Genetic analysis of the first 4 patients with beta-ureidopropionase deficiency.

beta-Ureidopropionase is the third enzyme of the pyrimidine degradation pathway and it catalyses the irreversible hydrolysis of N-carbamyl-ss-aminoisobutyric acid or N-carbamyl-ss-alanine to beta-aminoisobutyric acid or ss-alanine, ammonia, and CO2. Analysis of the beta-ureidopropionase gene (UPB1) of the first 4 patients presenting with a complete enzyme deficiency, revealed the presence of 2 splice-site mutations (IVS1-2A>G and IVS8-1G>A) and one missense mutation (A85E). RT-PCR analysis of the complete beta-ureidopropionase cDNA suggested that both splice-site mutations lead to a variety of alternative splice variants, with deletions of a single or several exons. The alanine at position 85 was not conserved in other eukaryotic beta-ureidopropionase protein sequences.

A pivotal role for beta-aminoisobutyric acid and oxidative stress in dihydropyrimidine dehydrogenase deficiency?

Dihydropyrimidine dehydrogenase (DPD) constitutes the first step of the pyrimidine degradation pathway in which the pyrimidine bases uracil and thymine are catabolised to beta-alanine and beta-aminoisobutyric acid (beta-AIB), respectively. The mean concentration of beta-AIB was approximately 5- to 8-fold lower in urine of patients with a DPD deficiency, when compared to age-matched controls. Comparable levels of 8-hydroxydeoxyguanosine (8-OHdG) were present in urine from controls and DPD patients at the age <2 year. In contrast, slightly elevated levels of 8-OHdG were detected in urine from DPD patients with an age >2 year, suggesting the presence of increased oxidative stress.

Determination of ITPase activity in erythrocyte lysates obtained for determination of TPMT activity.

The indication for the determination of both thiopurine methyltransferase (TPMT) and inosine triphosphate pyrophosphohydrolase is identical (i.e., adverse drug reactions toward mercaptopurines). Therefore, we tested whether or not our standard procedure to prepare erythrocyte lysates for measurement of TPMT activity, which includes treatment with Chelex 100 (a chelating resin), was suitable for the measurement of ITPase activity. It also was tested to see if ITPase activity differs in EDTA and Heparin anti-coagulated blood samples. We found that there was no difference between the ITPase activity in erythrocyte lysates prepared from EDTA or Heparin anti-coagulated blood. Treatment with a chelating resin or omission of magnesium from the assay procedure resulted in decreased and nearly absent ITPase activity, respectively. We conclude that untreated erythrocyte lysates obtained for determination of TPMT activity are suitable for determination of ITPase activity. However, after treatment with Chelex 100 the erythrocyte lysates become unsuitable for determination of ITPase activity.

Activity of pyrimidine degradation enzymes in normal tissues.

In this study, we measured the activity of dihydropyrimidine dehydrogenase (DPD), dihydropyrimidinase (DHP) and beta-ureidopropionase (beta-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 beta-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 beta-UP were also present in a variety of other tissues, especially in bronchus.

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 microM. The intra-assay variation and inter-assay variation of plasma with added 5FU (1 microM, 10 microM, 100 microM) 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.

Clinical findings and a therapeutic trial in the first patient with beta-ureidopropionase deficiency.

The clinical, neurophysiological and neuroradiological work-up as well as the results of a specific treatment trial are presented of the first patient diagnosed with beta-ureidopropionase deficiency (E.C. 3.5.1.6, McKusick 606673). The patient presented with an early-onset dystonic movement disorder, severe developmental delay with marked impairment of visual responsiveness in combination with severely delayed myelination in magnetic resonance imaging studies. In addition, there were partial optic atrophy, pigmentary retinopathy and mild cerebellar hypoplasia. The enzyme defect was expected to lead to intracerebral deficiency of beta-alanine which seems to be a neuromodulator at inhibitory synapses. Therefore, a therapeutic trial with supplementation of beta-alanine was undertaken over 1.5 years with no convincing clinical improvement.

The pharmacokinetic effect of discontinuation of mesalazine on mercaptopurine metabolite levels in inflammatory bowel disease patients.

