Differences in activities of antioxidant superoxide dismutase, glutathione peroxidase and prooxidant xanthine oxidoreductase/xanthine oxidase in the normal corneal epithelium of various mammals.

Under normal conditions, antioxidants at the corneal surface are balanced with the production of reactive oxygen species without any toxic effects. Danger from oxidative stress appears when natural antioxidants are overwhelmed leading to antioxidant/prooxidant imbalance. The aim of the present study was to examine the activities of enzymes contributing to the antioxidant/prooxidant balance in normal corneal epithelium of various mammals. The enzyme activities of antioxidant superoxide dismutase and glutathione peroxidase, as well as prooxidant xanthine oxidoreductase/xanthine oxidase were examined using biochemical methods. Results show that superoxide dismutase activity is high in rabbits and guinea pigs, whereas in pigs the activity is low and in cows it is nearly absent. In contrast, glutathione peroxidase activity is high in cows, pigs and rabbits, whereas in guinea pigs the activity is low. As far as prooxidant enzymes are concerned, elevated xanthine oxidoreductase/xanthine oxidase activities were found in rabbits, lower activities in guinea pigs, very low activity in cows and no activity in pigs. In conclusion, the above results demonstrate inter-species variations in activities of enzymes participating in antioxidant/prooxidant balance in the corneal epithelium. It is suggested that the levels of antioxidant and prooxidant enzymes studied in the corneal epithelium might be associated with the diurnal or nocturnal activity of animals. UV rays decompose hydrogen peroxide to damaging hydroxyl radicals and perhaps for this reason large animals with diurnal activity (cow, pig) require more effective peroxide removal (high glutathione peroxidase activity) together with the suppression of peroxide production (low superoxide dismutase activity, low xanthine oxidoreductase activity).

Oxidative stress and inflammation in pregnancy.

Pregnancy is a period when increased oxidative stress can be expected. We have focused especially on oxidative stress and inflammation in the period of pregnancy, when prenatal screening is usually performed. We determined advanced oxidation protein products (AOPPs), C-reactive protein (CRP) and anticardiolipin antibodies (ACA) IgG and IgM levels in the serum of 86 pregnant women in the 1st trimester and 102 pregnant women in the 2nd trimester. AOPP levels in the maternal serum of pregnant women were significantly higher in the 1st and 2nd trimesters than they were in that of non-pregnant women (p<0.0001, p<0.001, respectively). Maternal serum CRP levels, too, were increased compared with those in non-pregnant women (1st and 2nd trimester versus non-pregnant women p<0.05, p<0.005, respectively). Just as with AOPPs and CRP, the ACA IgG levels in pregnant women were significantly higher in both trimesters than they were in non-pregnant women (1st and 2nd trimesters versus non-pregnant women p<0.05, p<0.001, respectively). Maternal serum CRP levels correlated positively with AOPPs in the 2nd trimester (r = 0.504, p<0.05). The increased levels of AOPPs, CRP and ACA IgG in the 1st and 2nd trimesters may reflect a maternal response to inflammatory and oxidative stress in pregnant women.

[Advanced glycation and oxidation products in patients with atherosclerosis]. / Produkty pokrocilé glykace a oxidace u pacientu s aterosklerózou.

BACKGROUND: Oxidative stress can potentiate atherogenesis via modification of biological structures and formation of new compounds, e.g. advanced glycation end products (AGEs) and advanced oxidation protein products (AOPP). The aim of the study was to determine AGEs and AOPP in patients with atherosclerosis, effect of statin therapy and relationship to parameters of lipid metabolism. METHODS AND RESULTS: AGEs (carboxymethyllysine - ELISA and fluorescent AGEs - spectrofluorimetry) and AOPP (spectrophotometry) were assessed in 42 patients with atherosclerosis and 21 healthy controls. AGEs are significantly elevated in patients with atherosclerosis in comparison with healthy subjects (carboxymethyllysine 9.02+/-1.66 microg/g prot. vs 7.52+/-1.18 microg/g prot., p<0.001, fluorescent AGEs 4.39 x 103+/-1.15 x 103 AU/g prot. vs 3.78 x 103+/-0.52 x 103 AU/g prot., p<0.001). Mean AOPP concentrations are also slightly higher, but this elevation is not quite significant (95.0+/-42.9 micromol/l vs 79.7+/-28.2 micromol/l, p=0.096). AGEs and AOPP correlate significantly with each other and with selected lipids. Patients with atherosclerosis treated with statins have slightly lower CML, AGEs and AOPP (it did not reach the statistical significance). CONCLUSIONS: Advanced glycoxidation products are elevated in patients with atherosclerosis and are related to parameters of lipid metabolism. Glycoxidation might be possibly therapeutically influenced by statins; however, further clinical studies are required to confirm this hypothesis.

