Joachim Greilberger

Oxidative damage of albumin in advanced liver disease

Albumin has a number of biological functions and the serum albumin level is related to prognosis in advanced liver disease. Oxidative stress is believed to play an important role in the pathogenesis of liver failure. The aim of the present study was to characterize oxidative modification of albumin in patients with various degrees of liver failure and to investigate implications for its binding function. Patients with liver cirrhosis (n=10), acute-on-chronic liver failure (n=8) and healthy controls (n=15) were included in the study. Three fractions of albumin were separated by HPLC according to the redox state of cysteine-34 and detected by fluorescence as well as UV absorption. Carbonyl groups were measured as a marker of oxidative modification in plasma proteins and, by western blotting, on albumin. Progressive oxidative modification of albumin was found with increasing severity of liver failure indicated by an increased content of carbonyl groups and oxidation of cysteine-34. Fluorescence properties of albumin were altered by oxidation and, in patients with acute-on-chronic liver failure, by high plasma levels of bilirubin. This alteration of albumin fluorescence by bilirubin provides evidence for a preferred binding of bilirubin to the fully reduced form of albumin.

Malondialdehyde, carbonyl proteins and albumin-disulphide as useful oxidative markers in mild cognitive impairment and Alzheimer's disease

The question arises as to whether oxidative stress has a primary role in neurodegeneration or is a secondary end-stage epiphenomenon. The aim of the present study was to determine oxidative stress parameters like malondialdehyde (MDA), carbonyl proteins (CP) and Albumin-disulphide (Alb-SSR) and relate these parameters to the immune parameter neopterin, folic acid and vitamin B12 as vitamins and homocysteine in patients with neuro-degenerative diseases (NDD), namely mild cognitive impairment (MCI) and Alzheimers disease (AD) compared to an aged matched control group. MDA, CP and Alb-SSR were significantly increased in the NDD group compared to controls, but not vitamin B12, folic acid and neopterin. Significant correlations were found between CP and Alb-SSR, CP and MDA and between MDA and Alb-SSR including patients with NDD and the control group. These results support the hypothesis that oxidative damage to lipids and proteins is an important early event in the pathogenesis of neurodegenerative diseases.

Probiotic supplementation affects markers of intestinal barrier, oxidation, and inflammation in trained men; a randomized, double-blinded, placebo-controlled trial.

BackgroundProbiotics are an upcoming group of nutraceuticals claiming positive effects on athlete’s gut health, redox biology and immunity but there is lack of evidence to support these statements.MethodsWe conducted a randomized, double-blinded, placebo controlled trial to observe effects of probiotic supplementation on markers of intestinal barrier, oxidation and inflammation, at rest and after intense exercise. 23 trained men received multi-species probiotics (1010 CFU/day, Ecologic®Performance or OMNi-BiOTiC®POWER, n = 11) or placebo (n = 12) for 14 weeks and performed an intense cycle ergometry over 90 minutes at baseline and after 14 weeks. Zonulin and α1-antitrypsin were measured from feces to estimate gut leakage at baseline and at the end of treatment. Venous blood was collected at baseline and after 14 weeks, before and immediately post exercise, to determine carbonyl proteins (CP), malondialdehyde (MDA), total oxidation status of lipids (TOS), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6). Statistical analysis used multifactorial analysis of variance (ANOVA). Level of significance was set at p < 0.05, a trend at p < 0.1.ResultsZonulin decreased with supplementation from values slightly above normal into normal ranges (<30 ng/ml) and was significantly lower after 14 weeks with probiotics compared to placebo (p = 0.019). We observed no influence on α1-antitrypsin (p > 0.1). CP increased significantly from pre to post exercise in both groups at baseline and in the placebo group after 14 weeks of treatment (p = 0.006). After 14 weeks, CP concentrations were tendentially lower with probiotics (p = 0.061). TOS was slightly increased above normal in both groups, at baseline and after 14 weeks of treatment. There was no effect of supplementation or exercise on TOS. At baseline, both groups showed considerably higher TNF-α concentrations than normal. After 14 weeks TNF-α was tendentially lower in the supplemented group (p = 0.054). IL-6 increased significantly from pre to post exercise in both groups (p = 0.001), but supplementation had no effect. MDA was not influenced, neither by supplementation nor by exercise.ConclusionsThe probiotic treatment decreased Zonulin in feces, a marker indicating enhanced gut permeability. Moreover, probiotic supplementation beneficially affected TNF-α and exercise induced protein oxidation. These results demonstrate promising benefits for probiotic use in trained men.Clinical trial registryhttp://www.clinicaltrials.gov, identifier: NCT01474629

