P.K. De Bree

Purine Nucleoside Phosphorylase Deficiency Associated with Selective Cellular Immunodeficiency

We studied a 15-month-old girl who had normal T-cell and B-cell immunity at birth, after which a gradual decrease in T-cell immunity developed. This selective cellular immunodeficiency was inherited as an autosomal recessive trait: two older sisters had the same immunodeficiency. Adenosine deaminase activity was present in erythrocytes and lymphocytes of the patient, parents and a healthy brother. Purine nucleoside phosphorylase activity was not found in the patients erythrocytes and lymphocytes (the parents and brother had intermediate values, indicating that the enzyme deficiency too was inherited as an autosomal recessive trait). Analysis of serum and urine from the patient and of serum from her two deceased sisters showed high levels of inosine and guanosine in addition to hypouricemia and hypouricosuria. The bone marrow was megaloblastic, and the blood hypochromic microcytic. The patient had spastic tetraparesis. Intoxication of the T lymphocytes after birth by metabolic products may explain the progressive cellular immunodeficiency.

Combined deficiency of xanthine oxidase and sulphite oxidase: a defect of molybdenum metabolism or transport?

A child is described who presented in the neonatal period with feeding difficulties, severe neurological abnormalities, lens dislocation of the eyes and dysmorphic symptoms of the head. Routine laboratory investigations revealed a decreased serum urate and a positive sulphite reaction of the urine. Subsequent chromatographic examinations showed xanthinuria and increased excretion ofS-sulphocysteine and taurine to be present. In addition, high thiosulphate and low sulphate excretions in the urine were observed. Xanthine oxidase deficiency was demonstrated in a jejunal biopsy specimen, whereas the excretion of sulphur containing substances was considered to be characteristic of sulphite oxidase deficiency.This new combination of defects may be the result of malfunctioning of both enzymes, possibly caused by alterations in the essential molybdenum containing active centre of the enzymes, which they share in common.

Dihydropyrimidine Dehydrogenase Deficiency Leading to Thymine-uraciluria. An Inborn Error of Pyrimidine Metabolism

Three unrelated patients with excessive thymine-uraciluria due to dihydropyrimidine dehydrogenase deficiency are described. Excretory values (mmol/g creatinine) were: uracil 2.0-10.5, thymine 2.3-7.5, 5-hydroxymethyluracil 0.2-0.9. Orally administered (index patient) uracil and thymine were excreted for the greater part whilst dihydrouracil and S-dihydrothymine were mainly metabolised. Dihydropyrimidine dehydrogenase activities (nmol X h-1 X mg-1 protein) in leucocytes were 0.04, 0.01 and less than 0.01 in the patients, 0.31-1.66 in their parents, and 1.01-4.46 in controls (n = 4). The patients presented with a non-specific clinical picture of cerebral dysfunction.

Two-dimensional thin-layer chromatography for the screening of disorders of purine and pyrimidine metabolism.

A method is presented for the two-dimensional thin-layer chromatographic screening of purines, pyrimidines and their nucleosides in the urine. Prior to chromatography, isolation of these substances from the urine is performed by anion-exchange column chromatography. Purines and pyramidines are quantitatively eluted with formic acid 0.01 M and 4 M respectively. The results of recovery and stability experiments are given. Normal excretory patterns are presented. Also results in patients with various diseases are shown: ornithine transcarbamylase deficiency, adenosine deaminase deficiency, purine nucleoside phosphorylase deficiency, adenine phosphoribosyltransferase deficiency, xanthine oxidase deficiency and hypoxanthine-guanine phosphoribosyltransferase deficiency. Finally the pattern of a patient on treatment with allopurinol is given.

Diagnosis of inherited adenylosuccinase deficiency by thin-layer chromatography of urinary imidazoles and by automated cation exchange column chromatography of purines

Patients with inherited adenylosuccinase deficiency excrete large quantities of succinyloaminoimidazolecarboxamide riboside (SAICAR) and succinyloadenosine (SAdo). A two-dimensional thin-layer chromatography method for the detection of SAICAR is described. The method consists of isolation of imidazoles with a cation exchange resin; TLC on cellulose plates, solvent I, isopropanol-ammonia 10% (4:1) and II, butanol-acetic acid-water (4:1:1); detection with Pauly reagent. SAICAR gives rise to an isolated spot with a characteristic bluish color. Also a simple one-dimensional thin-layer chromatography method using urine without any pretreatment for screening of high risk populations is given. Four new cases could be diagnosed. Clinical and chemical data, including concentrations of SAICAR and SAdo in urine, plasma and cerebrospinal fluid, determined by cation exchange column chromatography, are presented.

