Colonic microbiota can promote rapid local improvement of murine colitis by thioguanine independently of T lymphocytes and host metabolism.

OBJECTIVE: Mercaptopurine (MP) and pro-drug azathioprine are 'first-line' oral therapies for maintaining remission in IBD. It is believed that their pharmacodynamic action is due to a slow cumulative decrease in activated lymphocytes homing to inflamed gut. We examined the role of host metabolism, lymphocytes and microbiome for the amelioration of colitis by the related thioguanine (TG). DESIGN: C57Bl/6 mice with or without specific genes altered to elucidate mechanisms responsible for TG's actions were treated daily with oral or intrarectal TG, MP or water. Disease activity was scored daily. At sacrifice, colonic histology, cytokine message, caecal luminal and mucosal microbiomes were analysed. RESULTS: Oral and intrarectal TG but not MP rapidly ameliorated spontaneous chronic colitis in Winnie mice (point mutation in Muc2 secretory mucin). TG ameliorated dextran sodium sulfate-induced chronic colitis in wild-type (WT) mice and in mice lacking T and B lymphocytes. Remarkably, colitis improved without immunosuppressive effects in the absence of host hypoxanthine (guanine) phosphoribosyltransferase (Hprt)-mediated conversion of TG to active drug, the thioguanine nucleotides (TGN). Colonic bacteria converted TG and less so MP to TGN, consistent with intestinal bacterial conversion of TG to so reduce inflammation in the mice lacking host Hprt. TG rapidly induced autophagic flux in epithelial, macrophage and WT but not Hprt-/- fibroblast cell lines and augmented epithelial intracellular bacterial killing. CONCLUSIONS: Treatment by TG is not necessarily dependent on the adaptive immune system. TG is a more efficacious treatment than MP in Winnie spontaneous colitis. Rapid local bacterial conversion of TG correlated with decreased intestinal inflammation and immune activation.

Collection and determination of nucleotide metabolites in neonatal and adult saliva by high performance liquid chromatography with tandem mass spectrometry.

Saliva contains a number of biochemical components which may be useful for diagnosis/monitoring of metabolic disorders, and as markers of cancer or heart disease. Saliva collection is attractive as a non-invasive sampling method for infants and elderly patients. We present a method suitable for saliva collection from neonates. We have applied this technique for the determination of salivary nucleotide metabolites. Saliva was collected from 10 healthy neonates using washed cotton swabs, and directly from 10 adults. Two methods for saliva extraction from oral swabs were evaluated. The analytes were then separated using high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS). The limits of detection for 14 purine/pyrimidine metabolites were variable, ranging from 0.01 to 1.0µM. Recovery of hydrophobic purine/pyrimidine metabolites from cotton tips was consistently high using water/acetonitrile extraction (92.7-111%) compared with water extraction alone. The concentrations of these metabolites were significantly higher in neonatal saliva than in adults. Preliminary ranges for nucleotide metabolites in neonatal and adult saliva are reported. Hypoxanthine and xanthine were grossly raised in neonates (49.3±25.4; 30.9±19.5µM respectively) compared to adults (4.3±3.3; 4.6±4.5µM); nucleosides were also markedly raised in neonates. This study focuses on three essential details: contamination of oral swabs during manufacturing and how to overcome this; weighing swabs to accurately measure small saliva volumes; and methods for extracting saliva metabolites of interest from cotton swabs. A method is described for determining nucleotide metabolites using HPLC with photodiode array or MS/MS. The advantages of utilising saliva are highlighted. Nucleotide metabolites were not simply in equilibrium with plasma, but may be actively secreted into saliva, and this process is more active in neonates than adults.

Low-dose azathioprine or mercaptopurine in combination with allopurinol can bypass many adverse drug reactions in patients with inflammatory bowel disease.

BACKGROUND: The thiopurine drugs, azathioprine and mercaptopurine (MP), are established treatments for IBD. However, therapeutic failure caused by adverse drug reactions occurs frequently. AIM: To study combination of allopurinol with reduced-dose thiopurine in an attempt to avoid adverse drug reactions in the treatment of IBD. METHODS: Patients with drug reactions to full-dose thiopurines were recruited for combination therapy in two IBD centres in this retrospective study. Dosing was guided by measuring thiopurine methyltransferase (for UK patients) or thioguanine nucleotides and methyl-6MP (Australian patients). Response was monitored by clinical activity indices. RESULTS: Of 41 patients, 25 had non-hepatic and 16 had hepatitic reactions. Clinical remission was achieved in 32 patients (78%) with a median follow-up of 41 weeks (range 0.5-400). Patients who did not respond to combination therapy tended to fail early with the same adverse reaction. The relative risk of having an adverse reaction with methyl-6MP in the top interquartile range was 2.7 (1.3-28) times that with methyl-6MP in the lower three quartiles (95% confidence interval). CONCLUSION: The combined experience from our centres is the largest reported experience of this combination therapy strategy in IBD, and the first to provide evidence for benefit in thiopurine and allopurinol co-therapy to avoid non-hepatitic adverse drug reactions.

