Dr Pedro Iglesias

José Carlos Castro

Juan José Díez

CLINICAL SIGNIFICANCE OF ANEMIA ASSOCIATED WITH PROLACTIN-SECRETING PITUITARY TUMORS IN MEN

Keywords: Anemia, hemoglobin, hypogonadism, hypopituitarism, men, testosterone

whether they are published or not. The documents may come

• An association between anemia and macroprolactinomas in male patients has been documented.

• Anemia has been related with hypogonadism and tumor size in these patients.

What's new

• Mild normocytic and normochromic anemia is found in about a third of men with prolactinomas.

• The presence of anemia in these patients seems to be associated with macroprolactinoma induced panhypopituitarism.

Introduction

Prolactinomas are the most common hormone-secreting pituitary tumors, accounting for about 60% of primary pituitary tumors. These tumors are more frequently seen in women (>70%) than in men [START_REF] Nabarro | Pituitary prolactinomas[END_REF][START_REF] Mindermann | Age-related and gender-related occurrence of pituitary adenomas[END_REF][START_REF] Delgrange | Sex-related difference in the growth of prolactinomas: a clinical and proliferation marker study[END_REF]. The female/male ratio varies depending on the tumor size, being 20:1 and 1:1 for micro-(≤10 mm) and macroprolactinomas (>10mm), respectively [START_REF] Melmed | Pituitary tumors secreting growth hormone and prolactin[END_REF].

It has been reported that hematopoiesis is compromised in men who have low concentrations of testosterone due to a pituitary adenoma, supporting a direct relationship between serum testosterone levels and hematopoiesis in men [START_REF] Ellegala | Anemia, testosterone, and pituitary adenoma in men[END_REF]. More recently, an association between anemia and macroprolactinomas in male patients has also been documented [START_REF] Shimon | Anemia in a cohort of men with macroprolactinomas: increase in hemoglobin levels follows prolactin suppression[END_REF]. In this setting, anemia was related with hypogonadism and tumor size and an improvement in hemoglobin concentrations have been reported after achieving normalization of PRL levels and increase in testosterone concentrations with appropriate therapy [START_REF] Shimon | Anemia in a cohort of men with macroprolactinomas: increase in hemoglobin levels follows prolactin suppression[END_REF].

To our knowledge the relationship between pituitary function and the presence of anemia has not been extensively evaluated in male patients bearing prolactinoma. Our aim was to assess the prevalence of anemia associated with prolactinoma and to analyze its relationship with pituitary function at diagnosis in male patients with prolactin-secreting pituitary adenomas.

Patients and methods

Patients

We retrospectively investigated the presence of anemia at diagnosis of PRL-secreting pituitary tumors in 26 men and followed-up in the last 35 years at Organization criteria (Hb level <13 g/dl in men) (7). We also assessed the presence or absence of hypopituitarism at diagnosis registering type and number of pituitary deficiencies. Central hypogonadism was defined when serum testosterone was lower than 300 ng/dl and low or lownormal gonadotropin levels [START_REF] Bhasin | Testosterone therapy in men with androgen deficiency syndromes: an Endocrine Society clinical practice guideline[END_REF]. Hb, hematocrit, and hormone measurements were performed in the laboratories of each hospital using standard automated blood count analyzer, radioimmunoassay, immunoradiometric assay or enzymoimmunometric assay methods.

Statistical analysis

For quantitative variables, results are expressed as mean ± SD for normally distributed data, and as median (interquartile range) for nonparametric data. Adjustment to normal distribution was tested by the Kolmogorov test. Categorical variables are described as percentages. For comparisons of means between two groups of subjects the Student t-test was used for normally distributed data, and the Mann-Whitney test was employed for nonparametric data. For ratio comparisons the chi-square test was used. Correlations between quantitative variables were assessed using Pearson's correlation analysis. Models of logistic regression analysis were used to assess the presence of anemia at diagnosis of prolactinomas as a function of several qualitative and quantitative variables (serum hormone concentrations).

Differences were considered significant when p<0.05.

Results

Clinical features and analytical

Prevalence of anemia

Mean hemoglobin at diagnosis of prolactinoma was slightly lower than in the last visit of the patients (13.8 ± 1.3 g/dl vs 14.3 ± 1.0 g/dl; ns). No difference in terms of WBC and platelet counts was found. At diagnosis of prolactinoma, normocytic and normochromic anemia was present in 9 (34.6%) patients. Mean Hb value in anemic and non-anemic patients was 12.2 ± 0.5 g/dl and 14.5 ± 0.7 g/dl (p<0.001), respectively. In anemic patients the degree of anemia was always mild (Hb >11 g/dl). Macroprolactinoma patients showed lower Hb level than patients bearing microprolactinomas (13.5 ± 1.2 g/dl vs 15.1 ± 0.9 g/dl, p<0.05) (figure 1). None of the four patients with microprolactinomas exhibited anemia. The presence of anemia was not associated with age of the patients or serum PRL levels at diagnosis of prolactinomas.

Anemia and hypogonadism

Central hypogonadism was diagnosed in 14 (53.8%) patients, all of them with macroprolactinomas. Serum testosterone levels were significantly lower in macro-than in microprolatinoma patients (176.5 ± 107.7 ng/dl vs 441.0 ± 142.8 ng/dl, p<0.001).The presence of anemia was significantly associated with the involvement of gonadal (p=0.013) axis. Hb was significantly lower in hypogonadic compared with normogonadic patients (13.3 ± 1.2 g/dl vs 14.6 ± 1.4 g/dl, p<0.05). In addition, Hb was positively correlated (p<0.05) with serum testosterone in the whole group of male patients. However, when considering only hypogonadic men, Hb value was only positively correlated with FSH (r=0.732; p=0.004). No correlation between Hb and testosterone and gonadotropins in the normogonadic male patients was found. No correlation between Hb and serum PRL levels was found.

