Polyneuropathy Reveals Origins of Decade-long Gastrointestinal Symptoms in a Patient With Undiagnosed Mitochondrial Neurogastrointestinal Encephalopathy Caused by a Novel Mutation.

Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is a rare autosomal recessive disease that manifests with multiorgan presentation characterized by gastrointestinal, extraocular, and both peripheral and central nervous system involvement. MNGIE is caused by mutation in the TYMP (thymidine phosphorylase) gene, resulting in loss of thymidine phosphorylase enzyme activity. This causes its substrates, thymidine and deoxyuridine, to accumulate in tissues and plasma, while also causing secondary alterations in mitochondrial DNA. To date, more than 80 mutations have been reported in this gene. We present herein the clinical, neuroimaging, electrodiagnostic, and molecular findings of a patient with MNGIE caused by a novel homozygous missense mutation (C1175T > G) of the TYMP gene.

Successful liver transplantation in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE).

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a fatal disorder characterized by progressive gastrointestinal dysmotility, peripheral neuropathy, leukoencephalopathy, skeletal myopathy, ophthalmoparesis, and ptosis. MNGIE stems from deficient thymidine phosphorylase activity (TP) leading to toxic elevations of plasma thymidine. Hematopoietic stem cell transplant (HSCT) restores TP activity and halts disease progression but has high transplant-related morbidity and mortality. Liver transplant (LT) was reported to restore TP activity in two adult MNGIE patients. We report successful LT in four additional MNGIE patients, including a pediatric patient. Our patients were diagnosed between ages 14 months and 36 years with elevated thymidine levels and biallelic pathogenic variants in TYMP. Two patients presented with progressive gastrointestinal dysmotility, and three demonstrated progressive peripheral neuropathy with two suffering limitations in ambulation. Two patients, including the child, had liver dysfunction and cirrhosis. Following LT, thymidine levels nearly normalized in all four patients and remained low for the duration of follow-up. Disease symptoms stabilized in all patients, with some manifesting improvements, including intestinal function. No patient died, and LT appeared to have a more favorable safety profile than HSCT, especially when liver disease is present. Follow-up studies will need to document the long-term impact of this new approach on disease outcome. Take Home Message: Liver transplantation is effective in stabilizing symptoms and nearly normalizing thymidine levels in patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and may have an improved safety profile over hematopoietic stem cell transplant.

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.

Metabolomic profiling of the effects of Curcumae rhizoma and Sparganii rhizome on stress-led blood stasis.

Blood stasis (BS) is a complex syndrome with blood flow retardation or cessation. The Traditional Chinese Medicine, Curcumae rhizome (CR) and Sparganii rhizome (SR), showed promising effects on this disease, and especially effective when used in combination. However, the detailed influence of the TCMs on the BSS disturbed metabolic pathways was still unclear. In this study, a BS model was constructed in SD rat and the TCMs were used individually or in combination to assess the effects. As a result, combination of CR and SR led to the improvement in hemorheology parameters of up to 80% in the BS model. Further analyzing using metabolomics showed several metabolic pathways, including center carbon metabolism, amino acid metabolism, etc., recovered to the normal levels after treatment. Informatively, tyrosine and thymidine exhibited potential importance in the BSS and its treatment process. From these results, the metabolic profiles of BS and the SR-CR treatment were provided, which may helpful for better understanding the BSS mechanism and the development of more effective therapies.

Long-Term Sustained Effect of Liver-Targeted Adeno-Associated Virus Gene Therapy for Mitochondrial Neurogastrointestinal Encephalomyopathy.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is caused by mutations in TYMP, the gene encoding the enzyme thymidine phosphorylase (TP). TP dysfunction results in systemic accumulation of the noxious TP substrates thymidine and deoxyuridine. Gene therapy using either a lentiviral vector or adeno-associated vector (AAV) has proven to be a feasible strategy, as both vectors restore biochemical homeostasis in a murine model of the disease. This study shows that the effect of an AAV containing the TYMP coding sequence transcriptionally targeted to the liver persists long term in mice. Although the vector copy number was diluted and AAV-mediated liver TP activity eventually reduced or lost after 21 months at the lowest vector doses, the effect was sustained (with a negligible decrease in TP activity) and fully effective on nucleoside homeostasis for at least 21 months at a dose of 2 × 1012 vg/kg. Macroscopic visual inspection of the animals' organs at completion of the study showed no adverse effects associated with the treatment. These results further support the feasibility of gene therapy for MNGIE.

