Preliminary Evidence for Enhanced Thymine Absorption: A Putative New Phenotype Associated With Fluoropyrimidine Toxicity in Cancer Patients.

BACKGROUND: Chemotherapy for colorectal, head and neck, and breast cancer continues to rely heavily on 5-fluorouracil and its oral prodrug capecitabine. Associations of serious fluoropyrimidine adverse effects have focused on inherited deficiency of the catabolic enzyme, dihydropyrimidine dehydrogenase. However, abnormal dihydropyrimidine dehydrogenase activity accounts for only about one-third of observed toxicity cases. Thus, the cause of most fluorouracil toxicity cases remains unexplained. METHODS: For this small cohort study, thymine (THY) 250 mg was administered orally to 6 patients who had experienced severe toxicity during treatment with 5FU or capecitabine. Plasma and urine were analyzed for THY and its catabolites dihydrothymine (DHT) and ß-ureidoisobutyrate. RESULTS: Of the 6 patients, 2 had decreased THY elimination and raised urinary THY recovery consistent with inherited partial dihydropyrimidine dehydrogenase deficiency, confirmed by DPYD sequencing. Unexpectedly, 3 patients displayed grossly raised plasma THY concentrations but normal elimination profiles (compared with a normal range for healthy volunteers previously published by the authors). DPYD and DPYS sequencing of these 3 patients did not reveal any significant loss-of-activity allelic variants. The authors labeled the phenotype in these 3 patients as "enhanced thymine absorption". Only 1 of the 6 cases of toxicity had a normal postdose plasma profile for THY and its catabolites. Postdose urine collections from all 6 patients had THY/DHT urinary ratios above 4.0, clearly separated from the ratios in healthy subjects that were all below 3.0. CONCLUSIONS: This small cohort provided evidence for a hypothesis that fluorouracil toxicity cases may include a previously undescribed pyrimidine absorption variant, "enhanced thymine absorption," and elevated THY/DHT ratios in urine may predict fluorouracil toxicity. A prospective study is currently being conducted.

A neonate with recurrent vomiting and generalized hypotonia diagnosed with a deficiency of dihydropyrimidine dehydrogenase.

Deficiency of dihydropyrimidine dehydrogenase (DPD) is a rare inborn error of pyrimidine metabolism. To date, only about 50 patients are known worldwide. The clinical picture is varied and is not yet fully described. Most patients are diagnosed at the age of 1-3 years. We present a patient diagnosed 8 weeks postpartum. The female patient presented in the first 3 days after birth with agitation, choking, and vomiting. Six weeks later, the patient presented again with vomiting and insufficient weight gain. Metabolic screening of urine showed a strongly increased excretion of uracil and thymine, with no other abnormalities. This suggested a deficiency of DPD which was confirmed by enzyme analysis in peripheral blood mononucleair (PBM) cells (patient: activity <0.01 nmol/mg/h; controls: 9.9 +/- 2.8 nmol/mg/h). The patient was homozygous for the IVS14+1G>A mutation.MRI of the brain showed some cerebral atrophy; myelinization appeared normal. Many patients with DPD-deficiency suffer from convulsions and mental retardation, some show microcephaly, feeding difficulties, autism, and hypertonia. Our patient showed feeding difficulties and in the second half-year she developed slight motor retardation and generalized hypotonia. Further observation of the development of the patient may shed more light on the relationship between clinical symptoms and DPD deficiency. DPD deficiency may present in newborns with vomiting and hypotonia as the main symptoms.

Metabolism of deoxycytidine, thymine, and deoxythymidine in the hamster.

The ability of growing and of mature Syrian hamsters to anabolize (to liver DNA) or catabolize (to 14CO2) graded amounts of [2-14C]deoxythymidine (TdR), thymine, or deoxycytidine (CdR) was measured in vivo. Of the three precursors, CdR labeled DNA most efficiently and, as expected, incorporation of all three into DNA was greater in younger animals. The catabolism of [2-14C]CdR to respired 14CO2 was dose dependent and showed no signs whatsoever of saturation, even with the highest dose (greater than 20 mumoles/g liver). In contrast, TdR and thymine were catabolized more slowly and saturation was approached with modest doses. The excretion of CdR in the urine was low and independent of dose, while excretion of TdR and thymine was greater and was dose dependent. Rats tested with an intermediate dose of CdR did not catabolize significant quantities to 14CO2, but did excrete considerably more [C]CdR into the urine than did hamsters. These and other findings suggest that, while the rat and the hamster metabolize thymine (and TdR as well) in a similar fashion, they metabolize CdR quite differently, probably because the hamster has a much higher level of nucleoside aminohydrolase which deaminates CdR and related compounds. Because the human also has a very high level of this enzyme, the hamster appears to be a superior animal model for the study of cytosine-containing compounds intended for human use.