Hyperlipoproteinemia impairs endothelium-dependent vasodilation.

Atherogenic lipoproteins can cause endothelial dysfunction in the initial stage of atherogenesis. In our study we examined 134 patients with defined hyperlipoproteinemia (non-HDL cholesterol>4.1 mmol/l or triglycerides>2.5 mmol/l or taking any of lipid lowering drugs)--94 men and 40 women. The subgroup of controls of comparable age contained 54 normolipidemic individuals--30 men and 24 women. Patients with hyperlipoproteinemia revealed significantly lower ability of endothelium-dependent flow-mediated vasodilation (EDV) measured on brachial artery (4.13+/-3.07 vs. 5.41+/-3.82 %; p=0.032) and higher carotid intima media thickness than normolipidemic controls (0.68+/-0.22 vs. 0.58+/-0.15 mm; p=0.005). In regression analysis, EDV correlated significantly with plasma concentrations of oxLDL (p<0.05) HDL-cholesterol (p<0.05), Apo A1 (p<0.05), ATI (p<0.01) and non-HDL cholesterol (p<0.05). Patients with hyperlipoproteinemia showed higher plasma levels of oxLDL (65.77+/-9.54 vs. 56.49+/-7.80 U/l; p=0.015), malondialdehyde (0.89+/-0.09 vs. 0.73+/-0.08 micromol/l; p=0.010) and nitrites/nitrates (20.42+/-4.88 vs. 16.37+/-4.44 micromol/l; p=0.018) indicating possible higher long-term oxidative stress in these patients.

Oxidative stress, inflammation and autoimmune reaction in type 1 and type 2 diabetes mellitus.

Diabetes mellitus (DM) is associated with oxidative stress, elevation of inflammatory markers and other mechanisms, which may contribute to accelerated atherosclerosis. The aim of the study was to determine prominent factors of these pathogenic processes in patients with DM, to examine their relationship in serum, and to find out the differences between DM1 and DM2. Advanced oxidation protein products (AOPP), C-reactive protein (CRP), pregnancy-associated plasma protein-A (PAPP-A), anticardiolipin antibodies (ACA) and anti-beta2-glycoprotein-1 antibodies (anti-beta2-GPI) were determined in 27 patients with DM1, 27 patients with DM2 and 23 healthy subjects. AOPP, CRP and anti-beta2-GPI were significantly elevated in DM2 in comparison with healthy subjects (p<0.01, p<0.0001, p<0.0001, respectively). In DM1, anti-beta2-GPI were elevated (p<0.0001) as well, but there was no increase of either AOPP or CRP. There was no difference in PAPP-A levels in DM1 or DM2 and healthy subjects. In DM 1, AOPP correlate significantly with anti-beta2-GPI (r = 0.68, p<0.05). In DM2, there is a significant correlation between anti-beta2-GPI and PAPP-A (r=0.45, p<0.05). Oxidative stress and inflammation are more expressed in DM2 and they are partly related. In DM1, oxidative stress seems to be in closer link to autoimmune reaction than to inflammation.

[Ferritin, oxidative stress and coronary atherosclerosis]. / Feritin, oxidacní stres a koronární ateroskleróza.