Determination of telbivudine in the plasma of chronic hepatitis B patients in long-term treatment by high-performance liquid chromatographic-tandem mass spectrometry.

Creatine kinase elevation is commonly reported in telbivudine-treated patients. However, little is known about the relationship between this adverse drug reaction and plasma concentration. In this study, a sensitive, rapid and safe quantitative bioanalytical method has been established by using LC-MS/MS for the determination of telbivudine in a clinical study of chronic hepatitis B patients. The assay was linear in a dynamic 10-10,000 ng/mL range (r2 > 0.999) and total analysis time was 6 min in this method. The validated method was applied to quantitatively determine plasma concentration in chronic hepatitis B patients during long-term telbivudine treatment. The results revealed that telbivudine concentration in the creatine kinase-elevated group (707.92-2788.78 ng/mL) was significantly higher than those with normal creatine kinase (412.63-1108.32 ng/mL). This method was adapted for therapeutic drug monitoring.

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.

Dried blood spot analysis without dilution: Application to the LC-MS/MS determination of BMS-986001 in rat dried blood spot.

Sample dilution is one major challenge in dried blood spot (DBS) bioanalysis. To resolve this issue, we applied a no-dilution strategy for DBS analysis by using a calibration curve with very wide linear range. We developed an LC-MS/MS DBS assay with a linear range of 5 orders of magnitude (50-5000,000ng/mL) for BMS-986001, an HIV drug under development, by simultaneously monitoring two selective reaction monitoring transitions of different intensity. The assay was validated and successfully applied to the analysis of DBS samples collected in a toxicology study in rats dosed with BMS-986001. All samples were analyzed without any dilution. We also compared the concentration data generated from the DBS method and a validated plasma assay for the same study. The two sets of data agreed well with each other, demonstrating the validity of this strategy for DBS analysis. This approach provides an effective and convenient way to eliminate complicated dilution for DBS and other sample collection techniques.

UPLC-MS/MS method for the simultaneous quantification of anti-HBV nucleos(t)ides analogs: Entecavir, lamivudine, telbivudine and tenofovir in plasma of HBV infected patients.

Hepatitis B infection affects two billion people worldwide and 350 million of these are chronically infected. Chronic hepatitis B virus is one of the most important cause of mortality and morbidity worldwide. If it is left untreated, about one-third of affected people will develop progressive and possibly fatal liver disease, like hepatic cirrhosis and primary hepatocellular carcinoma. Currently, five nucleos(t)ide analogs are approved for the treatment of chronic HBV infection. They are: lamivudine, adefovir dipivoxil, telbivudine, entecavir and tenofovir disoproxil fumarate. In this work, we developed and validated an UPLC-Tandem mass spectrometry assay method capable of monitoring lamivudine, telbivudine, tenofovir and entecavir plasma concentrations. Both standards and quality controls (high, medium and low) were prepared in human plasma. Each sample was added with internal standard (5'amino-5'deoxy-thymidine) and then drugs were extracted through a protein precipitation protocol with acetonitrile+0.1% formic acid and then dried. The extracts were resuspended in water and then injected into the chromatographic system. The chromatographic separation was performed on an Acquity UPLC HSS T3 1.8 µm 2.1 × 150 mm column, with a gradient of water and acetonitrile, both added with formic acid (0.05%). Accuracy, intra-day and inter-day precision at quality controls levels fitted all FDA guidelines for all analytes, while matrix effects and recoveries resulted stable between samples for each analyte. Finally, we tested this method by monitoring plasma concentrations in 30 HBV+ patients with good results. This simple analytical method could represent a useful tool for the management of anti-HBV therapy.

Maternal dietary uridine causes, and deoxyuridine prevents, neural tube closure defects in a mouse model of folate-responsive neural tube defects.

