Introduction

Penile erection is a neurovascular event that requires dilatation of cavernosal arteries, relaxation of smooth muscle, increased intracavernosal blood flow and normal veno-occlusive function.¹ Although laboratory-based diagnostic procedures such as cavernosometry, nocturnal penile tumescence testing and penile blood flow studies are available for diagnosing erectile dysfunction (ED), sexual questionnaires have gradually replaced them with the definition of 'patient-centered evaluation' by Lue et al.^{2, 3} However, this widely accepted approach can be insufficient in some patients and more specialized diagnostic procedures might be necessary. The penile color Doppler ultrasonography is the most reliable and least invasive evidence-based assessment of ED in such cases.² However, it is costly and its diagnosis of a physical disorder associated with cavernous venous occlusion dysfunction is lacking.⁴

With respect to the aforementioned facts, there is a need for a universally accepted gold standard technique for evaluating patients' erectile capacity, which is not subjective or patient dependent as self-administered questionnaires and expensive or sonographer dependent as Doppler/duplex ultrasound. Thus, the aim of this study was to look into a molecule in penile blood sample that shows correlation with the tumescence grade and/or penile Doppler ultrasound parameters.

Materials and methods

Patients evaluation

After receiving the approval of local ethical committee, a total of 58 patients complaining of ED were evaluated. A detailed psychosexual history was taken and physical examination was given to all the patients. Patients with history of phosphodiesterase inhibitor usage or genital trauma, genital abnormalities, Peyronie's disease, endocrinopathies (including diabetes mellitus), cardiovascular diseases and major psychiatric or neurological disorders were excluded.

Those patients were also evaluated with a penile Doppler ultrasound (LOGIQ 9; General Electric Company, Fairfield, CT, USA), 10–20 min after intracavernosal injection of 60 mg of papaverine hydrochloride. Patients were instructed to do manual stimulation and no visual stimulation equipment was provided. Mean peak-systolic velocity (PSV), end-diastolic velocity (EDV) and resistive index (RI) of cavernosal arteries were recorded. The grade of tumescence was estimated clinically (grades 0–4) by the same researcher (ECS). Grade 0 was no response after the injection, grade 1 was minimal tumescence and no rigidity, grade 2 was moderate tumescence and no rigidity, grade 3 was full tumescence and moderate rigidity, grade 4 was full rigidity. During screening period, patients with tumescence grades 0 and 1 were also excluded because of the difficulties and pain during cavernal blood sampling. Therefore, a total of 46 patients with tumescence grades 2–4 were prospectively enrolled into the study. Signed informed patient consent was obtained before this procedure.

Biochemical measurements

Approximately 10 cm³ of cavernosal blood samples were drawn after the penile Doppler ultrasound (approximately 15 min after the injection of papaverine) and stored at -80 °C until being analyzed. The levels of calcium (Ca²⁺), myeloperoxidase (MPO), malondialdehyde (MDA), nitrite, nitrate, vasoactive intestinal peptide (VIP) and cyclic guanosin monophosphate (cGMP) and the activity of superoxide dismutase (SOD) were measured.

MPO activity determination

The MPO activity was assayed by measuring the H₂O₂-dependent reduction of o-dianisidine. In its reduced form o-dianisidine has a brown color. This was measured spectrophotometrically at 410 nm. The results are given as U ml^-1. One unit of MPO activity was defined as the amount of enzyme caused absorbance change in 1 min at 410 nm and 37 °C.⁵

SOD activity determination

As an index of the antioxidative defense system, we measured the activities of plasma SOD. Total (Cu–Zn and Mn) SOD (EC 1.15.1.1) activity was determined according to the method of Sun et al.⁶ The principle of the method is based on the inhibition of nitroblue tetrazolium (NBT) reduction by the xanthine–xanthine oxidase system as a superoxide generator. Activity was assessed in the ethanol phase of the plasma after 1.0 ml ethanol/chloroform mixture (5/3, v/v) was added to the same volume of the sample and centrifuged. One unit of SOD was defined as the amount of enzyme causing 50% inhibition in the NBT reduction rate. SOD activity was also expressed as U ml^-1 for plasma.⁶

Measurement of plasma MDA levels

MDA, which is the end product of lipid peroxidation, was used for the assessment of superoxide radical production. MDA levels of plasma samples were determined according to the method of Yoshioka et al. In this method, MDA reacts with thiobarbituric acid reagent under acidic conditions to generate a pink-colored product and determined at 532 nm. The results were given as nmol ml^-1. Tetramethoxypropane (T9889; Sigma Chemical Co., St Louis, MO, USA) was used as an external standard.⁷

Measurement of plasma nitrate/nitrite (NO_x) levels

NO levels in plasma were determined spectrophotometrically, based on the reduction of NO₃^- to NO₂^- by VaCI₃. NO levels were measured by the Griess reaction. Sodium nitrite and nitrate solutions (1, 10, 50, 100 M) were used as standards. Plasma samples were deproteinized before assay. Samples were added to 96% cold ethanol (1/2, v/v) and then vortexed for 5 min. After incubation for 30 min at +4 °C, the mixture was centrifuged at 14 000 r.p.m. for 5 min and the supernatants were used for the Griess assay.⁸

Measurement of plasma VIP/cGMP/Ca²⁺ levels

Both VIP and cGMP levels were measured with enzyme immunoassay (Phoenix Pharmaceuticals Inc., Burlingame, CA, USA and Cayman Chemical, Ann Arbor, MI, USA, respectively) and the levels of Ca²⁺ were measured using Abbott-Aeroset autoanalyser (Toshiba, Chicago, IL, USA) with original kits.

