Modulation of ROS production in human leukocytes by ganglioside micelles

Recent studies have reported that exogenous gangliosides, the sialic acid-containing glycosphingolipids, are able to modulate many cellular functions. We examined the effect of micelles of mono- and trisialoganglioside GM1 and GT1b on the production of reactive oxygen species by stimulated human polymorphonuclear neutrophils using different spectroscopic methods. The results indicated that exogenous gangliosides did not influence extracellular superoxide anion (O2.-) generation by polymorphonuclear neutrophils activated by receptor-dependent formyl-methionyl-leucyl-phenylalanine. However, when neutrophils were stimulated by receptor-bypassing phorbol 12-myristate 13-acetate (PMA), gangliosides above their critical micellar concentrations prolonged the lag time preceding the production in a concentration-dependent way, without affecting total extracellular O2.- generation detected by superoxide dismutase-inhibitable cytochrome c reduction. The effect of ganglioside GT1b (100 µM) on the increase in lag time was shown to be significant by means of both superoxide dismutase-inhibitable cytochrome c reduction assay and electron paramagnetic resonance spectroscopy (P < 0.0001 and P < 0.005, respectively). The observed phenomena can be attributed to the ability of ganglioside micelles attached to the cell surface to slow down PMA uptake, thus increasing the diffusion barrier and consequently delaying membrane events responsible for PMA-stimulated O2.- production.

Modulation of ROS production in human leukocytes by ganglioside micelles.

Recent studies have reported that exogenous gangliosides, the sialic acid-containing glycosphingolipids, are able to modulate many cellular functions. We examined the effect of micelles of mono- and trisialoganglioside GM1 and GT1b on the production of reactive oxygen species by stimulated human polymorphonuclear neutrophils using different spectroscopic methods. The results indicated that exogenous gangliosides did not influence extracellular superoxide anion (O2·â») generation by polymorphonuclear neutrophils activated by receptor-dependent formyl-methionyl-leucyl-phenylalanine. However, when neutrophils were stimulated by receptor-bypassing phorbol 12-myristate 13-acetate (PMA), gangliosides above their critical micellar concentrations prolonged the lag time preceding the production in a concentration-dependent way, without affecting total extracellular O2·â» generation detected by superoxide dismutase-inhibitable cytochrome c reduction. The effect of ganglioside GT1b (100 µM) on the increase in lag time was shown to be significant by means of both superoxide dismutase-inhibitable cytochrome c reduction assay and electron paramagnetic resonance spectroscopy (P < 0.0001 and P < 0.005, respectively). The observed phenomena can be attributed to the ability of ganglioside micelles attached to the cell surface to slow down PMA uptake, thus increasing the diffusion barrier and consequently delaying membrane events responsible for PMA-stimulated O2·â» production.

Reduction of oxidative changes in human spermatozoa by exogenous gangliosides.

The effect of exogenous gangliosides, the sialic acid-containing glycosphingolipids, on oxidative changes in human spermatozoa was investigated. The incorporation of disialogangliosides or trisialogangliosides (GD1b and GT1b, respectively) into the iron/ascorbate promoter system for induction of lipid peroxidation decreased the release of malondialdehyde (MDA) from peroxidizing spermatozoa. The application of monosialogangliosides and disialogangliosides (GM1 and GD1a, respectively) did not have any effect under identical experimental conditions. GT1b, at a micromolar concentration, significantly inhibited the production of MDA, a breakdown product of lipid peroxide decomposition in spermatozoa of normozoospermic infertile men (P < 0.001; n = 51). An enhanced generation of MDA exhibited by the sperm population from the low-density Percoll fraction containing defective and/or immature spermatozoa was significantly reduced in the presence of GT1b. These results and the experiments on the influence of iron-chelating agent ethylenediamine tetraacetic acid (EDTA) as well as ferrous ion concentration itself on lipid peroxidation support the hypothesis that the protective effect of ganglioside against MDA generation could be the result of its chelating activity. Furthermore, superoxide anion release of phorbol myristate acetate-stimulated spermatozoa was significantly reduced in the presence of 50 and 100 micromol l(-1) GD1b (P < 0.05) and GT1b (P < 0.005). The inhibitory effect of 100 micromol l(-1) GT1b on spermatozoa from infertile normozoospermic men was statistically significant (P < 0.001; n = 21) and did not depend on the initial superoxide anion production. In conclusion, the protective action of GD1b and GT1b could be related to both scavenging of free radicals and metal-chelating properties, which might have relevance in the protection against oxidation-induced processes in human spermatozoa.

