Oxidative stress in chronic lymphoedema.

BACKGROUND: Chronic lymphoedema is one of the most frequent and debilitating complications after surgical and radiological tumour treatment. Prevention and therapy of lymphoedema is therefore an important problem of the rehabilitation of those patients. AIM: To investigate whether chronic lymphoedema results in increased oxidative stress. DESIGN: Prospective case-control study. METHODS: We obtained venous blood samples from patients (n=38) with chronic lymphoedema and determined biomarkers of prooxidative reactions and of antioxidative defense system in the erythrocytes or blood plasma: reduced and oxidized glutathione (GSH and GSSG), and lipid peroxidation products such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE). Healthy volunteers (n=90) and patients who had undergone surgical and/or radiotherapeutic treatment of tumours without consequent lymphoedema (n=20) acted as controls. RESULTS: The blood of patients with chronic lymphoedema contained lower concentrations of GSH and higher levels of GSSG and of MDA and HNE, compared with the control group. MDA was increased by about three-fold in the serum of the lymphoedema patients. Accelerated free radical formation and lipid peroxidation processes were further demonstrated by the liberation of MDA and HNE into the blood serum after manual lymph drainage. DISCUSSION: Our data demonstrate enhanced formation of reactive oxygen species (ROS) and accelerated lipid peroxidation processes in chronic lymphoedematous tissue. The strengthening of antioxidative defense mechanisms could be useful in the therapy of chronic lymphoedema.

Carotenoid oxidative degradation products inhibit Na+-K+-ATPase.

This study investigates the biological significance of carotenoid oxidation products using inhibition of Na+-K+-ATPase activity as an index. Beta-carotene was completely oxidized by hypochlorous acid and the oxidation products were analyzed by capillary gas-liquid chromatography and high performance liquid chromatography. The Na+-K+-ATPase activity was assayed in the presence of these oxidized carotenoids and was rapidly and potently inhibited. This was demonstrated for a mixture of beta-carotene oxidative breakdown products, beta-Apo-10'-carotenal and retinal. Most of the beta-carotene oxidation products were identified as aldehydic. The concentration of the oxidized carotenoid mixture that inhibited Na+-K+-ATPase activity by 50% (IC50) was equivalent to 10 microM non-degraded beta-carotene, whereas the IC50 for 4-hydroxy-2-nonenal, a major lipid peroxidation product, was 120 microM. Carotenoid oxidation products are more potent inhibitors of Na+-K+-ATPase than 4-hydroxy-2-nonenal. Enzyme activity was only partially restored with hydroxylamine and/or beta-mercaptoethanol. Thus, in vitro binding of carotenoid oxidation products results in strong enzyme inhibition. These data indicate the potential toxicity of oxidative carotenoid metabolites and their activity on key enzyme regulators and signal modulators.

Erythrocyte free radical and energy metabolism.

The erythrocyte is a highly specialized cell whose main functions are oxygen transport and the mediation of carbon dioxide transport. Energy production in the mature erythrocyte depends on glycolysis, with glucose as the principal substrate. Glycolysis and the oxidative pentose phosphate pathway generate NADH and NADPH to reduce methemoglobin, which is being continuously produced, and the antioxidant glutathione, which is present in high concentrations. Red blood cells are equipped with a highly effective antioxidant defense even without the glutathione system. Compared with other cell types, they possess high activities of the most important antioxidant enzymes. Most of the nonenzymatic antioxidant capacity of whole blood is likewise localized in the erythrocytes. Circulating red cells are mobile free radical scavengers and provide antioxidant protection to other tissues and organs. An imbalance between pro-oxidant reactions and antioxidant defense is described in patients with chronic renal failure. Oxidative stress increases as antioxidant defenses are weakened by pro-oxidant hemodialysis factors; it increases further still in renal anemia with a very low red cell count. Thus in terms of free radical metabolism, the only arguments remaining over the complete correction of renal anemia are those in favor, with none against.

