Background: Targeted urinary metabolic profiling of patients with type 2
diabetes mellitus (DM) and diabetic nephropathy (DN) along with healthy control was
conducted with urine being a non-invasive biofluid and ideal for the biomarker studies
related to kidney disorders.
Objective: Other markers of risk for DN in combination with microalbuminuria are
needed for optimal clinical management. The purpose of this study was to propose
new urinary metabolic markers involved in the pathophysiology the DN.
Method: Liquid chromatography coupled mass spectrometry (LC-MS) method was
developed using diamond hydride column for 43 diverse polarities of metabolites.
Multiple reaction monitoring (MRM) was performed using optimized ionization
modes for the 43 studied metabolites. Diabetics (n=26), diabetic nephropathy (n=27) patients, and healthy
controls (27) participated in the study and chemometric analysis was performed using Graphpad and noncommercial
software “Multibase excel add in” to identify significant metabolites. Pathway analysis is
performed using free software metaboanalyst.
Results: Screening of metabolites with the Bonferroni multiple comparison test was performed, which
suggested significantly higher urinary excretion of hippurate in the DN patients as compared to DM and
the control group. Significant positive correlation was found between hippurate and albumin across all the
three groups. Partial least square discriminant analysis (PLS-DA) was performed for DN and DM groups
and showed a good separation between DM patients and DN patients. Further, the pathway analysis suggested
the involvement of arginine and proline metabolism in the pathogenesis of DN.
Conclusion: This study suggests potential use of new LC-MS method for targeted analysis of metabolites
covering different pathways and proposes hippurate potential urinary marker metabolites for discrimination
of DM and DN groups.