Oxidative stress expression status associated to Helicobacter pylori virulence in gastric diseases.

OBJECTIVES: To analyze the status of expression of inflammation markers, antioxidant and oxidant enzymes in biopsies from patients diagnosed with gastritis, gastric ulcer (GU) and gastric cancer (GC) and the Helicobacter pylori virulence from these isolated biopsies in order to evaluate a possible association among these factors. METHODS: H. pylori genotype from isolated biopsies was performed by PCR. The pattern of expression of inflammation (TNF-alpha, IL-1beta, IL-8, IL-10 and IL-12), oxidant (iNOS and Nox1) and antioxidant markers (MnSOD, GPX and CAT) of biopsies from gastritis, GU, GC and control groups was performed by RT-PCR. RESULTS: Different from other gastric diseases studied here, gastritis is characterized by an oxidative stress with significant expression of TNF-alpha, IL-8, IL-12, iNOS and Nox and significant absence of MnSOD and GPX expression. Gastritis was the only condition where there was an association between TNF-alpha or IL-8 expression and H. pylori cagA+/vacAs1 genotype. In this case, TNF-alpha expression was about 3 times higher when compared to control subjects. CONCLUSION: In this study, only gastritis was found to be associated with significant oxidative stress marker expression of TNF-alpha and IL-8 that was also related to H. pylori virulence, suggesting that they are the main oxidant stress markers responsible to trigger an increase in ROS level that contributes to decrease the expression of the MnSOD and GPX.

Enzymatic method for branched chain alpha-ketoacid determination: application to rapid analysis of urine and plasma samples from maple syrup urine disease patients.

A new method for the determination of branched-chain alpha-ketoacid concentration using lactate dehydrogenase (E C 1.1.1.27) isozyme C4 (LDH C4) from mouse testes is proposed. The assay is performed on urine and plasma without previous treatment. Alpha-ketoglutarate and pyruvate are determined on the same sample using glutamate dehydrogenase (GDH, EC 1.4.1.2) and lactate dehydrogenase isozyme A4 (LDH5) respectively and subtracted from the total alpha-ketoacid concentration obtained with LDH C4. This value corresponds to the branched chain alpha-ketoacid. Results were linear within the concentration range 8 to 170 mumoles/L. Detection limit was 8 mumoles/L. Analytical recovery was higher than 91%. For microplate assays, recoveries were higher than 84% and the detection limit was 20 mumoles/L. Determinations performed with GDH, LDH A4 and LDH C4 allow differentiation of E3 deficiency from other clinical phenotypes of maple syrup urine disease. The method is simple and fast, and adaptation to microplates would allow screening of newborns.

Enzymatic method for branched chain alpha-ketoacid determination: application to rapid analysis of urine and plasma samples from maple syrup urine disease patients

A new method for the determination of branched-chain alpha-ketoacid concentration using lactate dehydrogenase (E C 1.1.1.27) isozyme C4 (LDH) C4) from mouse testes is proposed. The assay is performed on urine and plasma without previous treatment. Alpha-ketoglutarate and pyruvate are determined on the same sample using glutamate dehydrogenase (GDH,EC 1.4.1.2) and lactate dehydrogenase isozyme A4 (LDH5) respectively and subtracted from the total alpha-ketoacid concentration obtained with LDH C4. This value corresponds to the branched chain alpha-ketoacid. Results were linear within the concentration range 8 to 170 mumoles/L. Detection limit was 8 mumoles/L. Analytical recovery was higher than 91 per cent. For microplate assays, recoveries were higher than 84 per cent and the detection limit was 20 mumoles/L. Determinations performed with GDH, LDH A4 and LDH C4 allow differentiation of E3 deficiency from other clinical phenotypes of maple syrup urine disease. The method is simple and fast, and adaptation to microplates would allow screening of newborns.

Enzymatic method for branched chain alpha-ketoacid determination: application to rapid analysis of urine and plasma samples from maple syrup urine disease patients

A new method for the determination of branched-chain alpha-ketoacid concentration using lactate dehydrogenase (E C 1.1.1.27) isozyme C4 (LDH) C4) from mouse testes is proposed. The assay is performed on urine and plasma without previous treatment. Alpha-ketoglutarate and pyruvate are determined on the same sample using glutamate dehydrogenase (GDH,EC 1.4.1.2) and lactate dehydrogenase isozyme A4 (LDH5) respectively and subtracted from the total alpha-ketoacid concentration obtained with LDH C4. This value corresponds to the branched chain alpha-ketoacid. Results were linear within the concentration range 8 to 170 mumoles/L. Detection limit was 8 mumoles/L. Analytical recovery was higher than 91 per cent. For microplate assays, recoveries were higher than 84 per cent and the detection limit was 20 mumoles/L. Determinations performed with GDH, LDH A4 and LDH C4 allow differentiation of E3 deficiency from other clinical phenotypes of maple syrup urine disease. The method is simple and fast, and adaptation to microplates would allow screening of newborns. (AU)