Evaluation of the NOX5 protein expression and oxidative stress in sperm from asthenozoospermic men compared to normozoospermic men.

PURPOSE: NADPH oxidase 5 (NOX5), the main isoform of NOX in spermatozoa, has been recognized as the main active generators of reactive oxygen species (ROS), including superoxide anion (O 2 -. ) and hydrogen peroxide (H2O2). ROS have been shown to play important roles in many physiological and pathological conditions in spermatozoa. The present study aims to investigate the alterations of NOX5 protein expression and oxidative stress (OS) status in asthenozoospermic men compared to normozoospermic men. METHODS: Semen samples were collected from 25 asthenozoospermic men and 28 normozoospermic men. In this study, NOX5 protein expression was evaluated by Western blotting. An OS status was evaluated by measuring of ROS (O 2 -. and H2O2), DNA damage and plasma membrane integrity in spermatozoa. RESULTS: The protein expression of NOX5 (p < 0.0001) was remarkably higher in asthenozoospermic men in comparison to normozoospermic men. In addition, the percentages of intracellular O 2 -. (p < 0.0001), H2O2 (p < 0.0001) in viable spermatozoa, apoptotic sperm cells with altered plasma membrane (p < 0.001) and DNA damage (p = 0.001) were significantly increased in asthenozoospermic men compared to normozoospermic men. CONCLUSIONS: The present study provides evidence that the overexpression of NOX5 protein may induce excessive ROS production and oxidative stress damages to DNA and plasma membrane integrity in asthenozoospermic men.

Association between the cytotoxic T-lymphocyte antigen-4 mutations and the susceptibility to systemic lupus erythematosus; Contribution markers of inflammation and oxidative stress.

The cytotoxic T lymphocyte antigen-4 (CTLA-4) also known as CD152 (cluster of differentiation 152) is a crucial negative regulator of the immune system. This protein receptor provides negative signals in order to suppress T-cell activation and immune attack against self-antigens, although its role is unclear.  The ability of CTLA-4 to limit T cell-mediated immune response has made it a major target in treatment of tumors and autoimmune diseases such as systemic lupus erythematosus (SLE). In this study, we investigated whether CTLA-4 G-1661A and CTLA-4 T-1722C mutations are associated with SLE. So one hundred nine SLE patients and 101 gender and age-matched unrelated healthy controls were recruited for this case-control study. The promoter mutations were detected by PCR-RFLP, neopterin, malondialdehyde (MDA) and serum lipid concentration were determined by HPLC and enzyme assay, respectively. RESULT: We found that both codominant (AA vs. GG) and recessive (AA vs. GA+GG) CTLA-4 G-1661A mutation significantly decreased the risk of SLE by 1.7 and 3.7 times, respectively.  Interestingly, SLE patients with AA genotypes of CTLA-4 G-1661A have lower neopterin and MDA concentration compared with GA+GG genotypes. The overall distribution of CTLA-4 T-1722C genotypes and alleles in SLE patients were similar to those in control group. In conclusion, our findings showed, that there is an association between systemic inflammatory markers, oxidative stress and the CTLA-4 G-1661A GG+AG genotypes, MDA and neopterin which are the most conventional risk factors for coronary heart disease, therefore these mutations may be consider as a risk factor for susceptibility to heart disease in SLE patients.

Matrix metalloproteinase 9 polymorphisms and systemic lupus erythematosus: correlation with systemic inflammatory markers and oxidative stress.

Systemic lupus erythematosus (SLE) is an autoimmune disease that involves multiple organs and is characterized by persistent systemic inflammation. Among the effects of inflammatory mediators, the induction of matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) and oxidative stress has been demonstrated to be important in the development of SLE. In this study, the possible association between MMP-9 and MMP-2 functional promoter polymorphism, stress, and inflammatory markers with development of severe cardiovascular disease (CVD), high blood pressure (HBP), and lupus nephropathy (LN) in SLE patients was investigated. The present case-control study consisted of 109 SLE patients with and without CVD, HBP and LN and 101 gender- and age-matched unrelated healthy controls from a population in western Iran. MMP-2 -G1575A and MMP-9 -C1562T polymorphisms were detected by PCR-RFLP, serum MMP-2 and MMP-9, neopterin, malondialdehyde (MDA) and lipid levels were determined by ELISA, HPLC and enzyme assay, respectively. We found that MMP-9 -C1562 T and MMP-2 -G1575A alleles act synergistically to increase the risk of SLE by 2.98 times (p = 0.015). Findings of this study also demonstrated that there is a significant increase in the serum levels of MMP-2, neopterin and MDA and a significant decrease in serum level of MMP-9 in the presence of MMP-9-C1562 T and MMP-2 -G1575A alleles in SLE patients compared to controls. Further, SLE patients with MMP-9 (C/T + T/T) genotype had significantly higher serum concentrations of MMP-2, neopterin, MDA and LDL-C, but lower serum MMP-9 and HDL-C levels than corresponding members of the control group. MMP-9 (C/T + T/T) genotype increased risk of hypertension in SLE patients 2.71-fold. This study for the first time not only suggests that MMP-9 -C1562 T and MMP-2 -G1575A alleles synergistically increase the risk of SLE but also high serum levels of MDA, neopterin, and circulatory levels of MMP-2 and lower MMP-9 in SLE patients. This information may be important in the evaluation of SLE progression and in the elucidation of the mechanisms of the disease pathogenesis.

