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A Comparison of Multiple Urine Markers for Interstitial Cystitis
0022-5347/02/1676-2461/0 THE JOURNAL OF UROLOGY® Copyright © 2002 by AMERICAN UROLOGICAL ASSOCIATION, INC.®
Vol. 167, 2461–2469, June 2002 Printed in U.S.A.
A COMPARISON OF MULTIPLE URINE MARKERS FOR INTERSTITIAL CYSTITIS DEBORAH R. ERICKSON, SHARON X. XIE, VEER P. BHAVANANDAN, MARCIA A. WHEELER, ROBERT E. HURST, LAWRENCE M. DEMERS, LESLIE KUSHNER AND SUSAN K. KEAY* From the Division of Urology and Departments of Health Evaluation Sciences, Biochemistry and Medicine and Pathology, Pennsylvania State University, College of Medicine, Hershey, Pennsylvania, Section of Urology, Yale School of Medicine, New Haven, Connecticut, Department of Urology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, Department of Urology, Long Island Jewish Medical Center, New Hyde Park, New York, and Division of Infectious Diseases, University of Maryland School of Medicine, Baltimore, Maryland
ABSTRACT
Purpose: We measured several urine markers in 24-hour specimens from patients with interstitial cystitis and healthy controls. For each marker we determined whether the urine level was significantly different in interstitial cystitis and control cases, and whether the marker level correlated with the symptom score. Materials and Methods: Study participants included 36 female patients with interstitial cystitis and 36 age matched female volunteers. Multiple urine aliquots were obtained to measure the various markers. Results: Certain markers were significantly increased in interstitial cystitis, including antiproliferative factor, epidermal growth factor, insulin-like growth factor (IGF) binding protein-3 and interleukin (IL)-6. Markers significantly decreased in interstitial cystitis were heparinbinding epidermal growth factor-like growth factor, cyclic guanosine monophosphate and methylhistamine. Other markers were not significantly different in the interstitial cystitis and control groups, including total glycosaminoglycans, epitectin, hyaluronic acid, IL-8, IL-1 and nitrates plus nitrites. IGF-1 was undetectable in 24-hour urine samples but spot voided samples from the same interstitial cystitis population had IGF-1 levels similar to previously reported levels. The only significant association of marker with symptom score was a positive correlation of IL-6 with nocturia. For all markers the conclusions were the same whether the marker was normalized to creatinine or to 24 hours. Conclusions: This study confirmed several previously reported urine alterations in interstitial cystitis, including increased anti-proliferative factor, epidermal growth factor, IGF binding protein-3 and IL-6, and decreased heparin-binding epidermal growth factor-like growth factor and cyclic guanosine monophosphate. Of all markers studied anti-proliferative factor had the least overlap in the interstitial cystitis and control groups, and so it is the most likely candidate to become a diagnostic test. KEY WORDS: bladder; cystitis, interstitial; growth substances; biological markers; signs and symptoms
To our knowledge the etiologies of interstitial cystitis are unknown. The most prominent theories are bladder epithelial dysfunction, bladder mast cell activation and allergic or autoimmune processes.1 Objective markers of interstitial cystitis would be useful for diagnosing interstitial cystitis more precisely, selecting patients with specific pathophysiological conditions for focused research and treatments, and providing objective outcome measurements for treatment trials. Several alterations in interstitial cystitis urine have been reported but urine markers are not widely used for interstitial cystitis diagnosis or research.1, 2 A reason is that many markers have been reported and measuring all would be expensive. Also, some previously reported markers were not confirmed to be altered in interstitial cystitis cases when tested by different groups.2 These contradictory results may have been due to different assay methods, sample sizes, patient selection criteria, treatments or patient heterogeneity. Thus, an impor-
tant first step in choosing the most useful markers would be to compare multiple interstitial cystitis markers in the same urine specimens. In this study we tested 14 interstitial cystitis markers. Each marker was previously reported to be altered or was expected to be altered in interstitial cystitis cases. One of us obtained 24-hour urine specimens and frozen aliquots were sent to co-authors for blinded marker analysis. For each marker we tested 2 hypotheses, including 1) whether the marker level was significantly different in interstitial cystitis and control cases, and 2) whether the marker level correlated with the symptom score. We believed that comparing these answers for several different markers may have identified which if any potential markers appears promising as a diagnostic test in patients with interstitial cystitis-like symptoms. The relevant literature for these markers was summarized. Bladder epithelial abnormalities. A main theory of interstitial cystitis is that the bladder epithelium is deficient.2 In the previous literature several urine alterations are described that may reflect epithelial abnormalities, including increased hyaluronic acid,3, 4 decreased epitectin5 and decreased total glycosaminoglycans (GAGs).6 However, 3 subsequent studies using different GAG assay methods did not
Accepted for publication January 4, 2002. Supported by the Interstitial Cystitis Association (Fishbein Foundation), and in part by National Institutes of Health Grants R01 DK38311 and DK 49450. * Financial interest and/or other relationship with Merck and Roche. 2461
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COMPARISON OF MULTIPLE URINE MARKERS FOR INTERSTITIAL CYSTITIS
confirm that urine GAGs were decreased in interstitial cystitis cases.3, 7, 8 The reasons for epithelial deficiency in interstitial cystitis are not definitively proven but they may involve alterations in growth or other factors that influence epithelial proliferation. For example, interstitial cystitis urine contains an antiproliferative factor that inhibits the proliferation of normal human bladder epithelial cells in culture.9, 10 Other urine growth factor alterations reported include decreased heparin-binding epidermal growth factor-like growth factor and increased epidermal growth factor (EGF), insulin-like growth factor (IGF)-1 and IGF binding protein-3.10, 11 Methylhistamine. Another main theory of interstitial cystitis is that bladder mast cells are activated, releasing histamine and other mediators.1, 12 Urine histamine and methylhistamine may reflect bladder mast cell activation. These 2 markers were previously noted to be elevated in the urine of untreated patients with interstitial cystitis.13 Cytokines. Urine cytokine levels may reflect bladder inflammation in interstitial cystitis cases. Urine interleukin (IL)-6 and IL-8 were previously reported to be elevated in some patients with interstitial cystitis.14 –17 Urine IL-1 was elevated in cases of bacterial cystitis but not in those of interstitial cystitis.18 We decided to retest whether IL-1 is elevated in interstitial cystitis since IL-1 is induced by substance P19 and substance P may be involved in interstitial cystitis pathogenesis.20 Urine markers of nitric oxide. Decreased nitric oxide may contribute to interstitial cystitis by several possible mechanisms. Nitric oxide is a smooth muscle relaxant and vasodilator, and so decreased nitric oxide may lead to bladder spasms or the decreased bladder blood flow reported in interstitial cystitis cases.21 Nitric oxide also inhibits mast cell activation. Urine nitric oxide levels may be reflected by cyclic guanosine monophosphate (cGMP) since nitric oxide activates guanylyl cyclase, or by nitrates plus nitrites since nitric oxide is oxidized to these compounds. Wheeler16 and Smith22 et al previously reported that patients with interstitial cystitis had decreased urine cGMP and decreased nitric oxide synthase activity in centrifuged urine pellets. Nitric oxide synthase measurements were impractical in this study but cGMP and nitrates plus nitrites were included as indirect indicators of nitric oxide activity. MATERIALS AND METHODS
Subjects. The study was approved by the institutional review board of Pennsylvania State University College of Medicine. Study participants included 36 white female patients with interstitial cystitis and 36 healthy female controls, of whom 33 were white and 3 were Asian. Each control was age matched within ⫾ 5 years to a patient with interstitial cystitis. Mean age of the patients with interstitial cystitis was 56.6 years (range 22 to 86). The mean duration of symptoms was 8.1 years (range 1 to 28). Patients with interstitial cystitis had chronic disease and were on various treatments, and some had good symptom relief. However, at original presentation all patients met the symptom criteria established by the National Institute of Diabetes, Digestive and Kidney Diseases.1 All underwent cystoscopy under anesthesia with bladder distention and had diffuse glomerulations or Hunner’s ulcer. Of the patients 31 underwent bladder distention 6 months to 8 years before this study. The remaining 5 patients were studied 3 to 6 months after distention and were having active symptoms. Thus, no patient was in a post-distention remission period. A total of 28 patients were receiving conventional interstitial cystitis treatments (table 1). In 4 cases trimethoprim-sulfamethoxazole, phenazopyridine or tolterodine provided no symptom relief. Another 3 patients received no medications because symptoms were tolerable with only diet and/or urinary alkalinization, while 1
TABLE 1. Interstitial cystitis treatments used by patients in the study No. Pts. Using Treatment 7 3 3 4 3 1 1 1 1 1 1 1 1
Treatment Pentosan polysulfate Amitriptyline Hydroxyzine Pentosan polysulfate, hydroxyzine Pentosan polysulfate, amitriptyline Amitriptyline, hydroxyzine Amitriptyline, hydroxyzine, clonazepam Pentosan polysulfate, amitriptyline, hydroxyzine, clonazepam Pentosan polysulfate, gabapentin Pentosan polysulfate, hyoscyamine Amitriptyline, gabapentin Monthly intravesical cocktail (dimethyl sulfoxide, triamcinolone, heparin, sodium bicarbonate) Biwkly. intravesical hydrocortisone
