Neurourology and Urodynamics 32:969–973 (2013)

Correlation Between Cystometric Volumes, ATP Release,and pH inWomenWith Overactive Bladder

Versus Controls

Ying Cheng,1* Kylie J. Mansfield,2 Wendy Allen,1 Richard J. Millard,3

Elizabeth Burcher,4 and Kate H. Moore11Department of Urogynaecology, St George Hospital, University of New South Wales, Sydney, North South Wales, Australia

2Graduate School of Medicine, University of Wollongong, Wollongong, North South Wales, Australia3Department of Urology, Prince of Wales Hospital, University of New South Wales, Sydney, North South Wales, Australia4Department of Pharmacology, School of Medical Sciences, University of New South Wales, Sydney, North South Wales,

Australia

Aims: In the bladder, ATP is an important signaling molecule, which is released by bladder stretch and acid. Wehypothesized that ATP might play a unique role in patients with OAB, characterized by low bladder volumes at firstdesire to void (FDV) and maximal cystometric capacity (MCC) and symptoms of frequency/urgency [mild bladder painsyndrome (BPS)]. Our aim was to investigate the correlation between ATP release and urodynamic parameters, as wellas urine pH, in OAB patients. Methods: Routine cystometry was performed in a consecutive series of 249 women.The voided urodynamic fluid (VUF) was stored at �208C and ATP measured using bioluminescence. Catheter urinewas collected for pH measurement. Correlations between two factors were tested by linear regression analysis.Results: Subjects with urinary tract infection, voiding dysfunction, and detrusor overactivity (DO) were excluded. ForOAB patients (n ¼ 25), there was an inverse correlation between ATP concentration in VUF and FDV (r2 ¼ 0.25;P ¼ 0.01) but not MCC. This was not seen in controls (n ¼ 69). In OAB, but not controls, there was a significant reversecorrelation (r2 ¼ 0.16; P ¼ 0.047) between ATP in VUF and urine pH. Urine pH was not significantly correlated withMCC in either group. Conclusions: In OAB patients, ATP is an important factor for initial perception of need tourinate (as indicated by FDV). This is similar to our previous findings in patients with DO, suggesting that ATP maymediate initial afferent sensation in patients with bladder dysfunctions characterized by urgency. ATP release wasalso strongly affected by urine pH, in patients with OAB (at FDV). Neurourol. Urodynam. 32:969–973, 2013.� 2012 Wiley Periodicals, Inc.

Key words: acid; ATP release; bladder pain syndrome; overactive bladder; urgency; urine pH; urodynamics

INTRODUCTION

‘‘Urgency’’ is the key symptom associated with the overac-tive bladder (OAB).1 In patients with OAB, urgency commonlyoccurs in conjunction with urge urinary incontinence andmany of these ‘‘OAB wet’’ patients are subsequently diag-nosed with detrusor overactivity (DO). However, in more thanhalf of OAB patients, urgency is said to occur without inconti-nence.1 In these ‘‘OAB dry’’ patients, bladder pain or discom-fort during filling often accompanies urgency and is definedas the bladder pain syndrome (BPS). BPS covers a continuumof bladder symptoms comprising suprapubic pain related tobladder filling, pressure, and/or discomfort, accompanied bypersistent urge, frequency, and nocturia, in the absence of in-fection or other identifiable causes.2

Despite intense investigation, the etiology of this syndromeis unknown, diagnostic criteria are debated and treatment isempirical. BPS symptoms include frequency, urgency, and noc-turia, but the overriding symptom is suprapubic or pelvicpain, pressure or discomfort, perceived to be related to the uri-nary bladder; this sensation worsens as the bladder fills and isrelieved after urination.2 In a proportion of patients who goon to have cystodistension, petechial hemorrhages, or otherlesions can be seen which may lead to a diagnosis of intersti-tial cystitis (IC).3

The relief of symptoms after urination suggests that bladderstretch might be implicated in the development of the

sensations associated with urgency. Adenosine triphosphate(ATP) is a versatile signaling molecule released from urothelialcells, which is hypothesized to be involved in signaling thesensation of urgency.4 It is now well accepted that stretchcauses increased release of ATP, which then interacts withspecific purinergic receptors (P2X3) on suburothelial afferentnerves which are involved in transmitting sensation to thecentral nervous system.5

Alterations in purinergic signaling in patients with OABhave been reported. A recent study examined ATP releasedinto the bladder, during routine cystometric filling of womenunder investigation for incontinence.6 In subjects with idio-pathic DO, a disorder characterized by frequency, nocturia andurgency due to fear of leakage, with detrusor contractions

Karl-Erik Andersson led the peer-review process as the Associate Editor responsi-ble for the paper.Conflict of interest: none.Grant sponsor: National Health and Medical Research Council of Australia; Grantnumber: ID527502; Grant sponsor: Pfizer OAB-LUTS OAB-LUTS CompetitiveGrants Program; Grant number: WS428347.*Correspondence to: Dr. Ying Cheng, Department of Urogynaecology, Level 1, WRPitney Clinical Sciences Building, St. George Hospital, Gray St, Kogarah, NSW2217, Australia. E-mail: y.cheng@unsw.edu.auReceived 5 July 2012; Accepted 1 October 2012Published online 5 November 2012 in Wiley Online Library

(wileyonlinelibrary.com).