Antioxidant status and serum levels of selectins in pre-eclampsia.

A cross-sectional study was conducted in a university hospital, enrolling 40 patients with pre-eclampsia (case group) and 40 healthy normotensive pregnant women (control group). Plasma activity of antioxidants and some adhesion molecules involved in oxidative stress were measured and compared between the two groups, according to the patients' age. In patients over the age of 30 years, serum levels of L-selectin and E-selectin were lower in pre-eclamptic patients (p < 0.05); antioxidants, catalase and superoxide dismutase did not significantly differ between the two groups, while glutathione peroxidase was significantly higher in the normotensive group (p < 0.05). In patients under the age of 30 years, E-selectin was significantly higher in the pre-eclampsia group (p < 0.05), while P-selectin, catalase and superoxide dismutase were not significantly different between the two groups (p > 0.05). Total antioxidative activity was similar between pre-eclamptic and normotensive patients (p > 0.05). This study revealed no relationship between total antioxidant activity and pre-eclampsia.

Synergistic effects of BuChE non-UU phenotype and paraoxonase (PON1) 55 M allele on the risk of systemic lupus erythematosus: influence on lipid and lipoprotein metabolism and oxidative stress, preliminary report.

There is some evidence indicating lipid peroxidation can affect progression of atherosclerosis, cardiovascular diseases (CVDs) and glomerulonephritis in systemic lupus erythematosus (SLE) patients. Human butyrylcholinesterase (BuChE) and paraoxonase-1 (PON1) are two major bioscavenger enzymes that are associated with inflammation, oxidative stress and lipid metabolism. Hyperlipidemia, increase in lipid oxidation reactions and defects in antioxidant status may lead to increased oxidative stress and high frequency of CVDs in SLE. It has also been suggested that deficiency in the function of the antioxidant system and an increase in reactive oxygen release (ROS) may play an important role in the pathogenesis of SLE. This study is the first investigation to examine the association of BuChE phenotypes, PON1 (L55M; PON-55-M) polymorphism, the levels of malondialdehyde (MDA), neopterin, lipid-lipoprotein and activities of BuChE and arylesterase activity (ARE) of PON with severity of SLE. The present case-control study consisted of 109 SLE patients and 101 gender- and age-matched, unrelated healthy control subjects from the population of west Iran. We found that the PON-55-M allele and BuChE non-UU act synergistically to increase the risk of SLE by 2.5 times (1.03-6.7, p = 0.044). There was a significant negative correlation between severity of SLE with serum BuChE activity (R = -0.31, p < 0.001) and positive correlation with serum neopterin level. The SLE patients with the PON-55-M (M/L + M/M) allele or with BuChE non-UU phenotype had significantly lower serum ARE and BuChE activities than those with PON-55-L/L or BuChE-UU phenotypes, respectively. In addition, their serum levels of MDA, neopterin and LDL-C were significantly elevated, suggesting that these individuals are more susceptible to CVD. However, further studies are needed to shed more light on the contribution of the M allele of PON1 and non-UU phenotypes of BuChE in the development of SLE in different ethnicities.

Methylentetrahydrofolatereductase (rs1801133) polymorphism and psoriasis: contribution to oxidative stress, lipid peroxidation and correlation with vascular adhesion protein 1, preliminary report.

