Bikunin is a plasma proteinase inhibitor often associated with inflammatory conditions. It has a half-life of few minutes and itis rapidly excreted into urine as urinary trypsin inhibitor (UTI).UTI levels are usually low in healthy individuals but they increase up to 10 fold in both acute and chronic inflammatory diseases. The report describes a sensitive direct method for UTI quantitation that does not require any antibody for protein detection. UTI purification was performed by anion exchange chromatography (DEAE-Sephacel resin) followed by SDSPAGE. A calibration curve for protein quantitation was set up by using a highly purified UTI fraction. UTI identification and structural characterization was performed by Nano-LC-MS/MS analysis. The method was applied on urine samples from 9 patients with type 1 diabetes, 11 patients with type 2 diabetesand 28 healthy controls, matched for age and sex with patients, evidencing higher UTI levels in both groups of patients inrespect to controls (p<0.001 and p=0.001, respectively).Spearman’s correlation tests highlighted no association between UTI levels and age in each group tested. Owing to the elevated sensitivity and specificity, the described method seems to be suitable for large-scale clinical studies. Additionally, MS/MS analysisprospects the possibility of characterizing post-translational modification (PTM) sites potentially able to influence UTIlocalization, function and pathophysiological activity. Preliminary results suggest that UTI levels could represent auseful marker of chronic inflammatory condition in type 1 and 2 diabetes.
Urine Bikunin Quantitation and Structural Characterization in Type 1 and Type 2 Diabetes Patients
Nieddu G;
2013-01-01
Abstract
Bikunin is a plasma proteinase inhibitor often associated with inflammatory conditions. It has a half-life of few minutes and itis rapidly excreted into urine as urinary trypsin inhibitor (UTI).UTI levels are usually low in healthy individuals but they increase up to 10 fold in both acute and chronic inflammatory diseases. The report describes a sensitive direct method for UTI quantitation that does not require any antibody for protein detection. UTI purification was performed by anion exchange chromatography (DEAE-Sephacel resin) followed by SDSPAGE. A calibration curve for protein quantitation was set up by using a highly purified UTI fraction. UTI identification and structural characterization was performed by Nano-LC-MS/MS analysis. The method was applied on urine samples from 9 patients with type 1 diabetes, 11 patients with type 2 diabetesand 28 healthy controls, matched for age and sex with patients, evidencing higher UTI levels in both groups of patients inrespect to controls (p<0.001 and p=0.001, respectively).Spearman’s correlation tests highlighted no association between UTI levels and age in each group tested. Owing to the elevated sensitivity and specificity, the described method seems to be suitable for large-scale clinical studies. Additionally, MS/MS analysisprospects the possibility of characterizing post-translational modification (PTM) sites potentially able to influence UTIlocalization, function and pathophysiological activity. Preliminary results suggest that UTI levels could represent auseful marker of chronic inflammatory condition in type 1 and 2 diabetes.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