Anemia and hypopituitarism

Hypopituirarism was present in 20 (76.9%) patients. Partial hypopituitarism was identified in 13 (50%) patients, whereas total hypopituitarism was diagnosed in 7 (26.9%) patients. Hb value was significantly lower in patients with total hypopituitarism compared with partial hypopituitarism (12.4 ± 1.0 g/dl vs 14.0 ± 1.2 g/dl, p=0.007) (figure 2). A positive and significant (p=0.007) association between the presence of anemia and the number of affected pituitary axes was found. Hb level was positively correlated with both FT4 (r=0.675; p=0.002)

F

Discussion

Our results show that anemia is not an uncommon finding in men with prolactinomas. In fact, anemia (Hb <13 g/dl) at diagnosis of prolactinomas was documented in more than onethird (34.6%) of our patients. The degree of anemia was always mild (Hb >11 g/dl). Anemia was always linked to the presence of macroprolactinoma and more frequently associated with total hypopituitarism.

An association between anemia and pituitary adenoma has been identified, mainly in men [START_REF] Ellegala | Anemia, testosterone, and pituitary adenoma in men[END_REF][START_REF] Shimon | Anemia in a cohort of men with macroprolactinomas: increase in hemoglobin levels follows prolactin suppression[END_REF]. In a retrospective analysis performed in 197 patients (100 men) older than 50 years of age with pituitary adenomas 31 (46.3%) of 67 men with low serum concentrations of testosterone were anemic (hematocrit < 40% for men). More recently, a retrospective study in a group of 36 consecutive male patients with macroprolactinomas showed that 16 (44.4%) male patients presented with anemia (Hb<13 g/dl). The prevalence of anemia in our study was slightly lower (~35%), however, this percentage might reflect more accurately the prevalence of anemia in men with prolactinomas because in our study we did not exclude male patients with microprolactinomas. This subgroup of patients was 15.4% and none of them showed anemia.

Testosterone modulates Hb levels in men by means of stimulating hematopoiesis by several mechanisms, such as increasing erythropoietin production and the number of erythropoietin-responsive cells [START_REF] Coviello | Effects of graded doses of testosterone on erythropoiesis in healthy young and older men[END_REF]. In this setting, low testosterone levels have been associated

with anemia in both middle-aged and older men [START_REF] Yeap | Serum testosterone levels correlate with haemoglobin in middle-aged and older men[END_REF][START_REF] Ferrucci | Low testosterone levels and the risk of anemia in older men and women[END_REF]. On the other hand, mild anemia has been described in both primary and secondary hypogonadism [START_REF] Schubert | Late-onset hypogonadism in the aging male (LOH): definition, diagnostic and clinical aspects[END_REF][START_REF] Yin | Treating hypogonadism in younger males[END_REF]. Pituitary tumors are the most common causes of secondary hypogonadism in men. Among them, prolactinomas are the most frequent hormone-secreting pituitary tumors, accounting for about 60%. In a recent study Shimon et al. [START_REF] Shimon | Anemia in a cohort of men with macroprolactinomas: increase in hemoglobin levels follows prolactin suppression[END_REF] reported that anemia associated with macroprolactinomas in men was related to hypogonadism and tumor size, improving after appropriate therapy which normalized both PRL and testosterone. Our study confirmed these results, i.e., the presence of anemia was associated with the involvement of gonadal axis which was present in macroprolactinomas.

Apart from hypogonadism other hormonal deficiencies such as hypothyroidism and Anemia is diagnosed in 23-60% of hypothyroid patients and the prevalence of hypothyroidism in anemic patients is about 20%. Moreover, hypothyroidism associated anemia is usually reversible with increasing Hb after hormone replacement therapy with levothyroxine [START_REF] Fein | Anemia in thyroid diseases[END_REF][START_REF] Vitale | Screening for hypothyroidism in older hospitalized patients with anemia: a new insight into an old disease[END_REF].

Studies in vitro have shown that thyroid hormones enhance hypoxia-induced erythropoietin production [START_REF] Fandrey | Thyroid hormones enhance hypoxiainduced erythropoietin production in vitro[END_REF]. Something similar occurs in patients with adrenal insufficiency. Adrenal insufficiency is often accompanied by anemia. This association has been reported in both primary [START_REF] Burke | Adrenocortical insufficiency[END_REF] and secondary [START_REF] Andrioli | Isolated corticotrophin deficiency[END_REF] adrenal insufficiency. Cortisol stimulates erythroid colony growth and it has been suggested that this hormone might play a role in the physiological regulation of human erythropoiesis [START_REF] King | Modulation of human erythropoiesis by hydrocortisone in vitro[END_REF].

Our study is the first one that evaluates and analyzes the relationship between anterior pituitary function and Hb in a significant number of men with prolactinomas. Our findings suggest that the presence of anemia in these patients is a marker of hypopituitarism and not only of hypogonadism. This fact is important due primarily to the therapeutic implications related to the introduction of hormone replacement therapy at diagnosis. Our results are limited by the retrospective nature of the study and the relatively low number of included patients.

In conclusion, the presence of anemia, usually mild (Hb >11 g/dl), is not an uncommon finding (~35%) in men with a recent diagnosis of prolactinoma. This usually indicates the existence of a macroprolactinoma associated with panhypopituitarism.

