Mitochondrial neurogastrointestinal encephalopathy: a clinicopathological mimic of Crohn's disease.

BACKGROUND: Mitochondrial neurogastrointestinal encephalopathy (MNGIE), due to mutations in TYMP, often presents with gastrointestinal symptoms. Two sisters, initially managed for Crohn's disease based upon clinical, imaging and pathological findings, were later found to have MNGIE. The cases provide novel clinicopathological insight, for two further reasons: both sisters remain ambulant and in employment in their late 20s and 30s; diagnosis in one sister was made after a suspected azathioprine-precipitated acute illness. CASE PRESENTATION: A 25-year-old female presented with diarrhoea, vomiting, abdominal pain, and bloating. Faecal calprotectin, colonic biopsies and magnetic resonance enterography were consistent with a diagnosis of Crohn's disease. Azathioprine initiation preceded admission with a sore throat, headache, myalgia, and pyrexia. Withdrawal led to rapid clinical improvement. MRI brain revealed persistent, extensive white matter changes. Elevated plasma and urine thymidine and deoxyuridine, and genetic testing for TYMP variants, confirmed MNGIE. Testing of the patient's sister, also diagnosed with Crohn's disease, revealed identical variants. In this context, retrospective review of colonic biopsies identified histological findings suggestive of MNGIE. CONCLUSIONS: Azathioprine interference in nucleic acid metabolism may interact with the mitochondrial DNA depletion of MNGIE. Nucleotide supplementation, proposed for treatment by manipulating mitochondrial nucleoside pools, may require caution. The late onset and mild phenotype observed confirms presentation can occur later in life, and may reflect residual thymidine phosphorylase activity. Clinicians should consider measuring plasma thymidine levels in suspected Crohn's disease to rule out MNGIE, particularly if white matter abnormalities are identified on neuroimaging.

Hemodialysis in MNGIE transiently reduces serum and urine levels of thymidine and deoxyuridine, but not CSF levels and neurological function.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare, autosomal-recessive mitochondrial disorder caused by TYMP mutations presenting with a multisystemic, often lethal syndrome of progressive leukoencephalopathy, ophthalmoparesis, demyelinating neuropathy, cachexia and gastrointestinal dysmotility. Hemodialysis (HMD) has been suggested as a treatment to reduce accumulation of thymidine and deoxyuridine. However, all studies so far have failed to measure the toxic metabolites in cerebrospinal fluid (CSF), which is the crucial compartment for CNS damage.Our study is the first prospective, longitudinal investigation, exploiting detailed serial testing of predefined clinical and molecular outcome parameters (including serial CSF assessments) in a 29-year-old MNGIE patient undergoing 1 year of extensive HMD. We demonstrate that HMD only transiently restores increased serum and urine levels of thymidine and deoxyuridine, but fails to reduce CSF levels of the toxic metabolites and is ineffective to influence neurological function. These findings have direct important implications for clinical practice: They prevent a burdensome, long-term invasive, but ultimately probably ineffective procedure in future MNGIE patients.

Assessment of thymidine phosphorylase function: measurement of plasma thymidine (and deoxyuridine) and thymidine phosphorylase activity.

We describe detailed methods to measure thymidine (dThd) and deoxyuridine (dUrd) concentrations and thymidine phosphorylase (TP) activity in biological samples. These protocols allow the detection of TP dysfunction in patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Since the identification of mutations in TYMP, the gene encoding TP, as the cause of MNGIE (Nishino et al. Science 283:689-692, 1999), the assessment of TP dysfunction has become the best screening method to rule out or confirm MNGIE in patients. TYMP sequencing, to find the causative mutations, is only needed when TP dysfunction is detected. dThd and dUrd are measured by resolving these compounds with high-performance liquid chromatography (HPLC) followed by the spectrophotometric monitoring of the eluate absorbance at 267 nm (HPLC-UV). TP activity can be measured by an endpoint determination of the thymine formed after 1 h incubation of the buffy coat homogenate in the presence of a large excess of its substrate dThd, either spectrophotometrically or by HPLC-UV.

Radiation metabolomics. 2. Dose- and time-dependent urinary excretion of deaminated purines and pyrimidines after sublethal gamma-radiation exposure in mice.