DOI 10.1002/nau.22344

� 2012 Wiley Periodicals, Inc.

observed during urodynamic filling, an inverse correlation be-tween ATP in voided urodynamic fluid (VUF) and first desireto void (FDV) was found.6 A similar study reported an increasein stretch induced ATP release from strips of human bladdertaken from patients with DO.7 Similarly stretch-induced ATPrelease is enhanced from cultured urothelial cells fromIC patients8 and in bladder strips from PBS patients.9 ATPconcentration is higher in urine from IC patients than in con-trols.10 Furthermore, purinergic receptors (P2X and P2Y) arealtered in an animal model of IC11 and increased in patientswith IC.12 In summary, human data suggest that ATP releasein bladder is associated with some symptom(s) of urgency.

Stretch is not the only stimulus for ATP release in the blad-der. In animal studies, release of ATP has been induced by acidstimulation, in rat mucosal strips,13 pig mucosal strips14 andin porcine urothelial cell cultures.15 In human studies, symp-toms of IC have been relieved by installation of lignocaine inalkaline solution.16 There have been few systematic studies ofthe effect of urine pH on voiding parameters, although oneearlier study reported that females with DO had significantlylower urinary pH than controls.17 Thus, increased acidity ofthe urine may contribute to urgency, perhaps via ATP release.We therefore hypothesized that patients with OAB mighthave a lower urine pH than controls, which might contributeto their symptoms.

The present work measures ATP release in VUF fromwomen with OAB, characterized by small bladder capacity,frequency, and nocturia. These subjects had no detrusor con-tractions during filling and were without overt suprapubicpain.

MATERIALS AND METHODS

Cystometric Measurements in Patients

Routine twin channel cystometry was performed in a conse-cutive series of 246 women (aged 28–87 years) who presentedto a recognized urogynecology unit from 2008 to 2009 for in-vestigation of lower urinary tract syndrome (LUTS) withsymptoms of frequency, urgency, nocturia with or without in-continence. Patients whose primary complaint was suprapu-bic pain and low bladder volume were excluded fromurodynamic testing. Informed consent was obtained and thestudy was approved by the local human research ethics com-mittee (SESIAHS HREC 06/11). Firstly, the patient was asked toempty the bladder in private. A catheter (7 Gauge French) wasinserted and a catheter specimen of urine (CSU) was collectedto test for bacterial cystitis and for pH measurement. Sterilesaline (room temperature) was then infused into the bladderat a filling rate of 75 ml/min. During the filling, the bladdervolumes at FDV and maximum cystometric capacity (MCC)were noted, and the presence of any detrusor contractionsrecorded, as described.6 MCC was defined as the volume whenthe patient and urodynamacist decided that permission tovoid has been given. After the test, patients voided into aclean uroflow chamber. The volume voided (VV) was notedand a sample of the fluid was collected (referred to subse-quently as ‘‘VUF’’) and frozen at �208C.

According to the clinical symptoms and cystometric results,patients were characterized as having OAB with increasedbladder sensation (in keeping with their symptoms of urgen-cy) or idiopathic DO. Patients with urodynamic stress inconti-nence or normal bladder function acted as controls. Womenwith voiding dysfunction or bacterial cystitis were excludedfrom the study. Twenty-nine patients were diagnosed withOAB, that is, clinical symptom of urgency with FDV �200 ml,

MCC �400 ml, but with a stable bladder. The prevalence ofOAB (without DO) in all patients in this series was 11.8%(29 out of 246). Four OAB patients found to have bacterial cys-titis (107 c.f.u./L) from a CSU on the test day were excludedfrom the study. Note that the results from the current studyperformed in patients with DO (n ¼ 48) have been publishedelsewhere.6

The control group included 69 patients, who were eitherurodynamically normal (n ¼ 11) or classified as pure urody-namic stress incontinence (n ¼ 58) without urge symptoms,voiding dysfunction, or bacterial cystitis, that is, they had in-voluntary leakage of urine during increased abdominal pres-sure in the absence of detrusor contractions, or else had nodemonstrable urodynamic abnormality (generally with co-ex-istent prolapse.