Single bouts of exercise affect albumin redox state and carbonyl groups on plasma protein of trained men in a workload-dependent manner

The purpose of this study was to investigate the effect of single bouts of exercise at three different intensities on the redox state of human serum albumin (HSA) and on carbonyl groups on protein (CP) concentrations in plasma. Trained men [n = 44, maximal oxygen consumption (Vo(2max)): 55 +/- 5 ml.kg(-1).min(-1), nonsmokers, 34 +/- 5 years of age] from a homogenous population, volunteers from a police special forces unit, were randomly assigned to perform on a cycle ergometer either at 70% (n = 14), 75% (n = 14), or 80% (n = 16) of Vo(2max) for 40 min. Blood was collected before exercise, immediately after the exercise test (IE), and 30 min after each test (30M) and 30 h after each test (30H). The reduced fraction of HSA, human mercaptalbumin (HMA), decreased at all three exercise intensities IE and 30M, returning to preexercise values by 30H (P < 0.05). HMA was primarily oxidized to its reversible fraction human nonmercaptalbumin 1 (HNA1). CP concentrations increased at 75% of Vo(2max) IE and 30M with a tendency (P < 0.1) and at 80% Vo(2max) IE and 30M significantly, returning to preexercise concentrations by 30H (P < 0.01). These results indicate that the HSA redox system in plasma is activated after a single bout of cycle ergometer exercise at 70% Vo(2max) and 40 min duration. The extent of the HSA modification increased with exercise intensity. Oxidative protein damage, as indicated by CP, was only significantly increased at 80% Vo(2max) intensity in this homogenous cohort of trained men.

Oxidative stress and epididymal sperm transport, motility and morphological defects

OBJECTIVES Radical oxidative species (ROS) have an important effect on sperm quality and quantity. Oxidative stress (OS) occurs when production of potentially destructive reactive oxygen species (ROS) exceeds the bodys own natural antioxidant defenses, resulting in cellular damage. OS is a common pathology seen in approximately half of all infertile men. Increased ROS generation and reduced antioxidant capacity is negatively correlated with sperm concentration and motility in infertile men. For the first time, we used a more stable and reliable sensitive carbonyl protein (CP) detection method to determine ROS in seminal plasma than measuring ROS directly to clarify the effect of OS on spermatozoa in terms of protein dysfunction. This is the first report to measure CP in seminal plasma as an indicator of OS. Furthermore, for the first time we correlated the results of CP measurement with light microscopy in combination with ultrastructural analysis by electron microscopy. MATERIAL AND METHODS 20 patients with idiopathic oligoasthenoteratozoospermia (iOAT) and 10 fertile controls were enrolled in this study. CP values were measured by enzyme-linked immuno sorbent assay (ELISA) to detect the level of OS. Transmission electron microscope (TEM) was used to detect axonemal anomalies. RESULTS Compared to fertile controls, statistically highly significant higher degrees of abnormal sperm parameters (P<0.001) could be found in iOAT patients. CP values were highly significantly elevated in iOAT patients than in normal controls (P<0.001). A statistically highly significant difference in different axonemal anomalies were found between iOAT patients and normal controls (P<0.001). CP values have been found to be positively correlated with different axonemal anomalies (absence of axoneme (r(2)=0.841), missing of central singlet tubules (r(2)=0.702) and missing of outer doublet tubules (r(2)=0.869). A statistically negative correlation were found between different axonemal anomalies (absent axoneme (r(2)=-0.780), missing of central singlet tubules (r(2)=-0.611), and missing of outer doublet tubules (r(2)=-0.738) and forward progressive sperm motility. CONCLUSION High levels of CP can be measured in iOAT patients, indicating that OS could underlie the aetipopathogenesis of the syndrome. OS negatively affects flagellar axonemal structure with subsequent impairment of forward progressive sperm motility. This can put an attention for antioxidants as a therapy for iOAT syndrome and further research to find how to decrease ROS production.