BACKGROUND: In vitro studies suggest interactions between mesalazine (mesalamine) and thiopurines by thiopurine S-methyltransferase (TPMT) inhibition, influencing the balance of hepatotoxic 6-methylmercaptopurine ribonucleotide and immunosuppressive tioguanine (thioguanine) metabolites. AIM: To examine the in vivo pharmacokinetic interaction between mesalazine and mercaptopurine. METHODS: A prospective study was performed in quiescent inflammatory bowel disease patients using the combination of mercaptopurine and mesalazine. Laboratory parameters, 6-methylmercaptopurine ribonucleotide and tioguanine levels and thiopurine S-methyltransferase activity in erythrocytes were measured at stable medication, after mesalazine discontinuation and mesalazine reintroduction, further mercaptopurine was continued. RESULTS: Seventeen patients were participated. Mean mercaptopurine dose was 0.78 mg/kg/day and median of mesalazine dose was 3000 mg/day. After mesalazine discontinuation, mean tioguanine levels changed significantly from 262 to 209 pmol/8 x 10(8) red blood cell, increasing to 270 after reintroduction. Mean 6-methylmercaptopurine ribonucleotide levels were 1422, 2149 and 1503 pmol/8 x 10(8) red blood cell respectively. Mean 6-methylmercaptopurine ribonucleotide/tioguanine ratio increased significantly from 6.3 at baseline to 11.2. Mean baseline thiopurine S-methyltransferase activity was 0.58 pmol/10(6) red blood cell/h and stable. All patients had wild-type thiopurine S-methyltransferase genotypes however, leucocyte counts were stable. DISCUSSION: A significantly higher tioguanine levels and improving 6-methylmercaptopurine ribonucleotide/tioguanine ratio were found during mesalazine/mercaptopurine combination. Theoretically, mesalazine inhibits thiopurine S-methyltransferase activity. In vivo thiopurine S-methyltransferase activity did not change, however. CONCLUSION: Mesalazine has synergistic effects on mercaptopurine therapy, but the mechanism is unclear. Combining these drugs may be further indication for mesalazine in inflammatory bowel disease treatment.

Dihydropyrimidine dehydrogenase deficiency presenting at birth.

Dihydropyrimidine dehydrogenase (DPD) deficiency (McKusick 274270) is a clinically heterogeneous autosomal recessive disorder of pyrimidine metabolism. DPD is the enzyme that catalyses the first and the rate-limiting step in the catabolism of uracil, thymine and the analogue 5-fluorouracil. To date, more than 30 patients have been diagnosed with a complete enzyme deficiency. Here, we describe the fifth case with a complete DPD deficiency presenting at birth with severe neurological abnormalities. The patient was homozygous for the common splice-site mutation IVS14+1G > A.

Pharmacokinetics of bolus 5-fluorouracil: relationship between dose, plasma concentrations, area-under-the-curve and toxicity.

The pharmacokinetics of 5-fluorouracil (5FU) have been related to toxicity and antitumor activity, in particular for continuous infusion schedules, but to a lesser extent for frequently used bolus injections. The use of intensive sampling schedules limits the application of pharmacokinetics to optimize individual dosing or to define the ideal combination with other drugs. We therefore reanalyzed a pharmacokinetic study in order to develop a limited sampling schedule. Patients received escalating doses of 5FU at 500, 600 and 720 mg/m2 as a bolus until toxicity developed. Blood samples were analyzed until 24 h after administration. The area under the concentration time curve from 0-90 min (AUC(0-90)) was strongly correlated with dose and also with toxicity (p = 0.0009). The 5FU concentrations at 30 and 60 min were correlated to the AUC(30-240) and to that of the AUC(0-90) (r2 = 0.970). The use of limited sampling (30, 60, 90 min) in a patient given 353 mg/m2 5FU with severe toxicity at initial dosing at 500 mg/m2 revealed that the AUC(0-90) at 353 mg/m2 was higher than the normal AUC(0-90) for 500 mg/m2. This patient appeared to have an 8-fold lower activity of the 5FU degradation enzyme dihydropyrimidine dehydrogenase. Limited sampling will allow us to define potential aberrant kinetics of pharmacokinetic interaction of 5FU with other drugs being developed for treatment of colorectal cancer.

