J.A.J.M. Bakkeren

Heterozygosity for Homocystinuria in Premature Peripheral and Cerebral Occlusive Arterial Disease

Premature arteriosclerosis and thromboembolic events are well-known complications of homozygous homocystinuria due to cystathionine synthase deficiency. It is unknown whether heterozygosity for homocystinuria predisposes to premature vascular disease. We explored the frequency of excessive homocysteine accumulation after standardized methionine loading in 75 patients presenting with clinical signs of ischemic disease before the age of 50:25 with occlusive peripheral arterial disease, 25 with occlusive cerebrovascular disease, and 25 with myocardial infarction. In seven patients in each of the first two groups but in none of the patients in the third group, heterozygosity for homocystinuria was established on the basis of pathological homocysteinemia after methionine loading and cystathionine synthase deficiency in skin fibroblast cultures. Because the frequency of heterozygosity for homocystinuria in the normal population is 1 in 70 at the most, we conclude that this condition predisposes to the development of premature occlusive arterial disease, causing intermittent claudication, renovascular hypertension, and ischemic cerebrovascular disease.

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.

Studies on (Na+-Ka+)-activated ATPase. XX. Properties of (Na+-K+)-activated ATPase in rat liver

1. 1. The ouabain-sensitive (Na+-K+-activated ATPase enzyme system was present in rather low activity (0.37 mole/kg dry weight per h) in rat liver. 2. 2. Pretreatment with 1.5 M urea decreased the Mg2+-activated ATPase activity without significantly affecting the (Na+-K+-ATPase activity, thus causing the relative activity of the latter to rise from 13% to 37%. This permitted to determine the properties of the (Na+-K+)-ATPase system with greater accuracy. 3. 3. The (Na+-K+)-ATPase required for activation both Na+ (Km = 6 mM) and K+(Km = 0.9 mM). Rb+ could replace K+ in activating the enzyme (Km = 0.8 mM). Maximal activation of the (Na+-K+)-ATPase system required 2 mM Mg2+ at an ATP concentration of 2 mM. 4. 4. The pH optimum for (Na+-K+)-ATPase was 7.3, while the Mg2+-activated ATPase activity had a pH optimum of 8.7. 5. 5. The optimal temperature for (Na+-K+)-ATPase and for Mg2+-ATPase activity was 45°. 6. 6. The (Na+-K+)-ATPase was inhibited by the digitalis glycoside ouabain (pI50 = 3.9) and the Erythrophleum alkaloid erythropleine (pI50 = 5.1).

High-performance liquid chromatographic assay for identification and quantitation of nucleotides in lymphocytes and malignant lymphoblasts

A method for the identification and quantitation of nucleotide pools in lymphocytes and leukemic blasts is described. Separation of these metabolites was performed by anion-exchange high-performance liquid chromatography using a pH and concentration gradient consisting of several linear steps. The mono-, di- and triphosphates of adenosine, cytidine, guanosine, inosine, uridine and xanthosine could conveniently be separated together with NAD+, cyclic AMP, NADP+ and uridinediphosphoglucose (UDPG). In addition, data on the accuracy and precision of the method are given and its potentials for use in the analysis of nucleotide pools in leukemic lymphoblasts are illustrated.

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.

Dihydropyrimidinase deficiency: Confirmation of the enzyme defect in dihydropyrimidinuria

Dihydropyrimidinase (DHP, EC 3.5.2.2) is the second enzyme in the degradation pathway of uracil and thymine. It catalyses the degradation of both dihydrouracil and dihydrothymine to N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyric acid, respectively. So far, four cases of dihydropyrimidinuria (McKusick 222748) have been reported (Duran et al 1991; Henderson et al 1993; Bakkeren et al, personal communication, 1994; Ohba et al 1994). The patients show a variable clinical phenotype comprising seizures or epileptic attacks (3 out of 4 patients) mental retardation (2 patients), growth retardation (1 patient) and dysmorphic features (1 patient). Since these patients excrete large amounts of dihydrouracil and dihydrothymine and moderate amounts of uracil and thymine in their urine, they can easily be detected (Van Gennip et al 1993). On the basis of the characteristic urinary metabolite profile it is assumed that the disease is caused by a deficiency of DHP. The direct measurement of the activity of DHP in patients has been hampered by the fact that the enzyme is almost exclusively expressed in liver tissue. Here, we provide for the first time direct evidence at the enzyme level for a deficient activity of DHP in liver in a patient with dihydropyrimidinuria.

Linkage analysis in X-linked adrenoleukodystrophy and application in post- and prenatal diagnosis

SummaryWe have performed linkage analysis with the DNA markers DXS52 and the clotting factor VIII gene (F8C), in several large families with X-linked adrenoleukodystrophy (ALD). The tight linkage to DXS52 could be extended giving a maximal LOD score of 22.5 at 1 cM. F8C was also tightly linked to ALD with a maximal LOD score of 7.8 without recombination. Multipoint linkage analysis with the markers DXS304, DXS52, and F8C indicated that both the gene for ALD and for F8C are distal to DXS52. In four patients with ALD, no major structural rearrangement in the Xqter region was observed; in particular, there were no abnormalities in the vision blindness genes. DNA analysis appeared to be of use in determination of the carrier status of females at risk, for the determination of the origin of the mutation in a particular family, and for prenatal diagnosis.

An accurate and sensitive assay of [14C]octanoate oxidation and its application on tissue homogenates and fibroblasts

A procedure was developed to assay [14C]octanoate oxidation from the production of both 14CO2 and 14C-labeled acid-soluble products. Octanoic acid and its CoA and carnitine esters were eliminated from the acid-soluble products by alkaline hydrolysis of the esters and acidification and binding of the acid to Lipidex 1000. The method was evaluated with homogenates of various rat tissues and human muscles and with human fibroblasts. 14CO2 production was variable and comprised less than 3% of the total oxidation products with homogenates and 26 +/- 19% with fibroblasts. As compared to palmitate, oxidation rates of octanoate were higher in rat liver and heart homogenates, of the same magnitude in muscle homogenates, but lower in fibroblasts. The proportion of antimycin-insensitive oxidation was much lower with octanoate than with palmitate. Using the assay a case of medium-chain acyl-CoA dehydrogenase deficiency could be indicated.

Studies on (Na+-K+-activated ATPase. XXI. Changes in (Na+-K+)-activated ATPase activity and ouabain-sensitive 86Rb+ uptake rate in regenerating rat liver

1. 1. In view of the reported ability of the regenerating rat liver after partial hepatectomy to maintain a higher potassium and a lower sodium concentration than normal liver the activity of the cation pump in the regenerating rat liver was studied. 2. 2. The activity of the (Na+-K+)-activated ATPase sytem was increased. A maximal increase of about 57% was observed after 3 to 6 days of regeneration. 3. 3. The ouabain-sensitive 86Rb+ uptake was increased from 2 to 6 days after regeneration by about 54% maximally. 4. 4. The passive efflux of 86Rb+ was decreased by about 41% from 1 to 6 days after partial hepatectomy. The passive 22Na+ influx was not significantly changed. 5. 5. The increase in active cation transport, assisted by the decreased passive cation efflux, was held responsible for the simultaneous increase in potassium level and decrease in soidum level in regenerating rat liver.

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