Succinic acid enhanced quantitative determination of blood modified nucleosides in the development of diabetic nephropathy based on hydrophilic interaction liquid chromatography mass spectrometry.

The epigenetic modification of DNA or RNA may have relationship with the development of diabetic nephropathy (DN). We designed to develop a liquid chromatography mass spectrometry (LC-MS) coupled with mobile phase additive sensitization method for the analysis of the dissociative epigenetic modified nucleosides in serum of patients from different periods of DN with the health comparison. Serum samples were collected from 43 healthy volunteers and 156 patients, which divided into four groups. A succinic acid enhanced LC-MS/MS strategy was developed for the quantitative analysis of seven nucleosides in serum samples. The signal intensity of seven nucleosides increased three to seven times, respectively. A series of statistical analysis were carried out to demonstrate the dynamic changes of modified nucleosides in four groups. This method showed good specific, accuracy, and stability. The results were calibrated and presented through the formation of m6A/C, I/C, 5-mdC/C, 5-mC/C, pseU/U to eliminate the individual and age differences. The m6A/C, I/C, and pseU/U were found having the dynamic changes in four groups (p< 0.05). And these five modified nucleosides can also used as one parameter to distinguish these four groups by ROC and LDA analysis. Additionally, they were found to be connected with several clinical biochemical indexes through multiple linear regression analysis. Our LC-MS/MS method assay successfully quantifies the modified nucleosides in serum. And three modified nucleosides can act as the potential biomarkers to describe the development of DN and help to evaluate the diagnosis, treatment, and prognosis of DN in clinical.