Development of a method for urine bikunin/urinary trypsin inhibitor (UTI) quantitation and structural characterization: Application to type 1 and type 2 diabetes

Abstract

Bikunin is a plasma proteinase inhibitor often associated with inflammatory conditions. It has a half-life of few minutes and it is rapidly excreted into urine as urinary trypsin inhibitor (UTI). UTI levels are usually low in healthy individuals but they can increase up to tenfold in both acute and chronic inflammatory diseases. This article describes a sensitive method for both direct UTI quantitation and structural characterization. UTI purification was performed by anion exchange micro-chromatography followed by SDS-PAGE. A calibration curve for protein quantitation was set up by using a 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 diabetes, and 28 healthy controls, matched for age and sex with patients, evidencing higher UTI levels in both groups of patients with respect 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 allows UTI quantitation from very low quantities of specimen. Furthermore, as UTI concentration is normalized for creatinine level, the analysis could be also performed on randomly collected urine samples. Finally, MS/MS analysis prospects the possibility of characterizing PTM sites potentially able to affect UTI localization, function, and pathophysiological activity. Preliminary results suggest that UTI levels could represent a useful marker of chronic inflammatory condition in type 1 and 2 diabetes.

Levels of Urinary Trypsin Inhibitor and Structure of Its Chondroitin Sulphate Moiety in Type 1 and Type 2 Diabetes

“…The aim of the present study was to characterize the CS moiety of UTI in diabetic patients by fluorophore-assisted carbohydrate electrophoresis (FACE) analysis, applying a preanalytical step for UTI purification. Furthermore, the obtained results allowed us to validate previous preliminary data on both type 1 and 2 diabetes mellitus (T1DM and T2DM) suggesting UTI as a potential useful marker of these two chronic inflammatory conditions [ 32 ].…”

“…UTI was purified according to a method recently developed by our research group [ 32 ] with slight modifications ( Figure 2 ). Briefly, two elution fractions were obtained by anion exchange chromatography; the first one contained UTI and UTI derivatives, while the latter contained free highly charged GAGs, that is, HS and CS, as assessed by electrophoresis on cellulose acetate plates.…”

Ulinastatin attenuates diabetes-induced cardiac dysfunction by the inhibition of inflammation and apoptosis

“…A previous study reported higher levels of ulinastatin in patients with type 1 and type 2 DM compared with healthy controls ( 29 ). The increased ulinastatin levels observed in patients with DM may reflect a systemic inflammatory state triggered by hyperglycemia, representing a marker of chronic inflammation in DM.…”

The effect of ulinastatin on hyperglycemia in patients undergoing hepatectomy

“…Recent in vivo and in vitro studies have also indicated that UTI administration can inhibit the additional expression of inflammatory cytokines such as tumor necrosis factor (TNFa), interleukin (IL)-2 and IL-8 (which play important parts in dysglycemia), impairment of the bloodebrain barrier, as well as postoperative cognitive dysfunction [9e13]. Recent research has also suggested that UTI could be a useful marker of chronic inflammatory conditions in patients with type-1 or type-2 diabetes [14]. However, treatment of perioperative hyperglycemia by UTI administration has not been reported.…”