Dihydrothymine Research Articles

Partial protein binding of uracil and thymine affects accurate dihydropyrimidine dehydrogenase (DPD) phenotyping

Fluorouracil is among the most used antimetabolite drugs for the chemotherapeutic treatment of various types of gastrointestinal malignancies. Dihydropyrimidine dehydrogenase (DPYD) genotyping prior to fluorouracil treatment is considered standard practice in most European countries. Yet, current pre-therapeutic DPYD genotyping procedures do not identify all dihydropyrimidine dehydrogenase (DPD)-deficient patients. Alternatively, DPD activity can be estimated by determining the DPD phenotype by quantification of plasma concentrations of the endogenous uracil and thymine concentrations and their respective metabolites dihydrouracil (DHU) and dihydrothymine (DHT). Liquid chromatography - mass spectrometry (LC-MS) detection is currently considered as the most adequate method for quantification of low-molecular weight molecules, although the sample preparation method is highly critical for analytical outcome. It was hypothesized that during protein precipitation, the recovery of the molecule of interest highly depends on the choice of precipitation agent and the extent of protein binding in plasma. In this work, the effect of protein precipitation using acetonitrile (ACN) compared to strong acid perchloric acid (PCA) on the recovery of uracil, thymine, DHU and DHT is demonstrated. Upon the analysis of plasma samples, PCA precipitation showed higher concentrations of uracil and thymine as compared to ACN precipitation. Using ultrafiltration, it was shown that uracil and thymine are significantly (60–65 %) bound to proteins compared to DHU and DHT. This shows that before harmonized cut-off levels of DPD phenotyping can be applied in clinical practice, the analytical methodology requires extensive further optimization.

Quantification of uracil, dihydrouracil, thymine and dihydrothymine for reliable dihydropyrimidine dehydrogenase (DPD) phenotyping critically depend on blood and plasma storage conditions

Establishing dihydropyrimidine dehydrogenase (DPD) activity is highly important in determining the correct starting dose of fluoropyrimidines such as 5-fluorouracil and capecitabine. The concentration ratio of endogenous uracil with its metabolite dihydrouracil (DHU) is a well-known parameter that is linked to DPD activity. Concentration ratios such as thymine over its DPD-converted metabolite dihydrothymine (DHT) is less described and may serve as an alternative diagnostic biomarker for DPD activity.In this study, we describe the development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for the quantification of uracil, DHU, thymine, and DHT in human plasma. In addition, stability experiments were performed.Uracil and thymine were quantified up to 80.0 ng/mL and DHU and DHT up to 800 ng/mL. Intra- and inter-assay precision were maximum 8.0 % and 7.6 %. respectively. Also, recovery was adequate and significant matrix-effects and carry-over were excluded. Stability experiments showed that uracil concentrations increased with 27–52 % when stored for 1 or 2 h at ambient temperatures compared to cold storage. Thymine, DHU, and DHT concentrations remained stable, thymine after 1 h in plasma excluded, showing the DHT:T ratio might be a more robust marker for DPD activity than DHU:U.In conclusion, we present here a novel assay capable of quantifying uracil, thymine, DHU and DHT in a single analytical run. We provide additional data showing increased stability for DHU, thymine and DHT compared to uracil. This assay may be used as a diagnostic test in future studies, establishing the association of these endogenous biomarker concentrations with DPD activity and safety to treatment with fluoropyrimidines. In addition, future research should also be focused on reducing pre-analytical instability. Standardization in this field is essential to set proper reference values and to allow inter-study comparison on clinical outcomes.

A case-control study to assess the ability of the thymine challenge test to predict patients with severe to life threatening fluoropyrimidine-induced gastrointestinal toxicity.

A previous study suggested that a thymine (THY) challenge dose could detect aberrant pharmacokinetics in known cases of fluoropyrimidine toxicity compared with healthy volunteers. The preliminary data suggested that urine sampling also could detect this aberrant disposition. The aim of this case-control study was to assess the ability of the urinary THY challenge test to discriminate cases of severe gastrointestinal toxicity in a cohort of patients treated with 5-fluorouracil or capecitabine. Patients (n=37) received a 250mg (per os) dose of THY and a cumulative urine sample was collected for 0-4h. The urinary amounts of THY and metabolite dihydrothymine (DHT) were determined by liquid chromatography/mass spectrometry. Genomic DNA was analysed for DPYD gene variants. Renal function was estimated from blood creatinine levels. Cases (n=9) and noncases (n=23) of severe (grade≥3) gastrointestinal toxicity were defined based on Common Terminology Criteria for Adverse Events. The median THY/DHT ratios were 6.2 (interquartile range 2.9-6.4) in cases, including the 2 patients who were DPYD heterozygous carriers. However, this was not significantly different (P=.07) from the THY/DHT in noncases (median 2.6, interquartile range 2.8-4.2). Although creatinine clearance was lower (P=.001) in cases, renal function could not discriminate cases from noncases. However, logistic regression analysis using both of these explanatory variables could discriminate most cases (receiver operating characteristic area 0.8792, 95% confidence interval 0.72-1.00). The THY challenge test combined with a patient's renal function may be useful as a phenotypic diagnostic test to detect risk of life-threatening fluoropyrimidine gastrointestinal toxicity.

