R. A. De Abreu

Elevated urine, blood and cerebrospinal fluid levels of uracil and thymine in a child with dihydrothymine dehydrogenase deficiency

In the urine of a child with unexplained convulsions large amounts of uracil and thymine were detected by gas chromatography. Identification was performed by coupled gas chromatography-mass spectrometry. Quantitation of the urinary excretion by means of a sensitive high-performance liquid chromatographic (HPLC) method revealed a 1000-fold elevation compared to normal. Serum and cerebrospinal fluid levels of the two pyrimidine bases were about a hundred times higher than normal. In fibroblasts the activity of dihydrothymine dehydrogenase was determined by measuring the conversion of radioactive labelled thymine to dihydrothymine with HPLC of the reaction mixture. In the patients cells a complete deficiency of dihydrothymine dehydrogenase activity was found. Our patient is the first case described with such a proven enzyme deficiency.

Determination of extracellular and intracellular thiopurines and methylthiopurines by high-performance liquid chromatography

The thiopurine antimetabolites 6-thioguanine and 6-mercaptopurine are important chemotherapeutic drugs in the treatment of childhood acute lymphoblastic leukaemia. Measurement of metabolites of these thiopurines is important because correlations exist between levels of these metabolites and the prognosis in childhood acute lymphoblastic leukaemia. The reversed-phase method for the determination of extracellular thiopurine nucleosides and bases was previously developed and has been modified such that methylthiopurine nucleosides, bases, thioxanthine and thiouric acid can be measured also. The anion-exchange method enables the determination of intracellular mono-, di- and triphosphate (methyl)thiopurine nucleotides in one run. Extraction on ice with perchloric acid and dipotassium hydrogenphosphate results in good recoveries for (methyl)thiopurine nucleotides in lymphoblasts and peripheral mononuclear cells and for methylthioinosine nucleotides in red blood cells. Measurement of the low concentrations of mono-, di- and triphosphate thioguanine nucleotides in red blood cells (detection limit 20 pmol/10(9) cells) is possible after extraction with methanol and methylene chloride, followed by oxidation of thioguanine nucleotides with permanganate and fluorimetric detection.

Lipid peroxidation in homocysteinaemia

Homocysteinaemia due to cystathione synthase deficiency (CSD: McKusick 236200) is a rare autosomal recessive inborn error of methionine metabolism. The most life-threatening complications caused by CSD are thromboembolism and vascular abnormalities. Boers et al (1985) provided evidence that even mild homocysteinaemia, as seen in heterozygote CSD patients, is predisposing for development of premature peripheral and cerebral occlusive arterial disease

Concentration of nucleotides and deoxynucleotides in peripheral and phytohemagglutinin-stimulated mammalian lymphocytes: Effects of adenosine and deoxyadenosine

Concentrations of purine and pyrimidine ribonucleotides were measured with HPLC in lymphocytes of man, horse, pig and sheep and in rat thymocytes. The ATP concentration was highest in lymphocytes of all species and about 850 pmol/10(6) cells in human and equine lymphocytes, higher in porcine and lower in ovine lymphocytes and rat thymocytes. The GTP concentration was comparable in human, equine and porcine lymphocytes, but lower in ovine lymphocytes. ATP concentration was also measured in lymphocytes of man, horse and pig with a luciferin-luciferase assay. During culturing with or without phytohemagglutinin the ATP concentrations decreased in these lymphocytes. The concentrations of TTP and dATP were measured with a DNA polymerase assay. Phytohemagglutinin-stimulation increased the TTP concentration in lymphocytes of all three species, the dATP concentration only in human lymphocytes. ATP, TTP and dATP concentrations and thymidine incorporation were measured in phytohemagglutinin-stimulated lymphocytes after 24 and 48 h culturing in the presence of adenosine or deoxyadenosine. Adenosine increased the ATP concentration in porcine and equine, but not in human lymphocytes. Deoxyadenosine and adenosine did not affect the TTP concentration. Deoxyadenosine decreased the ATP concentration only in the presence of EHNA in human lymphocytes, but increased it in other conditions and in equine and porcine lymphocytes. Deoxyadenosine in the presence of EHNA increased the dATP concentration in human, equine and porcine lymphocytes 3-, 10-, and 9-fold, respectively, and decreased considerably thymidine incorporation. Deoxyadenosine without EHNA increased the dATP concentration 2-5-fold, decreased the thymidine incorporation in lymphocytes of man and horse, but stimulated incorporation in porcine lymphocytes about 5-fold. The latter results indicate that accumulation of dATP is not always associated with inhibition of cell proliferation.

