H. Van Lenthe

Genotype and phenotype in patients with dihydropyrimidine dehydrogenase deficiency

Abstract Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disease characterised by thymine-uraciluria in homozygous deficient patients and has been associated with a variable clinical phenotype. In order to understand the genetic and phenotypic basis for DPD deficiency, we have reviewed 17 families presenting 22 patients with complete deficiency of DPD. In this group of patients, 7 different mutations have been identified, including 2 deletions [295–298delTCAT, 1897delC], 1 splice-site mutation [IVS14+1G>A)] and 4 missense mutations (85T>C, 703C>T, 2658G>A, 2983G>T). Analysis of the prevalence of the various mutations among DPD patients has shown that the G→A point mutation in the invariant splice donor site is by far the most common (52%), whereas the other six mutations are less frequently observed. A large phenotypic variability has been observed, with convulsive disorders, motor retardation and mental retardation being the most abundant manifestations. A clear correlation between the genotype and phenotype has not been established. An altered β-alanine, uracil and thymine homeostasis might underlie the various clinical abnormalities encountered in patients with DPD deficiency.

Profound variation in dihydropyrimidine dehydrogenase activity in human blood cells: major implications for the detection of partly deficient patients

SummaryDihydropyrimidine dehydrogenase (DPD) is responsible for the breakdown of the widely used antineoplastic agent 5-fluorouracil (5FU), thereby limiting the efficacy of the therapy. To identify patients suffering from a complete or partial DPD deficiency, the activity of DPD is usually determined in peripheral blood mononuclear cells (PBM cells). In this study, we demonstrated that the highest activity of DPD was found in monocytes followed by that of lymphocytes, granulocytes and platelets, whereas no significant activity of DPD could be detected in erythrocytes. The activity of DPD in PBM cells proved to be intermediate compared with the DPD activity observed in monocytes and lymphocytes. The mean percentage of monocytes in the PBM cells obtained from cancer patients proved to be significantly higher than that observed in PBM cells obtained from healthy volunteers. Moreover, a profound positive correlation was observed between the DPD activity of PBM cells and the percentage of monocytes, thus introducing a large inter- and intrapatient variability in the activity of DPD and hindering the detection of patients with a partial DPD deficiency.

Clinical and biochemical findings in six patients with pyrimidine degradation defects

Darrow DC (1945) Congenital alkalosis with diarrhea. J Pediatr 26: 519-532. Gamble JL, Fahey KR, Appleton J, MacLachlan E (1945) Congenital alkalosis with diarrhea. J Pediatr 26: 509-518. Groli C (1986) Congenital chloride diarrhea: Antenatal ultrasonographic appearance. J Clin Ultrasound 14: 283-295. Holmberg C, Perheentupa J (1985) Congenital Na diarrhea: A new type of secretory diarrhea. J Pediatr 106: 56-61. Holmberg C, Perheentupa J, Launiala K, Hallman N (1977) Congenital chloride diarrhea; clinical analysis of 21 Finnish patients. Arch Dis Child 52: 255-267. Pasternak A, Perheentupa J, Launiala K, Hallman N (1967) Kidney biopsy findings in familial chloride diarrhea. Acta Endocrinol 55: 1-9. Perheentupa J, Eklund J, Kojo N (1965) Familial chloride diarrhea (congenital alkalosis with diarrhea). Acta Paediatr Scand 159 (supplement): 119-120. Petres RE, Redwine FO (1982) Ultrasound in the intrauterine diagnosis and treatment of fetal abnormalities. Clin Obstet Gynecol 25: 753-771. Yanagisawa M, Obe Y, Yabuta K (1968) A case ofcongenital alkalosis with diarrhea. Paediatr Universit Tokyo 16: 44-47.