Dihydropyrimidinuria: a new inborn error of pyrimidine metabolism

To date only a single defect of pyrimidine catabolism has been described, namely the deficiency of dihydropyrimidine dehydrogenase (Berger et al., 1984), an enzyme which catalyses the formation of dihydrouracil and dihydrothymine from the corresponding pyrimidines. Various neurological abnormalities have been described in this group of patients. The further catabolism of the dihydropyrimidines is effected by dihydropyrimidinase (5,6-dihydropyrimidine amidohydrolase; EC 3.5.2.2). A deficiency of this enzyme has not yet been described. We report a patient with dihydropyrimidinuria, who most likely suffered from dihydropyrimidinase deficiency. Advanced chromatographic methods were necessary for the analysis of the patients urine.

D-Glyceric acidemia in a patient with chronic metabolic acidosis

A patient is described with glyceric acidemia and glyceric aciduria. The main clinical problems in infancy were severe metabolic acidosis and failure to thrive. The patient needs permanent treatment with bicarbonate. Hyperglycinemia, as described in the first case discovered elsewhere, was not present. The glyceric acid was found to have the D-configuration, as analyzed by capillary gas chromatography of its di-O-acetyl-l-menthyl ester. The abnormality may result from a defect in serine metabolism.

Urinary and faecal excretion of metabolizes of tyrosine and phenylalanine in a patient with cystic fibrosis and severely impaired amino acid absorption

In a patient with severely impaired amino acid absorption and cystic fibrosis, the urinary excretion of metabolites of phenylalanine and tyrosine was found to be highly increased. Presumably, for the greater part, these metabolites are products of bacterial degradation of the amino acids in the intestinal lumen. The main urinary metabolites were phenylacetic (con j.+ free), benzoic (conj.+ free), p-hydroxyphenylacetic and p-hydroxybenzoic (conj.+ free) acids; the first two represent a considerable percentage of phenylalanine intake and the latter two that of tyrosine intake. Minor urinary metabolites, probably typical for tyrosine malabsorption, were p-hydroxyphenylpropionic and p-hydroxyphenylacrylic acids; the latter compound, however, occurs seldom or never. In the faeces were found small amounts of p-hydroxyphenylpropionic, p-hydroxyphenylacetic, p-hydroxybenzoic and p-hydroxyphenyllactic acids, all derived from tyrosine. Also small quantities of the phenylalanine metabolites phenylacetic and benzoic acid were present. Faecal excretion of the metabolites was clearly less than their urinary elimination. The analytical data obtained from urine and faeces indicate that the tyrosine ammonia-lyase pathway and possibly also the phenylalanine ammonia-lyase pathway are important metabolic routes of intestinal bacteria. The urinary excretion of phenyl acids and p-hydroxyphenyl acids may be an important parameter for phenylalanine and tyrosine malabsorption.

Urinary purines and pyrimidines in patients with hyperammonemia of various origins

Excretion patterns of pyrimidines and purines in patients with various types of hyperammonemia have been investigated by 2-dimensional thin-layer chromatography and high pressure liquid chromatography (HPLC). For the quantitative analysis of pseudouridine, uracil and uridine a new procedure has been developed, consisting of pre-fractionation with Dowex 1 X 8, followed by dual column HPLC on a strong anion-exchanger and a reverse phase column. Thymine has also been analyzed in the pre-fractionated urine by a new HPLC method using the reverse phase column in combination with a strong cation-exchange column. Quantitative data for urinary pyrimidines and uric acid in hyperammonemia are given. In patients with a defect in one of the urea cycle enzymes, the level of pyrimidine excretion was found to depend on plasma ammonia concentrations. In other hyperammonemic patients, an increased excretion of orotic acid, uracil and uridine has only been found in one of the two patients with lysinuric protein intolerance, all other patients showing normal excretion patterns. Elevated uric acid excretions have been found frequently in our patients with hyperammonemia, but they did not always coincide with high plasma ammonia levels. A possible explanation for the difference in the excretion levels of the various pyrimidines is discussed.

A patient with purine nucleoside phosphorylase deficiency: Enzymological and metabolic aspects

1. Enzymological and metabolic data in a patient with nucleoside phosphorylase (NP) deficiency are described. 2. Incubation of intact NP-deficient red cells with [14C]adenosine showed a rapid uptake and conversion to inosine. Almost no radioactivity was incorporated in the adenosine nucleotides and no hypoxanthine labeling could be detected. 3. Incubation with [14C]inosine resulted in a rapid conversion to IMP in the normal intact red cells but in an accumulation of inosine in the medium with the erythrocytes of the patient, proving again that a NP deficiency is present. 4. The high PRPP level found may result from impaired consumption due to lack of substrates for the salvage enzyme HGPRT. 5. Incubation with [14C]hypoxanthine and [14C]adenine showed that normal HGPRT and APRT activities were present in the NP-deficient red cells. 6. In serum and urine of the patient the levels of inosine and guanosine were considerably increased, while the serum and urinary levels of uric acid were very low. In the two deceased sisters NP deficiency was also strongly suggested by analyses of the serum purines, of stored deep frozen samples.