Rheumatology 2003; 42: 805-807
© 2003 British Society for Rheumatology
Letters to the Editor |
Antioxidant status and lipid peroxidation in seminal plasma and spermatozoa of patients with ankylosing spondylitis
1 Departments of Physical Medicine and Rehabilitation, Division of Rheumatology and
4 Urology, Firat University, Faculty of Medicine, Elazig and
2 Departments of Biochemistry and
3 Physiology, Inonu University, Faculty of Medicine, Malatya, Turkey
SIR, Ankylosing spondylitis (AS) is a chronic inflammatory disease that affects predominantly young male individuals [1]. Fertility and a normal sexual life may be interrupted with functional sequealae and disease-modifying anti-rheumatic drugs (DMARDs), especially sulphasalazine. This is one of the important DMARDs in the management of AS and a well known cause of drug-induced infertility [1, 2].
Reactive oxygen species (ROS) play an important role in the pathogenesis of inflammatory joint diseases like rheumatoid arthritis (RA) and AS, and we have previously reported increased malondialdehyde (MDA), xanthine oxidase (XO) and catalase (CAT) activity in the sera of patients with AS [3]. On the other hand, the effect of ROS and/or inadequate antioxidant enzyme activity on seminal fluid or spermatozoa in RA and AS is still unknown.
The aim of this study, therefore, was to investigate whether there is an effect of inflammation-induced antioxidants on the oxidant/antioxidant status of spermatozoa and seminal plasma in patients with AS who are not being treated with DMARDs.
Seven male patients (mean age 30.4±7.6 yr, range 18–40 yr), fulfilling the New York criteria for AS [4], and eight age-matched healthy subjects (mean age 27.6±6.7 yr, range 21–39 yr) were included in the study. None of the patients was receiving DMARDs, such as sulphasalazine, or had a history of infertility problems. Patients who smoked or used any other drugs such as polyunsaturated fatty acids (PUFA) or antidepressants (except NSAIDs informally used by the patients) that had known effects on oxidant/antioxidant metabolism were excluded. Controls were healthy, non-smoking hospital staff who volunteered to participate in the study and none had infertility problems. Four patients and four controls had fathered children.
Semen for analysis was obtained by masturbation after a 3–5-day period of sexual abstinence. A 0.5-ml aliquot of semen was submitted to a standard examination according to WHO criteria. Sperm count and motility parameters were measured.
Semen samples were centrifuged at 1000 g for 10 min at +10°C to separate spermatozoa and seminal plasma. Cell sediment was washed three times with 10-fold isotonic NaCl solution to remove remnants of seminal plasma. After each procedure, the sperm cell–saline mixture was centrifuged at 1000 g for 10 min at +4°C. Samples were stored at -80°C until analyses. After thawing, cell sediments were treated with 4-fold ice-cold deionized water, homogenized at 16 000 rpm for 1 min and centrifuged at 5000 g for 15 min to obtain a supernatant.
All the analyses were made in this supernatant phase and in the seminal plasma. CAT (EC1.11.1.6) activity was measured by the determination of the rate constant of hydrogen peroxide decomposition. Total (Cu-Zn and Mn) superoxide dismutase (SOD) (EC1.15.1.1) activity was determined by the modified method based on the inhibition of NBT reduction by the xanthine–xanthine oxidase system. Determination of MDA level was based on the coupling of MDA with thiobarbituric acid (TBA) at +95°C using a spectrofluorometer adjusted to excitation 525 nm and emission 547 nm. XO (EC1.2.3.2) activity was measured spectrophotometrically by the formation of uric acid from xanthine through the increase in absorbancy at 293 nm. Nitrite (NO2-) and nitrate (NO3-) were estimated as an index of NO production. Quantification of nitrate and nitrite was based on the Griess reaction using previously reported methods [5]. Protein determinations in the homogenates were made by Lowry's method [6].
All of the subjects also underwent colour Doppler sonography (CDS) examination of the testicular vessels to exclude subclinical varicocele or testicular pathologies.
Statistical analyses were performed using SPSS software. Differences among groups were compared using the Mann–Whitney U-test. A two-tailed P<0.05 was considered statistically significant.
Semen analyses were within normal limits in patients and controls according to the WHO reference values. Testicular CDS examinations of patients and controls were normal. Seminal plasma SOD activity of patients was significantly higher than that of controls (P<0.02).
Seminal plasma CAT activity, and MDA and NO levels were not significantly different between the patient and control group. Spermatozoa (cellular) CAT, SOD and XO activities, and NO levels were not significantly different between the groups (Table 1
).
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Previous studies have suggested that decreased seminal plasma antioxidant activity could be responsible for male infertility. Increased ROS levels and/or decreased seminal plasma ROS scavengers—antioxidant enzyme activity—in idiopathic infertility has been suggested as the cause of impairment in sperm function [7]. Mammalian spermatozoa membranes are rich in PUFA and are sensitive to oxygen-induced damage mediated by lipid peroxidation. Lipid peroxidation of sperm membrane or accumulation of products of lipid peroxidation has been considered to be the key mechanism of ROS-induced sperm damage, leading to loss of the germinating ability or infertility [7].
ROS in seminal fluid can be produced not only by spermatozoa but also by infiltrating leucocytes [7]. Although different results have been reported in various studies, Wendling et al. [8] and Biasi et al. [9] have shown an increase in the oxidative metabolism of the phagocyte system in AS after N-formylmethionyl-leucyl-phenylalanine (fMLP) stimulation. Results of a previous study also showed that the superoxide anion radical production was significantly higher in patients with AS than in healthy subjects, in the resting state or after stimulation with fMLP and phorbol-12-myristate-13-acetate [10]. In our study we were not able to perform a procedure such as Percoll washing to eliminate leucocyte contamination. Thus it is difficult to estimate the exact origin of increased SOD activity that we measured in seminal plasma of AS patients.
Consequently, our study demonstrates for the first time an increased SOD activity in seminal plasma of a small group of untreated AS patients. This may be considered as a reflection of increased oxidative stress mediated by the systemic inflammatory process and/or activated leucocytes infiltrating into the seminal fluid. Detailed studies with a larger series of patients are required.
Notes
Correspondence to: S. Ozgocmen, Universite mah., Zubeyde Hanim Cad. Untas apt. No:124 Daire:9, 23200 Elazig, Turkey. E-mail: sozgocmen{at}hotmail.com 
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