Activity of Pyrimidine Degradation Enzymes in Normal Tissues

In this study, we measured the activity of dihydropyrimidine dehydrogenase (DPD), dihydropyrimidinase (DHP) and s-ureidopropionase (s-UP), using radiolabeled substrates, in 16 different tissues obtained at autopsy from a single patient. The activity of DPD could be detected in all tissues examined, with the highest activity being present in spleen and liver. Surprisingly, the highest activity of DHP was present in kidney followed by that of liver. Furthermore, a low DHP activity could also be detected in 8 other tissues. The highest activity of s-UP was detected in liver and kidney. However, low UP activities were also present in 8 other tissues. Our results demonstrated that the entire pyrimidine catabolic pathway was predominantly confined to the liver and kidney. However, significant residual activities of DPD, DHP and s-UP were also present in a variety of other tissues, especially in bronchus.In this study, we measured the activity of dihydropyrimidine dehydrogenase (DPD), dihydropyrimidinase (DHP) and ß-ureidopropionase (ß-UP), using radiolabeled substrates, in 16 different tissues obtained at autopsy from a single patient. The activity of DPD could be detected in all tissues examined, with the highest activity being present in spleen and liver. Surprisingly, the highest activity of DHP was present in kidney followed by that of liver. Furthermore, a low DHP activity could also be detected in 8 other tissues. The highest activity of ß-UP was detected in liver and kidney. However, low UP activities were also present in 8 other tissues. Our results demonstrated that the entire pyrimidine catabolic pathway was predominantly confined to the liver and kidney. However, significant residual activities of DPD, DHP and ß-UP were also present in a variety of other tissues, especially in bronchus.

Radiochemical assay for determination of dihydropyrimidinase activity using reversed-phase high-performance liquid chromatography

Abstract A radiochemical assay was developed to measure the activity of dihydropyrimidinase (DHP) in human liver homogenates. The method is based on the separation of radiolabeled dihydrouracil from N-carbamyl-β-alanine by HPLC with on-line detection of radioactivity combined with detection of 14CO2 by liquid scintillation counting. The assay was linear with time and protein concentration. The minimum amount of radiolabeled products which could be determined proved to be 12 pmol using a purified stock solution of [2-14C]-5,6-dihydrouracil. This highly sensitive assay is especially suitable to identify patients with a dihydropyrimidinase deficiency.

Cytidine triphosphate (CTP) synthetase activity during cell cycle progression in normal and malignant T-lymphocytic cells

The role of cytidine triphosphate (CTP) synthetase (EC 6.3.4.2.) in the pyrimidine ribonucleotide metabolism of MOLT-3 human T-ALL cell line cells and normal human T lymphocytes during the cell cycle traverse was studied. Highly pure G1-phase samples and samples enriched in S-phase cells were obtained by counterflow centrifugation. The activity of CTP synthetase in situ, measured in pulse-chase experiments, was similar in the G1-phase and S-phase MOLT-3 cells. In contrast, in S-phase T lymphocytes, an increased activity of CTP synthetase was observed compared with G1-phase T lymphocytes. Nevertheless, the MOLT-3 samples showed an increased activity of CTP synthetase in comparison with either G1-phase or S-phase enriched samples of normal T lymphocytes. Therefore, the increased activity of CTP synthetase of MOLT-3 cells is a cell cycle-independent feature, whereas among normal T lymphocytes, the increase in activity of CTP synthetase that arises after a growth stimulus is more prominent in the S-phase.

Inhibition of beta-ureidopropionase by propionate may contribute to the neurological complications in patients with propionic acidaemia

Propionic acidaemia is due to a primary deficiency of propionyl-CoA carboxylase (EC 6.4.1.3) activity. The clinical picture is characterized by repeated relapses and neurological sequelae are common. Among the neurological complications, focal and general seizures as well as EEG abnormalities are often observed. During relapse substantial accumulation of propionate occurs in all body fluids. β-Ureidopropionase (UP, EC 3.5.1.6) is the third enzyme in the degradation pathway of uracil and thymine. It catalyses the degradation of both β-ureidopropionic acid and β-ureidoisobutyric acid to β-alanine and β-aminoisobutyric acid, respectively. A deficiency of UP or one of the other enzymes of pyrimidine degradation leads to a diminished production of β-alanine, a neurotransmitter amino acid. Diminished production of β-alanine also occurs in other pyrimidine degradation defects and is presumed to be a contributing factor in the neurological abnormalities seen in the patients with those defects (Van Gennip et al 1997). Propionate has been reported to inhibit UP in Euglena gracilis (Wasternack et al 1979). We wondered whether inhibition of UP by propionate or β-hydroxypropionate could be demonstrated in vitro in human liver and in vivo in patients with propionic acidaemia.