Succinylpurinemic autism: increased sensitivity of defective adenylosuccinate lyase towards 4-hydroxy-2-nonenal.

We studied the effect of trans-4-hydroxy-2-nonenal on the wild-type human adenylosuccinate lyase and on the enzyme from a patient compound-heterozygous for two missense mutations (P75A/D397Y; McKusick 103050.0003/103050.0004). Both the enzymes were inhibited by 10-50 microM trans-4-hydroxy-2-nonenal in a concentration-dependent manner by means of a mixed-type co-operative mechanism. A significantly stronger inhibition was noticed in the presence of the defective enzyme. Nonanal and trans-2,3-nonenal inhibited the enzymes to a less extent and at about 10-times higher concentrations. Hydroxylamine reversed the inhibition by trans-4-hydroxy-2-nonenal, trans-2,3-nonenal or nonanal in the case of the wild-type enzyme, but it was ineffective to reverse the inhibition by trans-4-hydroxy-2-nonenal on the defective enzyme. Dithiothreitol slightly decreased the inhibition exerted by trans-4-hydroxy-2-nonenal on both the wild-type and the defective adenylosuccinate lyase, while it did not produce practically any change in the presence of trans-2,3-nonenal or nonanal.

Improved antioxidative protection in winter swimmers.

Adaptation to oxidative stress is an improved ability to resist the damaging effects of reactive oxygen species, resulting from pre-exposure to a lower dose. Changes in uric acid and glutathione levels during ice-bathing suggest that the intensive voluntary short-term cold exposure of winter swimming produces oxidative stress. We investigated whether the repeated oxidative stress in winter swimmers results in improved antioxidative adaptation. We obtained venous blood samples from winter swimmers and determined important components of the antioxidative defense system in the erythrocytes or blood plasma: reduced and oxidized glutathione (GSH and GSSG), and the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (Cat). The control group consisted of healthy people who had never participated in winter swimming. The baseline concentration of GSH and the activities of erythrocytic SOD and Cat, were higher in winter swimmers. We interpret this as an adaptative response to repeated oxidative stress, and postulate it as a new basic molecular mechanism of increased tolerance to environmental stress.

Metabolism of 4-hydroxynonenal, a cytotoxic lipid peroxidation product, in thymocytes as an effective secondary antioxidative defense mechanism.

The metabolism of the aldehydic lipid peroxidation product, 4-hydroxynonenal (HNE),was studied in suspensions of mouse thymocytes. Thymocytes are characterized by low lipid peroxidation in comparison with other cell types notwithstanding their high content of arachidonic acid. In our study a very high capacity of HNE metabolism in thymocytes was observed: 27.7 nmol/mg w.w./min. That is about the same HNE degradation rate as determined in liver cells or small intestinal enterocytes, which are the cells with the by far highest capacity for the degradation of HNE and other aldehydic lipid peroxidation products in comparison with other cell types. The primary and secondary HNE metabolites in thymocytes were identified and quantified after the addition of 100 microM HNE to thymocyte suspensions: the glutathione-HNE conjugate, the hydroxynonenoic acid, the 1,4-dihydroxynonene, water, and the glutathione-dihydroxynonene conjugate. Furthermore, the HNE binding to proteins was measured. The very rapid HNE degradation in thymocytes besides the high amounts of lipophilic chain-breaking antioxidants is postulated to be an important secondary antioxidative mechanism and the main factor for the low accumulation of lipid peroxidation products in these cells.1668

Identification of metabolic pathways of the lipid peroxidation product 4-hydroxynonenal in in situ perfused rat kidney.