BACKGROUND: Psoriatic patients are at greater risk of oxidative stress and inflammation which is associated with abnormal plasma lipid metabolism and lipid peroxidation.There is not any information about the clinical significance of relation between methylentetrahydrofolatereductase (MTHFR) 677-T allele with malondialdehyde (MDA), lipids, apolipoproteins and vascular adhesion protein-1 (VAP-1) partakes in the migration process of lymphocytes into sites of inflammation. OBJECTIVES: This study is the first investigation to examine the association of MTHFR (rs1801133) C677T polymorphism, serum level of MDA, VAP-1, lipid-lipoprotein and apolipoproteins with the risk of psoriasis. METHODS: The present case-control study consisted of 100 psoriatic patients and 100 gender- and age-matched unrelated healthy controls from west population of Iran. MTHFR-C677T (rs1801133) polymorphisms were detected by restriction fragment length polymorphism (PCR-RFLP), VAP-1 by ELISA, apolipoproteins by immunoprecipitation, lipid and apolipoproteins by spectrophotometery and MDA by HPLC. RESULTS: We found that dominant/recessive model (CC + CT/TT) and T allele of MTHFR-677 alleles significantly 7.45 and 1.76 times increased risk of psoriasis, respectively. The psoriasis patients with MTHFR-677-T (C/T + T/T) allele had significantly higher serum MDA, VAP-1 and apolipoproteinsAPOB concentrations and ratio of APOB/APOA1 than the control subjects.The MTHFR-677-T allele frequencies in psoriasis patients were significantly higher than that in control group (28.5% vs. 18.5%; P = 0.018).We found a significant positive correlation between VAP-1 with MDA (P = 0.047) and LP (a) (P = 0.025). CONCLUSION: In the present study, we demonstrated for the first time that the psoriasis patients with MTHFR-677-T (C/T + T/T) allele had higher serum levels of MDA, VAP-1, APOB and ratio of APOB/APOA1 and dominant/recessive model (CC+CT/TT) and T allele of MTHFR-677 are significantly more common in psoriasis and increased risk of psoriasis by 7.45 and 1.76 fold, respectively. These data suggest that psoriasis patients carrying of TT genotypes and T allele of MTHFR-677 may be more susceptible to cardiovascular disease and myocardial infarction.

Paraoxonase 1 (PON1) 55 polymorphism, lipid profiles and psoriasis.

BACKGROUND: Paraoxonase 1 (PON1) is a serum high-density lipoprotein-bound enzyme with antioxidant function. It hydrolyses lipid peroxides, protecting low-density lipoproteins from oxidative modifications. Patients with psoriasis are at greater risk of oxidative stress, which is associated with abnormal plasma lipid metabolism. OBJECTIVES: In this study, association of the PON1 55 M allele with serum arylesterase (ARE) activity, malondialdehyde (MDA), lipid profiles and psoriasis was investigated. METHODS: The present case-control study consisted of 100 patients with psoriasis with and without cardiovascular diseases (mean age 35·3 years) and 100 sex- and age-matched unrelated healthy controls (mean age 35·7 years) from the population of western Iran. The PON1 55 Met>Leu polymorphism was detected by polymerase chain reaction-restriction fragment length polymorphism. Serum ARE activity, MDA, and lipid and apolipoprotein levels were determined spectrophotometrically, by high-performance liquid chromatography and by enzyme assay, respectively. RESULTS: The presence of the PON1 55 M allele was found to be associated with psoriasis (odds ratio = 1·96, P = 0·017). The patients with psoriasis with the PON1 M (M/L + M/M) allele had higher MDA levels (4·12 ± 0·88 vs. 2·24 ± 0·55 µmol L(-1) , P < 0·001), apolipoprotein B (APOB)/APOA1 ratio (0·91 ± 0·66 vs. 0·66 ± 0·35, P = 0·004), APOB (111 ± 38·7 vs. 88·3 ± 22·5 mg mL(-1) , P = 0·001) and lipoprotein(a) [LP(a)] (21·9 ± 18·4 vs. 15·8 ± 16·6 mg mL(-1) , P = 0·034), but lower ARE activity (39·6 ± 11 vs. 45·9 ± 11·8 U mL(-1) , P = 0·031) than the control subjects. ARE activity showed a significant positive correlation with APOA1 and a negative correlation with MDA concentration in patients with psoriasis. CONCLUSIONS: The PON1 55 M allele is a risk factor for psoriasis. Carriers of this allele have high levels of MDA, APOB and LP(a), a high APOB/APOA1 ratio and low ARE activity. These results indicate that oxidative stress, impairment of the antioxidant system and abnormal lipid metabolism may play a role in the pathogenesis and progression of psoriasis and its related complications. These data suggest that patients with psoriasis are more susceptible to vascular diseases.

Matrix metalloproteinase-2 functional promoter polymorphism G1575A is associated with elevated circulatory MMP-2 levels and increased risk of cardiovascular disease in systemic lupus erythematosus patients.