was in long-term remission after previous dimethyl sulfoxide treatment without maintenance. All study participants completed the University of Wisconsin interstitial cystitis symptom scale.23, 24 This scale includes 7 bladder related symptoms, namely bladder discomfort, bladder pain, arising at night to urinate, frequent voiding during the day, sleeping difficulty because of bladder symptoms, urinary urgency and burning sensation in the bladder. Each symptom is scored between 0 —none and 6 —a lot. Thus, correlations can be tested for each of the 7 individual symptoms or for a total symptom score. Urine specimens. Each subject collected urine for 24 hours in a plastic container with no preservatives. Participants were not asked to adhere to any specific dietary restrictions during urine collection. Urine was maintained refrigerated during collection and brought on ice to Hershey Medical Center. Urine volume was measured and aliquots of urine were centrifuged, frozen and stored at ⫺70C until shipment on dry ice to coauthors by overnight courier. Co-authors were blinded to which samples were interstitial cystitis or control specimens. Marker assays. Total urine GAGs were measured by one of us (R. E. H.) using a previously described method.6 Epitectin was measured by one of us (V. P. B.) using an enzyme-linked immunoassay (ELISA), as previously described.5 Hyaluronic acid was measured by one of us (V. P. B.). The previously used radioimmunoassay kit was no longer available, and so hyaluronic acid was measured by a competitive assay using biotinylated hyaluronic acid binding protein, similar to that described by Fosang et al.25 Anti-proliferative factor was measured by one of us (S. K. K.) using a thymidine uptake assay, as previously described.9 Heparin-binding epidermal growth factor-like growth factor, EGF, IGF-1 and IGF binding protein-3 were measured by one of us (S. K. K.) using ELISA, as previously described.11 Methylhistamine was measured at the core endocrine laboratory, Hershey Medical Center using a radioimmunoassay kit (Pharmacia, Uppsala, Sweden). This kit was used by El-Mansoury et al, who previously reported increased methylhistamine levels in interstitial cystitis cases.13 IL-6 was also measured at the core endocrine laboratory using a Quantikine ELISA kit (R & D Systems, Minneapolis, Minnesota).14 Cyclic GMP was measured by one of us (M. A. W.) by radioimmunoassay (Biomedical Technologies, Stoughton, Massachusetts), as previously described.22 Urine nitrate plus nitrite was also measured by one of us (M. A. W.), as previously described,16 and IL-8 was determined using an ELISA kit (Endogen, Woburne, Massachusetts). IL-1 was measured by one of us (L. K.) using a high sensitivity ELISA system (Amersham, Piscataway, New Jersey). Urine creatinine. Urine creatinine was measured at the clinical laboratory, Hershey Medical Center using a modular
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COMPARISON OF MULTIPLE URINE MARKERS FOR INTERSTITIAL CYSTITIS
autoanalyzer (Roche Diagnostics, Indianapolis, Indiana) that applies the picric acid reaction. Statistical analysis. Urine marker measurements were returned to Pennsylvania State University for statistical analysis, which was performed by one of us (S. X. X.). The Wilcoxon signed rank test was used to compare urine marker levels in patients with interstitial cystitis and controls. Since not all marker levels were normally distributed, results are reported as the median and interquartile range. Kendall’s tau correlation26 was calculated for each marker and symptom score. All statistical tests were 2-sided. All analyses were performed using commercially available software. RESULTS
Comparison of interstitial cystitis and control groups. Table 2 shows the median and interquartile ranges for urine markers in the interstitial cystitis and control groups. The markers significantly increased in interstitial cystitis cases were anti-proliferative factor activity, EGF, IGF binding protein-3 and IL-6. In contrast, heparin-binding epidermal growth factor-like growth factor, cGMP and methylhis-
tamine were significantly decreased in interstitial cystitis cases. Figures 1 to 7 show individual marker results. Markers not significantly different in the interstitial cystitis and control groups were total GAGs, epitectin, hyaluronic acid, nitrates plus nitrites, IL-1 and IL-8. Three patients had high IL-8 but since all others had normal levels, average IL-8 was not increased in the interstitial cystitis group. For all comparisons the results were the same whether the marker level was normalized to urine creatinine or to a 24-hour period. Of all markers tested anti-proliferative factor activity most clearly separated the interstitial cystitis and control groups. When anti-proliferative factor was reported as a binary variable with positive considered more than 2 standard deviations from the mean of an assay control, 33 of 36 patients with interstitial cystitis were positive but only 1 of 36 controls was positive. IGF-1 was undetectable in all except 1 patient with interstitial cystitis. To test whether 24-hour collection affected the results we tested frozen aliquots of 15 clean catch urine samples from the same interstitial cystitis patient population. IGF-1 in these specimens was similar to that in the
TABLE 2. Urine marker levels in interstitial cystitis and control groups Median (interquartile range) Marker Anti-proliferative factor activity (% change in thymidine incorporation) cGMP: pM./ml. pM./mg. creatinine nM./24 hrs. EGF: Ng./ml. Ng./mg. creatinine g./24 hrs. Epitectin: Ng./ml. Ng./mg. creatinine g./24 hrs. GAG: NM./ml. NM./mg. creatinine M./24 hrs. Hyaluronic acid: Ng./ml. Ng./mg. creatinine g./24 hrs. Heparin binding epidermal growth factorlike growth factor: Ng./ml. Ng./mg. creatinine g./24 hrs. IGF binding protein-3: Ng./ml. Ng./mg. creatinine g./24 hrs. IL-1: Pg./ml. Pg./mg. creatinine Ng./24 hrs. IL-6: Pg./ml. Pg./mg. creatinine Ng./24 hrs. IL-8: Pg./ml. Pg./mg. creatinine Ng./24 hrs. Methylhistamine: Ng./ml. Ng./mg. creatinine g./24 hrs. Nitrate: M./ml. M./mg. creatinine mM./24 hrs.
Interstitial Cystitis ⫺54
(22)
134 218 200
(133) (158) (148)
15.0 (21.8) 27 (16) 25.8 (22.5) 124 293 242
(200) (276) (274)
1.26 (2.19) 2.49 (2.30) 2.62 (2.37) 356 640 595
(314) (179) (243)
2.55 (1.55) 5 (5) 4.12 (4.06) 7.1 (10.8) 11 (13) 11 (13.6) 0.32 (0.37) 0.63 (0.89) 0.52 (0.81) 4.9 (3.9) 10.2 (13.7) 9.4 (9.0)
Control 16.5 206 329 338
(22) (209) (289) (274)
7.35 (4.8) 10 (10) 10.4 (5.8) 199 349 327
(311) (388) (380)
1.54 (1.62) 1.98 (1.97) 1.90 (1.70)
Difference ⫺68.5
p Value (Wilcoxon signed rank test)
(38.5)
⬍0.0001
(153) (290) (324)
0.0061 0.012 0.0052
9.1 15 12.7
(22.4) (23) (22.2)
⬍0.0001 ⬍0.0001 ⬍0.0001
⫺8.7 ⫺57 ⫺77
(333) (350) (347)
0.24 0.065 0.052
40 ⫺113 104
0.08 (2.24) ⫺0.12 (2.72) ⫺1.28 (2.57)
0.78 0.98 0.78
(206) (320) (284)
8.4 56 51
(324) (272) (399)
0.72 0.34 0.68
11.5 (8.05) 17 (19) 16.0 (11.7)
⫺7.3 ⫺12 ⫺11.9
(8.75) (20) (14.6)
⬍0.0001 ⬍0.0001 ⬍0.0001
1.3 3 2.9
(13.0) (11) (14.5)
0.055 0.0058 0.020
352 617 553
4.0 8 7.8
(4.6) (6) (5.3)
0.31 (0.79) 0.70 (1.5) 0.51 (1.4) 1.5 2.7 2.7
(0.4) (2.3) (1.7)
3.4 (11.2) 6.6 (19.5) 6.5 (18)
6.8 (8.5) 10.9 (20.4) 8.5 (15.7)
42.8 (53.1) 85.8 (83.9) 76.5 (85.2)
63.8 (62.2) 114 (65.4) 112 (80.8)
1.06 (0.77) 1.66 (1.22) 1.50 (1.06)
1.20 (1.27) 1.94 (1.52) 1.67 (1.14)
⫺0.012 (1.2) ⫺0.06 (1.7) ⫺0.07 (1.7) 3.4 7.2 6.7
(4.4) (14) (9.0)
⫺0.4 (11.8) 0.08 (16) 0.08 (16) ⫺20.4 ⫺29 ⫺16
(48) (76) (81)
0.096 (1.44) ⫺0.096 (2.29) ⫺0.24 (1.74)
0.66 0.75 0.69 ⬍0.0001 ⬍0.0001 ⬍0.0001 0.99 0.82 0.87 0.043 0.030 0.005 0.50 0.40 0.29
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COMPARISON OF MULTIPLE URINE MARKERS FOR INTERSTITIAL CYSTITIS
FIG. 1. Percent change in 3H-thymidine incorporation by human bladder epithelial cells incubated with interstitial cystitis (IC) or control (Ctr) urine specimens compared with cells incubated with culture medium alone. Horizontal line indicates median value. Vertical line indicates 25th and 75th percentiles.
previous report (data not shown).11 These results suggest that IGF-1 may bind to the plastic containers used to collect 24-hour urine specimens or IGF-1 may be unstable when stored in the refrigerator for 24 hours. Symptom scores. Table 3 lists median and interquartile ranges of University of Wisconsin symptom scores in the interstitial cystitis and control groups. The total University of Wisconsin score is the sum of the scores of the 7 individual interstitial cystitis symptoms. In the interstitial cystitis group urine marker levels were tested for correlations with symptom scores. No marker levels correlated with the total University of Wisconsin score. Only 1 correlation was identified for any marker and any of the 7 individual symptom scores. IL-6 positively correlated with nocturia score when normalized to creatinine and to 24 hours (r ⫽ 0.30, p ⫽ 0.013 and r ⫽ 0.27, p ⫽ 0.024, respectively).
FIG. 2. EGF in interstitial cystitis (IC) and control (Ctr) urine specimens. A, per mg. urine creatinine. B, per 24 hours. Horizontal line indicates median value. Vertical line indicates 25th and 75th percentiles.
DISCUSSION
In this study we compared several urine components in patients with interstitial cystitis and controls. All markers were measured in the same urine specimens obtained by a single investigator at a single institution. Some urine markers were altered in interstitial cystitis cases, while others were not. Urine glycoconjugates. Urine glycoconjugate alterations are thought to reflect bladder epithelial deficiency in interstitial cystitis cases. One of us (R. E. H.) originally reported that total GAGs were decreased in interstitial cystitis urine.6 Later studies using different assay methods did not confirm decreased total GAG in interstitial cystitis urine.3, 7, 8 The current study using the original assay procedure6 also did not confirm decreased total GAG levels in interstitial cystitis cases. A possible explanation was that the original study included new untreated patients with interstitial cystitis, while the current study included patients with interstitial cystitis who were on chronic treatment. It is also possible that different patient characteristics may have a role, particularly if the previous study contained more patients with more severe disease manifestations. Two of us (D. R. E. and V. P. B.) previously reported that interstitial cystitis urine had significantly decreased epitectin (MUC-1 glycoprotein).5 However, the current study did
not confirm a significant decrease. This discrepancy may have been related to assay variability since mean urine epitectin levels in this study were much lower than in the previous report in interstitial cystitis cases and controls (0.29 and 0.35 versus 3.9 and 6.4 g./mg. creatinine, respectively).5 This study also failed to confirm previous reports3, 4 that interstitial cystitis cases have increased urine hyaluronic acid. A possible explanation was that the 3 studies used different assay methods for hyaluronic acid. Another possible explanation was that our original report included untreated patients with interstitial cystitis,4 while the current study included chronically treated patients. Heterogeneity of the hyaluronic acid molecule may also be relevant. Wei et al previously reported that the high molecular weight fraction of hyaluronic acid was increased in interstitial cystitis urine.3 As a separate analysis, we compared the various hyaluronic acid fractions in some samples and also observed increased high molecular weight hyaluronic acid in interstitial cystitis cases (unpublished data). Despite these negative results when studying chronically treated patients with interstitial cystitis, we have not ruled out that GAGs or hyaluronic acid may be useful markers in new untreated patients. We are performing a prospective study to measure
COMPARISON OF MULTIPLE URINE MARKERS FOR INTERSTITIAL CYSTITIS
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FIG. 3. IGF binding protein-3 (BP3) in interstitial cystitis (IC) and control (Ctr) urine specimens. A, per mg. urine creatinine. B, per 24 hours. Horizontal line indicates median value. Vertical line indicates 25th and 75th percentiles.
