Erectile Dysfunction and NO: Variations of NO Penile Blood Levels Before and After Sildenafil Treatment

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ORIGINAL ARTICLE Erectile dysfunction and NO: variations of NO penile blood levels before and after sildenafil treatment

CA Palmerini1, A Zucchi2, F Fioretti2 and G Arienti1

1Laboratory of Biochemistry, Department of Internal Medicine, University of Perugia, Perugia, Italy and 2Department of Urology and Andrology, University of Perugia, Perugia, Italy

Erectile dysfunctions are not uncommon, especially in patients suffering from metabolic syndrome and from a number of circulatory and psychiatric problems. cGMP diesterase inhibitors, such as sildenafil, have proven to be beneficial in the treatment of many such conditions. Our patients, all of them complaining of erectile dysfunction, were treated with sildenafil (50 mg, thrice a week for 6 weeks). All patients reported beneficial effects and were not clinically distinguishable (interview and Doppler scores). We sampled blood for systemic circulation (cubital vein) and from penis (corpora cavernosa) before and after prolonged sildenafil treatment, and measured nitrate ( þ nitrite) levels in plasma and in red blood cells (RBCs). Hemoglobin is a powerful catalyst of NO oxidation to nitrate, and we thought that nitrate in RBC might be a more sensitive parameter than plasma nitrate. We found that the ratio of penile vs systemic blood plasma nitrate was similar in all patients before or after sildenafil treatment. On the other hand, the same parameter measured in RBC showed that, at the beginning of treatment, patients could be divided into two groups: one with a high ratio and the other with a low ratio. Therefore, clinically similar patients could be biochemically divided into two populations. The difference disappeared after treatment, thus hinting at a curative effect of the drug. The mechanisms underlying this behavior are still unknown and the clinical implication of two populations that can be distinguished by RBC nitrate is yet to be evaluated. International Journal of Impotence Research (2009) 21, 321–325; doi:10.1038/ijir.2009.33; published online 16 July 2009

Keywords: erectile dysfunction; nitrate; nitrite; nitric oxide; red blood cells; sildenafil