BACKGROUND: Folic acid prevents neural tube closure defects (NTDs), but the causal metabolic pathways have not been established. Serine hydroxymethyltransferase 1 (SHMT1) is an essential scaffold protein in folate-dependent de novo thymidylate synthesis in the nucleus. SHMT1-deficient mice provide a model to investigate folic acid-responsive NTDs wherein disruption of de novo thymidylate synthesis impairs neural tube closure. OBJECTIVE: We examined the effects of maternal supplementation with the pyrimidine nucleosides uridine, thymidine, or deoxyuridine with and without folate deficiency on NTD incidence in the Shmt1 mouse model. DESIGN: Shmt1(+/+) and Shmt1(-/-) female mice fed folate-replete or folate-deficient diets and supplemented with uridine, thymidine, or deoxyuridine were bred, and litters (n = 10-23 per group) were examined for the presence of NTDs. Biomarkers of impaired folate status and metabolism were measured, including plasma nucleosides, hepatic uracil content, maternal plasma folate concentrations, and incorporation of nucleoside precursors into DNA. RESULTS: Shmt1(+/-) and Shmt1(-/-) embryos from dams fed the folate-deficient diet were susceptible to NTDs. No NTDs were observed in litters from dams fed the folate-deficient diet supplemented with deoxyuridine. Surprisingly, uridine supplementation increased NTD incidence, independent of embryo genotype and dietary folic acid. These dietary nucleosides did not affect maternal hepatic uracil accumulation in DNA but did affect plasma folate concentrations. CONCLUSIONS: Maternal deoxyuridine supplementation prevented NTDs in dams fed the folate-deficient diet, whereas maternal uridine supplementation increased NTD incidence, independent of folate and embryo genotype. These findings provide new insights into the metabolic impairments and mechanisms of folate-responsive NTDs resulting from decreased Shmt1 expression.

Comparison of the absolute level of epigenetic marks 5-methylcytosine, 5-hydroxymethylcytosine, and 5-hydroxymethyluracil between human leukocytes and sperm.

5-Methylcytosine is one of the most important epigenetic modifications and has a profound impact on embryonic development. After gamete fusion, there is a widespread and rapid active demethylation process of sperm DNA, which suggests that the paternal epigenome has an important role during embryonic development. To better understand the epigenome of sperm DNA and its possible involvement in a developing embryo, we determined epigenetic marks in human sperm DNA and in surrogate somatic tissue leukocytes; the analyzed epigenetic modifications included 5-methyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, and 5-hydroxymethyl-2'-deoxyuridine. For absolute determination of the modification, we used liquid chromatography with UV detection and tandem mass spectrometry techniques with isotopically labeled internal standards. Our analyses demonstrated, for the first time to date, that absolute global values of 5-methyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, and 5-hydroxymethyl-2'-deoxyuridine in sperm are highly statistically different from those observed for leukocyte DNA, with respective mean values of 3.815% versus 4.307%, 0.797 versus 2.945 per 104 deoxynucleosides, and 5.209 versus 0.492 per 106 deoxynucleosides. We hypothesize that an exceptionally high value of 5-hydroxymethyluracil in sperm (>10-fold higher than in leukocytes) may play a not yet recognized regulatory role in the paternal genome.

Nonclinical Safety Profile of BMS-986001, a Nucleoside Transcriptase Inhibitor for Combination Retroviral Therapy.