The metabolism of the cytotoxic lipid peroxidation product 4-hydroxynonenal was studied in perfused rat kidney. We investigated the total capacity of the rat kidney to metabolize 4-hydroxynonenal (HNE) and quantified the metabolites in the venous effluents as well as in the excreted urine. A rapid utilization of HNE was demonstrated, due to its immediate reactions with cellular compounds and its metabolism. During the first 3 min more than 80% of the infused HNE was metabolized in the perfused kidney. Glutathione-HNE conjugate (GSH-HNE: 35%), the corresponding alcohol 1,4-dihydroxynonene (1,4-DHN: 12%), HNE-mercapturic acid conjugate (HNE-MA: 4%), 4-hydroxynonenoic acid (HNA: 7%), tricarboxylic acid (TCA-cycle metabolites), and water (32%) were identified as primary and secondary metabolic products. We postulated that the total capacity of rat kidney to metabolize 4-hydroxynonenal with about 160-190 nmol/g wet wt/min. (initial influent concentration was 100 nmol/ml HNE) and other aldehydic products of lipid peroxidation is in the same range as that in other organs, e.g., intestine with 22 nmol/g wet wt/min (initial 70 nmol/ml HNE) (Siems et al. 1995. Life Sci. 57: 785-789) and heart with about 50 nmol/g wet wt/min (initial 10 nmol/ml HNE) (Grune et al. 1994. Cell Biochem. Funct. 12: 143-147). Compared to other organs, liver and kidney seemed to be the most important organs for the elimination of the final products of metabolism. The importance of the kidney in the formation of HNE-mercapturic acid conjugate was demonstrated (Alary et al. 1995. Chem. Res Toxicol. 8: 34-39). The selective excretion of this final metabolite of aldehyde metabolism may be of central importance in the detoxification of a number of lipid peroxidation products.

Metabolic fate of 4-hydroxynonenal in hepatocytes: 1,4-dihydroxynonene is not the main product.

4-Hydroxynonenal (HNE) is a major aldehydic product of lipid peroxidation known to exert several biological and cytotoxic effects. The metabolic fate of this aldehyde was investigated in hepatocytes as a cell type with a rapid HNE degradation. The experiments were carried out in rat hepatocytes at 37 degrees C at initial HNE concentrations of 1 microM-that means in the range of physiological and pathophysiologically relevant HNE levels-, 5 microM or 100 microM, respectively. About 95% of 100 microM HNE was degraded within 3 min of incubation. At 1 microM HNE the physiological level of about 0.1 to 0.2 microM was restored already after 30 sec. As primary products of HNE in hepatocytes the glutathione-HNE- 1:1-adduct, the hydroxynonenoic acid and the corresponding alcohol of HNE, the 1,4-dihydroxynon-2-ene, were identified. In contrast to previous reports, the corresponding alcohol of the HNE, 1,4-dihydroxynon-2-ene, was not the main HNE metabolite by far. The sum of these three primary HNE products accounts for about two-thirds of the total HNE degradation after 3 min of incubation. Furthermore, the beta-oxidation of hydroxynonenoic acid including the formation of water was demonstrated. The quantitative share of HNE binding to proteins, contrary to its great functional importance, is low with about 3% of total HNE consumption after 3 min incubation. The glycine-cysteine-HNE, cysteine-HNE adducts, and the mercapturic acid from glutathione-HNE adduct are not formed. In total, almost 90% of HNE degradation could be balanced by the formation of different HNE metabolites. The fast metabolism underlines the role of HNE degrading pathways in hepatocytes as one important part of the antioxidative defense system in order to protect proteins from modification by aldehydic lipid peroxidation products.

Inhibition of NADPH oxidase-mediated superoxide radical formation in PMA-stimulated human neutrophils by 4-hydroxynonenal--binding to -SH and -NH2 groups.