Matrix metalloproteinase-2 (MMP-2) is a zinc dependent endonuclease that degrades type IV collagen, the major structural component of basement membranes. MMP-2 functional promoter polymorphism G1575A affects circulating level of MMP-2 and may be considered an important genetic determinant of cardiovascular disease (CVD) in systemic lupus erythematosus (SLE) patients. In this study, association between MMP-2 1575A allele with serum MMP-2, neopterin and lipid-lipoprotein levels and with SLE and developing CVD was investigated. The present case-control study consisted of 109 SLE patients with and without CVD (mean age, 35.6 years) and 101 gender- and age-matched, unrelated, healthy controls (mean age, 37.1 years) from the population in the west of Iran. MMP-2 1575G/A polymorphism was detected by polymerase chain reaction (restriction fragment length polymorphism) PCR-RFLP, serum MMP-2, neopterin and lipid levels were determined by enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC) and enzyme assay, respectively. The presence of MMP-2 G1575A allele was found to be associated with SLE and developed CVD (OR = 1.78, p = 0.029 and OR = 3.43, p = 0.025, respectively). The SLE patients with MMP-2 A (G/A + A/A) allele had higher MMP-2 activity (301 ± 166 vs. 194 ± 35.5, p = 0.002), neopterin (29.4 ± 39.4 vs. 7.3 ± 4.6, p = 0.005), LDL-C (120 ± 25.7 vs. 87 ± 39.3, p = 0.045) and lower HDL-C (39.6 ± 11 vs. 45.9 ± 11.8, p = 0.031) levels than the control subjects. There was a significantly positive correlation between MMP-2 level with neopterin, total cholesterol and TG levels and negative correlation with HDL-C level in SLE patients with CVD. MMP-2 G1575A allele may be a risk factor for SLE. The carriers of this allele have high levels of MMP-2, neopterin, total cholesterol and TG and lower levels of HDL, thus, they are more likely to develop heart disease.

Increased receptor for advanced glycation end products in spermatozoa of diabetic men and its association with sperm nuclear DNA fragmentation.

Although the majority of patients with diabetes have disorders in sexual function, associations between diabetes mellitus and sperm function at the molecular level are largely unknown. As receptor for advanced glycation end products plays a key role in many diabetic complications, we hypothesised that it may be involved in sperm nuclear DNA fragmentation. RAGE levels were determined using ELISA and western blot analysis in sperm samples from 32 diabetic and 35 nondiabetic men. Sperm DNA fragmentation was assessed using TUNEL assay. Diabetic men had significantly higher mean levels of RAGE protein (P < 0.001) and DNA fragmentation (P < 0.001) in spermatozoa. Sperm RAGE was directly correlated to sperm DNA fragmentation in diabetic men (r = 0.81, P < 0.001). The high positive correlation between RAGE levels and nuclear DNA fragmentation in spermatozoa of diabetic men suggests a central role of RAGE in disturbances in sexual function of diabetic men.

Adenosine deaminase activity in fertile and infertile men.

Adenosine deaminase (ADA; E.C.3.5.4.4) catalyses the deamination of adenosine to inosine. In the human reproductive system, the importance of enzymes that affect metabolism of adenosine, particularly adenosine deaminase, has been noticed. The purpose of this study was to determine the plasma activities of total adenosine deaminase (ADAT), and its isoenzymes, ADA1 and ADA2, in fertile and infertile men. Plasma activities of ADA and its isoenzymes were measured in 55 fertile men and 70 infertile men. There was a significant difference in the ADA1 and ADA2 activities between fertile and infertile individuals (P < 0.01). The activity of ADAT, ADA2 and ADA1 in infertile men was higher than that in fertile individuals. This alteration in ADA activity can lead to reduced adenosine levels, which may be involved in disturbing the fertility process.

Relationship between advanced glycation end products and increased lipid peroxidation in semen of diabetic men.

AIMS: Majority of diabetic male patients have disturbances in their reproductive systems. However, the mechanisms underlying these disturbances are largely unknown. Since advanced glycation end products (AGE) have a key role in oxidative stress and cell damage in diabetic complications, we hypothesize that AGEs may be involved sperm lipid peroxidation. METHODS: total AGEs in seminal plasma of 32 diabetic and 35 non-diabetic men was determined by spectrofluorimetric method and carboxy methyl lysine (CML) level was assayed using ELISA. Contents of lipid peroxidation in sperm and seminal plasma were determined by thiobarbituric acid reaction. Total antioxidant capacity (TAC) was measured by a colorimetric assay. RESULTS: total AGEs were found significantly higher in seminal plasma of diabetic men than non-diabetic group (p<0.001) whereas no significant differences in seminal plasma CML values between two groups was observed. Moreover, sperm and seminal plasma lipid peroxidation were significantly higher in diabetic subjects than non-diabetic men and a significantly lower TAC was detected in diabetic group compare to non-diabetics. CONCLUSIONS: these results showed an increment in AGEs in seminal plasma of diabetic subjects and may suggest a key role for glycation process and increased oxidative stress in reproductive system dysfunction.