Nucleoside reverse transcriptase inhibitors (NRTIs)/nucleotide reverse transcriptase inhibitors are key components of combination antiretroviral therapy for HIV infection. First-generation NRTIs are associated with mitochondrial toxicity in patients, mainly due to inhibition of human DNA polymerase γ (hDNA polγ) that manifests as adverse events such as lipodystrophy, lactic acidosis, myopathy, cardiomyopathy, or nephropathy in patients. In chronic nonclinical studies in rodents and nonrodents, eukaryotic (host) mitochondrial toxicity manifests as some drug-specific toxicities similar to human toxicity. BMS-986001, a novel thymidine analog with minimal hDNA polγ inhibition, has demonstrated antiretroviral activity in early clinical studies. The primary toxicity of BMS-986001 in rats and monkeys is bone marrow dyserythropoiesis with associated decreases in red blood cell mass. Additionally, at high doses, severe platelet reductions accompanied by cutaneous petechiae began during weeks 8 and 11 in 3 of 60 monkeys in chronic toxicity studies. In a 6-month study, platelet reductions required euthanasia of the 2 affected monkeys (300 mg/kg/d) at week 14, but with dose reduction (200 mg/kg/d) remaining monkeys had no platelet changes. One affected monkey (200 mg/kg/d) in a 9-month study completed dosing and its platelet counts recovered during a 1-month recovery. Formation of platelet-bound immunoglobulin in the presence of BMS-986001, together with rapid and complete platelet recovery in the absence of BMS-986001, suggested that platelet decreases in monkeys may be immune mediated. No findings indicative of mitochondrial toxicity were observed in rats or monkeys given BMS-986001, suggesting an improved safety profile compared to marketed NRTI or tenofovir disoproxil fumarate.

HPLC-UV analysis of thymidine and deoxyuridine in plasma of patients with thymidine phosphorylase deficiency.

We present a simple, fast and validated method for the determination of the two nucleosides thymidine (dThd) and deoxyuridine (dUrd) in plasma of patients with symptoms suggestive of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), using high performance liquid chromatography coupled with ultraviolet spectrophotometric detection (HPLC-UV). Plasma sample (100µL) pretreatment was based on simple deproteinization by 1.2M perchloric acid, using theophylline as internal standard (I.S.). HPLC-UV analysis was carried out on a Synergi 4µm Hydro-RP, 150×4mm I.D. column, at room temperature. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (20mM, pH 4.5) and acetonitrile (95:5, v/v), at an isocratic flow rate of 0.7mL/min. The UV detector was set at 267nm. The chromatographic run lasted 19min. Similar pyrimidine nucleotides and nucleosides do not interfere with the assay. Calibration curves were linear for both dThd and dUrd over a range of 0.5 to 5.0µg/mL. The limit of quantitation was 0.5µg/mL for both nucleosides and the absolute recovery was >90% for dThd, dUrd and the I.S. Both intra- and inter-assay precision and accuracy were lower than 10% at all tested concentrations. The proposed method was successfully applied to measure plasma concentrations of dThd and dUrd in two MNGIE patients. This assay simplifies both plasma pretreatment and chromatographic conditions of previously reported procedures and describes the first validated method for the determination of the two nucleotides in human plasma.

Randomized, placebo-controlled single-ascending-dose study to evaluate the safety, tolerability and pharmacokinetics of the HIV nucleoside reverse transcriptase inhibitor, BMS-986001, in healthy subjects.

The objectives of this study were to evaluate the safety, tolerability and pharmacokinetics (PK) of BMS-986001 as a single oral dose in healthy male subjects. Sixty-four healthy male subjects were randomized to receive a single dose of BMS-986001 or placebo in this single-blind, placebo-controlled, sequential ascending-dose study. There were eight treatment groups (10, 30, 100, 300, 600, and 900 mg fed; and 100 and 300 mg fasted) of eight subjects each (BMS-986001 n = 6/placebo n = 2). BMS-986001 was well tolerated, with no serious adverse events (AEs), deaths, or discontinuations due to AEs reported. AEs were experienced by 14.6% of subjects receiving BMS-986001; however, these did not appear to be dose related and were not considered to be related to study drug. BMS-986001 was rapidly absorbed and exhibited a linear dose-exposure relationship across the dose range studied. PK appeared similar whether administered with or without food. Administration of BMS-986001 as a single dose was generally safe and well tolerated. A linear dose-exposure relationship was seen across all doses studied, with no apparent food effect. Further clinical development is warranted.

Phase I, open-label, single-dose study to evaluate the pharmacokinetics and safety of telbivudine in children and adolescents with chronic hepatitis B.