An unusual pyridine nucleotide accumulating in erythrocytes: its identity and positive correlation with degree of renal failure.

We have investigated an unusual nucleotide that accumulates, with precursors, in the erythrocytes of patients in uraemia. This nucleotide is related chemically to the NAD breakdown product, N1-methyl-2-pyridone-5-carboxamide (Me2Py), found in high concentrations in the plasma of uraemic patients. Both Me2Py and the nucleotide accumulate to high concentrations in the blood during uraemia: our investigations of samples from renal out-patients have provided information on a plausible link between the two compounds.

Adenylosuccinate lyase deficiency--first British case.

A deficiency of adenylosuccinate lyase (ASDL) is characterised by the accumulation of SAICAriboside (SAICAr) and succinyladenosine (S-Ado) in body fluids. The severity of the clinical presentation correlates with a low S-Ado/SAICAr ratio in body fluids. We report the first British case of ADSL deficiency. The patient presented at 14 days with a progressive neonatal encephalopathy and seizures. There was marked axial and peripheral hypotonia. Brain MRI showed widespread white matter changes. She died at 4 weeks of age. Concentrations of SAICAr and SAdo were markedly elevated in urine, plasma and CSF and the SAdo/SAICAr ratio was low, consistent with the severe phenotype. The patient was compound heterozygous for 2 novel ADSL mutations; c.9 G>C (A3P) and c.572 C>T (R190X).

Mutation in the ITPA gene predicts intolerance to azathioprine.

Inosine triphosphate pyrophosphatase (ITPase) deficiency occurs with polymorphic frequencies in Caucasians and results in the benign accumulation of the inosine nucleotide ITP. In 62 patients treated with azathioprine for inflammatory bowel disease, the ITPA 94C>A deficiency-associated allele was significantly associated with adverse drug reactions (OR 4.2, 95% CI 1.6-11.5, p = 0.0034). Significant associations were found for flu-like symptoms (OR 4.7, 95% CI 1.2-18.1, p = 0.0308), rash (OR 10.3, 95% CI 4.7-62.9, p = 0.0213) and pancreatitis (OR 6.2, CI 1.1-32.6, p = 0.0485). Polymorphism in the ITPA gene thus predicts AZA intolerance. Alternative immunosuppressive drugs, particularly 6-thioguanine, should be considered for AZA-intolerant patients with ITPase deficiency.

Erythrocyte thiopurine methyl transferase assessment prior to azathioprine use in the UK.

BACKGROUND: Individuals with low activity of a key metabolic enzyme, thiopurine methyl transferase (TPMT), are more susceptible to azathioprine-induced myelosuppression. AIM: To determine the pattern of use of TPMT activity estimation, with respect to azathioprine use, by medical practitioners in the UK. DESIGN: Retrospective analysis of assay use. METHODS: We analysed all test results (n=3291), and patient and practitioner details, from inception of TPMT assay in 1990 to the end of December 2000, held at the Purine Research Laboratory, Guy's Hospital, London. Patient details were anonymized. Repeat analyses and requests from outside the UK were excluded. RESULTS: The male:female ratio was approximately equal and the mean age was 46.6 (range 0.5-97) years. Thirteen different medical specialities requested assays; Dermatology and Gastroenterology were the most frequent users, together accounting for 86% of requests. The numbers of centres requesting the assay varied widely both within and between different specialities. Some 80% of individuals had normal TPMT activity, 9% enzymic activity above normal, and 10% low activity. Fifteen had no detectable enzymic activity: 0.45% (1:220) of the study population. DISCUSSION: This incidence of undetectable enzyme activity is significantly higher than the previously reported level of 1:300 derived from smaller studies, and makes the economics of screening more attractive.

Abnormal development of hypoxanthine-guanine phosphoribosyltransferase-deficient CNS neuroblastoma.

Lesch-Nyhan syndrome encompasses a host of neurological symptoms, caused by a deficiency of the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGPRT). How the absence of this enzymes activity affects development of the nervous system is unknown. In this study, we examined the ability of N2aTG, a HGPRT-deficient neuroblastoma and its HGPRT-positive counterpart to proliferate and differentiate at various densities. In summary, N2aTG cells proliferated less and differentiated more than N2a cells, with the former cells exhibiting enhanced sensitivity to the effects of low-density culture. Given the homogeneity of this neuroblastoma cell line and its use in studies of neuronal development, the present study indicates that N2aTG cells may prove a suitable in vitro model for the study of non-dopaminergic neuronal development in Lesch-Nyhan syndrome.

Ecto-nucleotidase of cultured rat superior cervical ganglia: dipyridamole is a novel inhibitor.