4-Hydroxynonenal (HNE), a major lipid peroxidation product, effectively inhibits the superoxide radical formation by NADPH oxidase of phorbol myristate acetate (PMA)--stimulated human PMNL. The I50 value for the inhibition of NADPH oxidase-mediated superoxide radical formation by 4-hydroxynonenal was found to be 19 microM. The HNE inhibition involves the reaction with both -SH and -NH2 groups. Superoxide formation as final result of the NADPH oxidase cascade was almost completely restored by addition of dithiothreitol. In presence of hydroxylamine only a minor restoration of superoxide radical formation was found. A combination of dithiothreitol and hydroxylamine yielded the greatest recovery. Two other aldehydes with the same chain length as HNE but different binding to lysine, histidine and cysteine residues, trans-2,3-nonenal and nonanal, gave I50 values for the inhibition of NADPH oxidase-mediated superoxide formation rate of 110 microM or > 300 microM, respectively.

Increased levels of lipid peroxidation products malondialdehyde and 4-hydroxynonenal after perinatal hypoxia.

For quantitative evaluation of lipid peroxidation after perinatal hypoxia in umbilical arterial cord blood samples from 109 healthy, acidotic, and asphyctic neonates with a gestational age ranging from 26 to 41 wk, the levels of aldehydic lipid peroxidation products malondialdehyde (MDA) and 4-hydroxynon-2-enal (HNE) were measured. Furthermore, the concentrations of oxidized and reduced glutathione (GSSH and GSH) and the purine compounds hypoxanthine and uric acid were determined. With increasing gestational age MDA and HNE levels increased. Furthermore, an increased level of GSH was also found. After perinatal hypoxia the concentrations of MDA and HNE rose distinctly (p < 0.001), reflecting sensitively the extent of in vivo lipid peroxidation. HNE is proposed to be a new parameter for quantitative evaluation of posthypoxic cellular damage in the perinatal period. HNE is a more specific parameter for estimation of lipid peroxidation processes in comparison with MDA. Additionally, HNE is cytotoxic and mutagenic at nanomolar concentrations. The increased levels of both MDA and HNE were accompanied by a strong decrease of GSH concentrations (p < 0.001), indicating the rapid consumption of GSH via a glutathione peroxidase reaction but additionally the high reactivity of HNE with sulfhydryl groups. During oxygen deficiency, increased levels of hypoxanthine (p < 0.01) and uric acid (p < 0.05) were due to the accelerated degradation of purine nucleotides. The rate of purine degradation including xanthine oxidase reactions characterizes the extent of an important radical source during oxygen deficiency, contributing to peroxidation of polyunsaturated fatty acids and the formation of peroxidation of polyunsaturated fatty acids and the formation of secondary aldehydic lipid peroxidation products.

Inhibition of poly(ADP-ribose) formation by 4-hydroxynonenal in primary cultures of rabbit synovial fibroblasts.

The formation of poly(ADP-ribose) in primary cultures of rabbit synovial fibroblasts after treatment with active oxygen released by xanthine/xanthine oxidase is inhibited by addition of 1 and 10 microM 4-hydroxy-2,3-trans-nonenal (HNE). The endogenous formation of HNE by the xanthine/xanthine oxidase system is not responsible for the inhibitory effect of the aldehyde, owing to the low accumulation rate of the lipid peroxidation product in the system used. HNE is able to inhibit the isolated nuclear enzyme ADP-ribosyltransferase, as shown by an in vitro assay with an Ki of 4 mumol/litre. Therefore the molecular basis of HNE-mediated effects on cell proliferation, differentiation and transformation might be due to the inhibitory effect of poly(ADP-ribos)ylation.

4-hydroxynonenal inhibits Na(+)-K(+)-ATPase.

4-Hydroxynonenal binds rapidly to Na(+)-K(+)-ATPase, and this was accompanied by a decrease in measurable sulfhydryl groups and a loss of enzyme activity. The I50 value for Na(+)-K(+)-ATPase inhibition by 4-hydroxynonenal was found to be 120 microM. Although the sulfhydryl groups could be completely restored with beta-mercaptoethanol during the reaction of the Na(+)-K(+)-ATPase-HNE-adduct, the Na(+)-K(+)-ATPase activity was only partially restored by this reducing agent. A combination of hydroxylamine and beta-mercaptoethanol yielded the greatest recovery of enzyme activity, 85% of original. Thus, 4-hydroxynonenal binding to Na(+)-K(+)-ATPase led to an irreversible decrease of enzyme activity under the conditions employed. It is hypothesized that 4-hydroxynonenal reacts with sulfhydryls at sites on the enzyme that are inaccessible by beta-mercaptoethanol. Furthermore, evidence was obtained that 4-hydroxynonenal reacts with other amino acids such as lysine to form adducts that also interfere with protein function.