Pyrimidine degradation defects and severe 5-fluorouracil toxicity.

5-Fluorouracil (5FU) remains one of the most frequently prescribed chemotherapeutic drugs for the treatment of cancer. Recently, the pivotal role of the catabolic pathway of 5FU in the determination of toxicity towards 5FU has been highlighted. Patients with a (partial) dihydropyrimidine dehydrogenase deficiency proved to be at risk of developing severe toxicity after the administration of 5FU. A partial dihydropyrimidinase deficiency proved to be a novel pharmacogenetic disorder associated with severe 5FU toxicity.

Increased cytotoxicity of 2',2'-difluoro-2'-deoxycytidine in human leukemic cell-lines after a preincubation with cyclopentenyl cytosine.

The in vitro modulating effect of Cyclopentenyl cytosine (CPEC) on the metabolism of gemcitabine was studied in lymphocytic and myeloid leukemic cell-lines. In MOLT-3 cells, that were pretreated with CPEC, the incorporation of 2',2'-difluoro-2'-deoxycytidine triphosphate (dFdCTP) into DNA was significantly increased by 57-99% in comparison with cells that were only treated with gemcitabine. The increased incorporation of dFdCTP into DNA in CPEC pretreated cells was paralleled by an increase in apoptotic and necrotic cells of 17-34%. In HL-60 cells that were preincubated with CPEC, increased concentrations of the mono-/di- and triphosphate form of gemcitabine were observed, as well as an increased incorporation of dFdCTP into DNA (+773%). This increased incorporation was paralleled by a significant increase in apoptosis and necrosis. We conclude that CPEC enhances the incorporation of dFdCTP into DNA and thus increases the cytotoxicity of gemcitabine in lymphocytic and myeloid leukemic cell-lines.

Head imaging abnormalities in dihydropyrimidine dehydrogenase deficiency.

Dihydropyrimidine dehydrogenase (DPD) deficiency is a rare autosomal recessive disorder of pyrimidine metabolism. Patients may present with a wide range of neurological symptoms during the first years of life. Head imaging abnormalities have been reported only rarely and include diffuse cerebral atrophy and white-matter hyperintensity. The pathogenesis of the white-matter abnormalities is unknown, although environmental factors and altered energy metabolism may be involved. To further understanding of the spectrum of brain abnormalities associated with DPD deficiency, we report a 17-month-old girl, born to a consanguineous Pakistani couple, who had a history of encephalopathy, prolonged hypoventilation, developmental delay and failure to thrive. Head MRI showed prominent sulci and abnormal T2 prolongation in the cerebral white matter and brainstem. Thus, DPD deficiency may feature prominent brain abnormalities involving the cerebral white matter and brainstem. Anoxic stress may have contributed to the clinical presentation and brain findings in this case. In order to define more clearly the contribution of DPD deficiency to the pathogenesis of these MRI abnormalities, we recommend performing detailed analysis of urine pyrimidine metabolites in patients who have such findings.

Dihydropyrimidine dehydrogenase deficiency and acute neurological presentation.

Dihydropyrimidine dehydrogenase (DPD) deficiency has been linked to 5-fluorouracil toxicity, but patients may present a wide clinical spectrum. We describe a 1-year-old Tunisian girl with a dramatic onset of neurological symptoms suggesting the possible triggering role of environmental factors.

Pharmacokinetics of S-1, an oral formulation of ftorafur, oxonic acid and 5-chloro-2,4-dihydroxypyridine (molar ratio 1:0.4:1) in patients with solid tumors.

