Vaskresenija Lipovac

NADH-dependent oxidoreductase (diaphorase) activity and isozyme pattern of sperm in infertile men.

Diaphorase activities were measured in spermatozoa from 104 infertile men. The relationships between enzyme activity, sperm characteristics (density, motility, viability), and distribution of electrophoretic patterns were examined. There was a highly significant difference (p less than .001) between the sperm diaphorase activities in specimens with sperm concentration above 40 million per milliliter (0.685 +/- 0.5 mIU/10(6) sperm; n = 57) when compared with the diaphorase values of the oligospermic specimens with sperm density below 40 million per milliliter (1.53 +/- 1.5 mIU/10(6) sperm; n = 47). However, there was no evidence to support any relationship between sperm diaphorase activities, sperm motility, viability, and the distribution of three diaphorase electrophoretic patterns. This indicates that these parameters do not account for the differences between the diaphorase activity of spermatozoa of infertile men with sperm concentrations below and those above 40 million per milliliter.

ANTIOXIDATIVE EFFECT OF MELATONIN ON HUMAN SPERMATOZOA

The ability of melatonin to suppress experimentally induced lipid peroxidation (LPO) in sperm membrane was investigated in 41 samples of infertile men. Iron/ascorbate (0.04/0.2 mmol)-induced LPO was measured by the formation of malondialdehyde (MDA) using the thiobarbituric acid method. Sperm incubated in the presence of melatonin (2-6 mmol) exhibited a concentration-dependent decrease of MDA generated from hydroperoxide of the sperm plasma membrane in the presence of promoter system. Addition of 6 mmol of melatonin significantly reduced the rate of lipid peroxidation in sperm of unselected donors (mean +/- SE in control samples = 26.4 +/- 2.9 vs. 6.5 +/- 1.1 nmol MDA/108 sperm in melatonin-treated samples; n = 16, p <. 005). Inhibitory effect of melatonin was also significant in the presence of 0.015 mmol of ferrous ions (20.5 +/- 1.7 vs. 7.9 +/- 1.6 nmol MDA/108 sperm in melatonintreated samples; n = 7, p <. 02) and 0.005 mmol of ferrous ions (20.2 +/- 2.8 vs. 9.9 +/- 2.4 nmol MDA/ 108 sperm; n = 6, p <. 05). Comparing the effect of melatonin with that of Trolox, an analog of vitamin E, a similar effect at concentration of 0.1-0.2 mmol of Trolox was found (25.2 +/- 2.9 vs. 11.8 +/- 1.2 nmol MDA/108 sperm in Trolox-treated samples; n = 7, p <. 005). The obtained data of in vitro experiments show that melatonin is 40-fold less efficient than Trolox in achieving the 50% reduction in LPO (4 vs. 0.1 mmol). Since the physiological concentration of melatonin in human semen is at the nanomolar level, its antioxidative role in vivo is probably of minor importance.

Relationship of sperm superoxide dismutase-like activity with other sperm-specific enzymes and experimentally induced lipid peroxidation in infertile men

Summary. Superoxide dismutase‐like activity (SOD‐like), isoenzyme lactate dehydrogenase‐C4 (LDH‐C4) and NADH‐diaphorase activities in spermatozoa have been investigated from 58 normozoospermic and 27 oligozoospermic men. Significantly higher SOD‐like, LDH‐C4 and diaphorase activities (P<0.01, P<0.005 and P<0.0001, respectively) were detected in spermatozoa from oligozoospermic men, compared to the activities found in normozoospermic samples. SOD‐like activity (mean±SE) in oligozoospermic samples amounted to 8.3±1.6 U 10−8 spermatozoa, while in spermatozoa in normozoospermic men with a sperm concentration above 20 million of spermatozoa per ml amounted to 4.2±0.5 U 10−8. There was a close correlation between the SOD‐like activity and biochemical indicators of the presence of residual cytoplasm i.e. isoenzyme LDH‐C4 and NADH‐diaphorase (r = 0.53 and r = 0.66 in normozoospermic and r = 0.63 and r = 0.54 in oligozoospermic men, respectively). A positive relationship between SOD‐like activity and experimentally‐induced lipid peroxidation was detected in 54 infertile men (r = 0.30; P<0.05). These findings suggest that a higher level of superoxide dismutase‐like activity may reflect a defect in the development or maturation of spermatozoa and, thereby, a decreased fertility potential. Hence, determination of SOD‐like activity may give information on the state of maturity of human spermatozoa, while its role in the antioxidative protection remains to be determined.

