Pyrimidine catabolism: individual characterization of the three sequential enzymes with a new assay

Traut, Thomas W.; Loechel, Steve

doi:10.1021/bi00306a033

Cited by 72 publications

(40 citation statements)

References 28 publications

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“…Although human liver dihydropyrimidinase has not been purified, much lower Vmax/Km values for dihydrouracil and dihydrothymine were reported by Kikugawa and coworkers (1994) for purified rat liver, and by Brooks and coworkers (1983) for bovine liver dihydropyrimidinase (65 and 36 L/min/mg, respectively). Given the constant bodily flux of these pyrimidines through the catabolic pathway, it can be predicted that the dihydropyrimidine form will be constantly elevated because dihydropyrimidinase, rather than DPD, acts as the rate-limiting enzyme due to its lower substrate affinity and efficiency; this conclusion has been supported by others (Traut and Loechel, 1984;Naguib et al, 1985).…”

Section: Accepted M Manuscriptmentioning

confidence: 89%

Towards a test to predict 5-fluorouracil toxicity: Pharmacokinetic data for thymine and two sequential metabolites following oral thymine administration to healthy adult males

Duley

Shannon

et al. 2016

European Journal of Pharmaceutical Sciences

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a test to predict 5-fluorouracil toxicity: Pharmacokinetic data for thymine and two sequential metabolites following oral thymine administration to healthy adult males, (2015), doi: 10.1016/j.ejps.2015.10.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E DM A N U S C R I P T A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT2 AbstractThe fluoropyrimidine drugs 5-fluorouracil and its oral prodrug capecitabine remain first line therapy for solid tumours of the neck, breast and colon. However, significant and unpredictable toxicity affects about 10-25% of patients depending upon the mode of 5-fluorouracil delivery. The pharmacokinetics of thymine (5-methyluracil) may provide an approach for screening for 5-fluorouracil toxicity, based on the rationale that thymine is a close structural analogue of 5-fluorouracil and is catabolized by the same enzymatic pathway.Oral thymine loading tests were performed on 12 healthy volunteers. Each subject was given a single oral dose of 250 mg thymine in capsule form. Blood, urine and saliva samples were L/h/kg) exceeding normal human liver blood flow, suggesting low systemic bioavailability; urinary recovery of the thymine dose was low (<1%). Apparent formation rate-limited kinetics were observed for dihydrothymine, and the plasma concentration of dihydrothymine was consistently 10-fold higher than that of thymine. Plasma ß-ureidoisobutyrate concentrations, on the other hand, were similar to that of thymine. Genotyping confirmed that pathological mutations of the DPYD gene were absent. The urinary excretion ratio of thymine/dihydrothymine was informative of the maximum concentration. Saliva thymine was highly variable. These data are potentially useful as a basis for developing of a screening procedure to prospectively identify patients who are at risk of toxicity from fluoropyrimidine drugs.

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Section: Accepted M Manuscriptmentioning

confidence: 89%

Towards a test to predict 5-fluorouracil toxicity: Pharmacokinetic data for thymine and two sequential metabolites following oral thymine administration to healthy adult males

Duley

Shannon

et al. 2016

European Journal of Pharmaceutical Sciences

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“…Traut and Loechel [36] found that the concentration of H,Ura and N-carbamoyl-Palanine was 4 pM and 11 pM, respectively, at the steady state. If we adopt the K value of 55 at pH 7.0 (Table 2), the value of U -~/ U +~ is calculated to be 0.05.…”

Section: Discussionmentioning

confidence: 99%

Purification, characterization and inhibition of dihydropyrimidinase from rat liver