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

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.

Baseline diene conjugation in LDL lipids from newly diagnosed type 2 diabetic patients.

BACKGROUND: To determine whether the susceptibility of low-density lipoproteins (LDL) to oxidation was altered in subjects with newly diagnosed type 2 diabetes mellitus and impaired glucose tolerance using the measurement of LDL baseline diene conjugation (LDL-BDC) as an indicator of circulating mildly oxidized LDL in vivo. METHODS: LDL was isolated from sera of 26 newly diagnosed patients with type 2 diabetes mellitus (type 2 DM), 43 subjects with impaired glucose tolerance (IGT), and 67 subjects with normal glucose tolerance (NGT), who were matched for age, gender, body mass index and serum lipids. Twenty-seven healthy subjects with normal body mass index and normal values of lipids served as a control group. In all groups the LDL-BDC was compared with the production of conjugated dienes after copper-induced LDL oxidation, and with total antioxidative status. RESULTS: The LDL-BDC levels (micromol/l; mean +/- SD) were similar in the three glucose tolerance categories (NGT 66.5 +/- 6.9; IGT 67.9 +/- 6.5; type 2 DM, 68.1 +/- 7.1 p = 0.52) and there was no difference in comparison with the control group (68.9 +/- 4.2 micromol/l). Also, no significant differences between the groups were observed in the susceptibility to LDL oxidation in vitro or total antioxidative status. There was no correlation between glycemia and mildly oxidized LDL in vivo, nor with any of the parameters of the LDL oxidation in vitro. CONCLUSIONS: In the examined population of newly diagnosed diabetic patients with mild hyperglycemia, as evidenced by the level of glycated haemoglobin (HbA(1C)), a lack of changes in the susceptibility of LDL to oxidation both in vivo and in vitro was established.

Effect of creatine on erythrocyte rheology in vitro.

To evaluate how creatine influences erythrocyte deformability, we determined its effect on erythrocyte filterability in 9 subjects with insulin dependent diabetes (IDDM) without complications, 14 diabetics with uremia and 10 non-diabetic controls. The short-term incubation (15 min at 37 degrees C) of diabetic erythrocytes with 3 mM creatine improved cell filterability (assessed according to the Reid method) from IDDM subjects without complications by 28.4% and that from diabetics with uremia by 18.9%. No rheological effect of creatine was found in erythrocytes from non-diabetic controls. However, a significant protective effect against erythrocyte filterability impairment induced by treatment of red blood cells from non-diabetic controls with hydrogen peroxide was observed with 3 mM (p < 0.04) and 5 mM (p < 0.01) creatine, respectively. Measurement of the thiobarbituric acid (TBA) reactivity was used to assess hydrogen peroxide induced formation of malondialdehyde (MDA). We found that creatine inhibits hydrogen peroxide-induced erythrocyte MDA-formation in a dose dependent manner by 20.4%, 22.3% and 41.4% for 1, 3 and 5 mM creatine, respectively. These results suggest that creatine by its ability to inhibit erythrocyte lipid peroxidation may contribute to the maintenance of normal cell deformability.

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/10(8) 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/10(8) sperm in melatonin-treated samples: n = 7, p < .02) and (.005 mmol of ferrous ions (20.2 +/- 2.8 vs. 9.9 +/- 2.4 nmol MDA/ 10(8) 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/10(8) 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.

In vitro effect of zinc on superoxide anion production by polymorphonuclear leukocytes of diabetic patients.

We evaluated the ability of zinc to modulate the superoxide anion released by isolated, phorbol myristate acetate (PMA)-stimulated polymorphonuclear leukocytes (PMNs). In the presence of zinc at physiological level (20 micromol/l), PMNs released 20% less superoxide anions than untreated cells, whereas at higher zinc concentration (200 micromol/l), a 40%-increase in superoxide anion production was detected. The direction (suppression or stimulation) of superoxide anion generation was dependent upon extracellular zinc level and did not discriminate type 1 and 2 diabetic subjects from normal controls. The mechanism(s) of dose-dependent pro- and antioxidative effects of zinc on isolated PMNs in vitro as well as the implication of this finding for the modulatory effect of Zn on PMN-superoxide anion production in vivo need further investigation.