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

The 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 250mg thymine in capsule form. Blood, urine and saliva samples were collected pre-dose and up to 5h post-dose. Concentrations of thymine, and its catabolites dihydrothymine and ß-ureidoisobutyrate were analysed by HPLC-tandem mass spectrometry in plasma, urine and saliva. The pharmacokinetic data of healthy volunteers were analysed assuming a non-compartmental model. Thymine peaked quickly (30-45min) in plasma to a maximum concentration of 170±185µg/L (mean±SD). Clearance was high (mean 57.9L/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.

Lack of correlation between phenotype and genotype for the polymorphically expressed dihydropyrimidine dehydrogenase in a family of Pakistani origin.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in pyrimidine catabolism. DPD deficiency is associated with an increased risk of toxicity in cancer patients receiving 5-fluorouracil (5-PU) treatment. DPD deficiency causes an inborn error of metabolism called thymine-uraciluria that is in some instances associated with convulsive disorders and developmental delay in children. We have studied the molecular mechanism accounting for DPD deficiency in a Pakistani pedigree having 2-year-old child with thymine-uraciluria and exhibiting some degree of motor impairment and developmental delay. A common splice mutation was found in the patient's dihydropyrimidine dehydrogenase (DPYD) gene that produces a mutant mRNA resulting in the complete lack of DPD protein and activity in lymphocytes and primary fibroblast. This trait segregated in the family following a typical Mendelian distribution. Surprisingly, the patient's brother also had thymine-uraciluria and was homozygous for the splicing mutation but was clinically asymptomatic. Sequence tagged sites (STS) linkage analyses within 5 megabases of telomeric and centromeric DNA surrounding the DPYD gene revealed no allelic polymorphism between the two brothers. These results suggest that DPD deficiency might not be the only cause of the more severe clinical phenotypes observed in certain thymine-uraciluria patients and that an incomplete correlation between phenotype and genotype is present in the population.

Population and family studies of dihydropyrimidinuria: prevalence, inheritance mode, and risk of fluorouracil toxicity.

To evaluate the prevalence of dihydropyrimidinuria (DHPuria), we analyzed urine samples from 21,200 healthy Japanese infants, and found two cases of DHPuria without clinical symptoms. Based on this result, we estimated the prevalence to be approximately 1/10,000 births in Japan. In addition, we analyzed pyrimidine catabolism on a previously reported family with an adult DHPuria case. We newly identified the sister of the propositus as the second case of DHPuria in this family, because she excreted large amounts of dihydrouracil and dihydrothymine. The parents and the child of the propositus showed slight increases of dihydrouracil and dihydrothymine. This is the first family with 2 cases of DHPuria, indicating that DHPuria is an inherited condition. To determine the inheritance of DHPuria in this family and to examine the risk of 5-fluorouracil (5-FU) toxicity, a uracil loading test was performed on the parents. Urinary dihydrouracil concentrations in the parents after the loading were several times higher than those in normal control persons, the finding being consistent with DHPuria heterozygotes. This, along with data on the propositus, his sister, and his child, indicates that DHPuria is an autosomal recessive condition. In addition, DHPuria homozygotes may have a high risk of 5-FU toxicity, while the risk is relatively low in heterozygotes.

Human infections with thymine-requiring bacteria.

Clinical details are presented of 16 patients from whom thymine-requiring (thy-) mutants of pathogenic organisms were isolated; all had been treated with co-trimoxazole. The urine of six patients infected with thy- mutants contained levels of a thymine-like compound sufficient to support their growth. This might be the result either of the breakdown of pus cells or of thymine production by living bacteria that persist in stones or scar tissue, a suggestion supported by the observation of mutant growth "in satellitism" in vitro. Since 1975 we have isolated mutants from patients who have had short courses of co-trimoxazole, in contrast to those we reported upon previously, all except one of whom had had long courses. We are now isolating thy- mutants more frequently than hitherto. Secondary mutations to a low thymine requirement may now be occurring more rapidly, thereby allowing more mutant organisms to survive. The clinical significance of infection with thy- mutants is not yet clear, but evidence is accumulating that they are pathogenic. Alternative chemotherapy is suggested for patients from whom such mutants have been isolated.

beta-Aminoisobutyric acid as a marker of thymine catabolism in malignancy.

