International Journal of Impotence Research (2005) 17, 19–22 & 2005 Nature Publishing Group All rights reserved 0955-9930/05 $30.00 www.nature.com/ijir
Comparison of oxidative/antioxidative status of penile corpus cavernosum blood and peripheral venous blood E Yeni1*, M Gulum1, S Selek2, O Erel2, D Unal1, A Verit1 and M Savas1 1 Department of Urology, Faculty of Medicine, Harran University, Sanliurfa, Turkey; and 2Department of Biochemistry, Faculty of Medicine, Harran University, Sanliurfa, Turkey
The aim of the study is to determine and to compare the oxidative and antioxidative status of penile corpus cavernosum and peripheral venous blood. A total of 28 adult healthy males were included in the study. Whole blood was simultaneously withdrawn from penile corpus cavernosum and the cubital vein and their plasma separated. Total antioxidant capacity (TAC), vitamin C, total protein, albumin, uric acid, bilirubin and total peroxide (TP) levels of both plasma samples were measured and compared. While TAC, total protein, albumin, bilirubin and uric acid levels were higher, vitamin C levels were lower in cavernosal blood than that of peripheral blood. On the other hand, TP level was found to be higher in penile blood samples than that of peripheral blood. We thought that the normal erectile process of the penile cavernosal body leads to increased production of oxidants as in the mechanism of ischaemia–reperfusion; however, the increase of TAC can prevent development of oxidative injury. International Journal of Impotence Research (2005) 17, 19–22. doi:10.1038/sj.ijir.3901262 Published online 14 October 2004 Keywords: cavernosal blood; oxidative stress; peripheral blood; reactive oxygen species; total antioxidant capacity; total peroxide
Introduction Reactive oxygen species (ROS) such as superoxide radical anion (O2 ), hydroxyl radical and hydrogen peroxide are produced in metabolic and physiological processes, and under certain conditions harmful oxidative reactions may occur in organisms.1 Oxidative injury of ROS is prevented by exogenous antioxidants such as vitamins E and C, and also by endogenous antioxidants, such as scavenger enzymes (superoxide dismutase, catalase and glutathione peroxidase), bilirubin and uric acid. Under some conditions, increases in oxidants and decreases in antioxidants cannot be prevented, and the oxidative/antioxidative balance shifts towards the oxidative status. Consequently, oxidative stress, which has been implicated in over 100 disorders, develops.1,2 The human penile corpus cavernosum consists mainly of endothelial and smooth muscle cells; it K
*Correspondence: E Yeni, Department of Urology, Faculty of Medicine, Harran University, 63100 Sanliurfa, Turkey. E-mail:
[email protected] Received 27 January 2004; revised 18 June 2004; accepted 12 August 2004
can be regarded as a compartment comparable to the vascular system.3,4 As similar for other vascular tissues of the body, the oxidative/antioxidative system is active in the penile structure, and cavernosal blood levels of oxidant/antioxidant elements can be an indicator of cavernosal tissue metabolic status and function.2,5 Evidences from basic scientific studies indicate that oxidative stress mediated through O2 and other ROS may have a role in the impaired cavernosal function such as erectile dysfunction (ED),2,4,6,7 ischemic priapism8 and peyronie disease.2,9 To the best of our knowledge, there is no report comparing oxidative/antioxidative status of samples of cavernosal and peripheral venous blood. In this study we determined and compared oxidative and antioxidative status of penile cavernosal blood and peripheral venous blood in healthy men. K
Materials and methods Subjects A total of 28 volunteer adult healthy males were included in the study. All participants were
Oxidative/antioxidative status of cavernosal blood E Yeni et al 20
comprehensively evaluated using medical history, sexual score with the International Index of Erectile Function, physical examination and blood analysis. The participants who had erectile dysfunction, diabetes mellitus, hypertension, hypercholesterolaemia, smoking and intake of multivitamin drugs in the previous 2 weeks were not included in the study. Participants were asked to fast for 10–14 h before blood sampling.