Effect of hemodialysis on the antioxidative properties of serum.

In patients with chronic renal failure undergoing regular hemodialysis (HD), oxidative stress is involved in the development of dialysis-related pathologies. The aim of the study was to measure the effect of HD treatment on the general antioxidative status of serum with special consideration of the specific oxidizability of lipids and proteins. Indicators for the oxidative/antioxidative status of plasma were monitored at the beginning and at the end of a dialysis session on the arterial and venous side of the dialyzer. A decrease in the antioxidant status was accompanied by an increased oxidizability of proteins as well as lipids during HD treatment. During the first passage of the dialyzer, the lag time of lipid oxidation decreased from 114.0+/-19.8 to 81.5+/-18.9 min, the lag time of protein oxidation decreased from 105.0+/-24.6 to 72.9+/-21.3 min and the total antioxidative status decreased from 518+/-24 to 252+/-124 microM trolox equivalents. The carbonyl content of serum proteins was high in patients with end stage renal disease (ESRD) (3.9+/-1.1 vs. 0.9+/-0.1 nmol/mg in controls) but did not change significantly during dialysis procedure. Our data demonstrate that the susceptibility of serum lipids and proteins to oxidative modification is severely increased by HD treatment.

Protein Modification Responds to Exercise Intensity and Antioxidant Supplementation

PURPOSE To assess the effects of different exercise intensities and antioxidant supplementation on plasma protein modification. METHODS Trained men (n = 41) from a homogenous population were randomly assigned to perform cycle ergometer exercise either at 70% or 80% of individual .VO2max. Each intensity group was randomly assigned to receive either juice powder concentrate (JPC 70%, n = 11; JPC 80%, n = 10) or placebo (Plac 70%, n = 10; Plac 80%, n = 10) capsules for 28 wk. Four controlled exercise bouts and blood collections were conducted at baseline and study weeks 4, 16, and 28. Blood samples were drawn before (BE), immediately after (IE), and 30 min (30M) and 30 h (30H) postexercise. These samples were analyzed to estimate concentrations of carbonyl groups on plasma proteins (CP) and the redox state of human serum albumin (HSA). RESULTS In the Plac group, CP concentrations increased at 80% of .VO2max IE and 30M, returning to preexercise concentrations by 30H (P < 0.05). At both 16 and 28 wk, the Plac groups had significantly higher BE and 30H CP concentrations than the JPC groups (P < 0.05). The reduced fraction of HSA, human mercaptalbumin (HMA), decreased at all four exercise tests at both exercise intensities IE and 30M, returning to preexercise values by 30H (P < 0.05). Supplementation had no influence on HSA. CONCLUSIONS These results indicate that CP concentrations increase with 80% .VO2max intensity. The JPC group had lower baseline CP levels after 16 and 28 wk and no exercise-induced CP increase. HSA is reversibly shifted to a more oxidized state by recent intense exercise.

T-Cadherin Mediates Low-Density Lipoprotein-Initiated Cell Proliferation Via the Ca2+-Tyrosine Kinase-Erk1/2 Pathway

The GPI-anchored protein T-cadherin was found to be an atypical LDL binding site that is expressed in various types of cells, including endothelial cells, smooth muscle cells, and neurons. Notably, the expression of T-cadherin was reduced in numerous types of cancers, although it was up-regulated in tumor-penetrating blood vessels, atherosclerotic lesions, and during neointima formation. Despite these intriguing findings, our knowledge of the physiological role and the signal transduction pathways associated with this protein is limited. Therefore, T-cadherin was overexpressed in the human umbilical vein-derived endothelial cell line EA.hy926, the human embryonic kidney cell line HEK293, and LDL-initiated signal transduction, and its consequences were elucidated. Our data revealed that T-cadherin serves as a receptor specifically for LDL. Following LDL binding to T-cadherin, mitogenic signal transduction was initiated that involved activation of PLC and IP3 formation, which subsequently yielded intracellular Ca2+ mobilization. Downstream to these early phenomena, activation of tyrosine kinase(s) Erk 1/2 kinase, and the translocation of NFκB toward the nucleus were found. Finally, overexpression of T-cadherin in HEK293 cells resulted in accelerated cell proliferation in an LDL-dependent manner, although cell viability was not influenced. Because LDL uptake was not facilitated by T-cadherin, our data suggest that T-cadherin serves as a signaling receptor for LDL that facilitates an LDL-dependent mitogenic signal in the vasculature.