Telbivudine is a nucleoside analogue that has been approved for the treatment of chronic hepatitis B virus (HBV) infection in adults at 600 mg/day. We conducted a phase I, open-label, first-in-pediatrics study to investigate the safety and pharmacokinetics of a single dose of telbivudine in HBV-infected children and adolescents. Eligible patients were enrolled sequentially from older to younger groups, with evaluation of safety and available pharmacokinetic data after each stratum. Adolescent patients (>12 to 18 years) received a single dose of 600 mg telbivudine as an oral solution, while children aged 2 to 12 years received a single dose of 15 or 25 mg/kg of body weight up to a maximum of 600 mg. Telbivudine was well tolerated; all adverse events were mild, and none occurred in more than one patient. The plasma telbivudine concentration-versus-time profiles in adolescents given 600 mg were similar to the mean profile of healthy adults receiving the same oral dose. Children aged 2 to <6 and 6 to 12 years receiving a single 15-mg/kg dose showed similar plasma exposures. To predict the steady-state exposure, plasma concentration-versus-time profiles for patients aged 2 to 12 years (15 mg/kg) and >12 to 18 years (600 mg) were fitted to a two-compartment 1st-order, microconstant, lag time, 1st-order elimination pharmacokinetic (PK) model. This analysis predicted the following dosages to mimic exposures in healthy adults receiving 600 mg/day: 20 mg/kg/day for children 2 to 12 years and 600 mg/day for adolescents. Studies are ongoing to evaluate the efficacy of the recommended dose in pediatric patients. (This study has been registered at ClinicalTrials.gov under registration no. NCT00907894.).

Diagnosis of mitochondrial neurogastrointestinal encephalopathy disease in gastrointestinal biopsies.

A 14-year-old boy with mitochondrial neurogastrointestinal encephalopathy (MNGIE) disease had a lifelong history of failure to thrive and gastrointestinal symptoms including vomiting, pain, and diarrhea, leading to progressive cachexia. At the age of 9 years, after an extensive workup, the diagnosis of Crohn disease was strongly suspected, and he underwent colonoscopy with multiple biopsies. At 11 years of age, vision change and poor balance lead to a diagnosis of leukodystrophy by magnetic resonance imaging. Investigations for metachromatic leukodystrophy, adrenal leukodystrophy, and globoid cell leukodystrophy were all negative. A diagnosis of MNGIE disease was suspected when he continued deteriorating with gastrointestinal symptoms, multiple neurologic deficits, and encephalopathy. Markedly diminished thymidine phosphorylase activity and increased thymidine plasma levels confirmed the diagnosis of MNGIE. At autopsy, megamitochondria were observed by light microscopy in submucosal and myenteric ganglion cells and in smooth muscle cells of muscularis mucosae and muscularis propria, along the entire gastrointestinal tract from the esophagus to the rectum. Megamitochondria in ganglion cells were also observed in a retrospective review of the endoscopic intestinal biopsies taken at age 9 and 13 years and in the appendectomy specimen obtained 1 month before his demise. This study corroborates the presence of megamitochondria in gastrointestinal ganglion cells in MNGIE disease, better illustrates their detailed morphology, and describes for the first time similar structures in the cytoplasm of gastrointestinal smooth muscle cells. Pathologists should be able to recognize these structures by light microscopy and be aware of their association with primary mitochondriopathies.

Improved ruggedness of an ion-pairing liquid chromatography/tandem mass spectrometry assay for the quantitative analysis of the triphosphate metabolite of a nucleoside reverse transcriptase inhibitor in peripheral blood mononuclear cells.