BACKGROUND AND AIM: In the recent years several studies showed the association between body iron stores, represented by serum ferritin, and atherosclerosis. It was proposed that iron bound to ferritin catalyzes the formation of highly reactive forms of oxygen free radicals which subsequently cause the oxidative modification of atherogenic lipoproteins. Aim of our study was to compare serum ferritin concentrations and certain markers of oxidative stress in patients with and without coronarographically assessed coronary vascular disease. METHODS AND RESULTS: Measurements were performed in 216 subjects at the age of 35-60 years. The patient group included 76 patients with coronarographically assessed coronary vascular disease (CVD) (mean age 51.16 +/- 5.713 years) and 140 healthy controls (mean age 50.21 +/- 5.331 years). The plasma concentration of ferritin was higher in patients (169.04 +/- 63.899 micrograms/l) than controls (87.70 +/- 41.394 micrograms/l), p < 0.001. The group of patients revealed significantly lower plasma concentrations of anti-oxLDL antibodies, nitrites/nitrates, tocopherol and high density lipoprotein cholesterol (HDL-cholesterol) than controls; on the contrary patients had significantly higher concentrations of hemoglobin, thrombocytes and triacylglycerols. In the whole cohort of investigated subjects, ferritin correlated positively with retinol, body mass index (BMI), total-cholesterol, triacylglycerols, low density lipoprotein cholesterol (LDL-cholesterol), blood glucose, creatinine, uric acid, alaninaminotransferase (ALT), aspartateaminotransferase (AST), hematocrite, erythrocytes, with occurrence of CVD and with sex. Inverse correlation was observed between ferritin and HDL-cholesterol. CONCLUSIONS: Our observations are consistent with the hypothesis that high stored iron levels, measured by serum ferritin concentrations, may contribute to the oxidative stress and thus elevate the risk for development of CVD.

UV Rays, the prooxidant/antioxidant imbalance in the cornea and oxidative eye damage.

In this minireview, the factors involved in the development of corneal injury due to an increased amount of UVB rays are summarized. Experimental studies have shown that an increased number of UVB rays leads to a profound decrease in corneal antioxidants (high molecular weight, antioxidant enzymes as well as low molecular weight, mainly ascorbic acid) so that a prooxidant/antioxidant imbalance appears. The decrease of corneal antioxidant protective mechanisms results in oxidative injury of the cornea and causes damage of the inner parts of the eye by UVB rays and by reactive oxygen species generated by them.

Advanced glycation end products in clinical nephrology.

As a result of oxidative and carbonyl stress, advanced glycation end products (AGEs) are involved in the pathogenesis of severe and frequent diseases and their fatal vascular/cardiovascular complications, i.e. diabetes mellitus and its complications (nephropathy, angiopathy, neuropathy and retinopathy, renal failure and uremic and dialysis-associated complications), atherosclerosis and dialysis-related amyloidosis, neurodegenerative diseases, and rheumatoid arthritis. They are formed via non-enzymatic glycation which is specifically enhanced through the presence of oxidative and carbonyl stress, and their ability to form glycoxidation products in peptide and protein structures finally modulating or inducing biological reactivity. Food can be another source of AGEs; however, high serum AGEs in hemodialysis patients might reflect nutritional status better. Several methods of renal replacement therapy have been studied in connection with the AGE removal, but unfortunately the possibilities are still unsatisfactory even if high flux dialysis, hemofiltration, or hemodiafiltration give better results than conventional low flux dialysis. AGEs are currently being studied in the patients on peritoneal dialysis as their precursors can be formed in the dialysis fluid. AGEs can cause damage to the peritoneum and so a loss of ultrafiltration capacity. Many compounds give promising results in AGE inhibition (inhibition of formation of AGEs, inhibition of their action or degradation of AGEs), are tested for these properties, and eventually undergo clinical studies (e.g. aminoguanidine, OPB-9195, pyridoxamine, antioxidants, N-phenacylthiazolium bromide, antihypertensive drugs, angiotensin-converting enzyme inhibitors and angiotensin II receptor-1 antagonists).

Markers of inflammation in preeclampsia.

Advanced oxidation protein products (AOPP) represent terminal products of proteins exposure to free radicals. The aim of this study was to estimate the serum AOPP levels in preeclamptic patients together with ultrasensitive C-reactive protein and anticardiolipin antibodies (ACA) IgG and IgM. 21 women in the third trimester of pregnancy were included in the study--10 women with preeclampsia and 11 women with normal outcome of pregnancy. AOPP levels in preeclampsia were higher than those in normal pregnant women in the third trimester, but not statistically significantly. The comparison with AOPP levels in non-pregnant women has shown a significant increase (P<0.0001). CRP in preeclampsia was significantly increased in comparison with third trimester levels in normal pregnancy (P<0.001) as well as with non-pregnant women (P<0.0001). In preeclampsia, the ACA IgG levels were even significantly lower than in normal pregnant women in the same gestation age, but significantly higher than in non-pregnant women (P<0.001). No difference was found in ACA IgM in preeclampsia and normal third trimester pregnancy and non-pregnant women. A statistically significant negative correlation was found between AOPP and ACA IgG (r= - 0.708, P<0.05). The results indicate enhanced oxidative and inflammatory reaction of maternal organism to pregnancy, which is more pronounced in preeclampsia than in uncomplicated pregnancy.