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

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. 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 β-ureidoisobutyrate. 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. 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.

Simultaneous determination of thymine and its sequential catabolites dihydrothymine and β-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 β-ureidoisobutyric (UIB) acid in human plasma and urine has been developed and validated. Plasma or diluted urine (200μL) was mixed with 1mL of deuterated-THY (internal standard) in acetonitrile, then centrifuged, the supernatant evaporated, and the residue reconstituted in 150μL 0.1% (w/w) formic acid in water. Separation was performed on a Waters Symmetry® C8 column (150mm×3.9mm; 5μm 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 5.12min, 5.17min and 5.00min, respectively. Linearity of the calibrations was established from 2 to 500μg/L. The lower limit of quantification was 5μg/L in plasma, and 10μg/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 250mg THY dose. THY was rapidly absorbed and eliminated with an apparent biphasic log-linear profile. DHT and UIB demonstrated apparent formation-rate limited kinetics.

Biochemical mapping of human NEIL1 DNA glycosylase and AP lyase activities

Base excision repair of oxidized DNA in human cells is initiated by several DNA glycosylases with overlapping substrate specificity. The human endonuclease VIII homologue NEIL1 removes a broad spectrum of oxidized pyrimidine and purine lesions. In this study of NEIL1 we have identified several key residues, located in three loops lining the DNA binding cavity, important for lesion recognition and DNA glycosylase/AP lyase activity for oxidized bases in double-stranded and single-stranded DNA. Single-turnover kinetics of NEIL1 revealed that removal of 5-hydroxycytosine (5-OHC) and 5-hydroxyuracil (5-OHU) is ∼25 and ∼10-fold faster in duplex DNA compared to single-stranded DNA, respectively, and also faster than removal of dihydrothymine (DHT) and dihydrouracil (DHU), both in double-stranded and single-stranded DNA. NEIL1 excised 8-oxoguanine (8-oxoG) only from double-stranded DNA and analysis of site-specific mutants revealed that Met81, Arg119 and Phe120 are essential for removal of 8-oxoG. Further, several arginine and histidine residues located in the loop connecting the two β-strands forming the zincless finger motif and projecting into the DNA major groove, were shown to be imperative for lesion processing for both single- and double-stranded substrates. Trapping experiments of active site mutants revealed that the N-terminal Pro2 and Lys54 can alternate to form a Schiff-base complex between the protein and DNA. Hence, both Pro2 and Lys54 are involved in the AP lyase activity. While wildtype NEIL1 activity almost exclusively generated a δ-elimination product when processing single-stranded substrates, substitution of Lys54 changed this in favor of a β-elimination product. These results suggest that Pro2 and Lys54 are both essential for the concerted action of the β,δ-elimination in NEIL1.

Rapid gas chromatographic–mass spectrometric diagnosis of dihydropyrimidine dehydrogenase deficiency and dihydropyrimidinase deficiency

A rapid yet reliable chemical diagnosis for dihydropyrimidine dehydrogenase (DHPD) deficiency, and possibly dihydropyrimidinase (DHP) deficiency in cancer patients, prior to therapy with pyrimidine analogues such as 5-fluorouracil, is desired for prevention of severe side-effects by these drugs. We have reported the basic separation and quantitation technology for pyrimidine metabolites using gas chromatography–mass spectrometry. A proposal to use the number ( n) of standard deviations (SD) above the normal mean, as the index of the excessive urinary excretion of the metabolites appears not to be commonly used. When used, the values were too small, such as two or three, even in genetic disorders. Here, we applied the method to 11 urine specimens from proven cases including two DHP carriers and proved how specific the method is, because “ n”-values were markedly large for thymine (T), uracil (U) and/or dihydrothymine (DHT) and dihydrouracil (DHU). In three cases with DHPD deficiency, two were siblings, one with symptoms and the other without, n was 12 for T and 5.9 for U, and 5-hydroxymethyluracil was distinctly detected. These values indicate that the nature of genetic mutation relates closely to the degree of metabolite accumulation in pyrimidine disorders. In six patients with DHP deficiency, n was 8.4–12 for DHT and 7.2–11 for DHU. Many mutations are known for both genes and the assay of residual enzyme activity may be time-consuming or invasive especially for those with DHP deficiency. Thus, this noninvasive yet comprehensive urinalysis has great value for those without a family history, as the first trial, before DNA or the enzyme assay. Our findings again raise the question whether the metabolic block really causes the symptoms found in pyrimidine disorders.

