype.

Hyperphenylalaninemia Due to Dihydropteridine Reductase Deficiency: Diagnosis by Measurement of Oxidized and Reduced Pterins in Urine

Hyperphenylalaninemia due to dihydropteridine reductase deficiency results from the inability to maintain the aromatic amino acid hydroxylase cofactor, tetrahydrobiopterin, in its reduced or active form. Diagnosis of the disease is usually made by direct enzymatic assay on liver biopsies or in cultured skin fibroblasts. Evidence is presented that normal children and classic phenylketonuric children excrete mainly tetrahydrobiopterin in their urines, whereas children with dihydropteridine reductase deficiency excrete only oxidized forms of biopterin. Details of a rapid high performance liquid chromatographic assay for the measurement of the various forms of biopterin in urine are presented. This assay can be used to screen for suspected dihydropterine reductase mutants.

New Developments in Hyperphenylalaninemia

In recent years it has become apparent that hyperphenylalaninemia in newborn infants may be caused by a variety of defects in the enzyme phenylalanine hydroxylase or by an abnormality in either the enzyme dihydropteridine reductase (DHPR) or the synthesis of dihydrobiopterin (BH2) (Figure). Despite the rarity of the latter conditions1 there are important implications for management in making this diagnosis. It has been suggested that all new cases of hyperphenylalaninemia now be screened for these variants.2 The purpose of the Task Force was to define the feasibility of this course at the present time. BACKGROUND The hepatic system for conversion of phenylalanine to tyrosine is complex and consists of at least two essential enzymes, phenylalanine hydroxylase (catalyzing reaction 1) and dihydropteridine reductase (catalyzing reaction 2), and an essential, nonprotein cofactor, tetrahydrobiopterin3: (1) Phenylalanine + O2 + tetrahydrobiopterin → tyrosine + H2O + quinonoid dihydrobiopterin; (2) nicotinamide adenine dinucleotide (reduced form) + H+ + quinonoid dihydrobiopterin → nicotinamide adenine dinucleotide (oxidized form) + tetrahydrobiopterin. Most cases of hyperphenylalaninemia detected by newborn infant screening programs are caused by deficiencies of phenylalanine hydroxylase ranging from the severe "classic phenylketonuria (PKU)" to the more benign "hyperphenylalaninemic variant." The patients with classic PKU usually respond to a diet low in phenylalanine. However, of the estimated 400 cases of classic PKU detected annually in the United States, up to 3% may show progressive neurologic deterioration despite adequate dietary treatment. The elevated phenylalanine in these infants may be caused by either DHPR deficiency or a deficient synthesis of BH2 or to other still unidentified causes.

Differential Diagnosis of Variant Forms of Hyperphenylalaninemia

In 1975, a form of hyperphenylalaninemia was described in which neurologic deterioration occurs despite dietary control of the elevated blood phenylalanine levels.1 Subsequently, it was demonstrated that this condition can be caused by defects in the phenylalanine hydroxylase system other than in phenylalanine hydroxylase itself, ie, by a tissue deficiency of dihydropteridine reductase (DHPR)2,3 or of biopterin.4 Since then, research in the field of these newer, unresponsive variants of phenylketonuria (PKU) has moved ahead swiftly. The treatment for these forms of hyperphenyl-alaninemia, which have been estimated to account for about 10% of the total, is different from that used for classic PKU.

DIHYDROPTERIDINE REDUCTASE DEFICIENCY DIAGNOSIS BY ASSAYS ON PERIPHERAL BLOOD-CELLS

Human peripheral lymphocytes, granulocytes, and platelets contain the enzyme dihydropteridine reductase. A simple procedure and assay for diagnosing dihydropteridine reductase on peripheral blood is described. In twelve controls the level of enzyme activity in a lymphocyte-platelet preparation was 29.5±4.8 (SD) nmol NADH oxidised/min/mg protein. Two heterozygotes for dihydropteridine reductase deficiency had activities of 6·4 and 10·3 nmol NADH oxidised/min/mg protein, respectively.