Laboratory Measurements

The pH of the initial CSU was measured with pH meter(Eutech, Bukit Raja, Klang, Malaysia) accurate to 0.01. ATP invoided fluid was measured using a GloMax 20/20 biolumines-cence assay after construction of a standard curve.6 The ATPcontent of urine could not be accurately measured.

Statistic Analysis

ATP data were not normally distributed and were expressedas median (interquartile range, IQR). The pH data wereexpressed as mean � SEM. Statistical analyses were per-formed using GraphPad Prism 5.0 software (San Diego, CA).Correlations between two factors were tested by linear regres-sion analysis. The Mann–Whitney’s U-test was used for com-parison of ATP levels (nonparametric distribution) and theunpaired t-test was used for pH comparisons. Methods, defini-tions, and units conform to the standards recommended bythe International Continence Society except where specificallynoted.

RESULTS

Comparison of OAB and Control Patients

The clinical and experimental data from women with OABand from control women (urodynamic stress incontinence)are summarized in Table I. The median age of control womenwas not statistically significant different from that of patientswith OAB. The ATP concentrations measured in VUF were sim-ilar between the two groups. The mean pH of urine from OABpatients was no different from that of the controls.Both FDV and MCC were significantly lower in patients

with OAB compared with control women. It was noteworthythat patients with OAB reported that filling to the MCC on thetest day was painful.

Correlation Between ATP Concentration in Voided UrodynamicFluid and Cystometric Parameters

In control patients (Fig. 1A), there was no relationship be-tween ATP concentration in VUF and FDV. In contrast, therewas a strong inverse correlation between ATP concentrationand FDV in OAB patients (Fig. 1B). In other words, a higherATP concentration was related to a lower FDV in patientswith OAB.With respect to MCC, there were no correlations between

ATP concentration and MCC in either controls (Fig. 1C) orpatients with OAB (Fig. 1D). The relationship between ATP

970 Cheng et al.

Neurourology and Urodynamics DOI 10.1002/nau

concentration and voided volume (VV) was also examined,but no correlations were seen for either control or patientswith OAB (data not shown).

Relationship Between Urine pH and ATP Concentrationand Urodynamic Parameters

In controls, there was no correlation between ATP concen-tration in the urodynamic fluid and the pH of the urine sam-ple collected immediately prior to the urodynamic test(Fig. 2A). However, in women with OAB, there was a signifi-cant reverse correlation between ATP concentration and theurine pH. Thus, the lower the urine pH, the higher ATP concen-tration detected in the subject’s urodynamic fluid (Fig. 2B).

The relationship between urine pH and cystometric vol-umes was also explored. Urine pH did not show any correla-tion with FDV in either controls (Fig. 2C) or OAB patients(Fig. 2D). In control subjects, there was a correlation(P ¼ 0.0005) of urine pH with MCC, but this may have beendue to the effect of one extreme outlier, with pH of 8.8 and

MCC volume of 900 ml. After recalculation of the regressionequation following exclusion of this outlier, the correlationwas not significant (P ¼ 0.056; Fig. 2E). No relationshipbetween urine pH and MCC was seen in patients with OAB(Fig. 2F).

DISCUSSION

To our knowledge, this is the first report showing that theATP concentration in VUF of women with OAB without DO isinversely correlated with the FDV. That is, the smaller thebladder volume at FDV, the higher was the ATP release. Thissuggests that ATP is an important factor for the initial percep-tion of the need to urinate, in patients with OAB. This is simi-lar to our previous findings in patients with proven DO,6

suggesting that ATP may modulate initial afferent sensationin patients whose main complaint is urinary urgency. In con-trast, such a relationship between ATP release and FDV wasnot found in the control group, whose main complaint isstress incontinence.18

Fig. 1. Linear regression analysis of ATP concentration in voided bladder fluid and urodynamic volumes in (A,C)control (n ¼ 69) and (B,D) patients with OAB (n ¼ 25). In control women, there was an absence of relationship of

ATP concentration with (A) FDV and (C) MCC. In patients with OAB, there was a significant inverse correlation of

ATP with (B) FDV (P ¼ 0.01) but not with (D) MCC.