FiguresFigure 1 .Figure 2 .

 12 FiguresFigure1. Hemoglobin values at diagnosis in both macro-and microprolactinomas in male patients. The horizontal line corresponds to the hemoglobin level consistent with anemia.


MBBS Christopher Dowson

MBBS Arun Sahai

Prokar Jane Watkins

Shamim Dasgupta

Khan

MD Mohammad S Khan

Prof Prokar Dasgupta

The safety and efficacy of botulinum toxin-A in the management of bladder oversensitivity: A randomised double blind placebo controlled trial

Keywords: detrusor overactivity, bladder oversensitivity, botulinum toxin, overactive bladder, sensory urgency

The safety and efficacy of botulinum toxin-A in the management of bladder oversensitivity: A randomised double blind placebo controlled trial

Introduction

Overactive Bladder Syndrome (OAB) is defined by the International Continence Society (ICS) as "Urinary urgency, usually accompanied by frequency and nocturia, with or without urgency urinary incontinence, in the absence of urinary tract infection or other obvious pathology" [START_REF] Abrams | The standardisation of terminology of lower urinary tract function: report from the Standardisation Sub-committee of the International Continence Society[END_REF]. First line management is usually undertaken in the primary care setting and consists initially of fluid management and bladder retraining followed by a trial of anticholinergic medication if required. If these efforts fail to control symptoms then a referral to secondary care usually follows where the patient will usually undergo further investigations including urodynamic assessment. At this stage a distinction can be made between patients with evidence of aberrant detrusor contractions during filling cystometry and those without. In the absence of any associated pathological abnormality the former condition is termed Idiopathic Detrusor Overactivity (IDO) and the latter as Bladder Oversensitivity (BO), previously termed motor urgency and sensory urgency respectively [START_REF] Abrams | The standardisation of terminology of lower urinary tract function. The International Continence Society Committee on Standardisation of Terminology[END_REF]. The most recent ICS terminology report defines bladder oversensitivity as "An increased perceived bladder sensation during filling with specific cystometric findings of i) early first desire to void ii) an early strong desire to void, which occurs at low bladder volumes iii) a low maximum cystometric capacity iv) no abnormal increases in detrusor pressure" [START_REF] Haylen | An International Urogynecological Association (IUGA)/International Continence Society (ICS) joint report on the terminology for female pelvic floor dysfunction[END_REF]. In effect this divides patients with OAB into two distinct clinical groups. Both have the same symptomology i.e. frequency and urgency of micturition, but for one group this is correlated with an abnormal contraction of the bladder (IDO), whereas in the other it is not (BO).

Botulinum toxin-A (botn-A) has emerged as an effective second line therapy in the management of OAB and detrusor overactivity (DO) with a recent European consensus giving it a grade A recommendation for use in this condition [START_REF] Apostolidis | Recommendations on the use of botulinum toxin in the treatment of lower urinary tract disorders and pelvic floor dysfunctions: a European consensus report[END_REF]. Whist there is some experience of managing BO patients with botn-A reported in the literature they tend to form part of larger heterogeneous patient cohorts. To date there have been no randomised, placebo controlled trials specifically examining the effects of botn-A in this group of patients.

Materials & Methods

This study was a randomised, double blind placebo controlled trial conducted at a single centre. The trial was performed in line with Good Clinical Practice and had local ethical approval (registration no. 06/Q0704; ISRCTN no. 57577615). Patients were randomised to either 100U Botox® (Allergen, Irvine, California) or normal saline (placebo). A list was prepared by an independent statistician and held in the trials office of the pharmacy department. Eligible patients were assigned a study number in sequence and the pharmacy dispensed either the placebo or active treatment according to the list. The trial injections were collected and prepared by a trained nurse who had no contact with the patient, such that the investigators received 10ml of clear solution without knowing whether it contained 100U of Botox or placebo.

Patients (18-79y) who fulfilled the criteria for a diagnosis of BO, as defined in the introduction, were invited to participate in the trial. All patients had failed conservative and pharmacological therapy with at least one anticholinergic medication. Exclusion criteria included current or planned pregnancy, breast feeding, painful bladder syndrome, pre-existing neurological condition, evidence of bladder outflow obstruction, indwelling catheter, previous bladder surgery, previous urological use of botn-A and continued anticoagulation with heparin or warfarin. Urodynamic data were collected at weeks 0 and 12. These specifically included maximum cystometric capacity (MCC), first desire to void (FD), normal desire to void (ND), post void residual volume (PVR) and the presence/absence of DO. At week 0, 4 and 12, patients completed a three day voiding diary which recorded 24hr urinary frequency (UF), urinary urgency (UU) and urge urinary incontinence (UUI). They The study procedures were performed in the out-patient setting by a single surgeon.

Urodynamics were performed in accordance with the ICS guidelines [START_REF] Schafer | Good urodynamic practices: uroflowmetry, filling cystometry, and pressure-flow studies[END_REF]. Prior to any intervention all patients gave a urine sample to exclude infection (and pregnancy if appropriate) and all received a oral dose of ciprofloxacin 500mg. Lignocaine gel (Instillagel, Clinimed, UK) was applied to the urethra prior to cystoscopy. Intradetrusor, trigone sparing injections of either 100 U botn-A (Botox, Allergan) or normal saline (placebo) were performed with a flexible cystoscopic technique using a 4mm Olympus needle. Ten sites along the base, posterior and lateral walls of the bladder were injected with 1ml of solution containing either 10 U botn-A (Botox, Allergan) in normal saline or normal saline alone (placebo). Three small bladder biopsies were taken, from 1cm above and lateral to the left ureteric orifice, baseline Independent statistical advice was sought with regards data. A repeated measures generalised linear model was used to compare the pre-and post intervention variable outcomes using SPSS® v17.0.

