title: Role of urinary cations in the aetiology of bladder symptoms and interstitial cystitis.
abstract: OBJECTIVE
  To identify and characterise urinary cationic metabolites, defined as toxic factors, in patients with interstitial cystitis (IC) and in control subjects. To evaluate the cytotoxicity of the urinary cationic metabolite fraction of patients with IC vs control subjects and of individual metabolites in cultured urothelial cells.
  
  
  METHODS
  Cationic fractions (CFs) were isolated from the urine specimens of 62 patients with IC and 33 control subjects by solid-phase extraction. CF metabolites were profiled using C18 reverse-phase high performance liquid chromatography (RP-HPLC) with UV detection, quantified by area-under-the-peaks using known standards, and normalized to creatinine. RP-HPLC and liquid chromatography (LC)-mass spectrometry (MS)/tandem MS (MS/MS) were used to identify major CF peaks. HTB-4 urothelial cells were used to determine the cytotoxicity of CFs and of individual metabolites with and without Tamm-Horsfall protein (THP).
  
  
  RESULTS
  RP-HPLC analysis showed that metabolite quantity was twofold higher in patients with IC compared with control subjects. The mean (SEM) for control subjects vs patients was 3.1 (0.2) vs 6.3 (0.5) mAU*min/μg creatinine (P < 0.001). LC-MS identified 20 metabolites. Patients with IC had higher levels of modified nucleosides, amino acids and tryptophan derivatives compared with control subjects. The CF cytotoxicity was higher for patients with IC compared with control subjects. The mean (SEM) for control subjects vs patients was -2.3 (2.0)% vs 36.7 (2.7)% (P < 0.001). A total of 17 individual metabolites were tested for their cytotoxicity. Cytotoxicity data for major metabolites were all significant (P < 0.001): 1-methyladenosine (51%), 5-methylcytidine (36%), 1-methyl guanine (31%), N(4)-acetylcytidine (24%), N(7)-methylguanosine (20%) and L-Tryptophan (16%). These metabolites were responsible for higher toxicity in patients with IC. The toxicity of all metabolites was significantly lower in the presence of control THP (P < 0.001).
  
  
  CONCLUSIONS
  Major urinary cationic metabolites were characterised and found to be present in higher amounts in patients with IC compared with control subjects. The cytotoxicity of cationic metabolites in patients with IC was significantly higher than in control subjects, and control THP effectively lowered the cytotoxicity of these metabolites. These data provide new insights into toxic factor composition as well as a framework in which to develop new therapeutic strategies to sequester their harmful activity, which may help relieve the bladder symptoms associated with IC.
authors: Department of Urology, University of California (UC) San Diego Health System, San Diego, USA.
authors: C Lowell
authors: CL
authors: Parsons
authors: Timothy
authors: T
authors: Shaw
authors: Zoltan
authors: Z
authors: Berecz
authors: Yongxuan
authors: Y
authors: Su
authors: Paul
authors: P
authors: Zupkas
authors: Sulabha
authors: S
authors: Argade
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D016022:Case-Control Studies; D002412:Cations / Q000378:metabolism*; D002853:Chromatography, Liquid; D018856:Cystitis, Interstitial / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D005260:Female; D006801:Humans; D013058:Mass Spectrometry; D009705:Nucleosides / Q000378:metabolism*; D015203:Reproducibility of Results; D058949:Uromodulin / Q000378:metabolism; D019459:Urothelium / Q000378:metabolism / Q000473:pathology
chemical_list: D000596:Amino Acids; D002412:Cations; D009705:Nucleosides; C548612:UMOD protein, human; D058949:Uromodulin
keywords: LC-MS; cytotoxicity; interstitial cystitis; urinary cationic fraction; urinary metabolites