Kikugawa

Kaneko

Fujimoto-Sakata

et al. 1994

European Journal of Biochemistry

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Dihydropyrimidinase (DHPase) was purified 564-fold over the initial rat liver extract, using heat, ammonium sulfate fractionation, DEAE-Sepharose CL-6 B, carboxymethyl-Sepharose CLQB, hydroxyapatite and Sephacryl S-300 chromatography. The purified enzyme was shown to be homogeneous by gel electrophoresis both in the presence and absence of SDS. Its molecular mass, determined by gel filtration, was 215 kDa and the subunit mass was 54 kDa. DHPase catalyzed the reversible cyclization of 5,6-dihydrouracil (HJJra) to N-carbamoyl-P-alanine or 5,6-dihydrothymine (H,Thy) to N-carbamoyl-P-aminoisobutync acid.Authentic 5-bromo-5,6-dihydrouracil (BrH,Ura) and commercially available H,Thy were racemic. However, these 5-substituted 5,6-dihydropyrimidines were hydrolyzed by over 96% and 9 8%, respectively, by DHPase. These results suggest that dihydropyrimidinase has no stereo specificities for 5-substituents of H,Ura. The addition of H,Ura and H,Thy competitively inhibited the enzyme activity against BrH,Ura. However, the addition of N-carbamoyl-P-alanine or N-carbamoyl-j?-aminoisobutyric acid showed hyperbolic mixed-type inhibition, when BrH,Ura was used as the substrate. The values of the dissociation constants of BrH,Ura, N-carbamoyl-P-alanine and N-carbamoyl-Paminoisobutyric acid were 17 pM, 0.38 mh4 and 0.38 mM, respectively.DHPase from the rat liver contains 4 mol Zn2+/mol active enzyme, presumably one atomhubunit. Zn2+ also inhibited the hydrolysis of BrH,Ura by the enzyme. The K, for Zn" as an inhibitor of DHPase was 23 pM, and the maximum rate of inactivation was 0.057 min-' at 37OC. HJJra and H,Thy protected the enzyme activity from Zn2+ inactivation.Uracil is metabolized to P-alanine via 5,6-dihydrouracil (HJJra) and N-carbamoyl-P-alanine [l, 21. The enzymes of uracil catabolism are also active toward thymine, metabolizing it via dihydrothymine (H,Thy) and N-carbamoyl-Paminoisobutyric acid to (R)-P-aminoisobutyric acid [l, 3, 41. In mammals, P-alanine and (R)-P-aminoisobutyric acid are transported into mitochondria [5, 61, where they are further metabolized to acetyl-CoA and propionyl-CoA, respectively, by P-alanine-oxoglutarate aminotransferase, (R)-P-aminoCorrespondence to N. Tamaki, Laboratory of Nutritional Chemistry, Faculty of Nutrition, Kobe-Chin University, Nishi-ku, Kobe 651-21, JapanAbbreviations. DHPase, dihydropyrimidinase ; BrH,Ura, 5-bromo-5,6-dihydrouil: H,Ura, 5,6-dihydrouracil; H,Thy, 5,6-dihydrothymine.Enzymes. Dihydropyrimidinase, 5,6-dihydropyrimidine amidohydrolase (EC 3.5.2.2); 4-aminobutyrate aminotransferase, P-alanine-oxoglutarate aminotransferase (EC 2.6.1.19) ; (R)-3-amino-2-methylpropionate-pyruvate aminotransferase, ~-3-aminoisobutyrate-pyruvate aminotransferase (EC 2.6.1.40) ; methylmalonate semialdehyde dehydrogenase (EC 1.2.1.27) ; dihydropyrimidine dehydrogenase (EC 1.3.1 .l); carbamoyl-phosphate synthetase (EC 6.3.5.5) ; aspartate transcarbamoylase (EC 2.1.3.2) ; dihydroorotase (EC 3.5.2.3); RNA nucleotidyltransferase (EC 2.7.7.6) ; pyruvate kinase (EC 2.7.1.40); lactate dehydroge...

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“…The spectrophotometric methods for DPD is adequate for tissues with high enzymatic activity, but not sensitive enough for tissues with low enzymatic activities. The utiliza-80% suMval tion of radiolabeled substrates coupled to the separation of substrate and products by TLC is considerably more sensitive (18), however this chromatographic technique is much less efficient than HPLC. In this investigation, we have used radiolabeled thymine as the substrate, as opposed to radiolabeled uracil which was previously employed in the TLC method.…”

Section: Discussionmentioning

confidence: 99%

Pyrimidine base degradation in cultured murine C-1300 neuroblastoma cells and in situ tumors.

Tuchman

O’Dea²,

Ramnaraine³

et al. 1988

J. Clin. Invest.

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Dihydropyrimidine dehydrogenase (DPD), the initial, ratelimiting step in pyrimidine degradation, was studied in two cell lines of murine neuroblastoma (MNB-T1 and MNB-T2) that were derived from C-1300 MNB tumor carried in A/J mice. The MNB-T2 (low malignancy) cell line was originally derived from the in situ tumor and carried in tissue culture for more than 100 passages; the MNB-T1 (high malignancy) line consisted of a new sub-culture that was also established from the in situ MNB tumor. DPD activity was determined in cytosolic preparations of MNB utilizing high performance liquid chromatography to separate the radiolabeled substrate (12-'4Cjthy-mine) from [2-'4Cjdihydrothymine. The apparent affinity of DPD for NADPH in MNB cells (Km -0.08 mM) was identical to that of A/J mouse brain and liver. The DPD activity of the high malignancy (MNB-T1) cell line was 14.3% of that observed in the low malignancy (MNB-T2) line. In situ tumors formed after implantation of high malignancy (MNB-T1) cells into A/J mice had only 25.2% of the DPD activity observed in tumors derived from low malignancy (MNB-T2) cells. When MNB-T2 cells were injected into naive A/J mice, tumors developed in only 68% of animals, the tumor growth rate was slow and a mortality of 20% was observed. In contrast, tumors derived from injected MNB-T1 cells showed a faster growth rate and 100% mortality. Most MNB-T2 derived tumors were not lethal and ultimately resolved while the MNB-T1 derived tumors were invariably lethal. These studies support the concept that the levels of DPD activity in neoplastic cells are inversely related to their malignant expression and also provide a model to study differences between neuroblastoma cell lines derived from the same in situ tumor but which manifest different neoplastic behavior.

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Pyrimidine catabolism: individual characterization of the three sequential enzymes with a new assay

Cited by 72 publications

References 28 publications

Towards a test to predict 5-fluorouracil toxicity: Pharmacokinetic data for thymine and two sequential metabolites following oral thymine administration to healthy adult males

Towards a test to predict 5-fluorouracil toxicity: Pharmacokinetic data for thymine and two sequential metabolites following oral thymine administration to healthy adult males

Purification, characterization and inhibition of dihydropyrimidinase from rat liver

Pyrimidine base degradation in cultured murine C-1300 neuroblastoma cells and in situ tumors.

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