Gamma-radiation exposure of humans is a major public health concern as the threat of terrorism and potential hostile use of radiological devices increases worldwide. We report here the effects of sublethal gamma-radiation exposure on the mouse urinary metabolome determined using ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry-based metabolomics. Five urinary biomarkers of sublethal radiation exposure that were statistically significantly elevated during the first 24 h after exposure to doses ranging from 1 to 3 Gy were unequivocally identified by tandem mass spectrometry. These are deaminated purine and pyrimidine derivatives, namely, thymidine, 2'-deoxyuridine, 2'-deoxyxanthosine, xanthine and xanthosine. Furthermore, the aminopyrimidine 2'-deoxycytidine appeared to display reduced urinary excretion at 2 and 3 Gy. The elevated biomarkers displayed a time-dependent excretion, peaking in urine at 8-12 h but returning to baseline by 36 h after exposure. It is proposed that 2'-deoxyuridine and 2'-deoxyxanthosine arise as a result of gamma irradiation by nitrosative deamination of 2'-deoxycytidine and 2'-deoxyguanosine, respectively, and that this further leads to increased synthesis of thymidine, xanthine and xanthosine. The urinary excretion of deaminated purines and pyrimidines, at the expense of aminopurines and aminopyrimidines, appears to form the core of the urinary radiation metabolomic signature of mice exposed to sublethal doses of ionizing radiation.

Pharmacokinetics of 5-carboranyl-2'-deoxyuridine in rats.

The pharmacokinetics of 5-carboranyl-2'-deoxyuridine (CDU) after intravenous administration of 25 mg/kg was investigated in rats. The uptake of CDU into brain was also examined. Concentrations of CDU in plasma, urine, and brain were measured by reverse phase HPLC. Plasma concentrations of CDU declined in a biexponential fashion with a terminal half-life of 1.26 +/- 0.28 h. The plasma protein binding of CDU was linear and the average fraction bound to plasma proteins was 0.95 +/- 0.02. The total clearance of CDU was 0.69 +/- 0.20 L/h/kg whereas clearance of unbound drug was much greater (15.33 +/- 4.44 L/h/kg). Thus, the total clearance of the drug is limited, in part, by the high degree of plasma protein binding, resulting in a moderate total clearance. No unchanged CDU was detected in urine. Furthermore, there was no trace of CDU glucuronide in urine samples. The steady-state volume of distribution of CDU was 0.70 +/- 0.23 L/kg. The brain:total plasma CDU concentration ratios determined in two rats were 0.47 and 0.36, while the brain:unbound plasma CDU concentration ratios were 10.26 and 7.87. The results of this study suggest that it is possible to achieve significant levels of CDU in brain. The high degree of plasma protein binding restricted extensive distribution of this lipophilic compound. The results of this study suggest further investigations of CDU as a neutron sensitizer for boron neutron capture therapy (BNCT) are warranted.

Metabolism of anti-herpes agent 5-(2-chloroethyl)-2'-deoxyuridine in mice and rats.

The enzymatic splitting and metabolic elimination of anti-viral agent 5-(2-chloroethyl)-2'-deoxyuridine [CEDU] have been studied. For elucidation of structures of metabolites, several different kinds of extraction, purification and spectroscopic methods were used (Extrelut LC, TLC, HPLC, MS, NMR, IR, UV and CD). For mass spectral analysis, various ionization techniques (EI, CI and FAB-MS) were performed as complementary methods. After oral administration of [14C]-CEDU to mice and rats, the parent compound, 5-(2chloroethyl) uracil [CEU] and hydroxylated CEU metabolites were isolated and identified from urine and faeces by the above mentioned methods. The CEDU showed rapid phosphorolysis in vitro with thymidine phosphorylase Km 41.0 +/- 5.0; and uridine phosphorylase Km 10.0 +/- 1.5. The cleavage of the N-glycosidic bond of the nucleoside analogue and a new metabolic pathway of CEDU [stereoselective oxidation of 5-(2-chloroethyl) uracil] was observed in both species.

Biotransformation of 5-(2-chloroethyl)-2'-deoxyuridine in male NMRI mice.

When the selective anti-herpes agent [2-14C]-5-(2-chloroethyl)-2'-deoxyuridine [( 14C]CEDU) was administered as a single oral dose to mice, 87.9% of the radioactivity was excreted in the urine and 11.2% in the feces within 72 hr. Compounds accounting for 84% of the 14C radioactivity in the 0- to 24-hr urine were isolated by various chromatographic techniques and identified by MS, NMR, IR, and CD analysis. Approximately 25% of the radioactivity found in the urine was the parent compound (CEDU). According to the 14C-metabolites detected in the urine, one may infer that [14C]CEDU is metabolized, first, by cleavage of its N-glycosidic bond, resulting in the formation of 5-(2-chloroethyl)uracil (38.7%) and, second, by stereoselective hydroxylation of the alpha carbon atom of the haloalkyl side chain of 5-(2-chloroethyl)uracil, resulting in the formation of 5-(1-hydroxy-2-chloroethyl)uracil (29.6%). CEDU was absorbed rapidly from the gastrointestinal tract and the bloodstream, and did not show any particular accumulation in mouse tissues, as revealed by whole body autoradiography.