Blood withdrawal and analysis The participants were placed in a supine position and blood samples were withdrawn from a cubital vein and the cavernos body into heparinised tubes. Obtained blood samples were immediately stored on ice and centrifuged at 41C, 3000 rpm, for 10 min. The plasma was separated and stored at 801C until analysis. Total antioxidant capacity (TAC), vitamin C, total protein, albumin, uric acid, bilirubin and total peroxide (TP) levels of both plasma samples were measured at the same time.
added with stirring to make the final working reagent (250 mM ammonium ferrous sulphate, 100 mM xylenol orange, 25 mM H2SO4 and 4 mM BHT in 90% v/v methanol in a final volume of 100 ml). The blank reagent contained all the components of the solution except ferrous sulphate. Aliquots (200 ml) of plasma were mixed with 1800 ml FOX2 reagent. After incubation at room temperature for 30 min, the vials were centrifuged at 12 000 g for 10 min. Absorbance of the supernatant was then determined at 560 nm. TP content of plasma samples was determined as a function of the absorbance difference between test and blank tubes using a solution of H2O2 as standard. The coefficient of variation for individual plasma samples was less than 5%.
Measurement of individual antioxidants Serum total protein, albumin, uric acid and bilirubin levels were measured by using commercial kits (Abbott). Vitamin C concentration was measured by using FRASC method.11
Measurement of TAC TAC of plasma was measured using the ferric reducing/antioxidant power (FRAP) assay, developed by Benzie and Strain.10,11 Briefly, 1.5 ml of working prewarmed 371C FRAP reagent (10 v 300 mM acetate buffer, pH 3.6 þ 1 v 10 mM 2,4,6tripyridyl-S-triazine in 40 mM HCl þ 1 v 20 mM FeCl3) was vortex mixed with 50 ml test sample and standards. The test was performed at 371C. Absorbance at 593 nm was read against a reagent blank at a predetermined time (0–4 min) after sample–reagent mixing. Trolox was used as standard in the assay and the results were expressed as mmol Trolox equiv./l.12
Measurement of TP level TP concentrations were determined using the ‘FOX2’ method13 with minor modifications.14 The FOX2 test system is based on oxidation of ferrous ion to ferric ion by various types of peroxides contained within the plasma samples, to produce a colored ferric–xylenol orange complex whose absorbance can be measured. The FOX2 reagent was prepared by dissolving ammonium ferrous sulphate (9.8 mg) in 250 mM H2SO4 (10 ml), to give a final concentration of 250 mM ferrous ion in acid. This solution was then added to 90 ml of HPLC-grade methanol containing 79.2 mg butylated hydroxytoluene (BHT). Finally, 7.6 mg xylenol orange was International Journal of Impotence Research
Statistical analysis Statistical analysis of the data was carried out with the Statistical Package for the Social Sciences, version 11.5, for Windows (SPSS, Inc). To compare the peripheral and cavernos blood samples, the Student’s t-test for paired samples was applied, assuming a 95% confidence interval. Bivariate associations between continuous variables were assessed by the Pearson correlation test. A P-value less than 0.05 was considered statistically significant.
Results The results are given as mean7standard deviation of mean. The age distribution of the patients was in the range 21–72 y, and the mean was 49713 y. As seen in Table 1, TAC, total protein, albumin, bilirubin and uric acid levels were higher, and vitamin C levels were lower in cavernosal blood than that of peripheral blood. The TP level was found to be higher in penile blood samples than that of peripheral blood. As seen in Table 2, there were significant correlations between the samples of cavernosal and peripheral blood for TAC, albumin and uric acid parameters.
Oxidative/antioxidative status of cavernosal blood E Yeni et al 21
Table 1 comparison of blood samples of cavernosal body and cubital vein for oxidative and antioxidative parameters Cavernosal
Total peroxide (mmol H2O2/l) TAC (mmol Trolox equiv./l) Total protein (g/dl) Albumin (g/dl) Total bilirubin (mg/dl) Uric acid (mg/dl) Vitamin C (mmol/l)
Cubital vein
Mean
7s.d.
Mean
7s.d.