Effect of cisplatin and 6-bromo-6-deoxy-L-ascorbic acid on some biochemical and functional parameters in mice.

The results of the present study demonstrate that 6-bromo-6-deoxy-L-ascorbic acid (6-BrAA), an antioxidative derivative of ascorbic acid, is capable of lowering the toxicity of cisplatin, cis-diaminedichloroplatinum (cis-DDP), an anticancerogenic drug. The biological aspects and pharmacological significance of a combined treatment of these two substances were investigated in a mouse model. The results indicate that the effectiveness of 6-BrAA on biological response(s) is strongly dependent on the dose of cis-DDP. Injection of 10 mg/kg body weight (bw) of cis-DDP following pretreatment with 6-BrAA (480 mg/kg bw) enhances the tissue-protecting effect of 6-BrAA and reduces, to some extent, the ensuing nephro-, liver and spleen toxicity. On the other hand, 6-BrAA in animals treated with a higher dose of cis-DDP (15 mg/kg bw) leads to exacerbation of the toxic cis-DDP effect and concurrent loss of the protective potential of 6-BrAA with respect to tissue damage. The exact mechanism(s) of 6-BrAA protection and exacerbation of the toxic cis-DDP effect is unclear, although scavenging or generating of free radicals may play an important role. The results obtained may be of importance in planning the rational use of cis-DDP and 6-BrAA administration in the potential treatment of cancer.

In vitro inhibition of superoxide anion production and superoxide dismutase activity by zinc in human spermatozoa.

The in vitro effect of zinc on superoxide anion (O2-) generation and on SOD-like activity in spermatozoa of infertile men was investigated. The formation of superoxide anion was stimulated by NADPH and the level of superoxide anion was measured by the reduction of ferricytochrome c. Both Percoll-isolated (n = 14) and washed spermatozoa (n = 14) exposed to 1 mmol/L zinc (60 min, 37 degrees C), released less (p < 0.002 and p < 0.04, respectively) superoxide anions than did zinc-untreated spermatozoa. These results implicate a possible role for zinc as a scavenger of excessive superoxide anions produced by defective spermatozoa in semen after ejaculation. Additionally, zinc was found to dose-dependently inhibit superoxide dismutase (SOD)-like activity of spermatozoa in vitro. The inhibition of SOD-like activity by an equal concentration of zinc (1 mmol/L) was less pronounced in oligospermic (p < 0.002; n = 16) and asthenozoospermic (p < 0.0005; n = 20) than in normozoospermic samples (p < 0.0001; n = 20). This differential ability of zinc to inhibit SOD-like activity may be relevant to the physiological function of spermatozoa in fertilization. The evidence that zinc may elicit an inhibition of both superoxide anion production and SOD-like activity in human spermatozoa, indicate the existence of novel, zinc-related mechanism(s) involved in the oxidative events occurring after ejaculation, with a possible modulatory effect on germ cell function.

Paraoxonase and arylesterase activities in the serum of two hyperlipoproteinaemic patients after repeated extracorporal lipid precipitation.

The effect of heparin-induced extracorporal lipid precipitation (HELP) on the activities of paraoxonase (EC 3.1.8.1) and arylesterase (EC 3.1.1.2) was studied in serum of a patient with hyperlipoproteinaemia (A) and of a patient with non-insulin dependent diabetes mellitus and hyperlipoproteinaemia (B). The enzyme activities were measured spectrophotometrically (Tris-HCl buffer, pH 7.4, 37 degrees C) with paraoxon and phenylacetate as substrates of paraoxonase and arylesterase, respectively. Both patients underwent HELP applications once a week over a period of 7 weeks. Over that period no overall change was observed either in enzyme activities or in the lipid and protein content of the sera. However, each HELP session caused an immediate decrease of EDTA-insensitive arylesterase activity (on average 56% in A and 42% in B), while EDTA-sensitive arylesterase remained almost unaltered. Paraoxonase remained unchanged in A, but decreased in B (approximately 60%). Of the atherogenic lipoprotein parameters, the most pronounced decrease was found in VLDL-cholesterol and in triglycerides (on average 45% in A and 32% in B), while the anti-atherogenic HDL-cholesterol decreased < 10%. Possible implications of the effect of HELP on the enzyme activities studied remain to be explained.

In vitro effect of zinc on oxidative changes in human semen.