urine glycoconjugates in new patients and compare them with relevant bladder biopsy features. Urine anti-proliferative factor and growth factors. Antiproliferative factor activity was significantly increased in interstitial cystitis cases. When comparing all markers in this study, anti-proliferative factor showed the least overlap in the interstitial cystitis and control groups (fig. 1). When anti-proliferative factor was considered a binary variable, 33 of 36 patients with interstitial cystitis but only 1 control were positive. Since anti-proliferative factor most clearly separated the interstitial cystitis and control groups, it is currently the most promising diagnostic marker. The 3 urine growth factors that were altered in interstitial cystitis were decreased heparin-binding epidermal growth factor-like growth factor, increased EGF and increased IGF binding protein-3. These 3 results are consistent with those in earlier reports.10, 11 In regard to interstitial cystitis pathophysiology these changes may be related. When purified antiproliferative factor is added to cultured human urothelial cells, it decreases their production of heparin-binding epidermal growth factor-like growth factor and increases their production of EGF and IGF binding protein-3.27 In regard to use as interstitial cystitis markers these growth factors were significantly altered in interstitial cystitis cases but showed more overlap in the interstitial cystitis and control groups
FIG. 4. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) in interstitial cystitis (IC) and control (Ctr) urine specimens. A, per mg. urine creatinine. B, per 24 hours. Horizontal line indicates median value. Vertical line indicates 25th and 75th percentiles.
than anti-proliferative factor. Some of this overlap may have been related to treatment. We are including these markers in our ongoing prospective study. IGF-1 was not detectable in 24-hour urine samples collected for this study. The most likely explanations are that it bound to the plastic containers or it was not stable when stored in the refrigerator for 24 hours. Methylhistamine. Methylhistamine is a stable metabolite of histamine. Urine methylhistamine may reflect bladder mast cell activation in interstitial cystitis cases.12, 13 El-Mansoury et al previously reported that urine methylhistamine was much higher in patients with interstitial cystitis than in healthy controls.13 We noted the opposite result, that is patients with interstitial cystitis had lower urine methylhistamine than controls. Mean methylhistamine plus or minus standard error of mean in controls was much higher in the current than in the previous study (79 ⫾ 50.6 versus
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COMPARISON OF MULTIPLE URINE MARKERS FOR INTERSTITIAL CYSTITIS
FIG. 5. IL-6 in interstitial cystitis (IC) and control (Ctr) urine specimens. A, in pg./mg. urine creatinine. B, ng./24 hours. Horizontal line indicates median value. Vertical lines indicate 25th and 75th percentiles.
17.2 ⫾ 7.9 ng./ml.) and in interstitial cystitis cases it was much lower (74 ⫾ 73 versus 158 ⫾ 135 ng./ml.). In regard to controls a key difference in the 2 studies was that the previous study required all participants (patients with interstitial cystitis and controls) to maintain a special diet on the day of urine collection. The diet avoided foods and drinks high in biogenic amine precursors, such as cheese, beer, wine, caffeine and so forth. Our study participants were not asked to maintain a special diet. The large difference in control urine methylhistamine in these 2 studies suggests that diet may be a major contributor to urine methylhistamine in healthy individuals. In further support of this conclusion 2 other studies that measured urine methylhistamine in healthy controls using the same assay and no special diet showed control urine methylhistamine levels similar to those in our study.28, 29 In regard to interstitial cystitis the previous study included new untreated patients, while our study included patients with chronic disease. Some interstitial cystitis treatments, including amitriptyline, hydroxyzine and pentosan polysulfate, are thought to stabilize mast cells and prevent hista-
FIG. 6. cGMP in interstitial cystitis (IC) and control (Ctr) urine specimens. A, in pM./mg. urine creatinine. B, in nM./24 hours. Horizontal line indicates median value. Vertical lines indicate 25th and 75th percentiles.
mine release.12, 30 Since 26 of our patients were on at least 1 of these medications, it is the most likely explanation of the lower methylhistamine levels in our patients. It is also possible that our patients were restricting their diets to avoid biogenic amine precursors because such dietary advice is widely available for patients with interstitial cystitis. However, diet does not explain the discrepancy in methylhistamine levels in our patients with interstitial cystitis and those of the previous study because all participants in the previous study were on a restricted diet. Despite these negative results when comparing chronically treated patients with interstitial cystitis with controls, we have not ruled out the possibility that methylhistamine may be a useful marker in untreated patients with interstitial cystitis. We are performing a prospective study to measure methylhistamine in new untreated patients and compare urine methylhistamine with the density of mast cells on patient bladder biopsies. Cytokines. IL-6 is a pro-inflammatory cytokine produced by different cell types, including vascular endothelial cells, macrophages and fibroblasts. The current study and 3 earlier series showed increased urine IL-6 in interstitial cystitis cases.14, 15, 17 Despite these consistent findings IL-6 is unlikely to be useful as a diagnostic marker for interstitial
COMPARISON OF MULTIPLE URINE MARKERS FOR INTERSTITIAL CYSTITIS
FIG. 7. Methylhistamine (MH) in interstitial cystitis (IC) and control (Ctr) urine specimens. A, in ng./mg. urine creatinine. B, in mcg./24 hours). Horizontal line indicates median value. Vertical lines indicate 25th and 75th percentiles.
TABLE 3. University of Wisconsin symptom scores in interstitial cystitis and control groups Symptoms
Bladder discomfort Bladder pain Nocturia Daytime frequency Sleeping difficulty due to bladder symptoms Urge to urinate Burning sensation in bladder Totals
Median (interquartile range) Interstitial Cystitis
Control
4 3 5 5 4 5 3
(2.5–6) (1.5–5) (2–6) (3.5–6) (1.5–6) (3–6) (2–6)
0 0 0 (0–1) 0 (0–2) 0 0 0
27.5 (18–35)
0 (0–3)
cystitis because it lacks specificity for that disease. Urine IL-6 is also elevated in other urological diseases, including bladder cancer31 and bacterial cystitis.32 However, in interstitial cystitis cases urine IL-6 appears to correlate with symptom severity15, 17 and the degree of inflammation on histological testing.14 Therefore, it is possible that IL-6 may be a useful indicator of disease activity in patients already
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known to have interstitial cystitis. Interestingly some patients with interstitial cystitis had clearly elevated IL-6, while others had levels in the control range (fig. 5). This finding may have been related to treatment effects or differences in interstitial cystitis pathophysiology in individuals. IL-8 is a chemokine that attracts neutrophils and T lymphocytes. Wheeler et al reported that IL-8 was elevated in some patients with interstitial cystitis and associated with leukocyte esterase positive urine.16 Peters et al noted that patients with active interstitial cystitis had increased urine IL-8, while those in remission after bacillus Calmette-Guerin treatment had low IL-8.15 We identified no significant difference in median urine IL-8 in the interstitial cystitis and control groups. However, 3 patients with interstitial cystitis had high IL-8. These patients also had high IL-6 and IL-1. We are including these cytokines in our prospective study and plan to correlate them with inflammatory changes on bladder biopsy. IL-1 has many pro-inflammatory effects.18 Our study showed that urine IL-1 was similar in patients with interstitial cystitis and controls, consistent with a previous report that urine IL-1 was elevated in bacterial cystitis but not interstitial cystitis cases.18 Urine markers of nitric oxide activity. Nitric oxide is induced during inflammation. Lundberg et al measured nitric oxide gas in the bladder and observed that increased levels were associated with any type of inflammation, including infection, radiation, bacillus Calmette-Guerin cystitis and interstitial cystitis.33 The results of this Swedish study may not be relevant to American patients with interstitial cystitis, of whom most do not have severe inflammation on bladder biopsy.34 Wheeler16 and Smith22 et al previously measured other markers of the nitric oxide pathway, namely nitric oxide synthase, cGMP and nitrates plus nitrites. Urine nitric oxide synthase and cGMP increased in bacterial cystitis but decreased in interstitial cystitis cases. Nitrate plus nitrite levels were not significantly altered in patients with interstitial cystitis, probably because the major source of urine nitrate is dietary.16, 35 The current study showed the same results. Urine cGMP was decreased in interstitial cystitis cases, while nitrate plus nitrite was not. Cyclic GMP levels overlapped in the interstitial cystitis and control groups (fig. 6). This finding may have been related to treatment or to differences in interstitial cystitis pathophysiology in individuals. In our continuing study we plan to test whether the lowest levels are associated with specific features of interstitial cystitis. Symptom correlations. We also tested for correlations of marker levels with symptom scores. The University of Wisconsin scale includes 7 aspects of interstitial cystitis. We noted almost no correlations of marker levels with total or individual symptom scores. A likely explanation is that the patients in this study were chronically treated for interstitial cystitis and a given treatment may have different effects on symptoms than on marker levels. For example, hydroxyzine has sedative effects that may improve the symptoms of nocturia and sleeping difficulty regardless of any alterations in urine histamine. Another explanation is that different individuals with interstitial cystitis may have different pathophysiological conditions, as reflected by different markers. For example, IL-6 may indicate a specific type of bladder inflammation in a subset of interstitial cystitis cases. If other patients have severe pain due to interstitial cystitis pathophysiology that does not involve IL-6, IL-6 would not correlate with pain scores when all patients with interstitial cystitis were analyzed as a single group. Another explanation is that symptom perception is subjective and affected by other factors in addition to the interstitial cystitis. For these reasons it may be difficult to identify associations of objective markers with subjective symptoms when analyzing a group of patients. It may be more appropriate to follow individuals and determine whether changes in objective markers corre-
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COMPARISON OF MULTIPLE URINE MARKERS FOR INTERSTITIAL CYSTITIS
late with changes in symptom scores with time. Studies are currently underway to evaluate urine interstitial cystitis markers in new untreated patients and follow the markers before and after treatment.