[Levels of advanced oxidation protein products (AOPP) in the first trimester of pregnancy]. / Hladiny produktu pokrocilé oxidace proteinu (AOPP) v prvním trimestru tehotenství.

Advanced oxidation protein products (AOPP) may be sensitive biomarkers for protein damage mediated by reactive oxygen species. AOPP were measured in the serum of 41 pregnant women in the 8th-12th week of pregnancy. Parameters of prenatal screening in the first trimester (pregnancy-associated plasma protein A--PAPP-A and free beta human chorionic gonadotrophin--free beta HCG) and anticardiolipin antibodies (ACA) IgG and IgM were determined as well. A group of healthy blood donors--women and men was used for comparison. AOPP were determined spectrophotometrically according to Witko-Sarsat [24] (absorbance at 340 nm) and were expressed in chloramine units (mumol/l). Other analytes were determined by immunoanalytic methods. AOPP levels in pregnant women in the first trimester are significantly higher in comparison with blood donors--women (89.46 +/- 33.38 mumol/l vs 57.34 +/- 16.31 mumol/l, p < 0.0001) but there is no statistically significant difference between pregnant women and blood donors--men (89.46 +/- 33.38 mumol/l vs 78.60 +/- 44.01 mumol/l). AOPP level does not correlate either with the age of pregnant women or with the parameters of prenatal screening and ACA IgG and IgM. Higher levels of AOPP in the serum of pregnant women in comparison with women--blood donors may reflect an increase of oxidative stress in pregnancy.

Oxidative stress in the brain tissue of laboratory mice with acute post insulin hypoglycemia.

Malondialdehyde (MDA), Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and selenium-dependent glutathione peroxidase (GSPHx) are currently considered to be basic markers of oxidative stress. MDA is one of the end-products of the peroxidation of membrane lipids, whereas enzymes Cu,Zn-SOD and GSHPx belong to the natural antioxidants. The role of oxygen free radicals in the pathogenesis of many diseases is well documented. The aim of this study was to ascertain the influence of insulin-induced acute hypoglycemia on oxidative stress in the brain tissue. Hypoglycemia was induced in ICR mice by intraperitoneal administration of insulin at a dose 24 IU/kg. There was a correlation between the severity of hypoglycemia and the levels of MDA, Cu,Zn-SOD and GSHPx. The results showed that in severe hypoglycemia (serum glucose concentration below 1.0 mmol/l) the lipoperoxidation in brain tissue expressed as the level of MDA was higher in comparison with normoglycemic controls (glycemia around 3.7 mmol/l) as well as in comparison with the levels of MDA during moderate hypoglycemia (glycemia ranging between 1-2 mmol/l). This indicates the enhancement of lipoperoxidation in the brain tissue during severe hypoglycemia. However, both enzymes - Cu,Zn-SOD or GSHPx - did not show a similar tendency.

Antibodies against oxidized low density lipoproteins in pregnant women.