P
0.06 2.09 7.75 4.42 0.60 7.27 37.47
0.02 0.18 0.56 0.28 0.28 1.49 10.61
0.04 1.89 6.97 4.40 0.39 5.55 42.14
0.01 0.10 0.45 0.23 0.13 0.86 4.50
0.001 0.001 0.001 0.07 0.004 0.001 0.041
Table 2 Relationships between blood samples of cavernosal body and cubital vein for oxidative and antioxidative parameters TP P TAC Albumin Bilirubin Uric acid Vitamin C
r P r P r P r P r P r P
TAC
Albumin
Bilirubin
Uric acid
Vitamin C
0.128 0.591 0.553 0.004 0.683 o0.001 0.167 0.425 0.478 0.012 0.001 0.995
r ¼ coefficient of correlation; p ¼ significance. TAC ¼ total antioxidant capacity, TP ¼ total peroxide.
Discussion Penile erection is a haemodynamic process, involving increased arterial inflow and restricted venous outflow, coordinated with corpus cavernosum and penile arterial smooth muscle relaxation.15 Sympathetic contractile factors maintain flaccidity while parasympathetic factors induce smooth muscle relaxation and erection. It is generally accepted that nitric oxide (NO) is the principal agent responsible for relaxation of penile smooth muscle.16 NO is derived from two principal sources: directly from nonadrenergic noncholinergic parasympathetic nerves and indirectly from the endothelium lining cavernosal sinusoids and blood vessels in response to cholinergic stimulation.17,18 The erection process can be divided into the following six phases: flaccid, filling, tumescent, full erection, rigid erection and detumescent. During the rigid erection phase, almost no blood flows through the cavernos artery; however, the short duration prevents the development of ischaemia or tissue damage.15 Hydrogen peroxide and other derivatives of peroxides are produced during the physiological
processes and they increase in some conditions.19 With the restarting of arterial flow after the cessation in penile cavernosal artery, multiple ROS may be generated owing to sequential, univalent reductions of molecular oxygen to superoxide anion, hydrogen peroxide and hydroxyl radical during the normal erectile process. In this study, we found that the TP level was significantly higher in penile cavernosal blood than that of peripheral venous blood, as seen in Table 1. According to the reported studies, our result is the first report to be published in this area. Plasma comprises various antioxidant components to prevent free radical injury. The fastest and the most potent antioxidant reactant among them is vitamin C.20 In this study, we determined that vitamin C concentration of penile blood was lower than that of cubital vein blood. The decrease of vitamin C concentration may be due to the reactive response of vitamin C against the increasing TP levels in penile blood. This result is also another first report to be published in this area. Other major individual antioxidant components of plasma are albumin, uric acid and bilirubin. Albumin constitutes the main antioxidant component of serum. Uric acid serves as a potent antioxidant by radical scavenging and reducing International Journal of Impotence Research
Oxidative/antioxidative status of cavernosal blood E Yeni et al 22
activities. Its concentration in plasma is almost 10-fold higher than vitamin C. Bilirubin is a strong physiological antioxidant that may provide important protection against atherosclerosis, coronary artery disease and inflammation.21 We found that all mentioned antioxidant components were higher in cavernosal blood than those of peripheral blood. The increment may possibly be due to stasis of blood and haemoconcentration in the penile cavernosal structure. TAC of plasma mainly arises from albumin, uric acid, vitamin C and bilirubin. In this study, we found that TAC of penile cavernosal blood is higher than that of peripheral blood. According to the reported studies, that is the first result that compares TAC of penile cavernosal and peripheral venous blood samples of the same individuals. We found significant correlations between the blood samples of the penile cavernosal body and peripheral vein for TAC, albumin and uric acid parameters as seen in Table 2. According to these correlations, it can be said that plasma TAC of peripheral blood may reflect the status of TAC of penile cavernosal blood.
Conclusion According to our results, we concluded that the physiological erectile process is similar to the ischaemia–reperfusion mechanism. Thus, excessive generation of ROS occurs in cavernosal body; however, increased antioxidants can overwhelm this oxidative status in physiological conditions. We also thought that an imbalance of oxidative/ antioxidative status leads to an oxidative injury, which may have a role in the ethiopathogenesis of ED.
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International Journal of Impotence Research
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