CONCLUSIONS
This study was an initial step in the process of confirming and identifying specific uses for several previously reported interstitial cystitis urine markers. The study was limited because patients were chronically treated but it provides useful conclusions and suggestions for future study. In regard to a diagnostic marker for interstitial cystitis antiproliferative factor most clearly separated the interstitial cystitis and control groups with the least overlap. Therefore, at this time it appears that anti-proliferative factor is the most promising marker as a diagnostic test. Several other markers showed significant differences in interstitial cystitis and control groups but with overlap. In patients with interstitial cystitis there were decreased heparin-binding epidermal growth factor-like growth factor and cGMP, and increased EGF, IGF binding protein-3 and IL-6. For some of these markers the overlap may have been due to treatments that caused some markers in interstitial cystitis cases to return to the normal range. A future study of these markers in untreated patients may reveal more clear separation of the interstitial cystitis and control groups. Alternatively certain markers, for example IL-6, may have overlapped because they apply only to a subset of interstitial cystitis cases. A prospective study of urine markers and bladder biopsy findings in untreated cases is underway that may clarify this issue. Several other markers showed no significant differences in the interstitial cystitis and control groups. For IL-1 and nitrates plus nitrites the lack of a difference was consistent with earlier reports. However, GAGs, epitectin and hyaluronic acid have previously been reported to be altered in interstitial cystitis cases but this finding was not confirmed in the current study. For epitectin the most likely explanation was assay variability. For GAGs and hyaluronic acid the most likely explanations were treatment status and patient heterogeneity. This study did not confirm a previous report of elevated methylhistamine in interstitial cystitis. A difference is that in our series urine methylhistamine in patients with interstitial cystitis cases was lower than in the previous study, probably because most of our patients were on treatment that may stabilize mast cells. However, another important difference was that the methylhistamine level in controls with no dietary restrictions was much higher than the previous level in controls on a restricted diet. This observation emphasizes the importance of diet when considering urine methylhistamine. Future studies of untreated patients that consider diet may reveal that methylhistamine is useful as a diagnostic marker or for identifying a subset of patients with a specific pathophysiological condition. We noted essentially no correlations of marker levels with symptom scores in interstitial cystitis cases. The most likely explanations were differential effects of treatments on marker levels versus symptoms, heterogeneity of interstitial cystitis pathophysiology and heterogeneity in patient symptom perceptions. Future research must emphasize following marker levels and symptoms in individuals with time. Dr. Vernon Chinchilli and Yvonne Matthews provided advice on statistical issues in the original study design, Amy Matthews performed data programming, Lisa McCully prepared the manuscript, and Pui Yan Chiu, Nancy Herb, Sarah Ordille and Chen-O Zhang provided technical assistance.
REFERENCES
1. Hanno, P.: Interstitial cystitis and related diseases. Historical perspective. In: Campbell’s Urology, 7th ed. Edited by P. C. Walsh, A. B. Retik, E. D. Vaughan, Jr. and A. J. Wein. Philadelphia: W. B. Saunders Co., p. 631, 1998 2. Erickson, D. R.: Urine markers of interstitial cystitis. Urology, 57: 15, 2001 3. Wei, D. C., Politano, V. A., Selzer, M. G. and Lokeshwar, V. B.: The association of elevated urinary total to sulfated glycosaminoglycan ratio and high molecular mass hyaluronic acid with interstitial cystitis. J Urol, 163: 1577, 2000 4. Erickson, D. R., Sheykhnazari, M., Ordille, S. and Bhavanandan, V. P.: Increased urinary hyaluronic acid and interstitial cystitis. J Urol, 160: 1282, 1998 5. Erickson, D. R., Mast, S., Ordille, S. and Bhavanandan, V. P.: Urinary epitectin (MUC-1 glycoprotein) in the menstrual cycle and interstitial cystitis. J Urol, 156: 938, 1996 6. Hurst, R. E., Parsons, C. L., Roy, J. B. and Young, J. L.: Urinary glycosaminoglycan excretion as a laboratory marker in the diagnosis of interstitial cystitis. J Urol, 149: 31, 1993 7. Erickson, D. R., Ordille, S., Martin, A. and Bhavanandan, V. P.: Urinary chondroitin sulfates, heparin sulfate and total sulfated glycosaminoglycans in interstitial cystitis. J Urol, 157: 61, 1997 8. Akcay, T. and Konukoglu, D.: Glycosaminoglycans excretion in interstitial cystitis. Int Urol Nephrol, 31: 431, 1999 9. Keay, S., Zhang, C.-O., Trifillis, A. L., Hise, M. K., Hebel, J. R., Jacobs, S.-C. et al: Decreased 3H-thymidine incorporation by human bladder epithelial cells following exposure to urine from interstitial cystitis patients. J Urol, 156: 2073, 1996 10. Keay, S. K., Zhang, C., Shoenfelt, J., Erickson, D. R., Whitmore, K., Warren, J. W. et al: Sensitivity and specificity of antiproliferative factor, heparin-binding epidermal growth factor-like growth factor, and epidermal growth factor as urine markers for interstitial cystitis. Urology, 57: 9, 2001 11. Keay, S., Zhang, C.-O., Kagen, D. I., Hise, M. K., Jacobs, S. C., Hebel, J. R. et al: Concentrations of specific epithelial growth factors in the urine of interstitial cystitis patients and controls. J Urol, 158: 1983, 1997 12. Sant, G. R. and Theoharides, T. C.: The role of the mast cell in interstitial cystitis. Urol Clin North Am, 21: 41, 1994 13. El-Mansoury, M., Boucher, W., Sant, G. R. and Theoharides, T. C.: Increased urine histamine and methylhistamine in interstitial cystitis. J Urol, 152: 350, 1994 14. Erickson, D. R., Belchis, D. A. and Dabbs, D. J.: Inflammatory cell types and clinical features of interstitial cystitis. J Urol, 158: 790, 1997 15. Peters, K. M., Diokno, A. C. and Steinert, B. W.: Preliminary study on urinary cytokine levels in interstitial cystitis: does intravesical bacille Calmette-Guerin treat interstitial cystitis by altering the immune profile in the bladder? Urology, 54: 450, 1999 16. Wheeler, M. A., Smith, S. D., Saito, N., Foster, H. E., Jr. and Weiss, R. M.: Effect of long-term oral L-arginine on the nitric oxide synthase pathway in the urine from patients with interstitial cystitis. J Urol, 158: 2045, 1997 17. Lotz, M., Villiger, P., Hugli, T., Koziol, J. and Zuraw, B. L.: Interleukin-6 and interstitial cystitis. J Urol, 152: 869, 1994 18. Martins, S. M., Darlin, D. J., Lad, P. M. and Zimmern, P. E.: Interleukin-1B: a clinically relevant urinary marker. J Urol, 151: 1198, 1994 19. Lotz, M., Vaughan, J. H. and Carson, D. A.: Effect of neuropeptides on production of inflammatory cytokines by human monocytes. Science, 241: 1218, 1988 20. Pang, X., Marchand, J., Sant, G. R., Kream, R. M. and Theoharides, T. C.: Increased number of substance P positive nerve fibers in interstitial cystitis. Br J Urol, 75: 744, 1995 21. Irwin, P. and Galloway, N. T. M.: Impaired bladder perfusion in interstitial cystitis: a study of blood supply using laser Doppler flowmetry. J Urol, 149: 890, 1993 22. Smith, S. D., Wheeler, M. A., Foster, H. E., Jr. and Weiss, R. M.: Urinary nitric oxide synthase activity and cyclic GMP levels are decreased with interstitial cystitis and increased with urinary tract infections. J Urol, 155: 1432, 1996 23. Keller, M. L., McCarthy, D. O. and Neider, R. S.: Measurement of symptoms of interstitial cystitis: a pilot study. Urol Clin North Am, 21: 67, 1994 24. Goin, J. E., Olaleye, D., Peters K. M., Steinert, B., Habicht, K.
COMPARISON OF MULTIPLE URINE MARKERS FOR INTERSTITIAL CYSTITIS
25.
26. 27.
28.
29.
and Wynant, G.: Psychometric analysis of the University of Wisconsin Interstitial Cystitis Scale: implications for use in randomized clinical trials. J Urol, 159: 1085, 1998 Fosang, A. J., Hey, N. J., Carney, S. L. and Hardingham, T. E.: An ELISA plate-based assay for hyaluronan using biotinylated proteoglycan G1 domain (HA-binding region). Matrix, 10: 306, 1990 Daniel, W. W.: Applied Nonparametic Statistics. Boston: PWSKENT Publishing Co., 1978 Keay, S., Kleinberg, M., Zhang, C.-O., Hise, M. K. and Warren, J. W.: Bladder epithelial cells from patients with interstitial cystitis produce an inhibitor of heparin-binding epidermal growth factor-like growth factor production. J Urol, 164: 2112, 2000 Stephan, V., Zimmermann, A., Kuhr, J. and Urbanek, R.: Determination of N-methylhistamine in urine as an indicator of histamine release in immediate allergic reactions. J Allergy Clin Immunol, 86: 862, 1990 Beyer, K., Niggemann, B., Schulze, S. and Wahn, U.: Serum tryptase and urinary 1-methylhistamine as parameters for monitoring oral food challenges in children. Int Arch Allergy Immunol, 104: 348, 1994
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30. Chiang, G., Patra, P., Letourneau, R., Jeudy, S., Boucher, W., Green, M. et al.: Pentosanpolysulfate inhibits mast cell histamine secretion and intracellular calcium ion levels: an alternative explanation of its beneficial effect in interstitial cystitis. J Urol, 164: 2119, 2000 31. Seguchi, T., Yokokawa, K., Sugao, H., Nakano, E., Sonoda, T. and Okuyama, A.: Interleukin-6 activity in urine and serum in patients with bladder carcinoma. J Urol, 148: 791, 1992 32. Davidoff, R., Yamaguchi, R., Leach, G. E., Park, E. and Lad, P. M.: Multiple urinary cytokine levels of bacterial cystitis. J Urol, 157: 1980, 1997 33. Lundberg, J. O., Ehren, I., Jansson, O., Adolfsson, J., Lundberg, J. M., Weitzberg, E. et al: Elevated nitric oxide in the urinary bladder in infectious and noninfectious cystitis. Urology, 48: 700, 1996 34. Denson, M. A., Griebling, T. L., Cohen, M. B. and Kreder, K. J.: Comparison of cystoscopic and histological findings in patients with suspected interstitial cystitis. J Urol, 164: 1908, 2000 35. Smith, S. D., Wheeler, M. A., Lorber, M. I. and Weiss, R. M.: Temporal changes of cytokines and nitric oxide products in urine from renal transplant patients. Kidney Int, 58: 829, 2000
Vol. 167, 2461–2469, June 2002 Printed in U.S.A.