Recognition and kinetics for excision of a base lesion within clustered DNA damage by the Escherichia coli proteins Fpg and Nth.

Ionizing radiation and radiomimetic anticancer agents induce clustered DNA damages that are thought to lead to deleterious biological consequences, due to the challenge that clustered damage may present to the repair machinery of the cell. Specific oligonucleotides, containing either dihydrothymine (DHT) or 7,8-dihydro-8-oxoguanine (8-oxoG) opposite to specific lesions at defined positions on the complementary strand, have been used to determine the kinetic constants, K(M), k(cat), and specificity constants, for excision of DHT and 8-oxoG by the Escherichia coli base excision repair proteins, endonuclease III (Nth) and formamidopyrimidine glycosylase (Fpg), respectively. For excision of DHT opposite to 8-oxoadenine by Nth or Fpg proteins, or 8-oxoG opposite to 8-oxoG by Fpg, the major change in the specificity constant occurs when the second lesion on the complementary strand is one base to the site opposite to DHT or 8-oxoG. The specificity constants for excision of DHT or 8-oxoG by both proteins are reduced by up to 2 orders of magnitude when an abasic site or a strand break is opposite on the complementary strand. Whereas the values of K(M) are only slightly affected by the presence of a second lesion, the major change is seen as a reduction in the values of k(cat). The binding of Fpg protein to oligonucleotides containing 8-oxoG is inhibited, particularly when a single strand break is near to 8-oxoG on the complementary strand. It is inferred that not only the binding affinity of Fpg protein to the base lesion but also the rate of excision of the damaged base is reduced by the presence of another lesion, particularly a single strand break or an AP site on the complementary strand.

Clustered DNA Damage, Influence on Damage Excision by XRS5 Nuclear Extracts and Escherichia coli Nth and Fpg Proteins

Ionizing radiation and radiomimetic anticancer agents induce clustered DNA damage, which are thought to reflect the biological severity. Escherichia coli Nth and Fpg and nuclear extracts from XRS5, a Chinese hamster ovary Ku-deficient cell line, have been used to study the influence on their substrate recognition by the presence of a neighboring damage or an abasic site on the opposite strand, as models of clustered DNA damage. These proteins were tested for their efficiency to induce a single-strand break on a (32)P-labeled oligonucleotide containing either an abasic (AP) site, dihydrothymine (DHT), 7,8-dihydro-8-oxo-2'deoxyguanine, or 7, 8-dihydro-8-oxo-2'deoxyadenine at positions 1, 3, or 5 base pairs 5' or 3' to either an AP site or DHT on the labeled strand. DHT excision is much more affected than cleavage of an AP site by the presence of other damage. The effect on DHT excision is greatest with a neighboring AP site, with the effect being asymmetric with Nth and Fpg. Therefore, this large inhibition of the excision of DHT by the presence of an opposite AP site may minimize the formation of double-strand breaks in the processing of DNA clustered damages.

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.

Absence of Pyrimidine Salvage and Prevention of Thymineless Radiosensitization in Escherichia coli thyA Cells Fed Dihydrothymine or Thymine Glycol

Little information is available concerning the metabolic fate of radiation-induced thymine base damage products once they have been excised from DNA. The present study was an attempt to determine whether or not thymine-requiring mutants of Escherichia coli could grow on dihydrothymine (DHT) and thymine glycol (TG) by "salvaging" the altered thymines. A second test of thymine product utilization was prevention of thymineless radiosensitization. Results showed that very low growth of Thy- cells on DHT or TG could be explained by the presence of less than or equal to 1% contaminating thymine in the mixtures. Radiation dose-modification factors (DMFs) for thyA cells fed DHT or TG for 3 h were 1.38 +/- 0.28 and 1.26 +/- 0.24, respectively, whereas the DMF for 3 h thymine-starved cells was 1.63 +/- 0.05. The small (approximately 25%) amelioration of thymineless radiosensitization observed in DHT- or TG-fed cells could probably be explained by contaminating thymine in the medium. Although DHT is a normal metabolite in some cells, neither DHT nor TG could be used efficiently by thymine-requiring cells in the protocol presented.

