title: 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.
authors.affiliation: Dipartimento di Scienze Biomediche, University of Sassari, Sassari, Italy.
authors.forename: Antonio Junior
authors.initials: AJ
authors.lastname: Lepedda
authors.forename: Gabriele
authors.initials: G
authors.lastname: Nieddu
authors.forename: Silvia
authors.initials: S
authors.lastname: Rocchiccioli
authors.forename: Pietro
authors.initials: P
authors.lastname: Fresu
authors.forename: Pierina
authors.initials: P
authors.lastname: De Muro
authors.forename: Marilena
authors.initials: M
authors.lastname: Formato
mesh_terms: D000328:Adult; D000368:Aged; D000510:Alpha-Globulins / Q000737:chemistry* / Q000652:urine*; D000595:Amino Acid Sequence; D015415:Biomarkers / Q000652:urine; D002853:Chromatography, Liquid; D003922:Diabetes Mellitus, Type 1 / Q000652:urine*; D003924:Diabetes Mellitus, Type 2 / Q000652:urine*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D016014:Linear Models; D008875:Middle Aged; D008969:Molecular Sequence Data; D053719:Tandem Mass Spectrometry; D055815:Young Adult
chemical_list: D000510:Alpha-Globulins; D015415:Biomarkers; C016001:alpha-1-microglobulin
keywords: Acute phase protein; Bikunin/urinary trypsin inhibitor (UTI); Renal impairment; Systemic inflammation; Type 1 and 2 diabetes