RESUMO
Uranium [U(Ⅵ)] in the blood is known to form stable complexes with apotransferrin (apo-Tf), which plays an important role in mediating the cytotoxicity induced by U(Ⅵ) transported to cells. The present study aimed to establish an new in vitro screening model of U(Ⅵ) decorporation agents through exploring the capability of chelating agents competing with U(Ⅵ) binding to apo-transferrin based on enzyme-linked immunosorbent assay (ELISA). The optimal concentrations of apo-Tf coated antigen, Tf antibody, secondary antibody and U(Ⅵ) treatment were achieved and the stability and reproducibility of this method were validated by methodology study. Using this model, the ability of four chelating agents to mobilize the U(Ⅵ) binding to apo-Tf was evaluated, and the rank of competitiveness was catechol-3,6-bis(methyleiminodiacetic acid） (CBMIDA) ≈ Tiron > apo-Tf > DTPA-CaNa3 ≈ DTPA-ZnNa3. The efficacy of these chelating agents in removal of U(Ⅵ) was tested by animal experiments. The results showed that immediate administration of CBMIDA or Tiron after injection of U(Ⅵ) in mice significantly promoted urinary U(Ⅵ) excretion and reduced U(Ⅵ) accumulation in kidneys and femurs, while DTPA-CaNa3 and DTPA-ZnNa3 have no obvious effects as compared to U(Ⅵ)-exposed mice alone, which was consistent with the results of competitive ELISA method. The animal experiments conform to the rules of the Animal Research Ethics Committee of School of Pharmacy of Fudan University. These results show that the new proposed method is rapid, simple and convenient with good reproducibility and has the potential to be used for in vitro screening of U(Ⅵ) decorporation agents.
RESUMO
Objective: To determine the rate constants for the cleavage of pyrophosphate to phosphate by human serum apo-transferrin.Methods: The cleavage was followed by31P NMR spectroscopy.The data for concentration changes of pyrophosphate (as determined from its NMR peak intensity) with time,in the absence and the presence of MgCl2,at different pH values,were fitted to give second order rate constants.Results: The rate constants at 312 K were 8.83×10-4 L·mmol-1·h-1 at pH 6.85,9.59×10-4 L·mmol-1·h-1 at pH 7.4,and 1.38×10-3 L·mmol-1·h-1 at pH 8.15,for reactions of apo-transferrin (0.5-1.0 mmol/L) with 5 molar equivalence of pyrophosphate.The rate constant increased to 1.21×10-3 L·mmol-1·h-1 at pH 7.4,312 K when 2 mmol/L MgCl2 was added.Conclusion: The cleavage of pyrophosphate to phosphate by human serum apo-transferrin is very slow and follows the second-order kinetics.Mg2+ can slightly enhance the rate of the cleavage.
RESUMO
Objective: To determine the rate constants for the cleavage of pyrophosphate to phosphate by human serum apo-transferrin.Methods: The cleavage was followed by31P NMR spectroscopy.The data for concentration changes of pyrophosphate (as determined from its NMR peak intensity) with time,in the absence and the presence of MgCl2,at different pH values,were fitted to give second order rate constants.Results: The rate constants at 312 K were 8.83×10-4 L·mmol-1·h-1 at pH 6.85,9.59×10-4 L·mmol-1·h-1 at pH 7.4,and 1.38×10-3 L·mmol-1·h-1 at pH 8.15,for reactions of apo-transferrin (0.5-1.0 mmol/L) with 5 molar equivalence of pyrophosphate.The rate constant increased to 1.21×10-3 L·mmol-1·h-1 at pH 7.4,312 K when 2 mmol/L MgCl2 was added.Conclusion: The cleavage of pyrophosphate to phosphate by human serum apo-transferrin is very slow and follows the second-order kinetics.Mg2+ can slightly enhance the rate of the cleavage.