Dihydropyrimidine dehydrogenase deficiency: Neurological aspects

A family with dihydropyrimidine dehydrogenase (DPD) deficiency is presented. In 3 persons a complete deficiency, and in 3 others a partial deficiency was detected in cultured fibroblasts. Two homozygote subjects and 1 heterozygote subject suffered from epileptic manifestations, in one of these homozygote subjects also microcephaly was found. DPD deficiency might be an etiological factor in the clinical picture of these patients. An autosomal recessive mode of inheritance of this deficiency was found.

Synthesis of lens protein in vitro V. Isolation of messenger-like RNA from lens by high resolution zonal centrifugation☆

Recently Lingrel described the isolation of a 9 S fraction from mouse reticulocytes which exhibited messenger activity in a cell-free system from rabbit reticulocytes [ 1,2]. Also from other eukaryotic systems RNA with template activity could be isolated. Heywood described the isolation of messenger-like RNA from muscle polyribosomes. He was able to show that with this messenger and 80 S ribosomes from reticulocytes a protein could be synthesized which has the characteristics of myosin [3] . An RNA fraction isolated from myeloma appeared to function as template for the synthesis of a mouse Ig light chain in a heterologous cell-free reticulocyte system [4]. The lens is a unique tissue which predominantly produces a class of highly specific proteins: the crystallins. Like mRNA from reticulocyte [5-71 the lens messenger has been reported to be stable [8] In the present paper a high resolution zonal centrifugation technique is described which allows the isolation of two species of lens messengers differing by a number of criteria from the bulk of ribosomal RNA and tRNA.

Prevention of vitamin K deficiency in infancy by weekly administration of vitamin K

Vitamin K prophylaxis has been developed to prevent classic haemorrhagic disease of the newborn. Single vitamin K administration after birth has been reported to fail, resulting in late haemorrhagic disease of the newborn. The preventive effect of oral administration of vitamin K1 1 mg, repeated weekly during the first three months of life, was studied in 48 healthy breast‐fed infants, by determination of thrombotest, PIVKA‐II and vitamin K1 concentrations at the age of 4, 8 and 12 weeks. All infants showed normal thrombotcst values and PIVKA‐II was not detectable. Vitamin K1 concentrations were negatively correlated with the number of days elapsed since the most recent vitamin K administration. Six to seven days after the latest application, mean levels were 1223,927 and 748 pg/ml at ages 4, 8 and 12 weeks, respectively. In conclusion, weekly administration of vitamin K1 1 mg offers complete protection against vitamin K deficiency and does not result in an accumulation of vitamin K1 in the blood.

Calcium-phosphate metabolism in autosomal recessive idiopathic strio-pallido-dentate calcinosis and Cockayne's syndrome

In three siblings with autosomal recessive idiopathic strio-pallido-dentate calcinosis (SPDC) and in three other siblings with Cockaynes syndrome (CS) studies on plasma values of calcium and phosphate, intestinal calcium absorption, radiograms of the hands and studies on the influence of parathyroid hormone (PTH) on the renal threshold for phosphate revealed no abnormalities. In one of the SPDC patients and one of the CS patients the effect of PTH on the cyclic adenosine monophosphate (cAMP) concentrations in urine and cerebrospinal fluid (CSF) were determined. In both a normal response of urinary cAMP was noted. In the CS patient the response of CSF cAMP was also normal. The SPDC patient, however, had a significantly decreased response of CSF cAMP. It is suggested that a decreased sensitivity of the cerebral adenylate cyclase complex is involved in the etiology of autosomal recessive idiopathic SPDC. Subsequently this disorder could be considered as cerebral pseudohypoparathyroidism. The etiology of CS remains unknown.

Discrepancies in ribonucleotide concentrations in human lymphocytes isolated from heparinized and defibrinized blood

R.A. De Abreu a* **, G.J. Peters b, J.A.J.M. Bakkeren a and J.H. Veerkamp ’ ’ Centre for Pediatric Oncolow, S.E. Netherlands, Department of Pediatrics, St. Radboud Hospital, P.O. Box 9101, 65OOHB Nijmegen b Division of Biochemical Pharmacology, Department of Oncology, Free Vniversiq Hospital, P.O. Box 7057, 1007 MB Amsterdam and’ Department of Biochemistry, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen (The Netherlands

Concentration of Nucleotides in Peripheral Blood Lymphocytes of Various Mammalian Species

Assay of intracellular pools of nucleotides forms an important tool in the study of regulation of cellular metabolism. Concentrations of ribonucleotides are changed in blood cells of leukemic patients (1) and in those of patients with adenosine deaminase (ADA) or purine nucleoside Phosphorylase (PNP) deficiency (2–5). Treatment with deoxycoformycin results in marked changes of adenine nucleotide concentrations (6–8).