J. Schaub

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.

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.

A method for galactose-1-phosphate uridyltransferase assay and the separation of its isozymes by DEAE-cellulose column chromatography

A simplified radioactive assay for galactose-1-phosphate uridyltransferase (EC 2.7.7.12) using a small DEAE-cellulose column for the identification of the endproduct (uridine diphosphate galactose) is described. The enzyme activities in red blood cell hemolysates of normal subjects are in the range of 24 to 33 (average 30.3) mumol UDPgalactose produced per g hemoglobin per h and in fibroblasts 0.39 and 1.39 (average 0.71) nmol per mg protein per min. Furthermore, different isozymes of red blood cell galactose-1-phosphate uridyltransferase were separated on DEAE-cellulose columns. In the case of a normal genotype, most of the enzyme activity is eluted at the earlier fractions with the low molar phosphate buffer, whereas the Duarte variant appeared at later fractions with higher molar phosphate buffer.

3′‐hydroxysepiapterin in patients with dihydrobiopterin deficiency

In recent years it has been shown that the group of diseases known as phenylketonuria includes very rare and severe diseases caused by defects in biopterin metabolism [l-6]. These findings have focused interest on a new area of research that may also have great significance for neuroscience and future treatment of affective disorders. Tetrahydrobiopte~n (BH,) is a cofactor of the monooxygenases phenylalanine-4hydroxylase, tyrosine-3-hydroxylase and probably also tryptoph~-S-hydroxyla~, key enzymes in dopamine and serotonin production. Biopterin is formed from guanosine triphosphate in several steps, some of which are not known exactly. We report here the detection of 3’-hydroxysepiapterin (HS) in urine from a patient with defective dihydrobiopterin (BH,) biosynthesis. This finding could be of value for the understanding of biopterin biosynthesis and the more exact localization of the enzyme defect in biopterindeficient patients.

Separation of acid and neutral α-glucosidase isoenzymes from fetal and adult tissues, cultivated fibroblasts and amniotic fluid cells by deae-cellulose and sephadex G-100 column chromatography

Isoenzymes of alpha-glucosidases (EC 3.2.1.20) from various human organs and body fluids from fetuses and adults were separated by DEAE-cellulose column chromatography and gel filtration using Sephadex G-100. A minicolumn (0.35 X 2.5 cm) was used for the DEAE-cellulose column chromatography of extracts from tissues as well as cultivated cells of skin fibroblasts and amniotic fluid. The enzyme activity in the eluates was measured by the use of a methylumbelliferyl derivative as substrate and a very sensitive Microscope fluorimeter. In most tissue samples alpha-glucosidase was eluted mainly as a single peak when monitored at acid pH and as two peaks when the activity was measured at neutral pH in both columns. Another small peak representing alpha-glucosidase was found in fresh extracts of cultured cells on DEAE-cellulose columns. Neutral alpha-glucosidase especially in fibroblasts was extremely sensitive to storage at -20 degrees C. DEAE-cellulose column chromatography of plasma and amniotic fluid showed similar elution patterns of alpha-glucosidase. Differences were noticed in the elution pattern of urine from infants and adults. The tissue distribution and the different characteristics of the enzyme in samples of various origins and ages were discussed.

Characterization of galactose-1-phosphate uridyl-transferase and galactokinase in human organs from the fetus and adult

Some properties of galactose-1-phosphate uridyltransferase (EC 2.7.7.12) and galactokinase (EC 2.7.1.6) were investigated in human organs, i.e., in liver kidney, skeletal muscle, lung, spleen, heart and brain from fetuses as well as liver, kidney and skeletal muscle tissues from adults. (1) Galactose-1-phosphate uridyltransferase (transferase) is quite stable when stored below 4 degrees C, and can be frozen from a couple of months without noticeable loss of activity. Galactokinase is relatively stable as long as the cell structure is intact. In cell homogenates its activity decreases very fast, especially under freezing conditions. (2) The pH optimum of transferase in all human tissues is at pH values between 8.2 and 8.4 except in erythrocytes in which it is at a higher pH value. Maximal activity of galactokinase is observed at approximately 8.2 in all human tissues. (3) The Km values of transferase are similar in all human organs, and the values in fetal tissues are not significantly different from those in adult tissues. In the case of galactokinase also no distinct tissue variations are observed in Km values. However, galactokinase affinity for both substrates is considerably higher in adult organs than in fetal organs. (4) Transferase and galactokinase activity in human liver is resolved into two major components on DEAE-cellulose columns. It seems that transferase and galactokinase exist in human tissues as more than two isoenzyme constituents.

Lysosomal Enzyme Activities of Human Fetal Organs during Development

The developmental patterns of four lysosomal enzymes have been investigated in liver, kidney, lung, heart, spleen, muscle and brain tissues of human fetuses at varius gestational ages. The largest increment in the activity of all four enzymes, namely acid alpha-glucosidase, alpha-galactosidase, beta-galactosidase and acid phosphatase had been observed in kidney with a 6- to 12-fold increase between the second and third trimester of gestation. The activity of all liver and spleen enzymes also increased considerably during these periods. In muscle, however, only alpha-glucosidase and acid phosphatase showed an increase in the activity, and in lung, acid phosphatase and beta-galactosidase. Most of brain and heart enzymes, except acid phosphatase, did not change significantly during the observation period. The activities of these lysosomal enzymes were also measured in tissues of a normal adult individual, and aspects of the neonatal and postnatal development of these enzymes were discussed.

A simple and rapid thin-layer chromatographic method for the assay of intestinal disaccharidases.

Abstract Thin-layer Chromatographic method was applied to the assay of intestinal disaccharidases using disaccharide substrates after incubation with intestinal biopsy material. In comparison with the quantitative hexokinase-glucose-6-phosphate dehydrogenase method, thin-layer chromatography showed good correlation, as was demonstrated with results from 12 normal children and from 15 patients with celiac disease. The sensitivity of the thin-layer Chromatographic method is sufficient to indicate even small activities, moreover its simplicity and the short time required constitute advantageous features of this method.

Properties of human alkaline phosphatase and prenatal diagnosis of hypophosphatasia

The alkaline phosphatase activity in serum, leukocytes, amniotic fluid and cultured fibroblasts and amniotic fluid cells has been characterized by inhibition studies and by mini-electrophoresis.

A simple thin-layer chromatographic technique for the assay of intestinal dipeptide hydrolases from human mucosal biopsy material

Abstract Thin-layer chromatography was applied for the assay of dipeptide hydrolases using dipeptide substrates after incubation with intestinal biopsy material. The behavior of 11 dipeptide hydrolases under normal and pathological conditions was studied as was demonstrated with the results from 60 children divided in four groups: normals (I), those with untreated celiac disease (II), celiac disease after treatment (III) and those suffering from symptoms of malabsorption syndrome (IV). The advantage of thin-layer chromatography is its simplicity and universal application for all dipeptide hydrolases with small amounts of mucosal tissue.