Determination of 5-fluorouracil in plasma with HPLC-tandem mass spectrometry

In this article, we describe a fast and specific method to measure 5FU with HPLC tandem-mass spectrometry. Reversed-phase HPLC was combined with electrospray ionization tandem mass spectrometry and detection was performed by multiple-reaction monitoring. Stable-isotope-labeled 5FU (1,3–15N2–5FU) was used as an internal standard. 5FU was measured within a single analytical run of 16 min with a lower limit of detection of 0.05 μM. The intra-assay variation and inter-assay variation of plasma with added 5FU (1 μM, 10 μM, 100 μM) was less then 6%. Recoveries of the added 5FU in plasma were > 97%. Analysis of the 5FU levels in plasma samples from patients with the HPLC tandem mass spectrometry method and a HPLC-UV method yielded comparable results (r2 = 0.98). Thus, HPLC with electrospray ionization tandem mass spectrometry allows the rapid analysis of 5FU levels in plasma and could, therefore, be used for therapeutic drug monitoring.

No circadian variation of dihydropyrimidine dehydrogenase, uridine phosphorylase, beta-alanine, and 5-fluorouracil during continuous infusion of 5-fluorouracil.

Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases and it catalyses the reduction of thymine and uracil to 5,6-dihydrothymine and 5,6-dihydrouracil, respectively. In mammals, the degradation of uracil by DPD is the only pathway leading to the biosynthesis of β-alanine. Furthermore, DPD is also responsible for the breakdown of the widely used antineoplastic agent 5-fluorouracil (5FU), thereby limiting the efficacy of the therapy. 5FU is one of the few drugs that shows some antitumour activity against various otherwise untreatable tumours including carcinomas of the gastrointestinal tract, breast, ovary and skin. Furthermore, 5FU is one of the few drugs for which a limited clinical effect has been shown when applied as a single agent during the treatment of advanced colorectal cancer. In order to exert its cyto-toxic effect against cancer, 5FU must first be anabolised to the nucleotide level. 5FU can be converted into FUMP by a sequential, two-step reaction consisting of the initial addition of a ribose by uridine phosphorylase (UP) to yield 5-fluorouridine (5FUrd), followed by phos-phorylation to FUMP by uridine kinase. The direct conversion of 5FU to FUMP is catalyzed by orotate phosphoribosyl transferase (OPRT) which transfers the ribose-phosphate moiety from phosphoribosyl pyrophosphate (PRPP) to 5FU. Although the cytotoxic effects of 5FU are probably directly mediated by the anabolic pathways, the catabolic route plays a significant role since more than 80% of the administered 5FU is catabolised by DPD. It has been reported that the bioavailability, efficacy as well as host-toxicity of 5FU follows a circadian rhythm in rodents1 and cancer patients2. In the present study, we investigated whether a circadian variation could be observed of the activity of DPD, UP and the plasma concentration of β-alanine and 5FU in patients treated with continuous infusion of 5FU.

Subcellular localization of dihydropyrimidine dehydrogenase.

Although dihydropyrimidine dehydrogenase (DPD) has been purified and characterized from liver tissues of various mammals conflicting data exist on its subcellular localization. To determine the localization of DPD we prepared crude subcellular fractions of a rat liver homogenate by means of differential centrifugation. In the fractions obtained (heavy mitochondrial, light mitochondrial, microsomal and cytosolic) the activities of different marker enzymes were measured as well as the activity of DPD. These results showed that almost all of the activity of DPD was located in the cytosolic fraction. To exclude any particulate-associated DPD, a light mitochondrial fraction was subsequently subjected to equilibrium density gradient centrifugation. The distribution profile of the activity of DPD and the various marker enzymes indicated that DPD from rat liver was exclusively located in the cytosol since no significant activity of DPD could be detected in any subcellular organelle.