Pentoxifylline-mediated reduction of superoxide anion production by human spermatozoa

Summary. The release of superoxide anions from sperm suspensions prepared for in vitro fertilization was measured by superoxide dismutaseinhibitable reduction of ferricytochrome C in response to phorbol myristate acetate. The superoxide anion production was found to be within the range of 0.21‐3.76 nmoles O‐2 per 106 spermatozoa. Spermatozoa pre‐incubated in the presence of 3.7 or 10 mM of pentoxifylline for 30 min at 37 °C released less (P<0.001) superoxide anions than did the untreated‐control spermatozoa. It has been concluded that the inhibitory effect of pentoxifylline on superoxide anion production may be a factor contributing to the improvement of sperm preparation before insemination, and may, thereby, increase the fertility potential.

Evaluation of ascorbate and urate antioxidant capacity in human semen

Summary. The ascorbate/urate ratio in the seminal plasma was studied in 76 randomly chosen infertile men. The levels of ascorbate and urate were found to vary widely (range: 93–954 μmol 1−1 and 127–670 μmol 1−1, respectively), while the ascorbate/urate ratio was 1.03 ± 0.63 (mean ± SD), indicating almost equimolar concentrations of both compounds in more than 60% of the subjects investigated. No relationship of ascorbate with any biochemical marker of accessory sex gland secretions was observed, whereas an inverse correlation of urate with some prostatic markers, acid phosphatase (−0.37; P<0.001), zinc (−0.35; P<0.002) and citric acid (−0.33; P<0.003), was found. In vitro experiments were conducted on an artificial suspension containing ascorbate and urate at physiological levels and activated polymorphonuclear leukocytes in the normal range (as proposed by WHO) to determine the extent to which the presence of superoxide anion‐generating leukocytes contribute to the depletion of ascorbate and urate. The ascorbate level did not change in the presence of 0.2 × 106 leukocytes ml−1 while higher amounts of activated polymorphonuclear leukocytes initiated ascorbate oxidation, the intensity of which was in correlation with the extent of leukocyte contamination. After incubation (37 °C, 30 min) in the presence of 0.4, 1.0 and 1.5 × 106 cells ml−1, the average decline from the initial ascorbic acid level was 24, 43 and 49%, respectively. However, exposure of whole semen, instead of buffer, to oxidants released from the same amount of activated polymorphonuclears led to only 2.6, 11 and 22% decrease of the ascorbic acid, most probably due to the action of other superoxide anion scavenger compounds present in semen. Excessive superoxide anion production due to the presence of activated leukocytes had no influence on urate level either in the artificial suspension or in semen. The ability of ascorbate to afford protection against leukocyte‐associated superoxide anions in not hampered in the semen of infertile men, provided that leukocyte contamination does not exceed 1 × 106 cells ml−1. The possible role of urate in stabilizing the ascorbate antioxidant activity in seminal plasma should be further investigated.

Superoxide anion production and some sperm-specific enzyme activities in infertile men

Summary The relationship between superoxide anion production and the activities of sperm specific NADH‐diaphorase and isozyme LDH‐C4 has been investigated in 25 normozoospermic and 17 oligozoospermic semen samples. The samples were separated into high and low density sperm populations on discontinuous, two step (40%:80%) Percoll gradients prior to analysis. Superoxide anion generation was significantly higher (P<0.001) and diaphorase activity significantly increased (P<0.01) in both low and high density sperm fractions from oligozoospermic patients compared to the values found in normozoospermic men. A significant positive correlation (P<0.01) between diaphorase activity and superoxide anion production was established in the oligozoospermic samples of low density sperm fraction. A tendency of statistically insignificant increase in LDH‐C4 isozyme activity was observed in the oligozoospermic samples. The activity of this isozyme appears to have no association with superoxide anion generation or diaphorase activity. These findings seem to suggest that excessive superoxide anion production along with increased diaphorase activity point to biochemical abnormalities which may affect the functional status of sperms. Hence, determination of these parameters may provide useful data in male infertility of unknown etiology.