A COMPARISON OF MULTIPLE URINE MARKERS FOR INTERSTITIAL CYSTITIS DEBORAH R. ERICKSON, SHARON X. XIE, VEER P. BHAVANANDAN, MARCIA A. WHEELER, ROBERT E. HURST, LAWRENCE M. DEMERS, LESLIE KUSHNER AND SUSAN K. KEAY* From the Division of Urology and Departments of Health Evaluation Sciences, Biochemistry and Medicine and Pathology, Pennsylvania State University, College of Medicine, Hershey, Pennsylvania, Section of Urology, Yale School of Medicine, New Haven, Connecticut, Department of Urology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, Department of Urology, Long Island Jewish Medical Center, New Hyde Park, New York, and Division of Infectious Diseases, University of Maryland School of Medicine, Baltimore, Maryland
ABSTRACT
Purpose: We measured several urine markers in 24-hour specimens from patients with interstitial cystitis and healthy controls. For each marker we determined whether the urine level was significantly different in interstitial cystitis and control cases, and whether the marker level correlated with the symptom score. Materials and Methods: Study participants included 36 female patients with interstitial cystitis and 36 age matched female volunteers. Multiple urine aliquots were obtained to measure the various markers. Results: Certain markers were significantly increased in interstitial cystitis, including antiproliferative factor, epidermal growth factor, insulin-like growth factor (IGF) binding protein-3 and interleukin (IL)-6. Markers significantly decreased in interstitial cystitis were heparinbinding epidermal growth factor-like growth factor, cyclic guanosine monophosphate and methylhistamine. Other markers were not significantly different in the interstitial cystitis and control groups, including total glycosaminoglycans, epitectin, hyaluronic acid, IL-8, IL-1 and nitrates plus nitrites. IGF-1 was undetectable in 24-hour urine samples but spot voided samples from the same interstitial cystitis population had IGF-1 levels similar to previously reported levels. The only significant association of marker with symptom score was a positive correlation of IL-6 with nocturia. For all markers the conclusions were the same whether the marker was normalized to creatinine or to 24 hours. Conclusions: This study confirmed several previously reported urine alterations in interstitial cystitis, including increased anti-proliferative factor, epidermal growth factor, IGF binding protein-3 and IL-6, and decreased heparin-binding epidermal growth factor-like growth factor and cyclic guanosine monophosphate. Of all markers studied anti-proliferative factor had the least overlap in the interstitial cystitis and control groups, and so it is the most likely candidate to become a diagnostic test. KEY WORDS: bladder; cystitis, interstitial; growth substances; biological markers; signs and symptoms
To our knowledge the etiologies of interstitial cystitis are unknown. The most prominent theories are bladder epithelial dysfunction, bladder mast cell activation and allergic or autoimmune processes.1 Objective markers of interstitial cystitis would be useful for diagnosing interstitial cystitis more precisely, selecting patients with specific pathophysiological conditions for focused research and treatments, and providing objective outcome measurements for treatment trials. Several alterations in interstitial cystitis urine have been reported but urine markers are not widely used for interstitial cystitis diagnosis or research.1, 2 A reason is that many markers have been reported and measuring all would be expensive. Also, some previously reported markers were not confirmed to be altered in interstitial cystitis cases when tested by different groups.2 These contradictory results may have been due to different assay methods, sample sizes, patient selection criteria, treatments or patient heterogeneity. Thus, an impor-
tant first step in choosing the most useful markers would be to compare multiple interstitial cystitis markers in the same urine specimens. In this study we tested 14 interstitial cystitis markers. Each marker was previously reported to be altered or was expected to be altered in interstitial cystitis cases. One of us obtained 24-hour urine specimens and frozen aliquots were sent to co-authors for blinded marker analysis. For each marker we tested 2 hypotheses, including 1) whether the marker level was significantly different in interstitial cystitis and control cases, and 2) whether the marker level correlated with the symptom score. We believed that comparing these answers for several different markers may have identified which if any potential markers appears promising as a diagnostic test in patients with interstitial cystitis-like symptoms. The relevant literature for these markers was summarized. Bladder epithelial abnormalities. A main theory of interstitial cystitis is that the bladder epithelium is deficient.2 In the previous literature several urine alterations are described that may reflect epithelial abnormalities, including increased hyaluronic acid,3, 4 decreased epitectin5 and decreased total glycosaminoglycans (GAGs).6 However, 3 subsequent studies using different GAG assay methods did not
Accepted for publication January 4, 2002. Supported by the Interstitial Cystitis Association (Fishbein Foundation), and in part by National Institutes of Health Grants R01 DK38311 and DK 49450. * Financial interest and/or other relationship with Merck and Roche. 2461
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COMPARISON OF MULTIPLE URINE MARKERS FOR INTERSTITIAL CYSTITIS
confirm that urine GAGs were decreased in interstitial cystitis cases.3, 7, 8 The reasons for epithelial deficiency in interstitial cystitis are not definitively proven but they may involve alterations in growth or other factors that influence epithelial proliferation. For example, interstitial cystitis urine contains an antiproliferative factor that inhibits the proliferation of normal human bladder epithelial cells in culture.9, 10 Other urine growth factor alterations reported include decreased heparin-binding epidermal growth factor-like growth factor and increased epidermal growth factor (EGF), insulin-like growth factor (IGF)-1 and IGF binding protein-3.10, 11 Methylhistamine. Another main theory of interstitial cystitis is that bladder mast cells are activated, releasing histamine and other mediators.1, 12 Urine histamine and methylhistamine may reflect bladder mast cell activation. These 2 markers were previously noted to be elevated in the urine of untreated patients with interstitial cystitis.13 Cytokines. Urine cytokine levels may reflect bladder inflammation in interstitial cystitis cases. Urine interleukin (IL)-6 and IL-8 were previously reported to be elevated in some patients with interstitial cystitis.14 –17 Urine IL-1 was elevated in cases of bacterial cystitis but not in those of interstitial cystitis.18 We decided to retest whether IL-1 is elevated in interstitial cystitis since IL-1 is induced by substance P19 and substance P may be involved in interstitial cystitis pathogenesis.20 Urine markers of nitric oxide. Decreased nitric oxide may contribute to interstitial cystitis by several possible mechanisms. Nitric oxide is a smooth muscle relaxant and vasodilator, and so decreased nitric oxide may lead to bladder spasms or the decreased bladder blood flow reported in interstitial cystitis cases.21 Nitric oxide also inhibits mast cell activation. Urine nitric oxide levels may be reflected by cyclic guanosine monophosphate (cGMP) since nitric oxide activates guanylyl cyclase, or by nitrates plus nitrites since nitric oxide is oxidized to these compounds. Wheeler16 and Smith22 et al previously reported that patients with interstitial cystitis had decreased urine cGMP and decreased nitric oxide synthase activity in centrifuged urine pellets. Nitric oxide synthase measurements were impractical in this study but cGMP and nitrates plus nitrites were included as indirect indicators of nitric oxide activity. MATERIALS AND METHODS
Subjects. The study was approved by the institutional review board of Pennsylvania State University College of Medicine. Study participants included 36 white female patients with interstitial cystitis and 36 healthy female controls, of whom 33 were white and 3 were Asian. Each control was age matched within ⫾ 5 years to a patient with interstitial cystitis. Mean age of the patients with interstitial cystitis was 56.6 years (range 22 to 86). The mean duration of symptoms was 8.1 years (range 1 to 28). Patients with interstitial cystitis had chronic disease and were on various treatments, and some had good symptom relief. However, at original presentation all patients met the symptom criteria established by the National Institute of Diabetes, Digestive and Kidney Diseases.1 All underwent cystoscopy under anesthesia with bladder distention and had diffuse glomerulations or Hunner’s ulcer. Of the patients 31 underwent bladder distention 6 months to 8 years before this study. The remaining 5 patients were studied 3 to 6 months after distention and were having active symptoms. Thus, no patient was in a post-distention remission period. A total of 28 patients were receiving conventional interstitial cystitis treatments (table 1). In 4 cases trimethoprim-sulfamethoxazole, phenazopyridine or tolterodine provided no symptom relief. Another 3 patients received no medications because symptoms were tolerable with only diet and/or urinary alkalinization, while 1
TABLE 1. Interstitial cystitis treatments used by patients in the study No. Pts. Using Treatment 7 3 3 4 3 1 1 1 1 1 1 1 1
Treatment Pentosan polysulfate Amitriptyline Hydroxyzine Pentosan polysulfate, hydroxyzine Pentosan polysulfate, amitriptyline Amitriptyline, hydroxyzine Amitriptyline, hydroxyzine, clonazepam Pentosan polysulfate, amitriptyline, hydroxyzine, clonazepam Pentosan polysulfate, gabapentin Pentosan polysulfate, hyoscyamine Amitriptyline, gabapentin Monthly intravesical cocktail (dimethyl sulfoxide, triamcinolone, heparin, sodium bicarbonate) Biwkly. intravesical hydrocortisone