Introduction cyclase, thus determining the relaxation of cavernous and vascular smooth muscle and inducing Erection requires the intervention of neurological, erection. The mechanism of endothelial dysfunction vascular, hormonal and psychological factors. Erec- is multifactorial and the results so far published in tile dysfunction is usually associated with a number literature indicate that altered levels of NO may be of conditions: hypertension, hyperlipidemia, dia- important causal factors for this condition.4 betes, stress and cigarette smoke, all of which imply Oxidative stress has been considered to be a an increased risk of cardiovascular disease.1 possible cause for erectile dysfunction.4,5 It implies Endothelial-derived factors mediate the vasodila- an increase in the concentration of superoxide, tation necessary for erection to occur; among these, which rapidly reacts with NO forming peroxyni- NO is of chief importance.2 It acts through the trites, thus reducing NO availability. Peroxynitrites stimulation of cyclic GMP (cGMP) production. The show an elevated cytotoxicity6 because they can endothelium of lacunar spaces and the intima of peroxidate membrane lipids, thereby inhibiting a penile arteries contribute to the local release of NO,3 number of regulatory enzymes. which diffuses into trabecular smooth muscle cells Peroxynitrites, similar to NO, relax vasal muscular and stimulates the synthesis of cGMP by guanylate cells, although they are less powerful than NO to this effect. The kinetics of relaxation are much different, as the action of peroxynitrites is slower Correspondence: Professor G Arienti, Department of than that of NO. Moreover, superoxide has a direct Internal medicine, Laboratory of Biochemistry, Via del 7 Giochetto, 06122 Perugia, Italy. vasoconstrictor effect and opposes the physiologi- E-mail: [email protected] cal erection mechanism. Endothelial cell apoptosis, Received 20 February 2009; revised 27 May 2009; caused by both peroxynitrites and superoxide, 8–10 accepted 27 May 2009; published online 16 July 2009 further removes NO. Erectile dysfunction and NO CA Palmerini et al 322 The therapy of erectile dysfunction includes 0.1 M CuSO4 and 50 mg of Cd to transform nitrate phosphodiesterase inhibitors (PDE-5), such as silde- into nitrite. After centrifugation at 5 000 g for 2 min, nafil and other compounds.11 Recent evidence12–15 the supernatant was injected into the reaction suggests that a long-term treatment with these drugs chamber of the system in which nitrite was may improve erectile function by influencing penile converted into NO after a reaction with cysteine and systemic vascular parameters (endothelial re- and CuCl2. Gaseous NO was then measured using habilitation), although no molecular mechanisms electrochemical assay. All these procedures are have been proposed for this action. described in detail by Palmerini et al.17 In this study, we evaluate NO metabolites (nitrates and nitrites) in cavernous and in peripheral red Approval of the ethics committee blood cells and plasma before and after a prolonged The experimental protocol described in this paper treatment with sildenafil, in patients affected by has been approved by the ethical committee. erectile dysfunction associated with hypertension and, in some instances, also with diabetes. We propose that a prolonged treatment with cGMP phosphodiesterase inhibitors may help normalize Results NO metabolism. The characteristics of the patients who participated in this study are presented in Table 1, which also Materials and methods reports nitrate ( þ nitrite) concentration in penile and systemic RBCs and plasma before and after Patients treatment with sildenafil. In this paper, we evaluate 20 patients, all of them All the patients (46–75-year-old) coming to our being affected by erectile dysfunction and hyperten- department had erectile problems. All of them were sion and seven being diabetic for 2 years. Erection affected by hypertension and seven were diabetic as was obtained by injecting 10 mg of prostaglandin E1 well. All patients reported advantages from sildena- (Caverjet, Pfizer, New York, USA) into the cavernous fil treatment as shown by the international index of bodies. Blood was then drawn in EDTA (5 ml from erectile function questionnaire. The beneficial ef- the penis and 5 ml from the peripheral blood) to fects were more evident as treatment time increased. assess plasma and erythrocyte nitrate. Patients were Eco-Doppler assessment gave normal results either successively treated for 6 weeks with sildenafil before or after treatment. (50 mg Â 3 times a week). The drug was taken in the To avoid variations due to basic nitrate or nitrite evening, before bedtime. After treatment, blood was concentrations in the blood, we compared periph- again sampled from all patients to determine nitrate, eral blood with cavernous blood samples during as reported below. erection and expressed the results as the ratio Patients were evaluated before and after sildenafil between RBC or plasma nitrate in penile vs systemic treatment using the international index of erectile blood, before and after treatment with sildenafil function questionnaire16 (questions 1–5 and 15), (50 mg, thrice a week for 6 weeks). and the circulatory status in the penis was evaluated Data referring to RBC nitrate are reported in by eco-Doppler assessment. Figure 1. Before treatment with sildenafil, the patients could be divided into two subgroups. One of them had a low penile/systemic nitrate ratio, whereas the Preparation of plasma and red blood cells other one presented a high ratio. Treatment with the Plasma and erythrocytes were separated by centri- drug, as described above, resulted in a decrease in the fugation of 5 ml of blood for 10 min at 1 500 g. ratio in the group of patients whose ratio was high The concentration of hemoglobin (Hb) in intact and an increase in this parameter in the patients in red blood cell (RBC) was expressed as the concen- whom it was low. In other words, prolonged tration of oxyHb released after lysis, achieved by treatment with sildenafil produced similar ratios pelleting the cells and suspending them in the same (about 1.15) in all the patients. volume of distilled water. OxyHb was determined On the other hand, no similar results were found in by measuring light absorbance at 415 nm. Lysed the plasma, in which the penile/systemic nitrate ratio RBCs were freed of Hb (which would have interfered did not seem to be affected by sildenafil. Figure 2 with nitrate determination) by boiling at 100 1C for shows that plasma nitrate ratios were all similar 2 min. Samples were then centrifuged at 5 000 g for before treatment and were not sensitive to sildenafil. 10 min. The clear supernatant was collected and used for nitrite/nitrate determination. Discussion Nitrite/nitrate determination Plasma (50 ml) or the Hb-free samples prepared as NO, in the presence of oxygen and water, presents a described above (50 ml) were mixed with 200 mlof very short half-life, which makes its direct

International Journal of Impotence Research Erectile dysfunction and NO CA Palmerini et al 323

Table 1 Age, pathological condition, smoking habit and nitrate concentrations in penile and systemic blood of patients treated with sildenafil

Patient Age Pathology Smoker Red blood cells (nitrate mol mgÀ1 Hb) Blood plasma (nitrate mmol lÀ1)