Results

Between November 2007 and November 2009 a total of 28 patients were identified with a diagnosis of BO and invited to participate in this trial. Five patients declined with concerns regarding the risk of having to perform CISC. Of the 23 patients who were injected as per protocol -1 failed to attend follow up and 1 was re-diagnosed as interstitial cystitis (Consort diagram -figure 1). Complete data for 21 patients was available (treatment arm = 10, placebo = 11. At this stage an unplanned interim analysis was undertaken with advice from the local research governance board, due to concern that the majority of patients had reported no clinical benefit. Following data analysis it was determined that there was likely to be little benefit in continued recruitment and the trial was terminated.

Table 1 describes the baseline urodynamic parameters and OAB symptoms for the entire cohort and demonstrates a low MCC, FD and ND in addition to a high day time UF which is associated with UU on over 70% of occasions. The average age of the botn-A and placebo groups was not significantly different (49.6+/-19 and 46.7+/-17y, p=0.7). The ratio of males to females was 2:8 and 4:7 in the botn-A and placebo group respectively. Urodynamic variables: There was a significant rise in MCC, (the primary endpoint of the study), in patients receiving botn-A compared to placebo (p=0.009) (figure 2). In the botn-A arm there was an mean increase in FD (figure 3), ND and PVR although OAB Symptoms: Overall there was no significant difference observed in UF, UU or UUI at any time point in either group, and no trends were noted (figures 4-6).

Individually, only two patients felt their symptoms were improved by the injections, whilst for the remainder their symptoms scores remained unchanged.

QoL measures: There was significant improvement in IIQ-7 at 4 weeks which became non-significant by week 12. There was no significant improvement in UDI-6 at any stage.

Patient perception of bladder condition: No significant change was observed in

PPBC score throughout the study period.

These results are summarised in Table 2.

Adverse events: Three patients in the botn-A group were required to perform CISC and none of them reported any symptomatic improvement. No one in the placebo group required CISC. UTI was detected in 4 patients in the botn-A arm (2 associated with performing CISC) and 1 in the placebo arm.

A patient in the botn-A group reported a general malaise which lasted two months following injections with no obvious cause identified. The patient did not require any specific intervention.

Discussion

This study is the first randomised controlled trial examining the effects of botn-A exclusively in patients with BO. Despite enrolling only a third of the patients suggested by the power calculation the primary endpoint was achieved with the data demonstrating a significant increase in MCC and a trend towards improvement in FD and ND. This did not, however, translate into a meaningful clinical benefit as there was no overall difference detected in OAB symptoms, QoL (with the exception of IIQ-7 at week 4) or PPBC. Individually, only two (20%) patients in the treatment arm derived any benefit from the injections, whilst the remainder experienced no change in their symptoms.

Several studies include patients with BO in their cohorts. Schulte-Baukloh et al. reported a series of seven patients, who received 300U botn-A. At 12 weeks they observed a significant rise in MCC and FD with a decrease in UF, nocturia and pad usage [START_REF] Schulte-Baukloh | Botulinum-A toxin for treatment of overactive bladder without detrusor overactivity: urodynamic outcome and patient satisfaction[END_REF]. Cohen et al reported a randomised controlled trial comparing 100u and 150U of botn-A in a cohort including patients with BO or IDO, although the authors do not distinguish between them in their analysis. Instead patients were sub-divided into those with UUI ("wet") and those with no UUI ("dry"). They found a significant decrease in UF in the "dry" group and a significant decrease in UUI in the "wet" group. They found no significant change in MCC or PVR and they did not observe any significant difference between the two doses [START_REF] Cohen | Preliminary results of a dose-finding study for botulinum toxin-A in patients with idiopathic overactive bladder: 100 versus 150 units[END_REF]. Flynn et al. also found no significant change in MCC and UF in a trial which compared either 200u or 300u botn-A with placebo in a group of patients with BO and IDO. They found significant improvement in UUI and QoL measures [START_REF] Flynn | Outcome of a randomized, doubleblind, placebo controlled trial of botulinum A toxin for refractory overactive bladder[END_REF]. In an open label study Schmid et al described significant improvement in 100 patients with OAB (46 BO and 54 IDO) They did not find a difference in treatment response between those with BO and those with IDO [START_REF] Schmid | Experience with 100 cases treated with botulinum-A toxin injections in the detrusor muscle for idiopathic overactive bladder syndrome refractory to anticholinergics[END_REF]. A recent randomised, placebo controlled, dose response trial involving 313 patients demonstrated an improvement in symptoms and QOL measures overall. This study concluded that the presence of DO during filling cystometry did not influence outcome, although the study was not specifically designed to examine BO [START_REF] Chapple | Benefit/risk assessment[END_REF]. Overall these studies tend to demonstrate some clinical improvement, although the data are inconsistent, particularly with regards to changes in MCC and UF.