Oxidized low density lipoproteins (oxLDL) formed in vivo induce a humoral immune response. Oxidative modification of LDL renders it immunogenic and a heterogeneous population of specific anti-oxLDL antibodies is produced. These antibodies could represent a biological marker of oxidative stress and serve as markers of atherosclerosis. Autoantibodies against oxLDL (oLAb) have been detected in human subjects practically of every age. oLAb also appear in the blood of pregnant women. Some studies have shown that the levels of antibodies to oxLDL were elevated in women with established preeclampsia. The present study was aimed to estimate the oLAb IgG levels in the first and second trimester of pregnancy. Furthermore, we estimated the correlation between maternal serum (MS) levels of oLAb and alpha-1-fetoprotein (MS AFP), human chorionic gonadotrophin (MS HCG) and trophoblast-specific-beta-1-glycoprotein (MS SP1), because these proteins are determined as a part of prenatal biochemical screening for fetal congenital abnormalities. Our study deals with the oLAb changes in women with pregnancy-induced hypertension. We also investigated the correlation between oLAb IgG and anticardiolipin antibodies IgG (ACA) in the serum of pregnant women. We examined 40 pregnant women attending Institute for Mother and Child Care for their antenatal care as outpatients. Routine blood samplings between the 9-13th week of pregnancy and 16-18th week of pregnancy were performed as a part of biochemical prenatal screening for fetal congenital abnormalities (Group 1). Their mean age was 27 +/- 4.1 years. Furthermore, we examined 26 women in the second or third trimester with pregnancy-induced hypertension (Group 2). Group 2 was compared with 49 pregnant women in the second or third trimester who were normotensive (Group 3). We used commercial standardized ELISA kits for determination of oLAb IgG, ACA IgG, MS AFP and MS HCG, MS SP1 was analyzed by single radial immunodiffusion. We did not find any differences in the levels of oLAb IgG in the first and second trimester in the women of Group 1. The correlation between oLAb and ACA IgG was not statistically significant (Spearman coefficient r=0.22, p=0.1). The correlation between oLAb IgG with MS AFP, MS HCG and MS SP1 was not statistically significant. Weak negative correlation for AFP and HCG was suggested both in the first and in the second trimester. The levels of oLAb IgG in the group of women with pregnancy-induced hypertension were significantly lower than in the group of normotensive women (348 +/- 388 U/ml v.s. 579 +/- 400 mU/ml, p<0.01). We can conclude that the levels of oLAb do not differ in the first and second trimester of gravidity. However, we cannot exclude the possible influence of an inverse relationship between oLAb IgG titers and the synthesis of fetoplacental antigens. This finding is important especially in the context of the results of prenatal biochemical screening. Pregnancy-induced hypertension is associated with lower levels of oLAb. Weak cross-reactivity between oLAb and anticardiolipin antibodies may exist but there is a possibility that there are two different populations of antibodies reacting with various antigens.

[Advanced oxidation protein products in pregnancy]. / Produkty pokrocilé oxidace proteinu v tehotenství.

OBJECTIVE: Pregnancy and mainly its complications are associated with increased oxidative stress. Advanced oxidation protein products (AOPP) can serve as one of its markers. SETTING: First Institute of Medical Chemistry and Biochemistry and Institute for Clinical Biochemistry, First Medical Faculty, Charles University; Institute for Care of Mother and Child, Prague. METHODS: Together with parameters of prenatal screening, AOPP were measured in the serum of 23 pregnant women in the 2nd trimester of pregnancy. A group of healthy blood donors--women and men was used for comparison. AOPP were determined spectrophotometrically according to Witko-Sarsat (absorbance at 340 nm) and are expressed in chloramin units (mumol/l). RESULTS: Serum AOPP concentrations in pregnant women are significantly higher in comparison with blood donors--women (85.90 +/- 18.70 mumol/l vs 57.34 +/- 16.31 mumol/l, P < 0.0001) but there is no statistically significant difference between pregnant women and blood donors--men (85.90 +/- 18.70 mumol/l vs 78.60 +/- 44.01 mumol/l). AOPP level does not correlate either with the age of pregnant women or with the parameters of prenatal screening (human chorionic gonadotrophin--HCG, alpha-1-fetoprotein--AFP and trophoblast-specific--beta-1-glycoproteion--SP1). CONCLUSION: AOPP as a marker of oxidative stress is increased in the serum of pregnant women in comparison with women--blood donors but is similar as in men--blood donors which supports the hypothesis of hormonal influence. Nevertheless, AOPP do not correlate with the parameters of prenatal screening (HCG, AFP and SP1).

[Effect of fibrates on VLDL and LDL lipoprotein composition and parameters of their oxidation in hypertriglyceridemia]. / Vliv fibrátu na slození lipoproteinu VLDL a LDL a parametry jejich oxidace u hypertriglyceridémie.