PP PS SP SS PP/SP PS/SS PP PS SP SS PP/SP PS/SS

1 48 Hypertension No 1.4 1.6 1.2 1.3 1.17 1.23 23 22 21 20 1.10 1.10 2 60 Hypertension Yes 1.9 2.0 1.5 1.8 1.27 1.11 23 19 34 24 0.68 0.79 3 57 Hypertension No 2.1 2.5 1.1 2.1 1.31 1.19 33 27 30 30 1.10 0.90 4 58 Hypertension No 1.9 2.1 1.4 2.0 1.36 1.05 18 12 21 20 0.86 0.60 5 57 Hypertension No 1.9 2.4 1.3 2.0 1.46 1.20 21 19 20 16 1.03 1.14 6 70 Hypertension No 1.8 2.0 1.2 1.9 1.50 1.05 30 29 23 22 1.30 1.32 7 57 Hyypertension No 1.8 2.1 1.3 2.0 1.38 1.05 23 33 28 32 0.82 1.03 8 51 Hyypertension No 2.7 1.8 1.9 1.5 1.42 1.20 26 21 37 24 0.70 0.88 9 60 Hyypertension Yes 3.0 1.4 2.0 1.3 1.50 1.08 37 33 28 32 1.32 1.03 10 48 Hyypertension No 2.2 1.6 1.8 1.2 1.22 1.33 47 48 33 30 1.42 1.60 11 69 Hypertension Diabetes type II No 2.3 2.4 2.6 2.0 0.88 1.20 18 16 25 15 0.72 1.07 12 75 Hyypertension No 1.2 2.2 1.8 1.7 0.67 1.29 24 30 23 28 1.04 1.07 13 49 Hyypertension No 1.4 1.7 2.6 1.6 0.54 1.06 33 32 32 31 1.03 1.03 14 57 Hypertension Diabetes type II No 2.0 1.8 3.1 1.6 0.65 1.13 24 27 25 22 0.96 1.23 15 62 Hyypertension No 1.9 2.6 2.0 1.8 0.95 1.44 23 20 20 22 1.15 0.91 16 60 Hypertension Diabetes type II No 1.5 2.1 2.6 1.8 0.58 1.17 18 19 17 18 1.06 1.06 17 58 Hypertension Diabetes type II No 1.2 1.6 1.8 1.5 0.67 1.07 43 40 43 41 1.00 0.98 18 51 Hypertension Diabetes type II No 1.3 1.8 2.0 1.7 0.65 1.06 31 35 29 40 1.07 0.88 19 60 Hypertension Diabetes type II No 1.4 1.9 2.1 1.8 0.67 1.06 35 35 27 33 1.30 1.06 20 46 Hypertension Diabetes type II No 1.3 2.2 2.0 2.1 0.65 1.05 29 27 23 24 1.26 1.13

Abbreviations: Hb, hemoglobin; PP, penile blood before treatment; PS, penile blood after the treatment with sildenafil; SP, systemic blood before treatment; SS, systemic blood after the treatment with sildenafil. The treatment with sildenafil was performed during 6 weeks (50 mg thrice a week).

determination in biological samples impossible. NO rapidly transforms into nitrite by auto-oxidation,9 and, in the presence of oxyHb,18,19 rapidly converts into nitrate. The concentration of nitrate in the blood is usually several orders of magnitude higher that than of nitrite. For this reason, the contribution of nitrite in the variation of nitrate reported in this paper is negligible.20 The detection electrochemical system used in this study and previously described by us17 is much more sensitive that the commonly used Griess method.21 We measure NO, nitrite and nitrate after conversion of nitrite and nitrate to gaseous NO in the detection system. The origin of nitrate in the human body is not only limited to NO metabolism but may also depend on other metabolic and nutritional processes.20,21 For this reason, we compare the levels of plasma and Figure 1 Ratios of penile vs systemic red blood cell (RBC) nitrate RBC nitrates in the cavernous blood and systemic content before (squares) and after (circles) sildenafil treatment. blood of our patients. Penile blood RBC nitrate (as nmol per mg of hemoglobin) was The determination of nitrates in plasma (Figure 2) 1.81±0.48 before treatment and 1.99±0.32 after treatment. did not show any variations before or after sildenafil Systemic blood values were 1.89±0.51 and 1.74±0.27, respec- treatment. This finding may be in agreement with tively. Statistics: Student’s t-test between the ratios, before and 22 after treatment, was not significant. Patients were divided into previously reported results, in which the authors two groups: one had a low pre-treatment ratio and the other a high demonstrated that the amount of NO metabolites pre-treatment ratio. Analyses of variance were carried out and does not change after penis erection in cavernous post-hoc comparison (Scheffe´,atP ¼ 0.05) was singled out in three groups of means: low pre-treatment ratio: 0.69, high pre- blood with respect to systemic blood. treatment ratio 1.32 and post-treatment ratio: 1.15, in both groups It has been reported that chronic treatment with 12,13,15,23 of patients. sildenafil has a long-term curative effect.

International Journal of Impotence Research Erectile dysfunction and NO CA Palmerini et al 324 Conflict of interest

The authors declare no conflict of interest.

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