Two studies examining only patients with IDO demonstrate more consistent positive results. Sahai et al found an improvement in almost all variables studied at 4 and 12 weeks after 200u botox with reductions in UF (15.4 to 9.2), UU (11.7 to 3.6) and UUI (5.0 to 1.5) and an increase in MCC (181 to 313) at 12 weeks [START_REF] Sahai | Efficacy of botulinum toxin-A for treating idiopathic detrusor overactivity: results from a single center, randomized, double-blind, placebo controlled trial[END_REF]. Khan et al found UF, UU & UUI reduced significantly by 67%, 100% and 54% respectively at 4 weeks [START_REF] Khan | Complete Continence after Botulinum Neurotoxin Type A Injections for Refractory Idiopathic Detrusor Overactivity Incontinence: Patient-Reported Outcome at 4 Weeks[END_REF]. In these studies dramatic improvements are observed in the symptoms of the majority of patients. In this study only 20% of patients demonstrated a symptomatic improvement with no change (or trend towards change) in UF, UU and UUI in the remaining 80%. We observed a significant increase in MCC (primary endpoint) which did not correlate with a symptomatic improvement. On that basis we sought statistical advice on re-powering the study to a symptomatic endpoint but interim power calculations (Statmate™ Graphpad Software) suggested that even with the small number of patients recruited we should have been able to detect a significant difference of 4, 3 and 1.6 episodes per 24 hours for UF, UU and UUI.

Possible explanations for the observed lack of effect include insufficient dosage, although many of the studies above utilised 100U of Botn-A with positive to inject all areas of the bladder to "knock out" aberrant detrusor contractions. In true BO no DO exists and it is therefore possible that localised injections into the bladder base and trigone might be more appropriate, as this area contains the most dense sensory innervation in the bladder and injections here have been shown to be beneficial in non-neurogenic OAB [START_REF] Smith | Simplified bladder botulinum-toxin delivery technique using flexible cystoscope and 10 sites of injection[END_REF]. Furthermore trigonal injections have now been shown not to induce vesico-ureteric reflux, an early potential concern with this technique [14;15] In addition to an established motor effect botn-A has been shown in a number of human studies to have a modulatory effect on the sensory apparatus of the bladder. This includes inducing a decreased expression of the purinergic receptor P2X3, the vanilloid receptor TRPV1, prostaglandins and nerve growth factor [START_REF] Apostolidis | Decreased sensory receptors P2X3 and TRPV1 in suburothelial nerve fibers following intradetrusor injections of botulinum toxin for human detrusor overactivity[END_REF][START_REF] Chuang | Intravesical botulinum toxin A administration inhibits COX-2 and EP4 expression and suppresses bladder hyperactivity in cyclophosphamide-induced cystitis in rats[END_REF][START_REF] Liu | Urinary nerve growth factor levels are elevated in patients with detrusor overactivity and decreased in responders to detrusor botulinum toxin-A injection[END_REF], all of which have been implicated in the pathogenesis of IDO and BO [START_REF] Lowe | Increased nerve growth factor levels in the urinary bladder of women with idiopathic sensory urgency and interstitial cystitis[END_REF][START_REF] Ray | Loss of purinergic P2X receptor innervation in human detrusor and subepithelium from adults with sensory urgency[END_REF][START_REF] Kim | Nerve growth factor and prostaglandins in the urine of female patients with overactive bladder[END_REF]. These observations, however, could be a secondary effect due to reduction in bladder contraction/pressure rather than due to primary action of the toxin. More direct evidence of sensory modulation has been derived from animal models in which decreased release of the neurokinins, CGRP and SP, (which have also been implicated in the pathogenesis of OAB), were observed following intra-vesical instillation of botn-A [START_REF] Andersson | Tachykinins: role in detrusor overactivity?[END_REF][START_REF] Chuang | Intravesical botulinum toxin a administration produces analgesia against acetic acid induced bladder pain responses in rats[END_REF][START_REF] Lucioni | Botulinum toxin type A inhibits sensory neuropeptide release in rat bladder models of acute injury and chronic inflammation[END_REF]. Caution must be exercised when extrapolating these results to IDO or BO as these laboratory models do not reproduce the specific pathophysiology, which to date, remains unclear.

It remains to be seen whether BO and IDO represent distinct pathological entities.

Haylen et al. prospectively compared 77 patients with BO with 74 patients with IDO

and could find no significant clinical differences between the 2 groups except for a significantly increased incidence of UUI in the IDO group [START_REF] Haylen | Is sensory urgency part of the same spectrum of bladder dysfunction as detrusor overactivity?[END_REF]. A distinction has been patients diagnosed with IDO or BO. They found trigonal TRPV-1 in BO patients to be higher than that of the bladder body mucosa or that of control tissue. In contrast no differences were found in TRPV-1 levels from the trigone or bladder base in IDO patients [START_REF] Liu | The molecular basis of urgency: regional difference of vanilloid receptor expression in the human urinary bladder[END_REF].

Currently the diagnosis of BO depends on the absence of aberrant detrusor contractions during filling cystometry but this can be unreliable, lacking in reproducibility and there will undoubtedly be some overlap with IDO [27;28]. A cystometric distinction between the IDO and BO is unlikely to influence or provide any useful prediction of response to first line treatments [29;30]. In this study it's possible that these cystometric limitations have resulted in patients with IDO rather than BO being recruited, but given the positive outcomes for patients with IDO (as discussed above) we would have expected this to improve the overall symptomatic results. Despite these reservations the distinction between BO and IDO is important for future research and may be clinically relevant when counselling patients before botn-A as our data suggests the likelihood of a satisfactory outcome will not be as good for BO.