Tumour uptake of radiolabelled pyrimidine bases and pyrimidine nucleosides in animal models. IX. Radiolabelled 1-(2'-fluoro-2'-deoxy-beta-D-ribofuranosyl)uracil and 1-(2'-chloro-2'-deoxy-beta-D-ribofuranosyl)uracil.

The biodistribution of radiolabelled 1-(2'-fluoro-2'-deoxy-beta-D-ribofuranosyl)uracil (2'-FUdR) and 1-(2'-chloro-2'-deoxy-beta-D-ribofuranosyl)uracil (2'-ClUdR) was evaluated using in-vivo and in-vitro tumour models. 6-[3H]-2'-FUdR exhibited a maximum tumour:blood (T:B) ratio (Walker 256 carcinosarcoma) of 1.0 at 15 min after injection whereas 2-[14C]-2'-FUdR and 2'-[36Cl]-2'-ClUdR exhibited maximum T:B ratios (Lewis lung carcinoma) of 3.0 and 4.1 respectively at 120 min. Clearance of blood radioactivity after injection of 6-[3H]-2'-FUdR, 2-[14C]-2'-FUdR and 2'-[36Cl]-2'-ClUdR was rapid and best described by a biexponential function. The clearance half-lives of the short-lived components were calculated as 1.6 min (95.5%), 2.3 min (94.7%) and 1.2 min (96.0%) respectively. The clearance half-lives of the long-lived components were 23 h (4.5%) for 6-[3H]-2'-FUdR, 89 min (5.3%) for 2-[14C]-2'-FUdR, and 49 min (4.0%) for 2'-[36Cl]-2'-ClUdR. Less than 36% of the 14C radioactivity present in the urine 2 h after injection of 2-[14C]-2'-FUdR was associated with 2'-FUdR, whereas greater than 91% of the 36Cl radioactivity in the urine 2 h after injection of 2'-[36Cl]-2'-ClUdR was associated with 2'-ClUdR. Both 6-[3H]-2'-FUdR (less than 3.1 pmol/10(6) cells) and 6-[3H]-2'-ClUdR (1.0 +/- 0.2 pmol/10(6) cells) were incorporated into cultured Raji tumour cells in vitro to a lesser extent than the natural nucleosides uridine (137.2 +/- 3.8 pmol/10(6) cells) and 2'-deoxyuridine (23.5 +/- 2.5 pmol/10(6) cells) after a 20 min incubation.

[Isolation and identification of metabolites of 5-ethyl-2'-deoxyuridine from rat urine]. / Isolierung und Identifizierung der Metaboliten von 5-Ethyl-2'-desoxyuridin aus Rattenurin.

The metabolites of 5-ethyl-2'-deoxyuridine (Aedurid) have been isolated and purified from rat urine by preparative thin-layer and column chromatography. With the aid of 1H-NMR and mass spectroscopy and by comparison with an authentic sample, the major metabolite could be identified as 5-(1-hydroxyethyl)uracil. The minor metabolite was identified as 5-ethyluracil.

Individual changes of DNA catabolite excretion in the course of antitumor therapy of Hodgkin's disease.

In patients with morbus Hodgkin, treated primarily by the actino- and chemotherapy, the excretion was followed of DNA catabolites (deoxycytidine, deoxyuridine, thymidine and their sum) in the course of the therapy. The dynamics was studied of changes in the time interval of interest and attention was paid to its relation to the clinical and histological type of disease and to the successful character of the therapy defined by reaching a complete remission. The group of patients as a whole was characterized by an increased excretion of catabolites in the time interval of interest. No dependence was demonstrated between the catabolite excretion and extent of the disease similarly as between the excretion and successful character of the therapy. The dynamics of the changes in the time intervals of interest was neither remarkably nor continuously increased or decreased. The test of the excretion of pyrimidine deoxyribonucleosides possesses sufficient sensitivity for demonstrating laws in relation to the therapy during group evaluation. With respect to individual variability of values of particular patients and to the absence of the relations mentioned above the test is not suitable to indicate the individual response to the anticancer therapy.

Deoxypyrimidineuria in the course of chemotherapy in M. Hodgkin.

In patients with Hodgkin's disease the excretion of pyrimidine deoxyribonucleosides in urine was followed up within the course of 92 chemotherapeutical series with cytostatics of COOP group. In 14-day chemotherapy series, the excretion of deoxycytidine, deoxyuridine, thymidine, and their sum was significantly increased. In this paper the results are analyzed and a possibility of utilization of this finding for the judgment in the clinical course of the disease is considered.

Urine excretion of nucleosides in Hodgkin's disease treated by combined chemotherapy.

The authors have followed the excretion of pyrimidine doexyribonucleosides in the urine of patients with generalized stages of Hodgkin's disease. They proved an increased excretion of deoxyuridine and thymidine in these patients compared to healthy persons. In the course of 14-days of combined chemotherapy a statistically significant increase in the excretion of DNA catabolites followed was found.