TABLE I. Clinical and Experimental Data for Subjects in This Study

Control OAB P-value

Number 69 25 n/a

Age, years (mean/median, 95% CI) 53/49, 50–57 56/54, 50–62 0.47

Detrusor contraction at cystometry 0 0 n/a

Frequency, persistent urgency, nocturia 0 25 n/a

Stress incontinence 58 (11 were normal) 16 n/a

FDV, ml (mean/median, 95% CI) 251/239, 228–274 137/140, 117–159 <0.0001

MCC, ml (mean/median, 95% CI) 542/500, 460–616 324/315, 270–353 <0.0001

Urine pH (mean � SEM) 6.28 � 0.09 6.18 � 0.16 0.45

ATP concentration, nM (mean/median, 95% CI) 12.9/9.0, 10.1–15.7 14.8/12.9, 9.5–20.2 0.58

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Neurourology and Urodynamics DOI 10.1002/nau

Previous studies in animal IC models and human IC bladderstrips and cells, have shown an enhanced ATP release in re-sponse to stretch.7–9 Our patients are at the very mild end ofthe BPS spectrum and we see no differences in the intravesicalATP concentration between our groups. Studies examiningATP release in patients with IC are not comparable to ourstudy in that they utilized cell culture or tissue strip method-ology. The only comparable study is our similar study inpatients with urodynamically proved DO where we also didnot see a difference in the intravesical ATP concentration com-pared to controls. It is plausible that the enhanced ATP releasemay only be associated with the severe end of the BPS diseasespectrum.

The causes of urgency are unknown, although infection, ep-ithelial permeability, mast cells, heredity, and neuroinflam-mation, have been suggested. However, there is no singlecause which is involved in all patients experiencing urgency,whether their diagnosis is OAB, BPS, or IC. Recent progressincludes the finding of an agent antiproliferative factor, foundalmost exclusively in the urine of people with IC, whichappears to block the normal growth of urothelial cells.19

Inflammatory mediators from within the bladder wall wouldhave additive effects to stretch, to stimulate myelinated andunmyelinated afferent nerves, as described.20,21 Thus, we sug-gest that afferent nerves from subjects with urgency could besensitized and have a greater afferent discharge than controlsubjects’ bladders, for the same degree of stretch.Sensitization of afferent nerves could also involve the sub-

urothelial myofibroblasts and interstitial cells which havebeen shown to play an important role in modulation bladderafferent function through purinergic signaling pathways.22 Inpatients with OAB/urgency there may be increased sensitivityof suburothelial myofibroblasts and interstitial cells to theurothelial-derived ATP.As shown in animal studies in vitro, acid is a potent stimu-

lant of ATP release,13–15 providing the impetus for investiga-tion of urine acidity here in patients. There was no differencein the urine pH from controls and patients with OAB in thisstudy, but in the patients with OAB, there was a significantrelationship between lower urinary pH and higher ATP re-lease. Although the saline filling solution used at cystometryis relatively neutral, prior to the test the internal milieu of the

Fig. 2. Linear regression analysis of the effect of urine pH on ATP concentration in voided urodynamic fluid and

cystometric volumes in (A,C,E) control (n ¼ 69) and (B,D,F) and OAB patients (n ¼ 25). In control women, there

was an absence of relationship of urine pH with (A) ATP concentration; and (C) FDV; but (E) a significant inverse

correlation between urine pH and MCC was seen. Note that in (E), when an extreme outlier (boxed) is removed,

the analysis is changed (r2 ¼ 0.054, P ¼ 0.056). In patients with OAB, there was a significant inverse correlation

of urine pH with (B) ATP in voided urodynamic fluid but not with (D) FDV; or with (F) MCC.

972 Cheng et al.

Neurourology and Urodynamics DOI 10.1002/nau

bladder may be somewhat acidic, influenced by the urine pH.Factors such as stress, medication, allergic reactions, sexual in-tercourse, hormonal changes, and diet may exacerbate bladdersymptoms. Pain is intensified by exposure to agents in thediet including carbonated drinks, citrus fruits, coffee, alcohol,and chili.23–25

In patients with OAB who are subsequently found to havecystoscopic features of IC, the urothelium is damaged, so Hþ

ions would access the lamina propria of the bladder wall moreeasily, thus providing an additional stimulus for suburothelialafferent nerves. Note that in our OAB patients, both FDV (me-dian 140 ml) and MCC (median 315 ml) are experimental val-ues limited by the sensation of painful filling and it is wellknown that such patients tolerate much larger volumes undergeneral anesthesia.26

CONCLUSIONS

In conclusion, the ATP concentration of VUF was inverselyrelated to the volume at FDV, in patients with OAB. Further-more, this ATP concentration was also directly related to theacidity of the subject’s urine. Arising from this study, itappears that the urothelium of patients with OAB behavesdifferently from the controls in regards to stretch induced ATPrelease. Therefore, we are now examining how the degree ofstretch affects the magnitude of ATP release, by undertakingserial samplings of ATP in filling fluid at predetermined fillingvolumes.27

ACKNOWLEDGMENTS

We thank Katrina Parkin RN, for help with patient recruit-ment and cystometry testing.

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