S-1 is an oral formulation of ftorafur (FT), oxonic acid and 5-chloro-2,4-dihydroxypyridine (CDHP) at a molar ratio of 1:0.4:1. FT is a 5-fluorouracil (5-FU) prodrug, CDHP is a dihydropyrimidine dehydrogenase (DPD) inhibitor and oxonic acid is an inhibitor of 5-FU phosphoribosylation in the gastrointestinal mucosa and was included to prevent gastrointestinal toxicity. We determined the pharmacokinetics of S-1 in 28 patients at doses of 25, 35, 40 and 45 mg/m(2). The plasma C(max) values of FT, 5-FU, oxonic acid and CDHP increased dose-dependently and after 1-2 h were in the ranges 5.8-13 microM, 0.4-2.4 microM, 0.026-1.337 microM, and 1.1-3.6 microM, respectively. Uracil levels, indicative of DPD inhibition, also increased dose-dependently from basal levels of 0.03-0.25 microM to 3.6-9.4 microM after 2-4 h, and 0.09-0.9 microM was still present after 24 h. The pharmacokinetics of CDHP and uracil were linear over the dose range. The areas under the plasma concentration curves (AUC) for CDHP and uracil were in the ranges 418-1735 and 2281-8627 micromol x min/l, respectively. The t(1/2) values were in the ranges 213-692 and 216-354 min, respectively. Cumulative urinary excretion of FT was predominantly as 5-FU and was 2.2-11.9%; the urinary excretion of both fluoro-beta-alanine and uracil was generally maximal between 6 and 18 h. During 28-day courses with twice-daily S-1 administration, 5-FU and uracil generally increased. Before each intake of S-1, 5-FU varied between 0.5 and 1 microM and uracil was in the micromolar range (up to 7 microM), indicating that effective DPD inhibition was maintained during the course. In a biopsy of an esophageal adenocarcinoma metastasis that had regressed, thymidylate synthase, the target of 5-FU, was inhibited 50%, but increased four- to tenfold after relapse in subsequent biopsies. In conclusion, oral S-1 administration resulted in prolonged exposure to micromolar 5-FU concentrations due to DPD inhibition, and the decrease in uracil levels after 6 h followed the pattern of CDHP and indicates reversible DPD inhibition.

Linoleic acid supplementation of Barth syndrome fibroblasts restores cardiolipin levels: implications for treatment.

The object of this study was to investigate whether the levels of cardiolipin in cultured skin fibroblasts of patients with Barth syndrome (BTHS) can be restored by addition of linoleic acid to growth media. To this end, fibroblasts from controls and BTHS patients were grown in the presence or absence of linoleic acid. High-performance liquid chromatography-electrospray ionization tandem mass spectrometry was used for quantitative and compositional analysis of cardiolipin. Incubation of cells from both BTHS and controls with different concentrations of linoleic acid led to a dose- and time-dependent increase of cardiolipin levels. The increased levels of cardiolipin in fibroblasts of BTHS patients after treatment with linoleic acid indicate that an increased amount of linoleic acid in the diet might be beneficial to BTHS patients.

Reduced 5-FU clearance in a patient with low DPD activity due to heterozygosity for a mutant allele of the DPYD gene.

5-fluorouracil pharmacokinetics, dihydropyrimidine dehydrogenase-activity and DNA sequence analysis were compared between a patient with extreme 5-fluorouracil induced toxicity and six control patients with normal 5-fluorouracil related symptoms. Patients were treated for colorectal cancer and received chemotherapy consisting of leucovorin 20 mg m(-2) plus 5-fluorouracil 425 mg m(-2). Blood sampling was carried out on day 1 of the first cycle. The 5-fluorouracil area under the curve(0-->3h) in the index patient was 24.1 mg h l(-1) compared to 9.8+/-3.6 (range 5.4-15.3) mg h l(-1) in control patients. The 5-fluorouracil clearance was 520 ml min(-1) vs 1293+/-302 (range 980-1780) ml min(-1) in controls. The activity of dihydropyrimidine dehydrogenase in mononuclear cells was lower in the index patient (5.5 nmol mg h(-1)) compared to the six controls (10.3+/-1.6, range 8.0-11.7 nmol mg h(-1)). Sequence analysis of the dihydropyrimidine dehydrogenase gene revealed that the index patient was heterozygous for a IVS14+1G>A point mutation. Our results indicate that the inactivation of one dihydropyrimidine dehydrogenase allele can result in a strong reduction in 5-fluorouracil clearance, causing severe 5-fluorouracil induced toxicity.

