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Tamm‐ H orsfall protein‐associated nucleotides in patients with interstitial cystitis
Author(s) -
Argade Sulabha,
Shaw Timothy,
Su Yongxuan,
Parsons C. Lowell
Publication year - 2013
Publication title -
bju international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.773
H-Index - 148
eISSN - 1464-410X
pISSN - 1464-4096
DOI - 10.1111/j.1464-410x.2012.11691.x
Subject(s) - chemistry , high performance liquid chromatography , electrospray ionization , tandem mass spectrometry , chromatography , mass spectrometry , metabolite , interstitial cystitis , tamm–horsfall protein , urine , urinary system , biochemistry , medicine
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.Objective To identify and characterise T amm‐ H orsfall 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 .Patients, Subjects and Methods T HP 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. 2 HC l ( DMB )‐high performance liquid chromatography ( HPLC ). T HP ‐associated metabolites were isolated by S uperdex‐200 size‐exclusion chromatography ( SEC ) as a second peak ( SP 2) and the SP 2 was further fractionated into six major components by C 18 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 ).Results The THP ‐associated metabolites ( SP 2) were more prevalent in patients with IC than in the controls (40.4% vs 12.6%, P < 0.001) as determined by S uperdex‐200 SEC . Superdex‐200 SEC showed higher SP 2 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 SP 2‐positive patients with IC than those who were SP 2‐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 SP 2 metabolites as nucleotides, namely 5′‐ CMP , 3′‐ UMP , 3′‐ AMP , 3′‐ GMP , 2′‐ AMP and 2′‐ GMP . The total nucleotide content of SP 2 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). L C‐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. H R‐ESI ‐ TOFMS gave further confimation of the nucleotide sturctures with high mass accuracy.Conclusions 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 SP 2‐positive patients with IC , suggesting biological significance. This study provides additional evidence that THP is abnormal in patients with IC .