Serum paraoxonase and cholineseterase activities in individuals with lipid and glucose metabolism disorders

In patients with hyperlipaemia, serum paraoxonase activities were polymodally distributed with 75% individuals in the low activity mode. In the same patients the distribution of serum cholinesterase activities was unimodal, but asymmetrical. Patients with impaired glucose tolerance or non-insulin-dependent diabetes mellitus had slightly higher cholinesterase activities than patients with hyperlipaemia only.

Serum sialic acid in subjects with impaired glucose tolerance and in newly diagnosed type 2 diabetic patients

Abstract. Several general population studies and those carried out in diabetic patients with complications have pointed to serum sialic acid as a marker of inflammation in atherosclerosis. In this study we examined whether total sialic acid (TSA) was changed in the sera of 28 newly diagnosed subjects with type 2 diabetes (type 2 DM), 47 subjects with impaired glucose tolerance (IGT) and 72 subjects with normal glucose tolerance (NGT). The associations between sialic acid and other atherosclerotic risk factors such as lipid profile, baseline diene conjugates in low-density lipoproteins (LDL-BDC) and fasting insulin were also investigated. We found a trend to TSA increase in subjects with impaired glucose tolerance and a significant increase in TSA in newly diagnosed patients with type 2 DM (2.2±0.3 vs. 1.9±0.3 mmol/l; p<0.03) when compared to subjects with NGT. Lipid profile and LDL-BDC, as a marker of circulating oxidized LDL, did not differ among glucose tolerance categories. Significant associations between total sialic acid and 2-h post-load glucose level, fasting insulin, insulin sensitivity, HDL-cholesterol and log of triglycerides were found in the examined subjects. Multiple regression analysis showed significant correlations between serum sialic acid and 2-h post-load glucose levels and insulin sensitivity. This study indicates that measurement of TSA as a marker of subclinical inflammation may be valuable as an independent parameter in identifying subjects at higher risk of developing type 2 diabetes and those who might benefit from anti-inflammatory treatment.

Antioxidant properties of ganglioside micelles.

Antioxidant activity of gangliosides GM1 and GT1b in the Fenton type of reaction was investigated by EPR spectroscopy using DMPO as a spin trap. Hydroxyl radical spin adduct signal intensity was significantly reduced in the presence of gangliosides at their micellar concentrations. Mean micellar hydrodynamic diameter was not changed, whereas significant changes in negative Zeta potential values were observed as evidenced by Zetasizer Nano ZS. This study showed that the primary mode of ganglioside action was not due to direct scavenging of OH·, but rather to the inhibition of hydroxyl radical formation. This phenomenon is related to the ability of ganglioside micelles to bind oppositely charged ferrous ions, thus reducing their concentration and consequently inhibiting OH· formation.

Ganglioside GT1b protects human spermatozoa from hydrogen peroxide-induced DNA and membrane damage.

We have reported previously that various gangliosides, the sialic acid containing glycosphingolipids, provide protection against sperm injury caused by reactive oxygen species (ROS). In this study, we investigated the effect of treatment of human spermatozoa with ganglioside GT1b on hydrogen peroxide (H(2)O(2))-induced DNA fragmentation and plasma membrane damage. Single-cell gel electrophoresis (Comet assay) used in the assessment of sperm DNA integrity showed that in vitro supplemented GT1b (100 microm) significantly reduced DNA damage induced by H(2)O(2) (200 microm) (p < 0.05). Measurements of Annexin V binding in combination with the propidium iodide vital dye labelling demonstrated that the spermatozoa pre-treated with GT1b exhibited a significant increase (p < 0.05) in the percentage of live cells with intact membrane and decreased phosphatidylserine translocation after exposure to H(2)O(2). Flow cytometry using the intracellular ROS-sensitive fluorescence dichlorodihydrofluorescein diacetate dye employed to investigate the transport of the extracellularly supplied H(2)O(2) into the cell interior revealed that ganglioside GT1b completely inhibited the passage of H(2)O(2) through the sperm membrane. These results suggest that ganglioside GT1b may protect human spermatozoa from H(2)O(2)-induced damage by rendering sperm membrane more hydrophobic, thus inhibiting the diffusion of H(2)O(2) across the membrane.