Clinical features of galactokinase deficiency: a review of the literature.

Galactokinase deficiency (McKusick 230200) is a rare autosomal recessive inborn error of galactose metabolism. Cataract and, rarely, pseudotumor cerebri caused by galactitol accumulation seem to be the only consistently reported abnormalities in this disorder. We performed a literature search to obtain information on the clinical spectrum of galactokinase deficiency. A total of 25 publications were traced describing 55 galactokinase-deficient patients. Cataract was reported in most patients. Clinical abnormalities other than cataract were reported in 15 (35%) out of 43 cases on which information was available. However, all symptoms were reported infrequently and a causal relationship with the galactokinase deficiency is unlikely. As cataract and pseudotumor cerebri appear to be the sole complications of galactokinase deficiency, the outcome for patients with galactokinase deficiency is much better than for patients with classical galactosaemia (McKusick 230400), a more common autosomal recessive disorder of galactose metabolism caused by galactose-1-phosphate uridyltransferase (GALT; EC 2.7.7.12) deficiency. Long-term follow-up of patients with this disorder has shown that, in spite of a severely galactose-restricted diet, most patients develop abnormalities such as a disturbed mental and/or motor development, dyspraxia and hypergonadotropic hypogonadism. Endogenous production of galactose has been considered an important aetiological factor. Although damage may well occur in utero, available evidence suggests that damage will continue after birth. Inhibition of galactokinase may then be a promising approach for controlling damage in GALT-deficient patients.

Mild learning difficulties and offending behaviour--is there a link with monoamine oxidase A deficiency?

We have attempted to replicate the findings of Brunner et al., who described a large Dutch kindred where several males were of borderline intelligence and showed characteristically aggressive and sometimes dangerous or extremely antisocial behaviour. The genetic defect for this syndrome was assigned to the p11-p21 region of the X chromosome following linkage analysis in a single kindred. Subsequent sequencing of a candidate gene, monoamine oxidase A (MAO-A), at the position of maximum linkage revealed a causative mutation in the coding region of the MAO-A gene in position 936. In addition to identifying both the phenotype and the associated mutation found by Brunner et al., we also wished to test the hypothesis that mutations elsewhere in the MAO-A gene could cause the low intelligence quotient/personality disorder phenotype associated with low urinary catecholamine degradation products. Fifty-four male subjects similar in clinical characteristics to the affected males in the Dutch kindred were identified within secure mental health facilities in England and Wales. All were assessed using the antisocial personality disorder section of the SCID-II interview instrument, and information about their offending behaviour and family history was obtained from the medical notes. A blood and early-morning urine sample was obtained from each patient. Analysis of urinary excretion patterns of biogenic amines and their metabolites, represented as ratios of normetanefrine to vanillylmandelic acid, revealed two possible cases of MAO-A deficiency, which were found to be negative after resampling.

beta-Ureidopropionase deficiency: a novel inborn error of metabolism discovered using NMR spectroscopy on urine.

In this work, NMR investigations that led to the discovery of a new inborn error of metabolism, beta-ureidopropionase (UP) deficiency, are reported. 1D (1)H-NMR experiments were performed using a patient's urine. 3-Ureidopropionic acid was observed in elevated concentrations in the urine spectrum. A 1D (1)H-(1)H total correlation spectroscopy (TOCSY) and two heteronuclear 2D NMR techniques (heteronuclear multiple bond correlation (HMBC) and heteronuclear single-quantum correlation (HSQC)) were used to identify the molecular structure of the compound that caused an unknown doublet resonance at 1.13 ppm. Combining the information from the various NMR spectra, this resonance could be assigned to 3-ureidoisobutyric acid. These observations suggested a deficiency of UP. With 1D (1)H-NMR spectroscopy, UP deficiency can be easily diagnosed. The (1)H-NMR spectrum can also be used to diagnose patients suffering from other inborn errors of metabolism in the pyrimidine degradation pathway.