Degradation of pyrimidines in Euglena gracills III. Ratio of uracil to thymine degradation

In photoorganotrophically-grown cells of Euglena gracilis var. Z uracil (Ura) and thymine (Thy) were degraded in a fixed ratio of 1 : 4. The predominant degradation of Thy can be measured in vivo by the kinetics of Thy and Ura degradation, by the effect of catabolic intermediates at different concentrations and by double labelling experiments with [5− 3H]Ura and [2− 14C]Thy. In the latter type of experiment, 3H/ 14C of isolated dihydrouracil (DHUra) and dihydrothymine (DHThy) is respectively 1 10 of the starting promortion of precursors, suggesting a predominant degradation of Thy. Enhanced anabolic reactions of pyrimidine compounds can be produced by inhibition of degradation with dihydropyrimidines.

ESR of free-radical conversion in gamma-irradiated dihydrothymine.

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Thymine and Uracil Metabolism in the Epidermis

As compared with the viable epidermis, the stratum corneum contains very small amounts of both DNA and RNA (1, 2, 3, 4). This decrease in nucleic acid content is correlated with the disappearance of nuclei and ribosomes which occurs at the junction of the granular layer and stratum corneum. Enzymes necessary for the hydrolysis of these cellular organelles have been found in the epidermis (5, 6, 7, 8, 9), but the purine and pyrimidine bases which are major components of nucleic acids are either absent or are present in trace quantities (1, 4). Some purine metabolites have been found in the stratum corneum (1, 4), but no data is available on the pyrimidines. Since certain disorders of the epidermis such as psoriasis are associated with abnormalities of nucleic acid turnover (10, 11, 12,13), it was felt that more detailed information on the metabolism of pyrimidines by the epidermis would be of importance. The purpose of this report is to describe the fate of thymidine and uridine incorporated into epidermal nucleic acids in vwo} and to investigate the catabolism of their respective pyrimidine bases by epidermis in vitro. MATERIALS Methyl tritiated thymidine (8 c/mmole), thymine-2-C14 (II mc/mmole) and uridine-2-C14 (25 mc/mmole) were purchased from New England Nuclear Corp., and dihydrothymine (DHT), dihydrouracil (DHU), B-alanine, and B-aminoiso-butyric acid (BAIB) from the California Biochemical Corp. The B-ureidoisobutyric (BUIB) and B-ureidopropionic acid (BUP) were prepared as described by Fink et al (14).

The excretion of beta-aminoisobutyric acid following thymine and dihydrothymine administration to man.

SUMMARYA study of the excretion of β‐aminoisobutyric acid (BAIB) after oral administration of thymine and of dihydrothymine (DHT) to a series of human subjects including normal, splenectomised and leukemic individuals, suggests that the spleen may influence this excretion. The leukemic patients studied differ from normal in their lower excretion of BAIB after DHT ingestion.

A metabolic investigation of urinary β-aminoisobutyric acid excretion in man

Comparative effects of various orally administered β-aminoisobutyric acid (BAIB) precursors on urinary BAIB excretion in genetically determined high and low excretors were studied in man. Orally administered thymine gives rise to more urinary BAIB than equal amounts of dihydrothymine (DHT) or β-ureidoisobutyric acid (BUIB) which metabolically are closer to BAIB (thymine DHT ⇒ 4 BUIB → BAIB). However, this phenomenon is shown to be due to the poorer in vivo uptake of DHT and BUIB than thymine. At low levels of BAIB precursors (thymine, DHT, and BUIB), a marked difference in the urinary BAIB excretion rate occurs between low and high excretors; whereas, at high levels, this difference is almost eliminated. The data rule out the possibility of this difference resulting from a metabolic block in the thymine → BAIB pathway in low excretors. Several other possible explanations are pointed out. Comparative aspects of the thymine → BAIB pathway in man, rat, and the mouse are discussed. Although this pathway is functional in all three species, marked differences exist which serve to explain why urinary BAIB is found normally only in man. Experiments with other test substances (thymidine, cytosine, orotic acid, thiamine, and valine) indicate that thymine is probably the only significant source of urinary BAIB in man.