RATIONALE: Nucleotide analogs are highly polar and ionic, which impose great challenges on bioanalysis. Ion-pairing liquid chromatography/tandem mass spectrometry (LC/MS/MS) is the predominant reported approach for such compounds. Assay ruggedness of ion-pairing LC/MS/MS methods was often a challenge due to the potential contamination of the ion source of the mass spectrometer and LC column performance deterioration caused by ion-pairing reagents. METHODS: An ion-pairing reagent was only added to the reconstitution solution to minimize its exposure to the MS ion source. To achieve optimum sensitivity, high pH mobile phases and negative ion ESI were needed for the LC/MS/MS method. However, high pH mobile phases led to the accumulation of ion-pairing reagent on the analytical column, which was washed off with an acidic solution to restore the column performance. In addition, isopropanol was used as a mobile phase modifier to improve peak shape and sensitivity. RESULTS: The limit of detection was established at 1.0 ng/mL in the cell lysate. The calibration curve showed good linearity over the range of 1.0 to 100 ng/mL. The overall accuracy was no less than 87.7% based on four levels of quality control samples. Inter-run precision and intra-run precision across four analytical runs for low, geometric, medium and high QCs were less than 12.9. CONCLUSIONS: By identifying and addressing the root cause of the assay ruggedness problem, we have developed a rugged ion-pairing LC/MS/MS method for a triphosphate (TP) metabolite of BMS-986001 in peripheral blood mononuclear cells. The new method overcame challenges such as a rapid deterioration of the peak shape, increased carryover and extremely poor column life. The peak shape was well maintained throughout multiple analytical runs. This method has been successfully applied to a toxicology study in cynomolgus monkey.

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.

Biodistribution and uptake of 3'-deoxy-3'-fluorothymidine in ENT1-knockout mice and in an ENT1-knockdown tumor model.

UNLABELLED: (18)F-3'-Deoxy-3'-fluorothymidine ((18)F-FLT) is a PET tracer that accumulates in proliferating tissues. The current study was undertaken to determine whether equilibrative nucleoside transporter 1 (ENT1) is important for (18)F-FLT uptake in normal tissues and tumors. METHODS: ENT1-knockout (ENT1(-/-)) mice were generated and compared with wild-type (ENT1(+/+)) mice using small-animal (18)F-FLT PET. In addition, ENT1(+/+) mice were also injected with the ENT1 inhibitor nitrobenzylmercaptopurine ribonucleoside phosphate (NBMPR-P) at 1 h before radiotracer injection, followed by (18)F-FLT small-animal PET. Tissues of interest were analyzed for thymidine kinase 1 and nucleoside transporters by immunoblotting and immunohistochemistry, respectively, and plasma thymidine levels were analyzed by liquid chromatography-mass spectrometry. Human lung carcinoma A549 cells were stably transfected with pSUPER-producing short-hairpin RNA against human ENT1 (hENT1) or a scrambled sequence with no homology to mammalian genes (A549-pSUPER-hENT1 and A549-pSUPER-SC, respectively). Cultured transfected cells were characterized for hENT1 transcript levels and (18)F-FLT uptake using real-time polymerase chain reaction and (3)H-FLT uptake assays, respectively. Transfected A549 cells were grown as xenograft tumors in NIH-III mice, which were analyzed by (18)F-FLT small-animal PET. RESULTS: Compared with noninjected ENT1(+/+) mice, ENT1(+/+) mice injected with NBMPR-P and ENT1(-/-) mice displayed a reduced percentage injected dose per gram (%ID/g) for (18)F-FLT in the blood (84 and 81%, respectively) and an increased %ID/g for (18)F-FLT in the spleen (188 and 469%, respectively) and bone marrow (266 and 453%, respectively). ENT1(-/-) mice displayed 1.65-fold greater plasma thymidine levels than did ENT1(+/+) mice. Spleen tissue from ENT1(+/+) and ENT1(-/-) mice displayed similar thymidine kinase 1 protein levels and significant concentrative nucleoside transporter 1 and 3 staining. Compared with A549-pSUPER-SC cells, A549-pSUPER-hENT1 cells displayed 0.45-fold hENT1 transcript levels and 0.68-fold (3)H-FLT uptake. Compared with A549-pSUPER-SC xenograft tumors, A549-pSUPER-hENT1 xenograft tumors displayed 0.76-fold %ID/g values (ex vivo gamma-counts) and 0.65-fold maximum standardized uptake values (PET image analysis) for (18)F-FLT uptake at 1 h after tracer injection. CONCLUSION: Loss of ENT1 activity significantly affected (18)F-FLT biodistribution in mice and (18)F-FLT uptake in xenograft tumors, suggesting that nucleoside transporters are important mediators of (18)F-FLT uptake in normal and transformed cells.