was in long-term remission after previous dimethyl sulfoxide treatment without maintenance. All study participants completed the University of Wisconsin interstitial cystitis symptom scale.23, 24 This scale includes 7 bladder related symptoms, namely bladder discomfort, bladder pain, arising at night to urinate, frequent voiding during the day, sleeping difficulty because of bladder symptoms, urinary urgency and burning sensation in the bladder. Each symptom is scored between 0 —none and 6 —a lot. Thus, correlations can be tested for each of the 7 individual symptoms or for a total symptom score. Urine specimens. Each subject collected urine for 24 hours in a plastic container with no preservatives. Participants were not asked to adhere to any specific dietary restrictions during urine collection. Urine was maintained refrigerated during collection and brought on ice to Hershey Medical Center. Urine volume was measured and aliquots of urine were centrifuged, frozen and stored at ⫺70C until shipment on dry ice to coauthors by overnight courier. Co-authors were blinded to which samples were interstitial cystitis or control specimens. Marker assays. Total urine GAGs were measured by one of us (R. E. H.) using a previously described method.6 Epitectin was measured by one of us (V. P. B.) using an enzyme-linked immunoassay (ELISA), as previously described.5 Hyaluronic acid was measured by one of us (V. P. B.). The previously used radioimmunoassay kit was no longer available, and so hyaluronic acid was measured by a competitive assay using biotinylated hyaluronic acid binding protein, similar to that described by Fosang et al.25 Anti-proliferative factor was measured by one of us (S. K. K.) using a thymidine uptake assay, as previously described.9 Heparin-binding epidermal growth factor-like growth factor, EGF, IGF-1 and IGF binding protein-3 were measured by one of us (S. K. K.) using ELISA, as previously described.11 Methylhistamine was measured at the core endocrine laboratory, Hershey Medical Center using a radioimmunoassay kit (Pharmacia, Uppsala, Sweden). This kit was used by El-Mansoury et al, who previously reported increased methylhistamine levels in interstitial cystitis cases.13 IL-6 was also measured at the core endocrine laboratory using a Quantikine ELISA kit (R & D Systems, Minneapolis, Minnesota).14 Cyclic GMP was measured by one of us (M. A. W.) by radioimmunoassay (Biomedical Technologies, Stoughton, Massachusetts), as previously described.22 Urine nitrate plus nitrite was also measured by one of us (M. A. W.), as previously described,16 and IL-8 was determined using an ELISA kit (Endogen, Woburne, Massachusetts). IL-1 was measured by one of us (L. K.) using a high sensitivity ELISA system (Amersham, Piscataway, New Jersey). Urine creatinine. Urine creatinine was measured at the clinical laboratory, Hershey Medical Center using a modular
2463
COMPARISON OF MULTIPLE URINE MARKERS FOR INTERSTITIAL CYSTITIS
autoanalyzer (Roche Diagnostics, Indianapolis, Indiana) that applies the picric acid reaction. Statistical analysis. Urine marker measurements were returned to Pennsylvania State University for statistical analysis, which was performed by one of us (S. X. X.). The Wilcoxon signed rank test was used to compare urine marker levels in patients with interstitial cystitis and controls. Since not all marker levels were normally distributed, results are reported as the median and interquartile range. Kendall’s tau correlation26 was calculated for each marker and symptom score. All statistical tests were 2-sided. All analyses were performed using commercially available software. RESULTS
Comparison of interstitial cystitis and control groups. Table 2 shows the median and interquartile ranges for urine markers in the interstitial cystitis and control groups. The markers significantly increased in interstitial cystitis cases were anti-proliferative factor activity, EGF, IGF binding protein-3 and IL-6. In contrast, heparin-binding epidermal growth factor-like growth factor, cGMP and methylhis-
tamine were significantly decreased in interstitial cystitis cases. Figures 1 to 7 show individual marker results. Markers not significantly different in the interstitial cystitis and control groups were total GAGs, epitectin, hyaluronic acid, nitrates plus nitrites, IL-1 and IL-8. Three patients had high IL-8 but since all others had normal levels, average IL-8 was not increased in the interstitial cystitis group. For all comparisons the results were the same whether the marker level was normalized to urine creatinine or to a 24-hour period. Of all markers tested anti-proliferative factor activity most clearly separated the interstitial cystitis and control groups. When anti-proliferative factor was reported as a binary variable with positive considered more than 2 standard deviations from the mean of an assay control, 33 of 36 patients with interstitial cystitis were positive but only 1 of 36 controls was positive. IGF-1 was undetectable in all except 1 patient with interstitial cystitis. To test whether 24-hour collection affected the results we tested frozen aliquots of 15 clean catch urine samples from the same interstitial cystitis patient population. IGF-1 in these specimens was similar to that in the
TABLE 2. Urine marker levels in interstitial cystitis and control groups Median (interquartile range) Marker Anti-proliferative factor activity (% change in thymidine incorporation) cGMP: pM./ml. pM./mg. creatinine nM./24 hrs. EGF: Ng./ml. Ng./mg. creatinine g./24 hrs. Epitectin: Ng./ml. Ng./mg. creatinine g./24 hrs. GAG: NM./ml. NM./mg. creatinine M./24 hrs. Hyaluronic acid: Ng./ml. Ng./mg. creatinine g./24 hrs. Heparin binding epidermal growth factorlike growth factor: Ng./ml. Ng./mg. creatinine g./24 hrs. IGF binding protein-3: Ng./ml. Ng./mg. creatinine g./24 hrs. IL-1: Pg./ml. Pg./mg. creatinine Ng./24 hrs. IL-6: Pg./ml. Pg./mg. creatinine Ng./24 hrs. IL-8: Pg./ml. Pg./mg. creatinine Ng./24 hrs. Methylhistamine: Ng./ml. Ng./mg. creatinine g./24 hrs. Nitrate: M./ml. M./mg. creatinine mM./24 hrs.
Interstitial Cystitis ⫺54
(22)
134 218 200
(133) (158) (148)
15.0 (21.8) 27 (16) 25.8 (22.5) 124 293 242
(200) (276) (274)
1.26 (2.19) 2.49 (2.30) 2.62 (2.37) 356 640 595
(314) (179) (243)
2.55 (1.55) 5 (5) 4.12 (4.06) 7.1 (10.8) 11 (13) 11 (13.6) 0.32 (0.37) 0.63 (0.89) 0.52 (0.81) 4.9 (3.9) 10.2 (13.7) 9.4 (9.0)
Control 16.5 206 329 338
(22) (209) (289) (274)
7.35 (4.8) 10 (10) 10.4 (5.8) 199 349 327
(311) (388) (380)
1.54 (1.62) 1.98 (1.97) 1.90 (1.70)
Difference ⫺68.5
p Value (Wilcoxon signed rank test)
(38.5)
⬍0.0001
(153) (290) (324)
0.0061 0.012 0.0052
9.1 15 12.7
(22.4) (23) (22.2)
⬍0.0001 ⬍0.0001 ⬍0.0001
⫺8.7 ⫺57 ⫺77
(333) (350) (347)
0.24 0.065 0.052
40 ⫺113 104
0.08 (2.24) ⫺0.12 (2.72) ⫺1.28 (2.57)
0.78 0.98 0.78
(206) (320) (284)
8.4 56 51
(324) (272) (399)
0.72 0.34 0.68
11.5 (8.05) 17 (19) 16.0 (11.7)
⫺7.3 ⫺12 ⫺11.9
(8.75) (20) (14.6)
⬍0.0001 ⬍0.0001 ⬍0.0001
1.3 3 2.9
(13.0) (11) (14.5)
0.055 0.0058 0.020
352 617 553
4.0 8 7.8
(4.6) (6) (5.3)
0.31 (0.79) 0.70 (1.5) 0.51 (1.4) 1.5 2.7 2.7
(0.4) (2.3) (1.7)
3.4 (11.2) 6.6 (19.5) 6.5 (18)
6.8 (8.5) 10.9 (20.4) 8.5 (15.7)
42.8 (53.1) 85.8 (83.9) 76.5 (85.2)
63.8 (62.2) 114 (65.4) 112 (80.8)
1.06 (0.77) 1.66 (1.22) 1.50 (1.06)
1.20 (1.27) 1.94 (1.52) 1.67 (1.14)
⫺0.012 (1.2) ⫺0.06 (1.7) ⫺0.07 (1.7) 3.4 7.2 6.7
(4.4) (14) (9.0)
⫺0.4 (11.8) 0.08 (16) 0.08 (16) ⫺20.4 ⫺29 ⫺16
(48) (76) (81)
0.096 (1.44) ⫺0.096 (2.29) ⫺0.24 (1.74)
0.66 0.75 0.69 ⬍0.0001 ⬍0.0001 ⬍0.0001 0.99 0.82 0.87 0.043 0.030 0.005 0.50 0.40 0.29
2464
COMPARISON OF MULTIPLE URINE MARKERS FOR INTERSTITIAL CYSTITIS
FIG. 1. Percent change in 3H-thymidine incorporation by human bladder epithelial cells incubated with interstitial cystitis (IC) or control (Ctr) urine specimens compared with cells incubated with culture medium alone. Horizontal line indicates median value. Vertical line indicates 25th and 75th percentiles.
previous report (data not shown).11 These results suggest that IGF-1 may bind to the plastic containers used to collect 24-hour urine specimens or IGF-1 may be unstable when stored in the refrigerator for 24 hours. Symptom scores. Table 3 lists median and interquartile ranges of University of Wisconsin symptom scores in the interstitial cystitis and control groups. The total University of Wisconsin score is the sum of the scores of the 7 individual interstitial cystitis symptoms. In the interstitial cystitis group urine marker levels were tested for correlations with symptom scores. No marker levels correlated with the total University of Wisconsin score. Only 1 correlation was identified for any marker and any of the 7 individual symptom scores. IL-6 positively correlated with nocturia score when normalized to creatinine and to 24 hours (r ⫽ 0.30, p ⫽ 0.013 and r ⫽ 0.27, p ⫽ 0.024, respectively).
FIG. 2. EGF in interstitial cystitis (IC) and control (Ctr) urine specimens. A, per mg. urine creatinine. B, per 24 hours. Horizontal line indicates median value. Vertical line indicates 25th and 75th percentiles.
DISCUSSION
In this study we compared several urine components in patients with interstitial cystitis and controls. All markers were measured in the same urine specimens obtained by a single investigator at a single institution. Some urine markers were altered in interstitial cystitis cases, while others were not. Urine glycoconjugates. Urine glycoconjugate alterations are thought to reflect bladder epithelial deficiency in interstitial cystitis cases. One of us (R. E. H.) originally reported that total GAGs were decreased in interstitial cystitis urine.6 Later studies using different assay methods did not confirm decreased total GAG in interstitial cystitis urine.3, 7, 8 The current study using the original assay procedure6 also did not confirm decreased total GAG levels in interstitial cystitis cases. A possible explanation was that the original study included new untreated patients with interstitial cystitis, while the current study included patients with interstitial cystitis who were on chronic treatment. It is also possible that different patient characteristics may have a role, particularly if the previous study contained more patients with more severe disease manifestations. Two of us (D. R. E. and V. P. B.) previously reported that interstitial cystitis urine had significantly decreased epitectin (MUC-1 glycoprotein).5 However, the current study did
not confirm a significant decrease. This discrepancy may have been related to assay variability since mean urine epitectin levels in this study were much lower than in the previous report in interstitial cystitis cases and controls (0.29 and 0.35 versus 3.9 and 6.4 g./mg. creatinine, respectively).5 This study also failed to confirm previous reports3, 4 that interstitial cystitis cases have increased urine hyaluronic acid. A possible explanation was that the 3 studies used different assay methods for hyaluronic acid. Another possible explanation was that our original report included untreated patients with interstitial cystitis,4 while the current study included chronically treated patients. Heterogeneity of the hyaluronic acid molecule may also be relevant. Wei et al previously reported that the high molecular weight fraction of hyaluronic acid was increased in interstitial cystitis urine.3 As a separate analysis, we compared the various hyaluronic acid fractions in some samples and also observed increased high molecular weight hyaluronic acid in interstitial cystitis cases (unpublished data). Despite these negative results when studying chronically treated patients with interstitial cystitis, we have not ruled out that GAGs or hyaluronic acid may be useful markers in new untreated patients. We are performing a prospective study to measure
COMPARISON OF MULTIPLE URINE MARKERS FOR INTERSTITIAL CYSTITIS
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FIG. 3. IGF binding protein-3 (BP3) in interstitial cystitis (IC) and control (Ctr) urine specimens. A, per mg. urine creatinine. B, per 24 hours. Horizontal line indicates median value. Vertical line indicates 25th and 75th percentiles.
