María Dolores Fernández-Moreno

Determination of thiopurine methyltransferase activity in human erythrocytes by high-performance liquid chromatography : Comparison with the radiochemical method

The current article describes a new assay to measure thiopurine methyltransferase (TPMT) activity from red blood cells. This method is based on the measurement of the reaction product 6-methylmercaptopurine (6-MMP) by high-performance liquid chromatography (HPLC). 6-MMP is extracted by ethyl acetate with recoveries of 85%, 80%, 80%, and 92% for 50, 250, 500, and 1,000 ng/100 &mgr;L packed red blood cells, respectively. 6-MMP was identified and measured by a Zorbax CN column installed in an HPLC system. The chromatograms were resolved using a mobile phase consisting of 40 mmol/L sodium phosphate buffer (pH 3) and methanol in a gradient from 1% to 20% of methanol. Under these conditions 6-MMP is well resolved from substrates (6-mercaptopurine and S-adenosyl- l -methionine) and endogenous peaks. When the TPMT activity from 20 patients was measured by the HPLC-linked assay and the classic radiochemical method, a linear correlation was obtained between both procedures (y = 0.99 x + 0.33;x-axis, radiochemical assay;y-axis, HPLC-linked assay;r = 0.98). In conclusion, the current report describes a new, reliable, safe, and nonradioactive method to measure TPMT activity that is shorter and simpler than the previously described ones.

Infliximab therapy reverses the increase of allograft inflammatory factor-1 in serum and colonic mucosa of rats with inflammatory bowel disease.

Abstract Objective: Our purpose was to study the molecular basis of infliximab (IFX) effect on colon mucosa in a colitis model and to identify new biomarkers of mucosal healing. Methods: Healthy rats and rats which were subjected to experimental colitis induced by dextran sulfate sodium, with or without IFX treatment (in the short- and long-term), were studied along with forty-seven IBD patients. Colon mucosal integrity by periodic acid Schiff (PAS) staining, intestinal damage by immunohistochemistry (proliferating cell nuclear antigen, β-catenin, E-cadherin, phosphotyrosine, p-p38, allograft inflammatory factor-1 (AIF-1) and colonic mucosal apoptosis by TUNEL staining were evaluated in rats while serum and colon AIF-1 levels were determined in IBD patients. Results: In rats with colitis, IFX reestablished the epithelial barrier integrity, recovered mucus production and decreased colon inflammation, as verified by reduced serum and colon AIF-1 levels; colon and serum AIF-1 levels were also lower in inactive IBD patients compare to active ones. P38 activation after IFX treatment tended to induce differentiation/proliferation of epithelial cells along the colonic crypt-villous axis. Conclusions: These findings support AIF-1 as a new biomarker of mucosal healing in experimental colitis and suggest that p38 activation is involved in the mucosal healing intracellular mechanism induced by IFX treatment.

710 AZATHIOPRINE PRODUCES OXIDIZATION OF 53 KDA AND 67 KDA PROTEINS AND INDUCES MITOCHONDRIAL DEPENDENT APOPTOSIS IN GSH DEPLETED HEPG2 CELLS

Background and Aims: Results of human and animal studies have suggested that alterations in intestinal motility, small intestinal bacterial overgrowth and increased intestinal permeability are involved in the development of NAFLD. Serotonin and its receptor 5-HT3R are key factors in the regulation of intestinal motility and recently have also been suggested to be involved in regulating permeability. Therefore, the aim of the present study was to investigate the effect of the intestinal 5-HT3R antagonist tropisetron on the development of steatohepatitis in genetically obese ob/ob mice. Methods: Four week old ob/ob knockout and C57/Bl6 wild-type mice had ad libitum access to plain water or water containing tropisetron for 6 weeks. Markers of liver damage were assessed. In addition mRNA expression of TNFa and MyD88 were determined by real time RT-PCR and endotoxin levels in portal plasma were determined using a LAL test. Serotonin reuptake transporter (SERT) and occludin protein concentrations were determined by Western blot and the number of 5-HT-positive cells was measured by immunhistochemistry in the small intestine. Results: Treatment with tropisetron significantly inhibited the development of NASH in ob/ob mice, leading to a marked decrease in hepatic steatosis and inflammation. In addition, tropisetron treatment blocked hepatic MyD88 and TNFa expression in ob/ob mice down to a level of wild-type controls. In vehicle treated ob/ob mice portal endotoxin levels were significantly increased by ~10-fold in comparison to wildtype controls. In contrast, in tropisetron treated mice, endotoxin levels in portal plasma were even lower than in wild-type controls. Moreover, concentration of the tight junction protein occludin and of SERT in the duodenum of tropisetron treated ob/ob mice was markedly increased in comparison to all other groups. However, the number of 5-HT-positive cells in the duodenum was not affected by tropisetron treatment. Conclusion: Our data suggests that alterations in the intestinal serotonergic system and herein practically the 5-HT3R may be involved in the regulation of intestinal permeability and subsequently the development of NASH in obese ob/ob mice and that the 5-HT3R might be a new target for the therapy against NASH.