Statistical analysis

The correlation analyses were performed with Spierman correlation analysis. The overall comparison of cGMP between tumescence groups was performed with Jonckheere–Terpstra test and subanalyses with Mann–Whitney U-test. Statistical significance level was set at 0.05. Data analyses were carried out using the SPSS 12.0 statistical package software (SPSS Inc., Chicago, IL, USA).

Results

A total of 46 ED patients with at least a moderate response to papaverine were included. The median age of the patients was 49.310.2 (range 24–67). The descriptive statistics of patients were given at Table 1. The numbers of the patients who had grades 2, 3 and 4 tumescence were 13 (28.3%), 10 (21.7%) and 23 (50.0%), respectively. We could not find any significant correlation between penile Doppler ultrasound parameters and any of penile blood Ca²⁺, MPO, MDA, nitrite, nitrate, VIP levels and SOD activity (Table 2). However, there was a significant negative correlation between cGMP and mean PSV (=-0.533, P=0.001), as well as with mean RI (=-0.468, P=0.005) (Figures 1 and 2). On the other hand, a positive but statistically insignificant correlation between cGMP and mean EDV was detected (=0.322, P=0.059) (Figure 3). Mean cGMP levels were 3.3470.694 pmol ml^-1 (2.295–4.685), 3.1930.669 pmol ml^-1 (2.165–4.094) and 2.7420.690 pmol ml^-1 (1.290–4.011) in grades 2, 3 and 4 tumescence groups, respectively, and the difference among mean cGMP levels of these groups were statistically significant (P=0.027) (Figure 4). In subanalyses, there was no significant difference between grades 2 and 3 or grades 3 and 4 in terms of cGMP levels (P=0.883, 0.118, respectively). However, there was a significant difference between grades 2 and 4 tumescence groups (P=0.039). In addition, the age of patients showed very weak and nonsignificant correlation with penile Doppler ultrasound and cavernous blood sample biochemical analyses (Table 3).

Figure 1.

Scatter plot of mean peak-systolic velocity (PSV) and cyclic guanosin monophosphate (cGMP) level.

Full figure and legend (39K)

Figure 2.

Scatter plot of mean resistive index (RI) and cyclic guanosin monophosphate (cGMP) level.

Full figure and legend (41K)

Figure 3.

Scatter plot of mean end-diastolic velocity (EDV) and cyclic guanosin monophosphate (cGMP) level.

Full figure and legend (38K)

Figure 4.

The mean level of cyclic guanosin monophosphate (cGMP) in different tumescence groups (bars represent standard deviations).

Full figure and legend (65K)

Table 1 - The sociodemographic characteristics of the patients.

Full table

Table 2 - The results of the penile color Doppler ultrasonography and penile blood sample analysis of the patients included.

Full table

Table 3 - Correlations between penile Doppler ultrasound parameters and cavernous blood biochemical analyses were shown with -values.

Full table

Discussion

Although many self-reported measures have been developed and initially used in the assessment of a patient with ED, they can be problematic especially in patients with higher age, lower income level and/or educational status.⁹ In addition, these questionnaires can be insufficient in the evaluation of the complex ED patients and more specialized diagnostic procedures might be necessary.

The penile blood flow study, which consists of combined intracavernousal injection and stimulation and blood flow measurement by duplex ultrasound, is the most reliable and least invasive evidence-based assessment of ED.² However, bias coming from the expertise of the sonographer; the high percentage of variations in penile arterial anatomy and the differences in PSV, EDV or RI values between each cavernous artery can potentially alter the interpretation of duplex sonography.^{10, 11} The high cost of this relatively invasive test and the risk of priapism after the required injection of vasoactive substances are also limiting the widespread usage of Doppler/duplex ultrasound in diagnosing ED.

In this study, we analyzed the blood sample taken from artificially erected penis with papaverine, to find a biomolecule that correlates with the tumescence grade and/or penile Doppler ultrasound parameters. Such a molecule should give an opinion about patients' erectile capacity, which is not subjective or patient depended as self-administered questionnaires and expensive or sonographer depended as Doppler/duplex ultrasound.

