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Abstract
Diabetes mellitus is one of the most challenging health concerns of the 21st century. With at least 30% of the diabetic population remaining undiagnosed, the development of a diagnostic test, more convenient and reliable than those currently used, would be highly beneficial. Urine allows for non-invasive detection of biomarkers, and is a valuable source of biomarkers particularly for proteomic research. This project aimed at identifying and assessing potential urinary biomarkers for the diagnosis of Type 2 diabetes using proteomic techniques.
After determining an efficient method to desalt and concentrate urine samples, 2-DE was performed on a small set of diabetic and control samples. Comparison of electrophoretic patterns and identification of proteins using LC- MS/MS allowed the selection of several potential biomarkers. The two most prominent markers, transferrin and kininogen, were then assessed on a larger set of samples using immunodetection methods.
Urinary transferrin excretion was measured using a commercial ELISA kit in 56 Type 2 diabetic and matched control samples. As observed on 2-DE patterns, transferrin excretion was significantly increased in Type 2 diabetic patients, although only in microalbuminuric patients compared to both diabetic normoalbuminuric (mean difference = 183.7 μg/mmol; p = 0.005) and control normoalbuminuric (mean difference = 171.1 μg/mmol; p = 0.007) patients.
SDS-PAGE western blotting allowed the semi-quantification of several kininogen isoforms/fragments in 44 Type 2 diabetic and matched control samples. Significant under-expression of the kininogen isoforms/fragments located at 50-65 kDa, as observed in the diabetic 2-DE patterns, was not confirmed. However, variation in intensity ratio for the main kininogen bands (at 50-65 kDa and 100-120 kDa) was close to significance between the diabetic normoalbuminuric and control normoalbuminuric patients, suggesting that the several kininogen isoforms/fragments may be excreted in altered proportions in Type 2 diabetes.
This preliminary study, although limited by the small population size and the lack of thorough clinical diagnosis in the recruitment of patients, confirmed the usefulness of proteomics as a first step in urinary biomarker discovery. Results suggested that both transferrin and kininogen may be biomarkers of interest for the development of better diagnostic tests for Type 2 diabetes, although further investigations are required.