Although the primary endpoint was achieved with a clear and significant increase in MCC, it did not correlate with an improvement in patient symptoms. This primary end point was chosen during the design phase as it was felt to be relatively objective and that previous experience with IDO patients had shown association between a rise in MCC and symptomatic improvement [START_REF] Sahai | Efficacy of botulinum toxin-A for treating idiopathic detrusor overactivity: results from a single center, randomized, double-blind, placebo controlled trial[END_REF]. In retrospect, using a symptom as the primary endpoint, for example UU, would have been more appropriate. The authors accept that this data is limited by the small numbers of patients and the unplanned interim analysis. Nevertheless the lack of any symptomatic improvement in most patients in this study, despite the changes in their urodynamic parameters is significant and raises questions regarding the role of botn-A as a therapy for this specific sub-group of OAB, particularly as we are used to observing far more dramatic symptomatic improvements in the majority of patients in the IDO population. In a wider sense this study also highlights the need for continued research to attempt to understand and distinguish what distinct pathological entities OAB represents and to describe their specific aetiologies so that future therapies can be targeted more effectively.

Conclusion

The results of this double blind placebo controlled trial in the use of botn-A in BO are disappointing. Despite demonstrating a significant increase in MCC there was no overall improvement in OAB symptoms orl QoL. No clinical benefit was observed in 80% of patients reinforcing the need for continued research to identify and define the different pathophysiological processes which present as the OAB syndrome.

  to complete the short forms of the Urogenital Distress Inventory (UDI-6) and Incontinence Impact Questionnaire (IIQ-7) and the Patient Perception of Bladder Condition questionnaire (PPBC) to obtain a patient orientated assessment of the treatment. The primary endpoint of the study was a change in MCC. A sample size of 58 was calculated to achieve a 90% power to detect a difference of 30%. Secondary endpoints included other urodynamic parameters [first desire to void (FD), normal desire to void (ND), post void residual volume (PVR)], OAB symptoms, assessment of QoL and PPBC.

  and at week 12 for use in a separate study. All patients were given a three day course of ciprofloxacin 500mg twice daily. Patients were individually unblinded after 12 weeks once all study data had been collected. Those who had received placebo were offered 100U Botox in an open label fashion.

  did not achieve statistical significance. One patient in the treatment arm and two in the placebo arm demonstrated DO at week 12 which had not been observed at baseline.

  of botn-A. They observed an overall reduction in UF and UU.

  outcomes. A further possibility is injection technique. The goal in DO is

  molecular level, by Lui et al. who analysed bladder biopsies from

Figure 1 :

 1 Figure 1:-Consort Flowchart

Figure 2 :

 2 Figure 2: -Maximum cystometric capacity (MCC) at baseline and at 12 weeks following intradetrusor injections. There is a significant rise in the MCC in the botn-A group (p=0.009). No change is observed in the placebo arm.

Figure 3 :

 3 Figure 3: -First desire to void. An increase is observed in the botn-A arm although it does not reach statistical significance.

Figure 4 :

 4 Figure 4: -Episodes of urinary frequency at weeks 0, 4 & 12. No significant differences were detected and no trends were evident.

Figure 5 :

 5 Figure 5:-Episodes of urinary urge at weeks 0, 4 & 12. No significant differences were detected and no trends were evident.

Figure 6 :

 6 Figure 6: -Episodes of urge urinary incontinence at weeks 0, 4 & 12. There is no significant difference detected and no trends observed.

Figure 2 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :

 23456 Figure 2: -Maximum cystometric capacity (MCC) at baseline and at 12 weeks following intradetrusor injections. There is a significant rise in the MCC in the botn-A group (p=0.009). No change is observed in the placebo arm. 209x297mm (300 x 300 DPI)

Table 1 -

 1 Baseline cystometric and symptom scores for entire study cohort

		Mean	SD
	MCC	275.2 ml	70.6
	FD	98.4 ml	64.6
	ND	146.0 ml	56.8
	PVR	22.1 ml	25.2
	UF	14.2 voids/24hrs	4.4
	UU	11.6 voids/24hrs	5.9
	UUI	1.7 voids/24hrs	2.2
	MCC -maximum cystometric capacity; FD -first desire to void; ND -normal desire to void;
	PVR -post void residual volume; UF -urinary frequency; UU -urinary urgency; UUI -Urge
	urinary incontinence.

Table 2 -

 2 Comparison of the botn-A and placebo arms at baseline (BL) and 4 and/or

	12 weeks
					Difference between mean
			Botn-A Placebo		p
					change (95% CI)
	MCC	BL	259	290		0.33
		12	365	290	105 (30 -180)	0.009
	FD	BL	86	110		0.40
		12	149	108	65 (-41 to 172)	0.22
	ND	BL	119	171		0.03
		12	189	177	69 (-40 to 168)	0.22
	PVR	BL	21	23		0.82
		4	62	29	35 (-24 to 95)
		12	78	30	50 (-20 to 120)	0.22
	UF	BL	15.6	12.8		0.17
		4	14.7	12.1	-0.1 (-3.9 to 3.7)
		12	14.9	12.8	-0.9 (-4.9 to 3.1)	0.82
	UU	BL	12.4	10.8		0.57
		4	12.8	11.0	-0.9 (-4.0 to 4.4)
		12	12.5	11.8	-1.1 (-5.0 to 2.8)	0.78
	UUI	BL	1.2	2		0.38
		4	1.4	2.4	-0.1 (-2.5 to 2.2)
		12	1.1	2.9	-1.0 (-3.7 to 1.7)	0.52
	IIQ-7	BL	19.2	12.9		0.04

Botn-A -botulinum toxin-A; MCC -maximum cystometric capacity

; FD -first desire to void; ND -normal desire to void; PVR -post void residual volume; UF -urinary frequency; UU -urinary urgency; UUI -Urge urinary incontinence. IIQ-7 -Incontinence impact questionnaire short form; UDI-6 -Urogenital distress inventory short form; PPBC -Patient perception of bladder condition. Page 22

of 28 International Journal of Clinical Practice International Journal of Clinical Practice