NO, derived from vascular endothelial and neural sources, plays an essential role in the early steps of the normal cascade of relaxation of the penile vasculature and cavernous smooth muscle, and its action is mediated through the cGMP.^{12, 13, 14} In respect to this well-known cascade, we analyzed the association between penile Doppler ultrasound parameters and NO metabolites (nitrite and nitrate), cGMP and Ca²⁺ levels in penile blood. In addition, the level of VIP, which is known as an neuromediator of penile erection, was determined for the same reason.¹⁵ Furthermore, we also looked into a possible correlation between ultrasound findings and MDA, MPO levels or SOD activity, because oxidative stress to cavernous tissue has been suggested to be a contributory factor in ED in spite of few data on how it contributes.^{16, 17}

We could not find any significant correlation between penile Doppler ultrasound parameters and nitrite, nitrate or VIP levels, supporting the previous studies performed to evaluate the significance of these molecules in male sexual function.^{14, 15} Penile blood Ca²⁺, MPO, MDA levels and the SOD activity were not significantly correlated with either penile Doppler ultrasound findings or tumescence grade, as well. However, the levels of cGMP, which is a second messenger involved in smooth muscle relaxation, was significantly associated with ultrasound findings. This molecule activates cGMP-depended protein kinases and phosphorylates certain proteins and ion channels resulting in a drop in cytosolic free Ca²⁺ and smooth muscle relaxation.¹⁸ We observed a significant negative correlation between cGMP and PSV, as well as RI, but a positive, statistically insignificant correlation between cGMP and EDV. Moreover, lower cGMP levels were observed in corporal blood samples from penises with higher tumescence grade.

As cGMP is known to be positively associated with the quality of erection and the rationale of using phosphodiestherase-5 (PDE5) inhibitors is to increase and not to decrease it, these results may be confusing and readers may expect higher penile cGMP levels in patients with better erection quality. However, cGMP was measured in penile blood coming from cavernosal sinuses, whereas this molecule plays its erectile role inside smooth muscle cells. Unfortunately, there is no data available in the English literature that can explain or support these findings. However, we might hypothesize that the increased level of cGMP measured outside smooth muscle cells in patients with lower erectile quality might be due to an increased leak of this molecule from those cell membranes, or decreased uptake of it by smooth muscle cells within the cavernous bodies. In addition, lack of blood circulation during full erection may result in decreased levels of cGMP, whereas continuing blood circulation during decreased erection can bring some cGMP into penile circulation from outside sources and may result in 'falsified' increase in cGMP levels.

Taking blood sample from cavernosal sinuses after the injection of papaverine may increase some concerns about the reliability of our findings, and some of the readers might think that the blood sample should have been taken during flaccidity, to reflect the actual biochemical situation without the effect of papaverine. Phosphodiesterases are the enzymes involved in cyclic nucleotide breakdown and an increase in cyclic adenosine monophosphate (cAMP) and/or cGMP levels can be induced by inhibition of these enzymes in the smooth muscle cells of corpus cavernosum with papaverine which is a nonspecific inhibitor of these enzymes.¹⁹ Because these enzymes do not perform their role in the cavernosal sinuses, the levels of cGMP measured from the blood circulating inside these sinuses should not be affected from the injection of this agent. Furthermore, in the screening period, we observed that taking cavernous blood sample is difficult and very painful for the patients when the penis is not erected and consequently, we excluded the patients with tumescence grades 0 and 1. However, further studies are required to ascertain that our findings can also be observed in men without papaverine injection.

As the statistically significant correlation between cGMP levels and penile Doppler ultrasound parameters and tumescence grade was observed after intracavernosal injection of papaverine, we believe that our preliminary findings should be taken into consideration by other researchers, and studies assessing the sensitivity and specificity of cGMP must be performed in comparison to a healthy male population. Synchronous cGMP measurement in cavernosal and systemic blood samples can also help in interpreting our data. In addition, the possible association between erectile capacity and the levels of Ca²⁺, VIP, MDA, nitrite, nitrate and the SOD activity might be demonstrated with larger trials. The penile blood levels of other molecules such as potassium, cAMP or acetylcholine, which take place in the mechanism of erection, can be studied for the same purpose.

We do not think that the undeniable importance of penile Doppler ultrasound in the evaluation of ED will decrease with the clinical extension of our findings. However, after setting the maximum level of cGMP in penile blood for a satisfying erectile quality with studies including more patients, this molecule can be useful in assuring the results of Doppler ultrasound. In addition, the correlation between penile blood cGMP level and the outcomes of patients receiving PDE5 inhibitors can be analyzed to determine whether penile cGMP level can help physicians to identify the patients likely to respond this treatment.

Conclusion

Penile blood cGMP level showed a significant negative correlation with mean PSV, RI values and tumescence grade, whereas there was a positive, statistically insignificant correlation between cGMP and mean EDV values. These findings suggest that penile blood cGMP level can potentially be considered as a marker to predict the erectile capacity. However, further studies are required to elucidate the exact mechanism of the association between lower cGMP levels with better erection quality.

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Acknowledgements

This study was partially supported by Novartis Pharmaceuticals, Oncology Group, Turkey. We thank Associate Professor Isa Ozbey, and Melike Erol Demirbilek, for their invaluable contribution to this study, and to our laboratory assistant Murat Sezgin for his generous efforts in helping us.