4-hydroxynonenal is degraded to mercapturic acid conjugate in rat kidney.

The cytotoxic lipid peroxidation product 4-hydroxynonenal (HNE) was infused into rat kidney. During the first 2 min a rapid degradation of a 100 microM HNE solution was demonstrated. After 5 min the consumption rate of 4-HNE reached a steady state of about 75 nmoles/ml. The total HNE consumption rate was about 200 nmoles/g w.w./min. The excretion rate into urine was about 0.1% of total HNE consumption. It could be demonstrated that the HNE-mercapturic acid formation takes place in the kidney. The formation of the HNE-mercapturic acid contributes up to 6% to total HNE consumption. Within 10 min of perfusion 2% of the HNE-mercapturic acid were excreted into urine. The residual 98% flow back into the blood circulation.

Hypoxia and reoxygenation of brain endothelial cells in vitro: a comparison of biochemical and morphological response.

Reactive oxygen species are thought to be important for a variety of pathological processes in the brain. Endothelial cells have been proposed as both a significant source of oxidants and targets of oxidative damage. Therefore, lipid peroxidation (LPO) was investigated and compared to biochemical and morphological alterations in cultured pig brain capillary endothelial cells after hypoxia (120 min. 95% N2/5% CO2) and reoxygenation (30 min. 95% O2/5% CO2). The content of thiobarbituric acid reactive substances (TBARS) representing radical-induced LPO was 2.50 +/- 0.46 after hypoxia and 5.92 +/- 0.54 nmol/mg protein after reoxygenation (p < 0.05 each, vs. normoxic control 1.79 +/- 0.21). During hypoxia, ATP content decreased to 7.9 +/- 1.6 nmol/mg protein; lactate dehydrogenase activity in the incubation solution increased to 0.17 +/- 0.03 U/mg protein; (p < 0.05 vs. control 15.7 +/- 3.1 and 0.09 +/- 0.02, respectively). After hypoxia, morphological changes in lysosomes, multivesicular bodies and vacuoles were observed in contrast to normoxic cells. During reoxygenation, the ATP values were normalized; electron micrographs showed increasing amounts of lysosomes, multivesicular bodies, vacuoles, blebs and lipofuscin granula and lyzed cells. Comparing the biochemical and morphological observations, a sequence of disturbances occurred, in which energy depletion was accompanied and followed, respectively, by membrane destruction, cellular disintegration and an increase in LPO products. These results support the assumption that the damage of brain endothelial cells caused by hypoxia and reoxygenation is accompanied by peroxidation of membrane lipids.

Postanoxic formation of aldehydic lipid peroxidation products in human renal tubular cells.

The levels of the aldehydic lipid peroxidation products 4-hydroxynonenal and malondialdehyde and the levels of adenine nucleotides were measured during anoxia/reoxygenation studies with isolated human renal tubular cells in vitro. Energy depletion of renal cells was demonstrated by the decrease of ATP level. ATP could be restored completely and rapidly during postanoxic reoxygenation. 4-Hydroxynonenal and malondialdehyde levels increased during reoxygenation. In parallel, the breakdown of physiological 4-hydroxynonenal levels was estimated. The 4-hydroxynonenal formation rate was estimated from accumulation and metabolic breakdown rates of this compound.

Transitions of hepatic purine metabolism of Ehrlich ascites tumor bearing mice in different phases of tumor growth.