BACKGROUND: Meta-analyses of epidemiological studies proved that hypertriacylglycerolemia (HTAG) is an independent CHD risk factor. The VLDL lipoproteins, which are the main TAG carrier, are precursors of atherogenic LDL and their increased concentration is related to the decrease of antiatherogenic HDL, increased ratio of small, dense LDL and represents one of the causes of the endothelial dysfunction. According to some authors, HTAG is one of the factors of the oxidation stress. MATERIAL AND METHODS: 45 patients of the studied group received 200 mg of micronized fenofibrate per day for six weeks. Before the beginning and after the end of treatment, following examinations were carried out: concentration of plasma lipids, lipoproteins, and apolipoproteins, composition of fatty acids (FA) in main lipid plasma classes and LDL (phosphatidylcholine--PC, TAG, cholesteryl esters--CE) and lipoperoxidation in VLDL and LDL, isolated by preparative ultracentrifugation. RESULTS AND CONCLUSIONS: In plasma, the treatment of HTAG led to a significant decrease of TC, TAG and apo-B concentration and to the increase of cholesterol concentration in HDL and in both HDL2 and HDL3 subfractions. In isolated LDL particles we observed a decrease of the TAG portion (by 25%) together with significant lag phase prolongation (by 33%, P < 0.05) and peak time retardation (by 24%, P < 0.05). In VLDL particles the concentration of cholesterol became smaller (by 28%), TAG (by 26%), phospholipids (by 28%) (in all groups P < 0.005) and the lag phase became significantly longer (by 16%, P < 0.01). Treatment with fenofibrate significantly reduced the linoleic acid (18:2n-6) in PC and TAG plasma, CE and TG LDL, in a higher ratio of palmitoleic acid (16:1n-7) in CE LDL, oleic (18:1n-9) in PC LDL, in significant concentration of total monoenic FA in PC and CE LDL and to a significant increase of the concentration of myristic acid (14:0) in CE and myristic and stearic acids (18:0) in TAG LDL. From our results it is possible to conclude that the six-week long treatment of HTAG with micronized phenofibrate led to significant modification of LDL and VLDL composition accompanied by their lower lipoperoxidation indexes. These favourable changes in oxidability were accompanied with changes in the composition of FA in CE, TAG and PC plasma as well as LDL.

[Carbonyl stress and chronic renal failure]. / Karbonylový stres a chronické selhání ledvin.

Pathogenesis of many diseases and their complications is linked to oxidative stress. In the last two years, the attention has been paid also to carbonyl stress which is alosely related to oxidative stress. Carbonyl stress is characterized as an increase of reactive carbonyl compounds caused by their increased formation and/or decreased breakdown and excretion. Reactive carbonyl compounds can be formed from carbohydrates, lipids and amino acids both by oxidative and non-oxidative pathways, can be detoxified by several enzymes and excreated by kidneys depending on their function. Carbonyl compounds can form advanced glycation end-products (AGEs) and advanced lipoperoxidation end-products (ALEs), which are known to take part in the pathogenesis mainly of diabetic and uremic complications.

[Determination of advanced glycation end products]. / Soucasné moznosti stanovení produktu pokrocilé glykace.

Advanced glycation end products--AGEs take part in the pathogenesis of many diseases and their complications--e.g. diabetes mellitus, chronic renal failure, atherosclerosis, Alzheimer's disease. Their determination could be of importance for follow up of progression of these diseases. Determination of AGEs is very difficult due to the heterogeneity of this group of substances, lack of standards, long analytical procedures and necessity of equipment. There are several methods for determination of advanced glycation end products: immunochemical method--ELISA (enzyme linked immunosorbent assay) with the use of either polyclonal or monoclonal antibodies, fluorimetry using the characteristic fluorescence spectrum of AGEs, HPLC--high performance liquid chromatography, and MS--mass spectrometry. Mass spectrometry is the best and most precise method. When coupled with other methods, mass spectrometry is a unique research method, which can be used mainly for characterization of new AGEs. HPLC is very precise as well, but its drawback is time-consuming sample preparation. Fluorimetry and ELISA could be in the future used for the special diagnostic of complications of chronic diseases.

Reactive oxygen species (ROS)-generating oxidases in the normal rabbit cornea and their involvement in the corneal damage evoked by UVB rays.

