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Technical Briefs |
1
Clinical Biochemistry
4
Urology,
2
Obstetrics and Gynecology, Hospital "Santa Barbara", 13500-Puertollano (Ciudad Real), Spain;
3
Department of Physiology, University of Murcia School of Medicine, 30071 Murcia, Spain;
In recent years, there has been a growing interest in the role of oxygen toxicity and free radical (FR) reactions in fertility potential (1)(2). The generation of reactive oxygen species (ROSs) can produce peroxidative damage to cell membranes, and lipid peroxidation triggers the loss of membrane integrity (1). Because polyunsaturated fatty acids and phospholipids of the human spermatozoa are highly susceptible to peroxidation, FRs generated by spermatozoa may be involved in the production of spermicidal cytotoxic end products. The production of abnormal concentrations of ROSs is now thought to be engaged in many aspects of human male infertility, in which spermatozoa are rendered dysfunctional by lipid peroxidation and altered membrane function, morphology, and motility (3). Both seminal plasma and spermatozoa contain several antioxidant factors; however, these factors may be insufficient to prevent lipid peroxidation. The role of lipid peroxidation in FR damage to human sperm has been documented (1)(2)(3). These studies present evidence indicating that human spermatazoa are capable of producing FRs and that loss of sperm function in certain cases of male infertility is associated with excessive activity of this FR generation system (2). Because direct detection of FRs in vivo is difficult because of their high reactivity and short life, increases in lipid peroxides and/or decreases in antioxidant capacity have been used as indirect evidence of the involvement of FRs and as measures of the degree of oxidative stress (2)(4)(5).
Nitric oxide (NO) is a pluripotential molecule that acts as both an autocrine and paracrine mediator of homeostasis, and derangement of its metabolism can be linked with many pathophysiological events (6). Under conditions of oxidative stress, NO rapidly reacts with the FR superoxide anion (O2-) to form an peroxynitrite anion (OONO-). At physiological pH, OONO- decomposes into intermediates with reactivities similar to the hydroxyl radical (OH-) and NO (7). The reaction of OONO- with biological substrates is known to include the oxidation of reduced thiol (-SH) groups (8). These interactions may contribute to cell injury, and therefore, measurements of the end metabolic products of NO and -SH may be relevant to better understanding the role of oxidative stress in sperm motility. Because of the importance of these indicator measurements, we undertook the current study to provide reliable reference intervals for human seminal plasma.
With the consent of our hospital's Clinical Research Committee we analyzed ejaculates from 100 adult subjects with normophysiological spermiograms (9). None of the men had signs of genital tract inflammation, such as positive culture or signs of germ-free tract inflammation, varicocele, or cryptorchidism, and all were selected for absence of known organic disease and were carefully screened for infectious, malignant, and other serious disorders. The mean ages of the study participants was 34 years (range, 22–46 years), and all had similar life-styles and dietary habits.
Semen samples were collected in the laboratory by masturbation after 3 or 4 days of sexual abstinence, and semen analyses were performed after complete liquefaction at 37 °C. Sperm count, motility, morphology, and viability were analyzed by standard methods (9). The initial leukocyte concentration was <25 x 10/L in all samples; therefore, the FR contribution of neutrophils was very small (9). After centrifugation (2500g) for 10 min in a centrifuge cooled to 4 °C, seminal plasma was removed carefully. During the assay period, samples were stored at -80 °C until analysis (usually within 30 days) in trace element-free tubes to maintain the stability of the seminal plasma samples and to preclude any in vitro lipid peroxidation.
Seminal plasma samples were analyzed in duplicate, and the absorbance was measured on a microplate reader (BioWhittaker Microplate Reader 2001). Interassay precision was determined using 20 assays over a 30-day period of seminal plasma from a control subject frozen at -80 °C and thawed immediately before each analysis. Statistical analysis (mean, standard deviation, and Kolmogorov–Smirnov test) was carried out with the SPSS statistical package (SPSS Inc.). The results of lipoperoxides (LPOs), NO, -SH, and total antioxidant status were expressed as nmol/10 spermatozoa.
LPOs were analyzed using the LPO-586 assay (Bioxytech®, OXIS International; interassay precision, 4.98%). This assay is based on the reaction of a chromogenic reagent (N-methyl-2-phenylindole in acetonitrile), with LPOs at 45 °C. One molecule of LPO reacts with two molecules of N-methyl-2-phenylindole in acid medium (methanesulfonic acid) to yield a stable chromophore with maximal absorbance at 586 nm. When the sample is mixed with reagents, most of the proteins precipitate, and the LPOs are simultaneously extracted. After sample homogenization and centrifugation, LPOs were measured as described above (10).
The total antioxidant status was measured using a kit supplied by Randox Laboratories® (interassay precision, 4.63%). In this assay, metmyoglobin reacts with H2O2 to form the radical species ferrylmyoglobin. A chromogen (2,2'-azinobis-[ethylbenzthiazolinesulfonic acid]; ABTS®) is incubated with the ferrylmyoglobin to produce the radical cation species ABTS®, which has a relatively stable blue-green color measured at 600 nm. Antioxidants in the added sample cause suppression of this color production to a degree that is proportional to their concentrations (11).
The -SH (protein-bound and free content in plasma) was estimated spectrophotometrically by the method of Sedlak and Lindsay (12), which is based on that of Ellman reagent [5,5'-dithiobis-(2-nitrobenzoic acid); DTNB], and the interassay precision was 4.56%. DTNB and reduced glutathione were obtained from Sigma Chemical Co. The NO end products in vivo are nitrite and nitrate, which are analyzed by the Nitric Oxide Colorimetric Assay® (Boehringer Mannheim; interassay precision, 4.96%). In this assay, the nitrate present in the sample is reduced to nitrite by reduced NADPH in the presence of nitrate reductase and quantitated colorimetrically after the reaction with the Griess reagent (13).
The group of subjects studied here was a representative sample of the
reference population, according to the IFCC guidelines (14).
The distribution of LPO, total antioxidant status, -SH, and NO end
product values in human seminal plasma sperm followed a gaussian
frequency distribution, as verified by the Kolmogorov–Smirnov test.
Aberrant values were excluded according to the IFCC guidelines
(14). Semen characteristics and reference intervals
expressed as mean ± 2 SD are given in Table 1
.
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The incidence of ROS formation by spermatozoa has not yet been established (3). The plasma membrane of human sperm is highly susceptible to lipid peroxidation because of its high content of polyunsaturated fatty acids. Therefore, the massive ROS production by spermatozoa can cause oxidative changes to the membranes, including a loss of membrane integrity and fluidity (1). These factors, together with the capacity of these cells to generate FRs, render them particularly susceptible to oxidative stress. High concentrations of malondialdehyde production in fertile males who have low fertilization rates have been demonstrated (2)(3). In addition, the reduction of these concentrations, using antioxidant therapy, correlated with the improvement of fertilization rates (3). In conclusion, these indicators can be used for the routine monitoring lipid peroxidation and oxidative stress in human seminal plasma sperm and as biochemical indices for sperm quality.
Acknowledgments
This work was supported by Fondo de Investigaciones Sanitarias (Madrid, Spain; Grants: FIS 96/1631 and 98/0606).
Footnotes
* address correspondence to this author at: C/Ctra. de Almodovar, 17, 2° E, 13500-Puertollano (Ciudad Real), Spain
References
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