Butyrylcholinesterase K variants increase the risk of coronary artery disease in the population of western Iran.

The conflicting results of several studies suggest that there is an association between the butyrylcholinesterase-K variant (BCHE-K, G1615A/Ala539Thr) and the risk of developing coronary artery disease (CAD) in diabetes and non-diabetic subjects. The objective of this study was to determine whether the presence of the BCHE-K variant exacerbates the risk of CAD in patients from western Iran with and without type 2 diabetes mellitus (T2DM). This case-control study comprised 464 subjects undergoing their first coronary angiography. They were matched and randomly assigned into four groups: CAD+T2DM+ (CAD/T2DM), CAD+DM(-) (CAD/ND), CAD(-)DM+ (T2DM/NCAD) and CAD(-)DM(-)(control). The BCHE-K variant was detected by PCR-RFLP. The BCHE-K allele frequency in CAD patients with and without T2DM [total CAD (TCAD)] and separately for each group (CAD/T2DM and CAD/ND) was significantly higher than in the control group (21.1 % versus 13.3 % (p = 0.001), 22.4 % versus 13.3 % (p = 0.001) and 19.7 % versus 13.3 % (p = 0.015), respectively). The odds ratios (ORs) for the BCHE-K heterozygous and homozygous variants in TCAD subjects were 1.65 (95 % CI 1.17-2.3; p = 0.004) and 4.3 (1.05-19.4; p = 0.048); for CAD/T2DM individuals 1.76 (1.2-2.6; p = 0.004) and 4.73 (0.96-23.3; p = 0.052); and for CAD/ND patients 1.53 (1.05-2.3; p = 0.029) and 3.88 (0.8-19.7; p = 0.7), respectively. The OR of the BCHE-K allele was found to be 1.74 (1.1-2.4; p = 0.001) in TCAD subjects, 1.87 (1.12-1.48; p = 0.001) in the CAD/T2DM group and 1.59 (1.04-1.4; p = 0.016) in CAD/ND subjects. These data suggest that the BCHE-K allele increases the risk of CAD in the population (with and without DM) in western parts of Iran, and its presence intensifies the risk of CAD in T2DM. The fact that the BCHE-K allele, even in the heterozygous form, exacerbates the risk of CAD in this population, suggests that a specific therapeutic intervention should be considered for this particular group of patients.

Decreased polyunsaturated and increased saturated fatty acid concentration in spermatozoa from asthenozoospermic males as compared with normozoospermic males.

The lipid composition of the sperm membrane has been shown to exert a significant effect upon the functional quality of spermatozoa. We have studied fatty acid composition of the phospholipids in spermatozoa in asthenozoospermic and normozoospermic men and determined the ratio of polyunsaturated fatty acids (PUFAs) to saturated fatty acids of spermatozoa of these two groups. Fatty acid concentration of spermatozoa was determined in 15 asthenozoospermic and eight normozoospermic semen samples by thin layer chromatography and gas chromatography. The most abundant polyunsaturated and saturated fatty acids in normozoospermic samples were docosahexaenoic acid (DHA 22 : 6 omega3, 98.5 +/- 4.5 nmol per 10(8) spermatozoa, mean +/- SE) and palmitic acid (103 +/- 17 nmol per 10(8) spermatozoa) respectively. The mean +/- SE values of DHA and palmitic acid in asthenozoospermic samples were 53.9 +/- 11.6 and 145 +/- 14.7 nmol per 10(8) spermatozoa respectively. Compared with normozoospermic samples, asthenozoospermic samples showed lower levels of PUFA and higher amount of saturated fatty acids. The mean +/- SE ratios of sperm PUFA/saturated fatty acids in asthenozoospermic and normozoospermic samples were 0.66 +/- 0.06 and 1.45 +/- 0.16 (P < 0.001) respectively. This study demonstrates that spermatozoa of asthenozoospermic men have lower levels of PUFA compared with saturated fatty acids. This may be contributory to the poor motility noted in samples from these men.