The corneas of albino rabbits were irradiated (5 min exposure once a day) with UVB rays (312 nm) for 4 days (shorter procedure) or 8 days (longer procedure). The eyes were examined microbiologically and only the corneas of sterile eyes or eyes with non-pathogenic microbes were employed. Histochemically, the activities of reactive oxygen species (ROS)-generating oxidases (xanthine oxidase, D-amino acid oxidase and alpha-hydroxy acid oxidase) were examined in cryostat sections of the whole corneas. Biochemically, the activity of xanthine oxidoreductase/xanthine oxidase was investigated in the scraped corneal epithelium. UVB rays significantly changed enzyme activities in the corneas. In comparison to the normal cornea, where of ROS-generating oxidases only xanthine oxidase showed significant activity in the corneal epithelium and endothelium, D-amino acid oxidase was very low and alpha-hydroxy acid oxidase could not be detected at all, in the cornea repeatedly irradiated with UVB rays, increased activities of xanthine oxidase and D-amino acid oxidase were observed in all corneal layers. Only after the longer procedure the xanthine oxidase and D-amino acid oxidase activities were decreased in the thinned epithelium in parallel with its morphological disturbances. Further results show that the xanthine oxidase/xanthine oxidoreductase ratio increased in the epithelium together with the repeated irradiation with UVB rays. This might suggest that xanthine dehydrogenase is converted to xanthine oxidase. However, in comparison to the normal corneal epithelium, the total amount of xanthine oxidoredutase was decreased in the irradiated epithelium. It is presumed that xanthine oxidoreductase might be released extracellularly (into tears) or the enzyme molecules were denatured due to UVB rays (particulary after the longer procedure). Comparative histochemical and biochemical findings suggest that reactive oxygen species-generating oxidases (xanthine oxidase, D-amino acid oxidase) contribute to the corneal damage evoked by UVB rays.

Quinolinic acid-iron(ii) complexes: slow autoxidation, but enhanced hydroxyl radical production in the Fenton reaction.

Quinolinate (pyridine-2,3-dicarboxylic acid, Quin) is a neurotoxic tryptophan metabolite produced mainly by immune-activated macrophages. It is implicated in the pathogenesis of several brain disorders including HIV-associated dementia. Previous evidence suggests that Quin may exert its neurotoxic effects not only as an agonist on the NMDA subtype of glutamate receptor, but also by a receptor-independent mechanism. In this study we address ability of ferrous quinolinate chelates to generate reactive oxygen species. Autoxidation of Quin-Fe(II) complexes, followed in Hepes buffer at pH 7.4 using ferrozine as the Fe(II) detector, was found to be markedly slower in comparison with iron unchelated or complexed to citrate or ADP. The rate of Quin-Fe(II) autoxidation depends on pH (squared hydroxide anion concentration), is catalyzed by inorganic phosphate, and in both Hepes and phosphate buffers inversely depends on Quin concentration. These observations can be explained in terms of anion catalysis of hexaaquairon(II) autoxidation, acting mainly on the unchelated or partially chelated pool of iron. In order to follow hydroxyl radical generation in the Fenton chemistry, electron paramagnetic resonance (EPR) spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was employed. In the mixture consisting of 100 mM DMPO, 0.1 mM Fe(II), and 8.8 mM hydrogen peroxide in phosphate buffer pH 7.4, 0.5 mM Quin approximately doubled the yield of DMPO-OH adduct, and higher Quin concentration increased the spin adduct signal even more. When DMPO-OH was pre-formed using Ti3+ /hydrogen peroxide followed by peroxide removal with catalase, only addition of Quin-Fe(II), but not Fe(II), Fe(III), or Quin-Fe(III), significantly promoted decomposition of pre-formed DMPO-OH. Furthermore, reaction of Quin-Fe(II) with hydrogen peroxide leads to initial iron oxidation followed by appearance of iron redox cycling, detected as slow accumulation of ferrous ferrozine complex. This phenomenon cannot be abolished by subsequent addition of catalase. Thus, we propose that redox cycling of iron by a Quin derivative, formed by initial attack of hydroxyl radicals on Quin, rather than effects of iron complexes on DMPO-OH stability or redox cycling by hydrogen peroxide, is responsible for enhanced DMPO-OH signal in the presence of Quin. The present observations suggest that Quin-Fe(II) complexes display significant pro-oxidant characteristics that could have implications for Quin neurotoxicity.

Oxidative stress, metabolism of ethanol and alcohol-related diseases.