The in vitro effect of zinc on superoxide anion (O2-) generation and on experimentally induced lipid peroxidation (LPO) in spermatozoa of infertile men was investigated. Washed spermatozoa pre-incubated for 30 min at 37 degrees C in the presence of 1 or 3 mmol l-1 zinc, released less superoxide anions (P < 0.03 and P < 0.02, respectively; n = 9) than the untreated spermatozoa. Similar results were obtained using activated polymorphonuclear leukocytes (1 x 10(6) cells ml-1) in the presence of 1 or 3 mmol l-1 Zn (P < 0.001 and P < 0.0002, respectively; n = 9). The in vitro evidence of the inhibitory effect of zinc on O2- generation by human spermatozoa and leukocytes indicates that zinc may act in vivo as a scavenger of excessive O2- production by defective spermatozoa and/or leukocytes in semen after ejaculation. A significant stimulatory effect of Zn (3 mmol l-1) on iron-induced lipid peroxidation, measured by the formation of thiobarbituric acid reactive substances (TBARS), was detected in the spermatozoa of 16 normo- and 17 asthenozoospermic males (P < 0.0001 and P < 0.001, respectively). In 11 samples with sperm concentration 20.3 +/- 2.1 x 10(6) ml-1, exhibiting initial TBARS concentration two times higher than in normo- and asthenozoospermic samples (40.5 +/- 2.4 vs. 17.1 +/- 1.1 and 28.5 +/- 4.1 nmoles TBARS 10(-8) spermatozoa), no effect of zinc on the LPO rate was found. The observed inhibitory effect of zinc on superoxide anion regardless of the initial O2- level and stimulatory effect of zinc depending on the initial LPO rate in human spermatozoa suggests that this metal ion participates in the oxidative changes occurring after ejaculation and thus may modulate the properties of germ cells.

Evaluation of ascorbate and urate antioxidant capacity in human semen.

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 mumol l-1 and 127-670 mumol l-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 x 10(6) 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 degrees C, 30 min) in the presence of 0.4, 1.0 and 1.5 x 10(6) 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 is not hampered in the semen of infertile men, provided that leukocyte contamination does not exceed 1 x 10(6) cells ml-1. The possible role of urate in stabilizing the ascorbate antioxidant activity in seminal plasma should be further investigated.

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

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.

Superoxide anion scavenging capacity of human seminal plasma.

This study has investigated the antioxidant capacity of human seminal plasma due to the presence of both high and low molecular weight antioxidant factors. Methods for the measurement of superoxide dismutase-like activity (SOD-like) and total antioxidant status (TAS) were automated, and had a within-run coefficient of variation of 7.3% for SOD-like activity and 4.8% for TAS. In 69 semen samples from unselected infertile men, SOD-like activity in seminal plasma ranged from 2 to 16 U/ml, with a mean of 6.9 +/- 2.8 U/ml. As SOD-like activity was correlated positively with levels of citric acid (p < 0.0001), zinc (p < 0.0002) and acid phosphatase activity (p < 0.0005), and there was no correlation with fructose levels, our results suggest that prostatic secretions are an important source of superoxide anion scavengers. Evaluation of SOD-like activity in infertile men with accessory sex gland infections (n = 12) showed significantly lower activity (p < 0.003) compared to values found in 12 infertile men without signs of infection. The values obtained for total antioxidant status (equivalent to the antioxidant capacity of alpha-tocopherol analogue) ranged from 1.7 to 2.3 mmol/L, with a mean of 2.1 +/- 0.1 (n = 40), reflecting the protective activity of ascorbate, urate and albumin, and to a very low extent of glutathione and taurine. The data obtained by TAS assay correlated with fructose, a major marker of vesicular secretion (p < 0.005), suggesting that low molecular weight components with antioxidant capacity derive partly from the seminal vesicles. The results indicate that the relative contribution of antioxidant defence systems capable of counteracting the deleterious action of superoxide anions, depends on the secretory activity of accessory sex glands and is independent of excessive ROS production due to increased oxidative stress.

An increase in micropulmonary red cell mass in hyperlipidaemic patients.