	F o
	r
	P
	e e r
	R e v i e w
	O n
	209x297mm (300 x 300 DPI)	l y

Page 23 of 28 International Journal of Clinical Practice International Journal of Clinical Practice

	F o
	r
	P
	e e r
	R e v i e w
	O n
	l y

Acknowledgements

PDG & AS acknowledge financial support from the Department of Health via the National Institute for Health Research (NIHR) comprehensive Biomedical Research Centre award to Guy's & St Thomas' NHS Foundation Trust in partnership with King's College London and King's College Hospital NHS Foundation Trust. They also acknowledges the support of the MRC Centre for Transplantation. The authors are grateful for the statistical support and advice from Irene Rebollo Mesa PhD, Research Fellow in Biostatistics and Bioinformatics, MRC Centre for Transplantation, King's College London. All authors are investigators for Allergan.


MD MSc FRCP FRCP Martin R Cowie
email: m.cowie@imperial.ac.uk

Rebecca Lucas

Edin Fesc

Rgn Msc

Iron replacement therapy in chronic heart failure

Keywords: New York Association Functional Class, EF: Ejection fraction, Hb: plasma haemoglobin concentration, Hct: haematocrit, EPO: erythropoietin, IV: intravenous

Anaemia is common in patients with chronic heart failure (HF), particularly if there is concomitant renal dysfunction. It is part of a cardio-renal-anaemia syndrome, in which each component impacts on the other in a vicious cycle.

Prevalence varies according to the definition used but a recent meta-analysis reported anaemia in over one-third of patients with chronic HF [START_REF] Groenveld | Anemia and mortality in heart failure patients. A systematic review and meta-analysis[END_REF]. The majority of studies in this meta-analysis used the WHO definition of anaemia (haemoglobin <12g/dl in women and <13g/dl in men). Anaemia prevalence increases with age and with HF severity, but averaged 37% in the studies included in the meta-analysis [START_REF] Groenveld | Anemia and mortality in heart failure patients. A systematic review and meta-analysis[END_REF].

Anaemia in patients with HF is associated with worse symptoms and decreased exercise capacity [START_REF] Kalra | Effect of anemia on exercise tolerance in chronic heart failure[END_REF]. It has also been shown to be independently associated with increased risk of hospitalisation and mortality [START_REF] Tang | Anemia in chronic heart failure[END_REF], both in patients with left ventricular systolic dysfunction and those with preserved ejection fraction [START_REF] Groenveld | Anemia and mortality in heart failure patients. A systematic review and meta-analysis[END_REF].

Outcome studies have not yet established that anaemia is a cause of HF progression rather than simply a marker of disease severity. Nonetheless, evidence is accumulating that there is benefit from correction of anaemia.

The multiple causes of anaemia in HF include haemodilution, chronic inflammation, chronic kidney disease and iron deficiency (for example, from NSAID-induced gastrointestinal bleeding). ACE inhibitors and angiotensin receptor blockers might also play a part by reducing erythropoietin (EPO) activity.

When other causes of anaemia have been excluded, HF anaemia is generally labelled as anaemia of chronic disease. Renal dysfunction and pro-inflammatory cytokine activation appear to contribute to this anaemia in the majority of patients (4), resulting in reduced EPO production or EPO resistance and defective iron utilisation, the latter via increased hepatic production of hepcidin protein (Figure).

Iron deficiency appears to be more important than previously thought as a contributor to anaemia in HF. Although absolute iron deficiency is reported in a minority of cases (<30%) (3) functional iron deficiency may be more common. Poor utilisation of seemingly adequate iron stores was identified in one study as an important factor associated with anaemia in HF [START_REF] Opasich | Blunted erythropoietin production and defective iron supply for erythropoiesis as major causes of anaemia in patients with chronic heart failure[END_REF].

Over the past decade several studies have been undertaken to assess the benefit of correcting anaemia in HF (Table ). Initial small single-centre studies reported some improvement in functional capacity and quality of life with use of EPO or its analogues together with supplementary iron (6,7) although a small multicentre study showed no significant effect on exercise tolerance [START_REF] Ponikowski | Effect of darbepoetin alfa on exercise tolerance in anemic patients with symptomatic chronic heart failure[END_REF] and the larger STAMINA-HeFT randomised trial of 319 patients found no significant clinical benefits from darbepoetin [START_REF] Ghali | on behalf of the STAMINA-HeFT group. Randomized double-blind trial of darbepoetin alfa in patients with symptomatic heart failure and anemia[END_REF]. A meta-analysis of seven randomised trials of erythropoiesis-stimulating agents (ESAs) in HF [START_REF] Van Der Meer | Erythropoietin treatment in patients with chronic heart failure: a meta-analysis[END_REF] reported reduced risk of HF hospitalisation. However, no HF trial has yet shown ESA benefit on mortality. Furthermore, the long-term safety of treatment is not proven. This is important in view of adverse cardiovascular effects (thrombotic risk and hypertension) associated with high haemoglobin levels after ESA treatment in chronic kidney disease (CKD) patients. Most recently, the TREAT trial in CKD patients with diabetes reported that treatment with darbepoetin (target haemoglobin 13g/dl) was associated with increased risk of stroke [START_REF] Pfeffer | for the TREAT investigators. A trial of darbepoetin alfa in type 2 diabetes and chronic kidney disease[END_REF]. The risk of cardiovascular side effects has led to guideline recommendations not to aim for normalisation of haemoglobin levels when using ESAs in CKD [START_REF]Anaemia management in people with chronic kidney disease[END_REF][START_REF]Clinical practice guidelines[END_REF].

