874 THE ROLE OF URINARY CATIONS IN INTERSTITIAL CYSTITIS

You have accessJournal of UrologyInfections/Inflammation of the Genitourinary Tract: Interstitial Cystitis1 Apr 2013874 THE ROLE OF URINARY CATIONS IN INTERSTITIAL CYSTITIS C. Lowell Parsons, Timothy Shaw, Paul Zupkas, and Sulabha Argade C. Lowell ParsonsC. Lowell Parsons San Diego, CA More articles by this author , Timothy ShawTimothy Shaw San Diego, CA More articles by this author , Paul ZupkasPaul Zupkas San Diego, CA More articles by this author , and Sulabha ArgadeSulabha Argade San Diego, CA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.443AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder epithelial dysfunction has been shown in interstitial cystitis (IC) patients. Urinary cations have been reported to injure GAG layer causing an epithelial leak and are toxic to cultured urothelial cells, thus they have been called toxic factors (TF). The current study was conducted to isolate and identify these TFs from crude urine employing solid-phase extraction (SPE), quantified in both IC patients and control subjects by reverse phase-high performance liquid chromatography (RP-HPLC). The major components of TF were isolated and analyzed by mass spectrometry and evaluated for their potential toxicity to cultured urothelial cells. METHODS Crude urine samples from controls and IC patients were fractionated using Oasis® MCX SPE Cartridge (Waters Corp.). The cationic fraction (CF) containing basic compounds were profiled by C18 RP-HPLC, quantified by area under the peaks and normalized to creatinine. Major individual peaks were isolated and identified by liquid chromatography mass spectrometry (LC-MS and LC-MS/MS). Human HTB4 urothelial cells were plated 20,000 cells/well and used to determine the toxicity of the MCX CF using CellTiter 96® AQueous One Solution Cell proliferation assay (MTS) (Promega). The identified major metabolites that were commercially available were also tested for cytotoxicity. RESULTS MCX CF was isolated from urine of 33 control subjects and 62 IC patients. The mean ± SEM was 3.13 ± 0.23 vs 6.32 ±0.51mAU*min/μg creatinine, respectively (p = 0.00001). The cytotoxicity assay of MCX CF showed much higher toxicity for IC patients compared to control 35% vs -3% (p <0.00001). The major cationic metabolites identified by LC-MS were, amino acids: L-Tryptophan and its derivatives, L-histidine, and L-Phenylalanine; Nucleosides: 1-methyladenosine, N2, N2-dimethylguanosine, 5-methylcytosine; and nucleic acid base: 1-methyl guanine. The HPLC profiles of controls show L-Tryptophan as single major metabolite while IC patient profiles show presence of several major metabolites. Three major metabolites were tested for cytotoxicity; the results showed 1-methyladenosine 57%, 1-methylguanine 21%, L-tryptophan 15%; all were significant (p<0.00001) compared to control 0% and urea 0%. CONCLUSIONS We have isolated and identified major cationic metabolites in IC patients using a simple protocol and reported its cytotoxicity to urothelial cells. The biological significance of these findings is substantial. The identification of these toxic factors provides both new insights into the root cause of IC and a framework for the development of new therapeutic strategies. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e360-e361 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information C. Lowell Parsons San Diego, CA More articles by this author Timothy Shaw San Diego, CA More articles by this author Paul Zupkas San Diego, CA More articles by this author Sulabha Argade San Diego, CA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...