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

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 (BH 2) 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 BH 2 deficiency after oral administration of L- erythro-5,6,7,8-tetrahydrobiopterin (BH 4), BH 2 (both 8μmol/kg body weight) and L-sepiapterin (2.8 and 4.2 μmol/kg in the 2 patients, respectively). BH 4 and partially even BH 2 decreased serum Phe also in the patient with DHPR deficiency. BH 4, intravenously, had no effect on serum Phe of the classical PKU patients and of the controls. d- erythro-5,6,7,8-Tetrahydroneopterin (NeH 4) had no effect on serum Phe in one patient with BH 2-deficiency. The patients with BH 2 deficiency excreted large amounts of neopterin, some dihydroneopterin and dihydroxanthopterin (XH 2), but no trace of biopterins, indicating a BH 2 synthetase deficiency in both patients. The patient with DHPR deficiency excreted remarkable amounts of BH 2 and XH 2 and traces of biopterin and neopterin. After BH 4 administration, excessive amounts of BH 2 and XH 2 were excreted. Untreated PKU patients excreted more pterins than did patients under PKU diet or normal controls; after BH 4 administration, PKU patients and controls excreted large amounts of BH 4, BH 2, biopterin. The results demonstrate that BH 4, BH 2, L-sepiapterin and NeH 4, in admixture with ascorbic acid (10 mg/kg body weight) can be absorbed easily upon oral administration. The PKU diet of patients with BH 4-deficiency can be replaced by oral BH 4 supplementation. The two patients with BH 2 synthetase deficiency need about 4μmol BH 4 kg −1 d −1 (1.25 mg BH 4 · 2 HC1 kg -1 d −1), and the patient with DHPR deficiency needs about 8 μmol BH 4 kg -1 d -1. Substitution of neurotransmitter precursors (dopa, 5-hydroxytryptophan and carbidopa) cannot be discontinued. Screening of all newborn hyperphenylalaninemics for BH 4 deficiency is suggested by a single oral administration of BH 4 · 2 HCl, 8 μmol kg −1, 1 h before a meal, and measurement of serum Phe before and 4 or 6 h after loading.

Urinary dihydroxanthopterin in the diagnosis of malignant hyperphenylalaninemia and phenylketonuria

Assessment of urinary dihydroxanthopterin is proposed as a simple method of recognition of patients with malignant hyperphenylalaninemia (MHPA). High levels of urinary dihydroxanthopterin are found in untreated patients with phenylketonuria (PKU) or with dihydropteridine reductase (DHPR) deficiency. After dietary control of the serum phenylalanine level in PKU, the urinary dihydroxanthopterin falls to near normal levels. In DHPR deficiency urinary dihydroxanthopterin levels are high even when serum phenylalanine levels are in the range achieved on dietary treatment. Low levels would be expected in patients with defects in tetrahydrobiopterin synthesis even before dietary treatment. Confirmation of the diagnosis of different forms of MHPA then requires more detailed studies, but dietary treatment of other PKU patients can proceed with confidence.

Dihydropteridine Reductase Deficiency Associated With Severe Neurologic Disease and Mild Hyperphenylalaninemia

A deficiency of hepatic dihydropteridine reductase (DHPR) activity was found in a neurologically impaired infant with mild hyperphenylalaninemia and normal levels of hepatic phenylalanine hydroxylase. DHPR is required for the regeneration of tetrahydrobiopterin, an essential cofactor in aromatic amino acid hydroxylation, a necessary step in the biosynthesis of the neurotransmitters, dopamine and serotonin. Evidence for decreased synthesis of these transmitters in this patient was provided by the finding of reduced levels of homovanillic acid and 5-hydroxyindole acetic acid, metabolites of dopamine and serotonin, respectively, in the cerebrospinal fluid and urine. Treatment with dopamine and serotonin precursors, L-3,4 dihydroxyphenylalanine and 5-hydroxytryptophan, respectively, was associated with improvement in temperament and motor tone and less frequent seizures. However, there was no improvement in gross motor function or language development.

Identification of Dihydropteridine Reductase in Human Platelets

Normal human platelets were shown to contain the enzyme dihydropteridine reductase. The enzyme was not found in a variety of other cells of hematogenous origin. Partial purification and kinetic and physical data indicated that the platelet enzyme is similar to that previously characterized from liver. Dihydropteridine reductase is important for the regeneration of tetrahydrobiopterin, a required cofactor in hydroxylation reactions involved in biogenic amine formation. The presence of the enzyme may indicate that some synthesis de novo of serotonin and/or catecholamines occurs in platelets, as opposed to a purely storage and transport function. In addition, screening for hyperphenylalaninemia due to dihydropteridine reductase deficiency may become feasible by assaying platelets for enzyme activity.