Urine from untreated patients with various tumours and controls has been examined for the excretion of beta-aminoisobutyric acid and uric acid. The patients were classified into four groups: I, beta-aminoisobutyric acid and uric acid both normal; II, beta-aminoisobutyric acid normal, uric acid elevated; III, beta-aminoisobutyric acid elevated, uric acid normal; IV, beta-aminoisobutyric acid and uric acid both elevated. Uric acid was used as an indicator for tissue-breakdown. Pseudouridine being a specific parameter for t-RNA degradation was estimated for comparison. Increased urinary concentrations of beta-aminoisobutyric acid were frequently found in tumour patients, especially in patients with leukaemia and non-Hodgkin lymphoma. Tissue breakdown being the cause of the beta-aminoisobutyric aciduria could only be considered in part of the patients. Moreover, strongly elevated ratios of beta-aminoisobutyric acid to uric acid were found. Urinary patterns of pyrimidines and purines were determined in order to exclude other abnormalities. The results are discussed in relation to thymine metabolism and renal function.

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.

Development and characterization of a monoclonal antibody with cross-reactivity towards uracil and thymine, and its potential use in screening patients treated with 5-fluorouracil for possible risks.

BACKGROUND: Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in catabolism of pyrimidines including 5-fluorouracil. There have been efforts to isolate a monoclonal antibody that will bind selectively to pyrimidine and can be used to measure the concentration of pyrimidine in blood and/or in urine that may reflect the activity of dihydropyrimidine dehydrogenase. However, the monoclonal antibodies selective to pyrimidine have not been available. METHODS: Using 1-carboxymethyl-uracil as a hapten, in which steric conformation of uracil is thought to be well maintained, extensive screening was done to isolate a monoclonal antibody specific to uracil. RESULTS: We established the first monoclonal antibody that reacted with uracil and thymine but not with pseudouridine, dihydrouracil, dihydrothymine, cytosine, uridine, or N-carbamyl-beta-alanine at the concentration of 100 microg/ml. CONCLUSIONS: The monoclonal antibody can be used to develop a simple screening assay for patients with dihydropyrimidine dehydrogenase deficiency. This may increase the safety of 5-fluorouracil treatment.

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 patient's cells a complete deficiency of dihydrothymine dehydrogenase activity was found. Our patient is the first case described with such a proven enzyme deficiency.

Possible prediction of adverse reactions to fluorouracil by the measurement of urinary dihydrothymine and thymine.

Dihydropyrimidine dehydrogenase (DPD) deficiency with a defect of the pyrimidine catabolic pathway has recently become the focus of considerable attention, due to the severe 5-fluorouracil (5-FU) toxicities occurring in DPD deficiency patients. Studies also suggest that 5-FU toxicities could occur in another pyrimidine metabolic disorder, dihydropyrimidinuria (DHPuria). This study shows that urinary dihydrothymine (DHT) and thymine (THY) are useful indexes for detection of DPD deficiency and DHPuria. We measured urinary DHT and THY in 276 Japanese adults to establish reference ranges. When males and females were compared, both DHT and THY levels were found to be significantly higher in females. The reference ranges (mean +/- SD with logarithmic values) for males were found to be 1.56-5.70 micromol/g of creatinine for DHT and 0.40-1.47 micromol/g of creatinine for THY. The reference ranges for females were found to be 1.89-8.33 micromol/g of creatinine for DHT and 0.58-2.30 micromol/g of creatinine for THY. In addition to this study we analyzed a DPD deficiency case and a DHPuria case. In the DPD deficiency case, the THY concentrations of all urine samples were out of the reference range. However, uracil levels in most of the samples were within the normal range. The DHPuria case excreted large amounts of DHT and dihydrouracil, both out of the normal range.

Dietary supplement of G-rutin reduces oxidative damage in the rodent model.

Dietary supplement of bioflavonoid rutin and calorie restriction were examined to investigate possible reduction in oxidative DNA damage, protein oxidation, and lipid peroxidation in animals. Rats were fed ad libitum 20% casein semipurified diet or a diet that was supplemented by 2.5% water soluble rutin derivative, 4(G)-alpha-glucopyranosylrutin (G-rutin) for 18 days. Two other groups of rats were fed the respective diets at 60% of the mean food intake of the ad libitum fed animals for the same period. Urinary excretion of thymine glycol and thymidine glycol, indices of DNA base damage in the whole body, were significantly low in the G-rutin-supplemented groups. The protein carbonyl contents, a measure of protein oxidation, were significantly low in the liver of G-rutin-supplemented groups and calorie-restricted groups. The data indicate that G-rutin provides an antioxidant defense in rodents against free radical-caused oxidative damage of DNA and proteins.

A screening method for dihydropyrimidine dehydrogenase deficiency with colorimetric detection of urinary uracil.