Meta-analysis of the HF trials reported no increased mortality or adverse events with ESAs (10) but the experience in CKD has led to caution among cardiologists and use of ESAs in HF anaemia (an unlicensed indication in Europe) has declined. Ongoing trials, including the large RED-HF trial of darbepoetin, which has all-cause mortality or HF hospitalisation as its primary endpoint, should provide useful data.

Meanwhile, the evidence base is moving on and there is increasing interest in the idea of iron replacement on its own for correcting anaemia in HF, with data suggesting that using IV iron alone to increase haemoglobin levels can improve cardiac and renal function. Some HF studies have also investigated iron replacement in patients with iron deficiency in the absence of anaemia.

The iron replacement studies in HF anaemia have used IV iron. Oral iron is often poorly tolerated and so may not be taken reliably; in addition, it may not increase iron stores because of hepcidin-induced reduction in duodenal iron absorption (4) as well as malabsorption associated with gut oedema. IV iron is more effective and there are no compliance issues.

Several small studies assessing this new strategy in HF anaemia suggested that treatment can increase haemoglobin, improve exercise capacity and symptoms [START_REF] Bolger | Intravenous iron alone for the treatment of anemia in patients with chronic heart failure[END_REF], reduce NTproBNP level and inflammatory status (C-reactive protein level) and reduce hospitalisations [START_REF] Toblli | Intravenous iron reduces NT-probrain natriuretic peptide in anemic patients with chronic heart failure and renal insufficiency[END_REF]. In the FERRIC-HF trial [START_REF] Okonko | Effect of intravenous iron sucrose on exercise tolerance in anemic and nonanemic patients with symptomatic chronic heart failure and iron deficiency[END_REF], in which IV iron was given to HF patients with iron deficiency with or without anaemia, treatment was associated with improved exercise capacity (mean of 60s greater treadmill duration, P=0.08, and 2.2ml/kg/min of peak oxygen consumption, P=0.01) and was well tolerated.

The largest iron study to date is the FAIR-HF trial [START_REF] Anker | for the FAIR-HF trial investigators. Ferric carboxymaltose in patients with heart failure and iron deficiency[END_REF] in which 459 patients with HF due to LV systolic dysfunction and iron deficiency (with or without anaemia) received IV iron or placebo. Active treatment was associated with improvement at week 24 in the Patient Global Assessment (odds ratio for improvement 2.5) and in the New York Heart Association functional class (odds ratio for improvement by one class 2.4). As with the ESA trials, none of the iron studies so far has had hard endpoints and further randomised studies are needed. IRON-HF is one ongoing trial of iron therapy that includes HF hospitalisations and mortality among the secondary endpoints [START_REF] Beck-Da-Silva | Rationale and design of the IRON-HF study: a randomized trial to assess the effects of iron supplementation in heart failure patients with anemia[END_REF].

There has in the past been concern about use of IV iron because of the history of anaphylaxis with iron dextran. The HF trials used newer non-dextran-containing formulations (iron sucrose or ferric carboxymaltose) with reduced risk of producing hypersensitivity reactions. Ferric carboxymaltose has the additional advantage that higher doses can be given: the total iron requirement can be given in one dose, avoiding the need for patients to make repeated visits for treatment. Also, unlike the other IV iron products, it does not require an initial test dose.

At our centre we have audited our use of IV iron (ferric carboxymaltose) in patients with heart failure and anaemia. In a 12 month period we have given single dose IV iron replacement in 30 patients, without any adverse effect. The patients had moderate to severe symptoms (mostly NYHA Class III) despite optimal medical management with ACE inhibitors, beta-blockers, and aldosterone antagonists and 40% had cardiac resynchronisation therapy also. Median Hb prior to IV iron therapy was 10.5 g/dl, with median estimated GFR of 44 ml/min/m 2 , serum iron 6.8 µmol/l (normal range: 12.6-26), ferritin 46 µg/l (normal range: 32-284), and transferrin saturation 9.5% (normal range: 20-45%). The change in mean Hb was 1g/dl after IV iron therapy, with no change in estimated GFR. Serum iron rose to 11.3 µmol/l, ferritin to 274 µg/l and transferrin saturation to 22%. [START_REF] Dickstein | ESC guidelines for the diagnosis and treatment of acute and chronic heart failure 2008[END_REF][START_REF] Hunt | focused update incorporated into the American College of Cardiology / American Heart Association 2005 guidelines for the diagnosis and management of heart failure in adults[END_REF] but anaemia is common in HF and its cause and correction should be considered. Specific causes should be investigated and iron status checked. In many cases anaemia is likely to be related to the cardio-renal-anaemia syndrome and treatment with IV iron can be considered. Clinical trials to date and practical experience indicate that with the modern formulations IV iron replacement is useful and well tolerated. However, we need trial data on clinical outcomes to assess the true value of treating HF anaemia, and further investigation of iron replacement in patients who are iron deficient but do not have anaemia.

Correction of anaemia is not yet established as routine therapy in HF guidelines

  -HF subanalysis suggests that treatment might also improve renal function(Ponikowski P. et al, reported at the 2010 Annual Scientific Meeting of the Heart Failure Association of the European Society of Cardiology, Berlin).

Acknowledgements: None

Page 7 of 10 International Journal of Clinical Practice International Journal of Clinical Practice

International Journal of Clinical Practice

International Journal of Clinical Practice