Tamm-Horsfall protein-associated nucleotides in patients with interstitial cystitis

WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: The nucleotides associated with Tamm-Horsfall protein (THP) identified in this study are 2'(3') isomers, which are uncommon and very little is known about their biochemical pathway and role in interstitial cystitis (IC). The current study provides additional evidence of our earlier finding that THP is abnormal in IC patients. This can adversely affect THP's protective function, and suggests that THP plays a role in the pathogenesis of the disease.

To identify and characterise Tamm-Horsfall protein (THP)-associated metabolites present in patients with interstitial cystitis (IC). Identification of these metabolites would give us insight into the complex interaction of THP with urinary metabolites and its effect on the protective function of THP.

THP was isolated from the urine specimens of 146 patients with IC and 87 control subjects, and was analysed for total sialic acid (SA) content by 1,2-diamino-4,5-methylenedioxybenzene. 2HCl (DMB)-high performance liquid chromatography (HPLC). THP-associated metabolites were isolated by Superdex-200 size-exclusion chromatography (SEC) as a second peak (SP2) and the SP2 was further fractionated into six major components by C18 reverse phase-HPLC. Ion-pair HPLC analysis was performed to identify and quantify THP-associated metabolites. The metabolite structures were confirmed by reversed-phase HPLC combined with electrospray ionization (ESI), liquid chromatography-tandem mass spectrometry (LC-MS and LC-MS/MS) and by high-resolution ESI-time of flight mass spectrometry (HR-ESI-TOFMS).

The THP-associated metabolites (SP2) were more prevalent in patients with IC than in the controls (40.4% vs 12.6%, P < 0.001) as determined by Superdex-200 SEC. Superdex-200 SEC showed higher SP2 content in patients with IC than in controls, as determined by area under the peak/100 μg of THP. The mean (sem), for patients was 84.3 (8.1) vs 18.0 (2.4) milli-absorption unit*min, for controls (P < 0.001). Total SA content of THP was much lower in SP2-positive patients with IC than those who were SP2-negative. The mean (sem) was 41.6 (3.2) vs 92.6 (2.2) nmol/mg THP, respectively (P < 0.001). Ion-pair HPLC identified SP2 metabolites as nucleotides, namely 5'-CMP, 3'-UMP, 3'-AMP, 3'-GMP, 2'-AMP and 2'-GMP. The total nucleotide content of SP2 was significantly higher in patients with IC than in controls. The mean (sem) was 25.3 (2.9) vs 2.2 (0.2) μg/mg THP, respectively (P < 0.001). LC-MS and LC-MS/MS confirmed the nucleotides based on retention time on HPLC, and mass to charge ratio (m/z) of the parent ion and the daughter ions. HR-ESI-TOFMS gave further confimation of the nucleotide sturctures with high mass accuracy.

In the THP of patients with IC, there is a direct correlation between reduced SA levels and high prevalence of nucleotides associated with it. The THP of patients with IC has a much higher content of these nucleotides than control, and these unique nucleotide isomers identified are very consistent in all SP2-positive patients with IC, suggesting biological significance. This study provides additional evidence that THP is abnormal in patients with IC.