Short-term supplementation with alpha-ketoglutaric acid and 5-hydroxymethylfurfural does not prevent the hypoxia induced decrease of exercise performance despite attenuation of oxidative stress.

Reactive oxygen species are thought to partly be responsible for the hypoxia induced performance decrease. The present study evaluated the effects of a broad based antioxidant supplementation or the combined intake of alpha-ketoglutaric acid (α-KG) and 5-hydroxymethylfurfural (5-HMF) on the performance decrease at altitude. 18 healthy, well-trained males (age: 25±3 years; height: 179±6 cm; weight: 76.4±6.8 kg) were randomly assigned in a double-blind fashion to a placebo group (PL), a α-KG and 5-HMF supplementation group (AO1) or a broad based antioxidant supplementation group (AO2). Participants performed 2 incremental exercise tests to exhaustion on a cycle ergometer; the first test under normoxia and the second under hypoxia conditions (simulated altitude, FiO2=13% ~ 4 300 m). Supplementation started 48 h before the hypoxia test. Maximal oxygen uptake, maximal power output, power output at the ventilatory and lactate threshold and the tissue oxygenation index (NIRS) were measured under both conditions. Oxidative stress markers were measured before the supplementation and after the hypoxia test. Under hypoxia conditions all performance parameters decreased in the range of 19-39% with no differences between groups. A significant change from normoxia to hypoxia (p<0.001) and between groups (p=0.038) were found for the tissue oxygenation index. Post hoc test revealed significant differences between the PL and both, the AO1 and the AO2 group. The oxidative stress parameter carbonyl protein changed from normoxia to hypoxia in all participants and 4-hydroxynonenal decreased in the AO1 group only. In conclusion the results suggest that short-term supplementation with an antioxidant does not prevent the performance decrease at altitude. However, positive effects on muscle oxygen extraction, as indicated by the tissue oxygenation index, might indicate that mitochondrial functioning was actually influenced by the supplementation.

Neopterin and 7,8-dihydroneopterin interfere with low density lipoprotein oxidation mediated by peroxynitrite and/or copper.

Low density lipoprotein (LDL) oxidation within the artery wall likely represents a key event in the formation of atherosclerotic lesions. Oxidatively modified LDL particles exert chemotactic properties on macrophages, and the uncontrolled uptake of modified LDL by macrophages leads to the formation of lipid-loaded foam cells, a hallmark of early stage atherosclerosis. Human macrophages stimulated by interferon- n generate reactive oxygen species (ROS), neopterin, and 7,8-dihydroneopterin. Higher concentrations of neopterin were found in atherosclerosis, and earlier studies have provided evidence that these neopterin derivatives are able to interfere with reactive species. We therefore investigated whether they also modulate LDL oxidation mediated by Cu(II) and/or peroxynitrite (ONOO m ). By means of UV-absorption recording the formation of conjugated dienes in the course of lipid oxidation as well as by measuring the relative electrophoretic mobility of oxidized LDL, we found that neopterin is capable of enhancing ONOO m - as well as Cu(II)-mediated LDL oxidation, whereas 7,8-dihydroneopterin mainly protects LDL from oxidation. However, in case of Cu(II)-mediated LDL oxidation, an initial prooxidative effect of 7,8-dihydroneopterin could be observed. We hypothesize that 7,8-dihydroneopterin may chemically reduce Cu(II) to Cu(I) thereby increasing its oxidative capacity. After total reduction of Cu(II), excess 7,8-dihydroneopterin may block the oxidative potential of Cu(I) and thus decrease the oxidation of LDL. These findings confirm the general behavior of pteridines in redox processes and suggest an in vivo contribution to the process of LDL oxidation.