Urinary Metabolomics Identifies a Molecular Correlate of Interstitial Cystitis/Bladder Pain Syndrome in a Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network Cohort

Abstract

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a poorly understood syndrome affecting up to 6.5% of adult women in the U.S. The lack of broadly accepted objective laboratory markers for this condition hampers efforts to diagnose and treat this condition. To identify biochemical markers for IC/BPS, we applied mass spectrometry-based global metabolite profiling to urine specimens from a cohort of female IC/BPS subjects from the Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network. These analyses identified multiple metabolites capable of discriminating IC/BPS and control subjects. Of these candidate markers, etiocholan-3α-ol-17-one sulfate (Etio-S), a sulfoconjugated 5-β reduced isomer of testosterone, distinguished female IC/BPS and control subjects with a sensitivity and specificity >90%. Among IC/BPS subjects, urinary Etio-S levels are correlated with elevated symptom scores (symptoms, pelvic pain, and number of painful body sites) and could resolve high- from low-symptom IC/BPS subgroups. Etio-S-associated biochemical changes persisted through 3-6months of longitudinal follow up. These results raise the possibility that an underlying biochemical abnormality contributes to symptoms in patients with severe IC/BPS.

Fig. 1

PCA-DA distinguishes an IC/BPS participant subgroup by a) negative and b) positive ESI-MS. Score plots of PCA-DA analysis of urine from IC/BPS participants (red) were clustered separately from non-IC/BPS controls (gray). Quality control samples (black) were prepared by pooling equal aliquots from each IC/BPS and non-IC/BPS sample, and run at random intervals. Each data point represents the average of triplicate runs. (c) Loading plot of PCA components 1 and 2 determined by negative ESI-MS. (d) Distribution of values from the loadings plot indicates two distinct subgroups within the IC/BPS population. The D1 loadings values resolve two distinct IC/BPS subgroups, with 19 out of the 40 (47.5%) samples in subgroup 1 (red), and 21 out of the 40 (52.5%) in subgroup 2 (blue).

Fig. 2

Targeted analysis confirms urinary etiocholan-3a-ol-17-one sulfate as a IC/BPS subgroup biomarker. (a) Targeted analysis reveals a significantly higher ratio of m/z 369¹⁸ to m/z 369²⁰ in IC/BPS subgroup 2 compared to subgroup 1 and non-IC/BPS controls (p = 0.0003 and 0.0022, respectively, t-test). Likewise, levels of m/z 369 were higher in subgroup 1 compared to non-IC/BPS controls (p = 0.0071, t-test). (b) High-resolution positive ion ESI mass spectrum is consistent with the empiric formula C₁₉H₃₀O₅S for a [M − H]⁻ species with a difference of − 2.18 ppm. (c) High-resolution negative ion tandem ESI spectrum of the candidate biomarker is consistent with the empiric formula C₁₉H₃₀O₅S ([M − H]⁻ at m/z 369.1745) that fragments to give a peak at m/z 96.9597, consistent with the presence of a sulfate group with the empiric formula SO₄H. (d) Matching GC-EI-MS spectra of this sulfatase-treated, TMS-derivatized analyte compared to a commercially available reference standard. Retention time for both the analyte and standard was 21.8 min.

Fig. 3

Urinary Etio-S is associated with IC/BPS subjects. (a) ROC curve analysis on the discovery set of 40 healthy participants and 40 IC/BPS participants' Etio-S levels and GUPI scores. The area under the curve (AUC) value of 0.87 is statistically significant from the null hypothesis (p < 0.001). (b) ROC curve analysis on validation samples using urine Etio-S levels and GUPI scores. These results were confirmed in three independent experiments.

Fig. 4

IC/BPS participants exhibit urinary sulfometabolomic shifts. (a) Representative MS–MS scan of Etio-S indicates loss of a sulfate group at m/z 97. Using this information, a precursor scan was designed to yield a metabolomic dataset selective for sulfoconjugated metabolites. (b) The characteristic m/z 97 loss, generated by sulfoconjugated aliphatic alcohols. (c) A PCA-DA plot comparing the differences in sulfoconjugated metabolites from IC/BPS and control urines shows the distinct differences in the urinary sulfometabolome of IC/BPS subjects. (d) The distribution of values from the loading plot arising from shifts in multiple sulfoconjugated molecules. These results were confirmed in three independent experiments.

Fig. 5

IC/BPS associated sulfometabolomic shifts are temporally sustained. (a) Profiling sulfoconjugated urinary molecules reveals persistent long-term IC/BPS-associated metabolic shifts. The PCA-DA plot indicates that the metabolic shifts in the IC/BPS sulfometabolome are sustained at longitudinal time points. (b) Values from the D1 loadings plot show that metabolic shifts in the urinary sulfometabolome can stratify subjects into subgroups, indicating its correlative capacity for IC/BPS. Each data point represents the average of triplicate runs.