Analysis of the oxidative damage of DNA,RNA, and their metabolites induced by hyperglycemia and related nephropathy in Sprague Dawley rats

Abstract

We used a sensitive and accurate method based on isotope dilution high-performance liquid chromatography–triple quadrupole mass spectrometry (ID-LC-MS/MS) to determine the levels of 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxo-dGsn) and 8-oxo-7,8-dihydroguanosin (8-oxo-Gsn) in various tissue specimens, plasma, and urine of hyperglycemic Sprague Dawley rats induced by streptozotocin (STZ). The oxidative DNA and RNA damages were observed in various organs and the amounts of 8-oxo-dGsn and 8-oxo-Gsn derived from DNA and RNA were increased with hyperglycemic status. In contrast to the results of the nucleic acid samples derived from tissues, the levels of 8-oxo-Gsn in urine and plasma were significantly higher compared with that of 8-oxo-dGsn, which most likely reflected the RNA damage that occurs more frequently compared with DNA damage. For the oxidative stress induced by hyperglycemia, 8-oxo-Gsn in urine may be a sensitive biomarker on the basis of the results in urine, plasma, and tissues. In addition, high levels of urinary 8-oxo-Gsn were observed before diabetic microvascular complications. Based on that the 8-oxo-dGsn was associated with diabetic nephropathy and RNA was more vulnerable to oxidative stress compared with DNA. We also propose that 8-oxo-Gsn is correlated with diabetic nephropathy and that 8-oxo-Gsn in urine could be a useful and sensitive marker of diabetic nephropathy.     

A single HPLC-PAD-APCI/MS method for the quantitative comparison of phenolic compounds found in leaf, stem, root and fruit extracts of Vaccinium angustifolium

A method was developed for the analysis of Vaccinium angustifolium Ait. (Lowbush blueberry), which is a widely used natural health product, particularly for the treatment of diabetic symptoms. While the anthocyanin content of the fruit has been well characterized, the chemistry of the vegetative parts used in supportive therapy for diabetes has been largely ignored. Using a metabolomics-based approach for compound identification with an emphasis on phenolic metabolites, a single HPLC-PAD-APCI/ MS method was developed for the separation and quantitation of the major metabolites found in the 95% ethanol extracts of leaf, stem, root and fruit. The leaf extract contained high concentrations of chlorogenic acid (approximately 100 microg/mg extract) and a variety of quercetin glycosides that were also detected in the fruit and stem extracts. Flavan-3-ol monomers (+)-catechin and (-)-epicatechin were found in all plant parts but their procyanidin dimers were exclusively identified in the stem and root. The accuracy and precision of the presented method were corroborated by low intra- and inter-day variations in quantitative results in all plant part extracts. Further validation of the extraction and analytical protocols focused on identified compounds with reputed anti-diabetic activity, revealing recoveries greater than 80% and detection limits of 0.12-2.73 microg/mL.