The concentrations of purine nucleotides, nucleosides and nucleobases in hepatocytes taken from healthy control mice and from Ehrlich ascites tumor bearing mice have been measured. The level of adenine nucleotides in the hepatocytes was higher during the proliferating phase in comparison with the resting phase of tumor growth and with control mice hepatocytes. The tracerkinetic studies on purine catabolism have been carried out on liver cells at different periods of tumor growth. The dynamics of radioactive tracers was mathematically modelled by a system of differential equations for both the concentrations and the specific radioactivities of the metabolites. Large differences in metabolic flux rates between control hepatocytes and hepatocytes in different phases of tumor growth were observed. The final purine degradation of hepatocytes was found to be accelerated in the resting phase but not in the proliferating phase of tumor growth. This result is in accordance with increased ATP concentration of liver itself in the proliferating phase of tumor growth. A conclusion is possible that the liver does not supply nucleosides and nucleobases for the Ehrlich ascites tumor cells during the proliferating phase of tumor growth.

Metabolism of 4-hydroxynonenal, a cytotoxic lipid peroxidation product, in Ehrlich mouse ascites cells at different proliferation stages.

The aldehydic lipid peroxidation product 4-hydroxynonenal (HNE) is cytotoxic at high concentrations (in the range of 100 microM); at low concentrations, it disturbs cell proliferation and exhibits genotoxic effects, and in the submicromolar range, HNE is chemotactic and stimulates phospholipase C. HNE is rapidly metabolized in eukaryotic cells. Here the metabolism of HNE was studied in suspensions of Ehrlich mouse ascites cells at different periods of the tumor age. The main products of HNE which were identified in the Ehrlich ascites cells, were glutathione-HNE-conjugate, hydroxynonenoic acid, and 1,4-dihydroxynonene. The formation of glutathione conjugates following the addition of HNE was higher in early phase cells when compared with cells in the late phase of tumor growth. That was in accordance with the increased consumption of the reduced form of glutathione. Ehrlich ascites tumor cells at the proliferation phase were able to reduce a higher amount of exogenous-added HNE, compared with cells at the stationary phase.

Uric acid and glutathione levels during short-term whole body cold exposure.

Ten healthy subjects who swim regularly in ice-cold water during the winter (winter swimming), were evaluated before and after this short-term whole body exposure. A drastic decrease in plasma uric acid concentration was observed during and following the exposure to the cold stimulus. We hypothesize that the uric acid decrease can be caused by its consumption after formation of oxygen radicals. In addition, the erythrocytic level of oxidized glutathione and the ratio of oxidized glutathione/total glutathione also increased following cold exposure, which supports this hypothesis. Furthermore, the baseline concentration of reduced glutathione was increased and the concentration of oxidized glutathione was decreased in the erythrocytes of winter swimmers as compared to those of nonwinter swimmers. This can be viewed as an adaptation to repeated oxidative stress, and is postulated as mechanism for body hardening. Hardening is the exposure to a natural, e.g., thermal stimulus, resulting in an increased tolerance to stress, e.g., diseases. Exposure to repeated intensive short-term cold stimuli is often applied in hydrotherapy, which is used in physical medicine for hardening.

Postischemic accumulation of the lipid peroxidation product 4-hydroxynonenal in rat small intestine.

4-Hydroxynonenal (HNE) as an indicator of lipid peroxidation was determined in rat jejunal mucosa. HNE was extracted as the dinitrophenylhydrazone derivative from the tissue, partially separated from other carbonyl compounds by thin-layer chromatography and measured by HPLC. During reperfusion of the small intestine following an ischemic period of 60 minutes a marked increase of the tissue concentration of HNE was observed. The mucosal HNE level passed a maximum value of 3.0 +/- 0.5 microM 10 min after the onset of reperfusion in comparison with 0.7 +/- 0.2 microM as initial value. The increased tissue level of the highly cytotoxic 4-hydroxyalkenal is suggested to be involved in the reperfusion induced morphological and biochemical changes of the small intestine.