Alcohol-induced oxidative stress is linked to the metabolism of ethanol. Three metabolic pathways of ethanol have been described in the human body so far. They involve the following enzymes: alcohol dehydrogenase, microsomal ethanol oxidation system (MEOS) and catalase. Each of these pathways could produce free radicals which affect the antioxidant system. Ethanol per se, hyperlactacidemia and elevated NADH increase xanthine oxidase activity, which results in the production of superoxide. Lipid peroxidation and superoxide production correlate with the amount of cytochrome P450 2E1. MEOS aggravates the oxidative stress directly as well as indirectly by impairing the defense systems. Hydroxyethyl radicals are probably involved in the alkylation of hepatic proteins. Nitric oxide (NO) is one of the key factors contributing to the vessel wall homeostasis, an important mediator of the vascular tone and neuronal transduction, and has cytotoxic effects. Stable metabolites--nitrites and nitrates--were increased in alcoholics (34.3 +/- 2.6 vs. 22.7 +/- 1.2 micromol/l, p < 0.001). High NO concentration could be discussed for its excitotoxicity and may be linked to cytotoxicity in neurons, glia and myelin. Formation of NO has been linked to an increased preference for and tolerance to alcohol in recent studies. Increased NO biosynthesis also via inducible NO synthase (NOS, chronic stimulation) may contribute to platelet and endothelial dysfunctions. Comparison of chronically ethanol-fed rats and controls demonstrates that exposure to ethanol causes a decrease in NADPH diaphorase activity (neuronal NOS) in neurons and fibers of the cerebellar cortex and superior colliculus (stratum griseum superficiale and intermedium) in rats. These changes in the highly organized structure contribute to the motor disturbances, which are associated with alcohol abuse. Antiphospholipid antibodies (APA) in alcoholic patients seem to reflect membrane lesions, impairment of immunological reactivity, liver disease progression, and they correlate significantly with the disease severity. The low-density lipoprotein (LDL) oxidation is supposed to be one of the most important pathogenic mechanisms of atherogenesis, and antibodies against oxidized LDL (oxLDL) are some kind of epiphenomenon of this process. We studied IgG oxLDL and four APA (anticardiolipin, antiphosphatidylserine, antiphosphatidylethanolamine and antiphosphatidylcholine antibodies). The IgG oxLDL (406.4 +/- 52.5 vs. 499.9 +/- 52.5 mU/ml) was not affected in alcoholic patients, but oxLDL was higher (71.6 +/- 4.1 vs. 44.2 +/- 2.7 micromol/l, p < 0.001). The prevalence of studied APA in alcoholics with mildly affected liver function was higher than in controls, but not significantly. On the contrary, changes of autoantibodies to IgG oxLDL revealed a wide range of IgG oxLDL titers in a healthy population. These parameters do not appear to be very promising for the evaluation of the risk of atherosclerosis. Free radicals increase the oxidative modification of LDL. This is one of the most important mechanisms, which increases cardiovascular risk in chronic alcoholic patients. Important enzymatic antioxidant systems - superoxide dismutase and glutathione peroxidase - are decreased in alcoholics. We did not find any changes of serum retinol and tocopherol concentrations in alcoholics, and blood and plasma selenium and copper levels were unchanged as well. Only the zinc concentration was decreased in plasma. It could be related to the impairment of the immune system in alcoholics. Measurement of these parameters in blood compartments does not seem to indicate a possible organ, e.g. liver deficiency.

[New markers of advanced damage caused by oxidative and carbonyl stress]. / Nové markery pokrocilého poskození oxidacním a karbonylovým stresem.

Oxidative stress, which is characterized as dysbalance between free radicals and antioxidants in favour of radicals, participates in the pathogenesis of many diseases and their complications. Carbonyl stress is closely related to oxidative stress and is described as increase of reactive carbonyl compounds caused by their increased formation or decreased degradation and clearance. Both oxidative and carbonyl stresses cause damage to proteins--they lead to formation of advanced oxidation protein products--AOPP, advanced glycation end-products--AGEs and advanced lipoperoxidation end-products--ALEs. These compounds have several biological effects--e.g. stimulation of secretion of cytokines, adhesive molecules and growth factors and take part in the development of complications of diabetes mellitus and chronic renal failure.