urine glycoconjugates in new patients and compare them with relevant bladder biopsy features. Urine anti-proliferative factor and growth factors. Antiproliferative factor activity was significantly increased in interstitial cystitis cases. When comparing all markers in this study, anti-proliferative factor showed the least overlap in the interstitial cystitis and control groups (fig. 1). When anti-proliferative factor was considered a binary variable, 33 of 36 patients with interstitial cystitis but only 1 control were positive. Since anti-proliferative factor most clearly separated the interstitial cystitis and control groups, it is currently the most promising diagnostic marker. The 3 urine growth factors that were altered in interstitial cystitis were decreased heparin-binding epidermal growth factor-like growth factor, increased EGF and increased IGF binding protein-3. These 3 results are consistent with those in earlier reports.10, 11 In regard to interstitial cystitis pathophysiology these changes may be related. When purified antiproliferative factor is added to cultured human urothelial cells, it decreases their production of heparin-binding epidermal growth factor-like growth factor and increases their production of EGF and IGF binding protein-3.27 In regard to use as interstitial cystitis markers these growth factors were significantly altered in interstitial cystitis cases but showed more overlap in the interstitial cystitis and control groups
FIG. 4. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) in interstitial cystitis (IC) and control (Ctr) urine specimens. A, per mg. urine creatinine. B, per 24 hours. Horizontal line indicates median value. Vertical line indicates 25th and 75th percentiles.
than anti-proliferative factor. Some of this overlap may have been related to treatment. We are including these markers in our ongoing prospective study. IGF-1 was not detectable in 24-hour urine samples collected for this study. The most likely explanations are that it bound to the plastic containers or it was not stable when stored in the refrigerator for 24 hours. Methylhistamine. Methylhistamine is a stable metabolite of histamine. Urine methylhistamine may reflect bladder mast cell activation in interstitial cystitis cases.12, 13 El-Mansoury et al previously reported that urine methylhistamine was much higher in patients with interstitial cystitis than in healthy controls.13 We noted the opposite result, that is patients with interstitial cystitis had lower urine methylhistamine than controls. Mean methylhistamine plus or minus standard error of mean in controls was much higher in the current than in the previous study (79 ⫾ 50.6 versus
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COMPARISON OF MULTIPLE URINE MARKERS FOR INTERSTITIAL CYSTITIS
FIG. 5. IL-6 in interstitial cystitis (IC) and control (Ctr) urine specimens. A, in pg./mg. urine creatinine. B, ng./24 hours. Horizontal line indicates median value. Vertical lines indicate 25th and 75th percentiles.
17.2 ⫾ 7.9 ng./ml.) and in interstitial cystitis cases it was much lower (74 ⫾ 73 versus 158 ⫾ 135 ng./ml.). In regard to controls a key difference in the 2 studies was that the previous study required all participants (patients with interstitial cystitis and controls) to maintain a special diet on the day of urine collection. The diet avoided foods and drinks high in biogenic amine precursors, such as cheese, beer, wine, caffeine and so forth. Our study participants were not asked to maintain a special diet. The large difference in control urine methylhistamine in these 2 studies suggests that diet may be a major contributor to urine methylhistamine in healthy individuals. In further support of this conclusion 2 other studies that measured urine methylhistamine in healthy controls using the same assay and no special diet showed control urine methylhistamine levels similar to those in our study.28, 29 In regard to interstitial cystitis the previous study included new untreated patients, while our study included patients with chronic disease. Some interstitial cystitis treatments, including amitriptyline, hydroxyzine and pentosan polysulfate, are thought to stabilize mast cells and prevent hista-
FIG. 6. cGMP in interstitial cystitis (IC) and control (Ctr) urine specimens. A, in pM./mg. urine creatinine. B, in nM./24 hours. Horizontal line indicates median value. Vertical lines indicate 25th and 75th percentiles.
mine release.12, 30 Since 26 of our patients were on at least 1 of these medications, it is the most likely explanation of the lower methylhistamine levels in our patients. It is also possible that our patients were restricting their diets to avoid biogenic amine precursors because such dietary advice is widely available for patients with interstitial cystitis. However, diet does not explain the discrepancy in methylhistamine levels in our patients with interstitial cystitis and those of the previous study because all participants in the previous study were on a restricted diet. Despite these negative results when comparing chronically treated patients with interstitial cystitis with controls, we have not ruled out the possibility that methylhistamine may be a useful marker in untreated patients with interstitial cystitis. We are performing a prospective study to measure methylhistamine in new untreated patients and compare urine methylhistamine with the density of mast cells on patient bladder biopsies. Cytokines. IL-6 is a pro-inflammatory cytokine produced by different cell types, including vascular endothelial cells, macrophages and fibroblasts. The current study and 3 earlier series showed increased urine IL-6 in interstitial cystitis cases.14, 15, 17 Despite these consistent findings IL-6 is unlikely to be useful as a diagnostic marker for interstitial
COMPARISON OF MULTIPLE URINE MARKERS FOR INTERSTITIAL CYSTITIS
FIG. 7. Methylhistamine (MH) in interstitial cystitis (IC) and control (Ctr) urine specimens. A, in ng./mg. urine creatinine. B, in mcg./24 hours). Horizontal line indicates median value. Vertical lines indicate 25th and 75th percentiles.
TABLE 3. University of Wisconsin symptom scores in interstitial cystitis and control groups Symptoms
Bladder discomfort Bladder pain Nocturia Daytime frequency Sleeping difficulty due to bladder symptoms Urge to urinate Burning sensation in bladder Totals
Median (interquartile range) Interstitial Cystitis
Control
4 3 5 5 4 5 3
(2.5–6) (1.5–5) (2–6) (3.5–6) (1.5–6) (3–6) (2–6)
0 0 0 (0–1) 0 (0–2) 0 0 0
27.5 (18–35)
0 (0–3)
cystitis because it lacks specificity for that disease. Urine IL-6 is also elevated in other urological diseases, including bladder cancer31 and bacterial cystitis.32 However, in interstitial cystitis cases urine IL-6 appears to correlate with symptom severity15, 17 and the degree of inflammation on histological testing.14 Therefore, it is possible that IL-6 may be a useful indicator of disease activity in patients already
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known to have interstitial cystitis. Interestingly some patients with interstitial cystitis had clearly elevated IL-6, while others had levels in the control range (fig. 5). This finding may have been related to treatment effects or differences in interstitial cystitis pathophysiology in individuals. IL-8 is a chemokine that attracts neutrophils and T lymphocytes. Wheeler et al reported that IL-8 was elevated in some patients with interstitial cystitis and associated with leukocyte esterase positive urine.16 Peters et al noted that patients with active interstitial cystitis had increased urine IL-8, while those in remission after bacillus Calmette-Guerin treatment had low IL-8.15 We identified no significant difference in median urine IL-8 in the interstitial cystitis and control groups. However, 3 patients with interstitial cystitis had high IL-8. These patients also had high IL-6 and IL-1. We are including these cytokines in our prospective study and plan to correlate them with inflammatory changes on bladder biopsy. IL-1 has many pro-inflammatory effects.18 Our study showed that urine IL-1 was similar in patients with interstitial cystitis and controls, consistent with a previous report that urine IL-1 was elevated in bacterial cystitis but not interstitial cystitis cases.18 Urine markers of nitric oxide activity. Nitric oxide is induced during inflammation. Lundberg et al measured nitric oxide gas in the bladder and observed that increased levels were associated with any type of inflammation, including infection, radiation, bacillus Calmette-Guerin cystitis and interstitial cystitis.33 The results of this Swedish study may not be relevant to American patients with interstitial cystitis, of whom most do not have severe inflammation on bladder biopsy.34 Wheeler16 and Smith22 et al previously measured other markers of the nitric oxide pathway, namely nitric oxide synthase, cGMP and nitrates plus nitrites. Urine nitric oxide synthase and cGMP increased in bacterial cystitis but decreased in interstitial cystitis cases. Nitrate plus nitrite levels were not significantly altered in patients with interstitial cystitis, probably because the major source of urine nitrate is dietary.16, 35 The current study showed the same results. Urine cGMP was decreased in interstitial cystitis cases, while nitrate plus nitrite was not. Cyclic GMP levels overlapped in the interstitial cystitis and control groups (fig. 6). This finding may have been related to treatment or to differences in interstitial cystitis pathophysiology in individuals. In our continuing study we plan to test whether the lowest levels are associated with specific features of interstitial cystitis. Symptom correlations. We also tested for correlations of marker levels with symptom scores. The University of Wisconsin scale includes 7 aspects of interstitial cystitis. We noted almost no correlations of marker levels with total or individual symptom scores. A likely explanation is that the patients in this study were chronically treated for interstitial cystitis and a given treatment may have different effects on symptoms than on marker levels. For example, hydroxyzine has sedative effects that may improve the symptoms of nocturia and sleeping difficulty regardless of any alterations in urine histamine. Another explanation is that different individuals with interstitial cystitis may have different pathophysiological conditions, as reflected by different markers. For example, IL-6 may indicate a specific type of bladder inflammation in a subset of interstitial cystitis cases. If other patients have severe pain due to interstitial cystitis pathophysiology that does not involve IL-6, IL-6 would not correlate with pain scores when all patients with interstitial cystitis were analyzed as a single group. Another explanation is that symptom perception is subjective and affected by other factors in addition to the interstitial cystitis. For these reasons it may be difficult to identify associations of objective markers with subjective symptoms when analyzing a group of patients. It may be more appropriate to follow individuals and determine whether changes in objective markers corre-
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COMPARISON OF MULTIPLE URINE MARKERS FOR INTERSTITIAL CYSTITIS
late with changes in symptom scores with time. Studies are currently underway to evaluate urine interstitial cystitis markers in new untreated patients and follow the markers before and after treatment.