It is known that pulmonary microcirculation rheology is partly affected by plasma levels of lipoproteins, but only a few data are available for humans. Therefore, in a sample of 30 normal volunteers and 90 patients with various types of primary hyperlipoproteinaemia, the plasma levels of total cholesterol (Chol), low density cholesterol (LDL), the high density cholesterol (HDL), triglyceride (Tg) and fibrinogen (Fib) were measured in conjunction with determinations of plasma viscosity (PV) and the pulmonary capillary red cell volume (RCVc). RCVc was estimated from measurements of the vascular component of the single-breath-diffusing lung capacity for carbon monoxide, using our own modification of the Roughton-Forster's method. By stepwise regression analysis, the variation in RCVc was almost completely accounted for (r2 = 0.87) by variations in PV, Chol, Tg and the anthropometric confounding factors. The proposed explanations for increased pulmonary capillary red cell mass (up to 151% of the predicted value) in hyperlipidaemic patients included the hypothesis of increased pulmonary microhaematocrit, which agrees with the observed in-vitro lipoprotein-dependent increase in erythrocyte aggregability.

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

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.

Effect of pentoxifylline on experimentally induced lipid peroxidation in human spermatozoa.

We demonstrated previously that pentoxifylline in millimolar concentrations can inhibit superoxide anion production by human spermatozoa. In the present study we have examined the effects of the same concentrations of pentoxifylline on experimentally induced lipid peroxidation, as measured by malondialdehyde formation in the thiobarbituric (TBA) assay. Under the experimental conditions used, preincubation of spermatozoa with pentoxifylline led to a significant dose-dependent stimulation (p < 0.005) of malondialdehyde production amounting to 10.77 +/- 2.35%, 13.45 +/- 2.99% and 17.4 +/- 1.99% (mean +/- SEM) for 1.9, 3.7 and 11.2 mmol/l pentoxifylline, respectively. In the presence of 11.2 mmol/l pentoxifylline, an increase in iron-catalysed lipid peroxidation potential was detected in samples of spermatozoa from 29 infertile men, regardless of their initial levels of malondialdehyde. The results of this study indicate that pentoxifylline might further augment the ferrous ion-stimulated decomposition of pre-accumulation lipid hydroperoxides in the sperm plasma membrane and thus promote malondialdehyde generation in the TBA assay. It is concluded that the stimulatory effect of pentoxifylline on iron-induced lipid peroxidation may have an adverse effect on the quality of sperm suspensions prepared for in vitro fertilization, a possibility which should be investigated further.

Side-effects of peptidoglycan monomer (PGM) treatment in mice.

The influence of single (4 mg/mouse) and multiple (1 mg/mouse per day for 5 consecutive days) injections of PGM on some hepatic enzymes, lipid peroxide generation in serum and liver, sialic acid concentration in serum and spleen and hepatic lysosomal membrane permeability was investigated. The studies performed showed that a single injection of PGM in vivo changed temporarily the permeability of lysosomal membranes, lipid peroxidation products and sialic acid concentration, and when administered in vitro modulated superoxide anion production and did not affect the activity of lysosomal membrane enzymes. Multiple injections of PGM did not cause significant changes in the examined parameters. Although the metabolic changes were time-limited and from the toxicological point of view, provoked transient effects, the results obtained may be of importance when using PGM in combined chemo-immunotherapy.

Effect of sialic acid on glycation-induced fluorescence of albumin.

The aims of this study were to determine whether in vitro nonenzymatic glycation of proteins by sialic acid (sialylation) induces the generation of fluorescence, and whether the presence of this keto sugar may affect the generation of fluorescence induced by other sugars. Incubation of bovine serum albumin (BSA; 1.5 mM) with sugars (50 mM in 0.2 M phosphate buffer, pH 7.4, at 37 degrees C) resulted in a time-dependent increase of formaldehyde release (moles/moles of protein). On the 17th day of incubation, the value was 0.53 +/- 0.06, 0.78 +/- 0.15 and 1.23 +/- 0.18 for sialic acid, fructose and glucose respectively, compared with 0.37 +/- 0.05 for BSA. The fluorescence intensity (arbitrary units/mg protein) was higher after 17 days of incubation with fructose (16.9 +/- 1.8) than with glucose (12.7 +/- 1.3), while no significant increase was noted with sialic acid compared with BSA (3.8 +/- 0.4). Fluorescence intensity increase by incubation with glucose (50 mM) was significantly reduced by sialic acid (20 mM) after both 10 (P < 0.001) and 14 (P < 0.001) days of incubation, while inhibition was weaker after 14 (P < 0.05) than after 10 (P < 0.001) days when fructose (50 mM) was used as the glycating agent. This indicates that sialic acid can be potentially used to limit the damage from adverse glycation-induced processes.