Protease profiling of different biofluids in type 1 diabetes mellitus

Objectives: We aimed to disclose the proteolytic events underlying type 1 diabetes and related complication through protease profiling in the bodily fluids serum, urine and saliva. Design and methods: Zymography followed by LC-MS/MS was performed for protease identification and quantitative comparison of proteolytic activity between healthy, type 1 diabetic patients with no complications and with retinopathy and nephropathy. Western blotting was also accomplished for MMP-9 and MMP-2 identification and expression analysis. Results: Only MMP-2 and MMP-9 were observed in serum with significantly increased levels and activity observed in diabetic patients. In urine and saliva other proteases besides MMPs were identified by MS and presented disease-dependent activity variations. Among these are complex MMP-9/Neutrophil gelatinase-associated lipocalin, aminopeptidase N, azurocidin and kallikrein 1 with more activity noticed in type 1 diabetes patients with nephropathy and/or retinopathy. Conclusion: Our data highlight the usefulness of urine and saliva for the monitoring of type-1 diabetes-related proteolytic events, where aminopeptidase N, azurocidin and kallikrein 1 appear as promising screening targets for type 1 diabetes-related complications. (C) 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.