Mary P FitzGerald, Matthew J Hejna, Michael J Tagge, Judith Senka, Susan O McGuire.
Loyola University Medical Center, Maywood, IL, USA

Introduction and Objectives: Urinary heparin-binding epidermal growth factor (HB-EGF) and ‘antiproliferative factor’ (APF) have been suggested as promising markers for the presence of IC. Our objectives were to determine whether similar findings were present in patients with symptomatically diagnosed PBS and to explore whether HBEGF/APF levels varied within PBS patients with symptom severity or with menstrual cycle phase.

Methods: We included ten menstruating PBS patients with PBS symptoms and monthly menses, ten nonmenstruating PBS patients with PBS symptoms and no menses, and ten asymptomatic controls with no PBS symptoms and monthly menses. Subjects donated midstream clean-catch urine samples weekly during a menstrual cycle. Samples were centrifuged and stored in aliquots at –80⁰C. HB-EGF concentration was determined by a direct capture method utilizing triplicate urine samples in an Immulon 4 HB Elisa plate, incubated overnight at 4⁰C. HB-EGF signal was revealed with a monoclonal antibody to HB-EGF (1:250, R&D) followed by goat anti-mouse IgG horseradish peroxidase (Santa Cruz). The signal was developed with an ABTS kit from KPL and measured spectrophotometrically at 405nm. HB-EGF concentration was determined by linear regression using a standard curve generated with carrier-free HB-EGF (R&D). APF activity was determined by using a standard MTS proliferation assay with T24 bladder carcinoma cells. T24 cells were plated in growth media containing 10% serum at a density of 10,000 cells per well in a 96-well plate, cells were allowed to attach overnight then changed to a serum free medium. Urine aliquots were adjusted to an osmolarity of 300mOms, sterile filtered and then added to the cell culture wells, at a concentration of 1/3 urine to 2/3 serum free media and allowed to grow for a further 36 hours. Cells were rinsed with buffer and fresh media containing MTS solution, readings were taken at 1, 2, 3, and 4 hours after addition of MTS. Data were expressed as a percentage of control, serum-free wells. We analyzed data to determine the effect of cycle week and diagnosis across the three patient populations by repeated measures ANOVA, controlling for age.

Results: There was considerable variability in HBEGF values during repetitive testing of individual patients, but mean HBEGF values were significantly lower in the PBS groups (median 29, range 24-51ng/mL) than in asymptomatic controls (median 36, range 27-47ng/mL;p=0.045). HBEGF did not vary significantly with age or with the week of menstrual cycle, nor with symptom ratings. There was no difference in T24 proliferation rates when patients with PBS were compared to controls. However, urine from two PBS patients demonstrated marked inhibition of T24 cell proliferation.

Conclusions: Our study supports and extends the findings of others concerning HBEGF as a possible marker for IC/PBS. In PBS patients, the mean of more than one urinary HBEGF value may prove to be a more discriminatory tool than single HBEGF values. Although two patients with PBS demonstrated considerable inhibition of T24 cell proliferation, consistent with the presence of APF, the urine from PBS patients overall did not inhibit proliferation and we were unable to extend prior findings with respect to APF and IC, to patients with symptomatically diagnosed PBS.

Funded by: NIH:DK066076

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