Based on studies of agonist potencies on intact rat superior cervical ganglia, it has been suggested that this ganglion possesses distinct receptors for purine and pyrimidine nucleotides. However, the potency of an agonist is dependent upon whether it is susceptible to extracellular metabolism by the tissue. The aim of this investigation was to study the metabolism of uridine or adenosine nucleotides and nucleosides and the effects of dipyridamole and an ecto-ATPase inhibitor ARL 67156 (6-N, N-diethyl-D-beta-gamma-dibromomethylene-ATP) on their metabolism. Adenosine- and uridine-5'-triphosphates (ATP and UTP) were catabolised by cultured rat superior cervical ganglia, to their di- and monophosphates. Both ATP and UTP breakdown was significantly inhibited by dipyridamole (10 mcM), whereas ARL 67156 (100 mcM), was a weaker inhibitor of ATP degradation and inhibited UTP breakdown by approximately 40%. Metabolism of ATP and UTP by cultured rat superior cervical ganglia was reduced after treatment with cytosine-beta-arabinoside, suggesting that non-neuronal cells along with neuronal cells contribute to their breakdown. In conclusion, these results indicate that rat superior cervical ganglia possess ecto-nucleotidases capable of catabolising purine and pyrimidine nucleotides to their nucleosides, and that dipyridamole is a potent inhibitor of ecto-nucleotidase activity.

Abnormalities in cellular adhesion of neuroblastoma and fibroblast models of Lesch Nyhan syndrome.

Lesch Nyhan syndrome is a neurological paediatric condition characterized by mental retardation, choreathotosis and self-mutilation. Biochemically, this condition has been attributed to a deficiency in the purine enzyme, hypoxanthine guanine phosphoribosyltransferase, however, the way this affects the development of the nervous system is still unknown. Ma et al.(15) and Stacey et al.(25) found that hypoxanthine guanine phosphoribosyltransferase-deficient neuroblastoma, differentiated significantly more than cells with this enzyme. Here, we report that adhesion of hypoxanthine guanine phosphoribosyltransferase-deficient neuroblastoma as well as fibroblasts from patients with Lesch Nyhan syndrome, exhibited dramatically enhanced adhesion compared to control cells. This increase in adhesion was dependent upon the cell type, density of the cells and upon the substrate used. Development of the nervous system is dependent on adhesion, in particular in the processes of migration, nucleation, differentiation and fasciculation. Our results suggest that the increased adhesion of hypoxanthine guanine phosphoribosyltransferase-deficient neuroblastoma and fibroblasts in vitro underpins the neuropathology of Lesch Nyhan syndrome.

Uridine and its nucleotides: biological actions, therapeutic potentials.

There are many disorders of pyrimidine metabolism and those that involve an alteration in uridine metabolism have neurological and systemic effects, which provide insights into the biological activity of uridine and its analogues. Studies of the metabolism and actions of pyrimidines have uncovered a wealth of information on how these endogenous metabolites modulate cell physiology. In this article, the roles for the pyrimidine nucleoside uridine and its nucleotide derivatives in the regulation of a number of biological systems are examined and benefits of further studies are outlined. An understanding of how uridine and its nucleotides modulate such vastly complicated biological systems should ultimately lead to the development of new ways for modulating human physiology in both normal and diseased states. Likely targets for therapy include the respiratory, circulatory, reproductive, and nervous systems, and the treatment of cancer and HIV infection.

The allopurinol load test lacks specificity for primary urea cycle defects but may indicate unrecognized mitochondrial disease.

Thirty-three children ranging from 2 weeks to 12 years of age were selected for allopurinol loading, 16 on the basis of an increased urinary ourotate excretion detected by routine organic acid analysis (group A), and 17 for clinical reasons suggesting a urea cycle defect (group B). The allopurinol load test proved positive in 13 of 16 patients from group A, mean peak orotate 64.0 mumol/mmol creatinine (upper limit of reference range, 13.2) and 11 of 17 patients from group B, mean peak orotate 41.0 mumol/mmol creatinine (upper limit of reference range, 13.2). Thorough investigation of these patients including urinary and plasma amino acid analysis and, in 17 cases, liver biopsy for histology and measurement of ornithine carbamyltransferase (OCT) and carbamyl-phosphate synthetase (CPS) activity failed to identify any evidence of a urea cycle disorder. However, muscle biopsies performed in 11 patients showed some evidence of mitochondrial disease in four cases, two defined on the basis of reduced respiratory chain enzyme activity and two on the basis of mtDNA abnormalities. These findings indicate that an increased excretion of orotate in sick children may not be uncommon and that a positive allopurinol load test result may not indicate a specific inherited urea cycle defect. In addition, these results raise the interesting possibility that defective ureagenesis may be a feature of mitochondrial disease in some individuals.