Dihydropyrimidine dehydrogenase deficiency has a neurological involvement as a common symptom among reported cases. No major symptom except that exists for DHPDH deficiency. On the other hand, relationship between neurological involvement and metabolic disorder is still obscure. For the purpose of looking for more patients with DHPDH deficiency, a screening method for DHPDH deficiency is introduced. Urinary uracil was determined colorimetrically. This method is not so complicated and less time consuming as previous method such as liquid chromatography. With this method, it is possible to detect about 1 mmol/l (12 mg/dl) of uracil, which is sensitive enough for the screening for DHPDH deficiency. Interfering substance in urine were negligible. Addition of albumin to normal urine dose not affect the result but proteinuria results in false positive. The urine from 83 epileptic children were screened with this method, but no patients were found.

Diagnosis of dihydropyrimidine dehydrogenase deficiency in a neonate with thymine-uraciluria.

Dihydropyrimidine dehydrogenase deficiency is an inborn error of pyrimidine metabolism characterised by thymine-uraciluria, convulsive disorders and developmental delay in paediatric patients, and an increased risk of toxicity from 5-fluorouracil treatment. This report is of the first patient with dihydropyrimidine dehydrogenase deficiency diagnosed in Hong Kong. The patient was a 2-day-old male neonate of Pakistani origin who presented with convulsions. Diagnosis was made by gas chromatographic-mass spectrometric detection of thymine-uraciluria and by molecular detection of a G to A point mutation in a 5'-splicing site leading to skipping of exon 14 in the DPYD gene of chromosome location 1q22. The results showed that the patient and his mother were homozygous and the father heterozygous for the splice site mutation. The mother also had thymine-uraciluria but was clinically asymptomatic.

[A case of gastric cancer with decreased dihydropyrimidine dehydrogenase activity].

We analyzed the pyrimidine metabolite in the urine of a patient with severe mucositis and hand and foot syndrome, who was administered 5-fluorouracil for recurrence of gastric cancer. From our analysis, it was suggested that the patient had decreased dihydropyrimidine dehydrogenase activity. Dihydropyrimidine dehydrogenase activity is usually measured in peripheral blood mononuclear cells, but this time it was estimated from the analysis of uracil, dihydrouracil, thymine, and dihydrothymine in the urine. We concluded that urinary analysis of the pyrimidine metabolism is effective as screening for the prediction and prevention of 5-fluorouracil toxicity.

Simultaneous determination of thymine and its sequential catabolites dihydrothymine and ß-ureidoisobutyrate in human plasma and urine using liquid chromatography-tandem mass spectrometry with pharmacokinetic application.

To study in vivo activities of dihydropyrimidine dehydrogenase, dihydropyrimidinase, and ureidoproprionase, a sensitive, accurate, selective and precise method for the determination of the endogenous pyrimidine thymine (THY) and its successive metabolites dihydrothymine (DHT) and ß-ureidoisobutyric (UIB) acid in human plasma and urine has been developed and validated. Plasma or diluted urine (200 µL) was mixed with 1 mL of deuterated-THY (internal standard) in acetonitrile, then centrifuged, the supernatant evaporated, and the residue reconstituted in 150 µL 0.1% (w/w) formic acid in water. Separation was performed on a Waters Symmetry C8 column (150 mm × 3.9 mm; 5 µm particle size), with gradient elution using a mobile phase of 0.1% (w/w) formic acid in water (phase A), and 15% (v/v) methanol in acetonitrile (phase B). The detection system was an Applied Biosystems model 3200 tandem mass spectrometer with atmospheric pressure chemical ionisation, and multiple reaction monitoring mode using the transitions: THY (m/z: 127.1-110.0), DHT (m/z: 129.1-68.9), UIB (m/z: 147.1-86.0), and deuterated-THY (m/z: 131.1-114.0). THY, DHT, and UIB eluted at 5.12 min, 5.17 min and 5.00 min, respectively. Linearity of the calibrations was established from 2 to 500 µg/L. The lower limit of quantification was 5 µg/L in plasma, and 10 µg/L in urine for THY, DHT and UIB. Ion-suppression had negligible effect. A pilot pharmacokinetic study analysed plasmas and urines from 2 healthy male subjects who each received an oral 250 mg THY dose. THY was rapidly absorbed and eliminated with an apparent biphasic log-linear profile. DHT and UIB demonstrated apparent formation-rate limited kinetics.

A mild phenotype of dihydropyrimidine dehydrogenase deficiency and developmental retardation associated with a missense mutation affecting cofactor binding.

OBJECTIVES: Evaluation of a non-synonymous mutation associated with dihydropyrimidine dehydrogenase (DPD) deficiency. DESIGN AND METHODS: DPD enzyme analysis, mutation analysis and molecular dynamics simulations based on the 3D-model of DPD. RESULTS: The substitution Lys63Glu is likely to affect the FAD binding pocket within the DPD protein and contributes to a near-complete DPD deficiency in a patient with developmental retardation. CONCLUSIONS: Like other DPD variants attenuating FAD binding, Lys63Glu should be included in screening for DPD deficiency.