CONCLUSIONS
This study was an initial step in the process of confirming and identifying specific uses for several previously reported interstitial cystitis urine markers. The study was limited because patients were chronically treated but it provides useful conclusions and suggestions for future study. In regard to a diagnostic marker for interstitial cystitis antiproliferative factor most clearly separated the interstitial cystitis and control groups with the least overlap. Therefore, at this time it appears that anti-proliferative factor is the most promising marker as a diagnostic test. Several other markers showed significant differences in interstitial cystitis and control groups but with overlap. In patients with interstitial cystitis there were decreased heparin-binding epidermal growth factor-like growth factor and cGMP, and increased EGF, IGF binding protein-3 and IL-6. For some of these markers the overlap may have been due to treatments that caused some markers in interstitial cystitis cases to return to the normal range. A future study of these markers in untreated patients may reveal more clear separation of the interstitial cystitis and control groups. Alternatively certain markers, for example IL-6, may have overlapped because they apply only to a subset of interstitial cystitis cases. A prospective study of urine markers and bladder biopsy findings in untreated cases is underway that may clarify this issue. Several other markers showed no significant differences in the interstitial cystitis and control groups. For IL-1 and nitrates plus nitrites the lack of a difference was consistent with earlier reports. However, GAGs, epitectin and hyaluronic acid have previously been reported to be altered in interstitial cystitis cases but this finding was not confirmed in the current study. For epitectin the most likely explanation was assay variability. For GAGs and hyaluronic acid the most likely explanations were treatment status and patient heterogeneity. This study did not confirm a previous report of elevated methylhistamine in interstitial cystitis. A difference is that in our series urine methylhistamine in patients with interstitial cystitis cases was lower than in the previous study, probably because most of our patients were on treatment that may stabilize mast cells. However, another important difference was that the methylhistamine level in controls with no dietary restrictions was much higher than the previous level in controls on a restricted diet. This observation emphasizes the importance of diet when considering urine methylhistamine. Future studies of untreated patients that consider diet may reveal that methylhistamine is useful as a diagnostic marker or for identifying a subset of patients with a specific pathophysiological condition. We noted essentially no correlations of marker levels with symptom scores in interstitial cystitis cases. The most likely explanations were differential effects of treatments on marker levels versus symptoms, heterogeneity of interstitial cystitis pathophysiology and heterogeneity in patient symptom perceptions. Future research must emphasize following marker levels and symptoms in individuals with time. Dr. Vernon Chinchilli and Yvonne Matthews provided advice on statistical issues in the original study design, Amy Matthews performed data programming, Lisa McCully prepared the manuscript, and Pui Yan Chiu, Nancy Herb, Sarah Ordille and Chen-O Zhang provided technical assistance.
REFERENCES
1. Hanno, P.: Interstitial cystitis and related diseases. Historical perspective. In: Campbell’s Urology, 7th ed. Edited by P. C. Walsh, A. B. Retik, E. D. Vaughan, Jr. and A. J. Wein. Philadelphia: W. B. Saunders Co., p. 631, 1998 2. Erickson, D. R.: Urine markers of interstitial cystitis. Urology, 57: 15, 2001 3. Wei, D. C., Politano, V. A., Selzer, M. G. and Lokeshwar, V. B.: The association of elevated urinary total to sulfated glycosaminoglycan ratio and high molecular mass hyaluronic acid with interstitial cystitis. J Urol, 163: 1577, 2000 4. Erickson, D. R., Sheykhnazari, M., Ordille, S. and Bhavanandan, V. P.: Increased urinary hyaluronic acid and interstitial cystitis. J Urol, 160: 1282, 1998 5. Erickson, D. R., Mast, S., Ordille, S. and Bhavanandan, V. P.: Urinary epitectin (MUC-1 glycoprotein) in the menstrual cycle and interstitial cystitis. J Urol, 156: 938, 1996 6. Hurst, R. E., Parsons, C. L., Roy, J. B. and Young, J. L.: Urinary glycosaminoglycan excretion as a laboratory marker in the diagnosis of interstitial cystitis. J Urol, 149: 31, 1993 7. Erickson, D. R., Ordille, S., Martin, A. and Bhavanandan, V. P.: Urinary chondroitin sulfates, heparin sulfate and total sulfated glycosaminoglycans in interstitial cystitis. J Urol, 157: 61, 1997 8. Akcay, T. and Konukoglu, D.: Glycosaminoglycans excretion in interstitial cystitis. Int Urol Nephrol, 31: 431, 1999 9. Keay, S., Zhang, C.-O., Trifillis, A. L., Hise, M. K., Hebel, J. R., Jacobs, S.-C. et al: Decreased 3H-thymidine incorporation by human bladder epithelial cells following exposure to urine from interstitial cystitis patients. J Urol, 156: 2073, 1996 10. Keay, S. K., Zhang, C., Shoenfelt, J., Erickson, D. R., Whitmore, K., Warren, J. W. et al: Sensitivity and specificity of antiproliferative factor, heparin-binding epidermal growth factor-like growth factor, and epidermal growth factor as urine markers for interstitial cystitis. Urology, 57: 9, 2001 11. Keay, S., Zhang, C.-O., Kagen, D. I., Hise, M. K., Jacobs, S. C., Hebel, J. R. et al: Concentrations of specific epithelial growth factors in the urine of interstitial cystitis patients and controls. J Urol, 158: 1983, 1997 12. Sant, G. R. and Theoharides, T. C.: The role of the mast cell in interstitial cystitis. Urol Clin North Am, 21: 41, 1994 13. El-Mansoury, M., Boucher, W., Sant, G. R. and Theoharides, T. C.: Increased urine histamine and methylhistamine in interstitial cystitis. J Urol, 152: 350, 1994 14. Erickson, D. R., Belchis, D. A. and Dabbs, D. J.: Inflammatory cell types and clinical features of interstitial cystitis. J Urol, 158: 790, 1997 15. Peters, K. M., Diokno, A. C. and Steinert, B. W.: Preliminary study on urinary cytokine levels in interstitial cystitis: does intravesical bacille Calmette-Guerin treat interstitial cystitis by altering the immune profile in the bladder? Urology, 54: 450, 1999 16. Wheeler, M. A., Smith, S. D., Saito, N., Foster, H. E., Jr. and Weiss, R. M.: Effect of long-term oral L-arginine on the nitric oxide synthase pathway in the urine from patients with interstitial cystitis. J Urol, 158: 2045, 1997 17. Lotz, M., Villiger, P., Hugli, T., Koziol, J. and Zuraw, B. L.: Interleukin-6 and interstitial cystitis. J Urol, 152: 869, 1994 18. Martins, S. M., Darlin, D. J., Lad, P. M. and Zimmern, P. E.: Interleukin-1B: a clinically relevant urinary marker. J Urol, 151: 1198, 1994 19. Lotz, M., Vaughan, J. H. and Carson, D. A.: Effect of neuropeptides on production of inflammatory cytokines by human monocytes. Science, 241: 1218, 1988 20. Pang, X., Marchand, J., Sant, G. R., Kream, R. M. and Theoharides, T. C.: Increased number of substance P positive nerve fibers in interstitial cystitis. Br J Urol, 75: 744, 1995 21. Irwin, P. and Galloway, N. T. M.: Impaired bladder perfusion in interstitial cystitis: a study of blood supply using laser Doppler flowmetry. J Urol, 149: 890, 1993 22. Smith, S. D., Wheeler, M. A., Foster, H. E., Jr. and Weiss, R. M.: Urinary nitric oxide synthase activity and cyclic GMP levels are decreased with interstitial cystitis and increased with urinary tract infections. J Urol, 155: 1432, 1996 23. Keller, M. L., McCarthy, D. O. and Neider, R. S.: Measurement of symptoms of interstitial cystitis: a pilot study. Urol Clin North Am, 21: 67, 1994 24. Goin, J. E., Olaleye, D., Peters K. M., Steinert, B., Habicht, K.
COMPARISON OF MULTIPLE URINE MARKERS FOR INTERSTITIAL CYSTITIS
25.
26. 27.
28.
29.
and Wynant, G.: Psychometric analysis of the University of Wisconsin Interstitial Cystitis Scale: implications for use in randomized clinical trials. J Urol, 159: 1085, 1998 Fosang, A. J., Hey, N. J., Carney, S. L. and Hardingham, T. E.: An ELISA plate-based assay for hyaluronan using biotinylated proteoglycan G1 domain (HA-binding region). Matrix, 10: 306, 1990 Daniel, W. W.: Applied Nonparametic Statistics. Boston: PWSKENT Publishing Co., 1978 Keay, S., Kleinberg, M., Zhang, C.-O., Hise, M. K. and Warren, J. W.: Bladder epithelial cells from patients with interstitial cystitis produce an inhibitor of heparin-binding epidermal growth factor-like growth factor production. J Urol, 164: 2112, 2000 Stephan, V., Zimmermann, A., Kuhr, J. and Urbanek, R.: Determination of N-methylhistamine in urine as an indicator of histamine release in immediate allergic reactions. J Allergy Clin Immunol, 86: 862, 1990 Beyer, K., Niggemann, B., Schulze, S. and Wahn, U.: Serum tryptase and urinary 1-methylhistamine as parameters for monitoring oral food challenges in children. Int Arch Allergy Immunol, 104: 348, 1994
2469
30. Chiang, G., Patra, P., Letourneau, R., Jeudy, S., Boucher, W., Green, M. et al.: Pentosanpolysulfate inhibits mast cell histamine secretion and intracellular calcium ion levels: an alternative explanation of its beneficial effect in interstitial cystitis. J Urol, 164: 2119, 2000 31. Seguchi, T., Yokokawa, K., Sugao, H., Nakano, E., Sonoda, T. and Okuyama, A.: Interleukin-6 activity in urine and serum in patients with bladder carcinoma. J Urol, 148: 791, 1992 32. Davidoff, R., Yamaguchi, R., Leach, G. E., Park, E. and Lad, P. M.: Multiple urinary cytokine levels of bacterial cystitis. J Urol, 157: 1980, 1997 33. Lundberg, J. O., Ehren, I., Jansson, O., Adolfsson, J., Lundberg, J. M., Weitzberg, E. et al: Elevated nitric oxide in the urinary bladder in infectious and noninfectious cystitis. Urology, 48: 700, 1996 34. Denson, M. A., Griebling, T. L., Cohen, M. B. and Kreder, K. J.: Comparison of cystoscopic and histological findings in patients with suspected interstitial cystitis. J Urol, 164: 1908, 2000 35. Smith, S. D., Wheeler, M. A., Lorber, M. I. and Weiss, R. M.: Temporal changes of cytokines and nitric oxide products in urine from renal transplant patients. Kidney Int, 58: 829, 2000