Prevention of free fatty acid-induced lipid accumulation, oxidative stress, and cell death in primary hepatocyte cultures by a Gynostemma pentaphyllum extract.

Hepatocytes of a primary cell culture that are exposed to high glucose, insulin, and linoleic (LA) acid concentration respond with lipid accumulation, oxidative stress up to cell death. Such alterations are typically found in patients with non-alcoholic fatty liver disease (NAFLD). We used this cellular model to study the effect of an ethanolic Gynostemma pentaphyllum (GP) extract in NAFLD. When hepatocytes were cultured in the presence of high insulin, glucose, and LA concentration the extract completely protected the cells from cell death. In parallel, the extract prevented accumulation of triglycerides (TGs) and cholesterol as well as oxidative stress. Our data further demonstrate that GP stimulates the production of nitric oxide (NO) in hepatocytes and affects the molecular composition of the mitochondrial phospholipid cardiolipin (CL). We conclude that GP is able to protect hepatocytes from cell death, lipid accumulation, and oxidative stress caused by diabetic-like metabolism and lipotoxicity. Therefore, GP could be beneficial for patients with diabetes mellitus and NAFLD.

Dronedarone prevents microcirculatory abnormalities in the left ventricle during atrial tachypacing in pigs.

BACKGROUND AND PURPOSE: Atrial fibrillation induces ischaemic microcirculatory flow abnormalities in the ventricle, contributing to the risk for acute coronary syndromes. We evaluated the effect of dronedarone on ventricular perfusion during rapid atrial pacing (RAP). EXPERIMENTAL APPROACH: Coronary and fractional flow reserve (CFR/FFR) were measured in the left anterior descending artery in 29 pigs. Six received RAP, six received RAP with dronedarone (RAP/D), seven received dronedarone alone, four received RAP with amiodarone (RAP/A), and six received neither (sham). In ventricular tissue, oxidative stress/ischaemia-related gene and protein expression was evaluated by RT-PCR and Western blotting; Isoprostanes were measured by GC-MS procedures. KEY RESULTS: CFR was decreased in the RAP group, compared with other groups. FFR was not different between groups. Effective refractory period was reduced in RAP compared with RAP/D. RAP-activated PKC phosphorylation tended to be decreased by dronedarone (P= 0.055) RAP induced NOX-1 and NOX-2 protein and the mRNA for hypoxia-inducible factor-1α (HIF-1α). Dronedarone reduced the pacing-dependent increase in the expression of NOX-2 protein and of HIF-1α mRNA. The oxidative stress marker, F(2)-isoprostane, was increased by RAP and this increase was attenuated by dronedarone. Other oxidative stress/ischaemia-related genes were induced by RAP compared with sham and were decreased by dronedarone treatment. In HL1 cells, dronedarone significantly inhibited the increased phosphorylation of PKCα after oxidative stress, with an almost significant effect (P= 0.059) on that after RAP. CONCLUSIONS AND IMPLICATIONS: Dronedarone abolished RAP-induced ventricular microcirculatory abnormalities by decreasing oxidative stress/ischaemia-related gene and protein expression in the ventricle.

Detection of monohydroxyeicosatetraenoic acids and F2-isoprostanes in microdialysis samples of human UV-irradiated skin by gas chromatography-mass spectrometry.

UV irradiation of the human skin leads to induction of oxidative stress and inflammation mediated by reactive oxygen radicals, lipid peroxidation, liberation of arachidonic acid from membrane phospholipids and formation of prostaglandins and leucotrienes. We investigated "lipid mediators", such as F(2)-isoprostanes (8-iso-PGF(2alpha), 9alpha,11alpha-PGF(2alpha)) and monohydroxyeicosatetraenoic acids (HETEs) in the dermal interstitial fluid obtained by a cutaneous microdialysis technique. Defined areas on the volar forearm of 10 healthy volunteers were exposed to UVB irradiation (20-60 mJ/cm(2)). Microdialysis membranes were cutaneously inserted beneath the irradiated area. The probes were perfused with isotonic saline solution, and microdialysate samples were collected at 20-min intervals up to 4-5 h. Oxidized arachidonic acid derivatives (2-, 3-, 5-, 8-12- and 15-HETEs, 8-iso-PGF(2alpha) and 9alpha,11alpha-PGF(2alpha)) could be detected and quantified in microdialysates of normal skin in the picomole (HETEs) and femtomole (isoprostanes) range and after UVB irradiation using sensitive gas chromatography-mass spectrometry/negative ion chemical ionization. UVB irradiation enhanced the levels of 8-iso-PGF(2alpha) after 24 h significantly, whereas the HETE levels were slightly increased within shorter time intervals (3 h after UVB irradiation). Further investigations have to show whether these new findings are relevant to validate therapeutic strategies for topical and systemic UV prevention agents or for monitoring of specific therapeutic strategies in inflammatory skin disorders.

Detection of enhanced monohydroxyeicosatetraenoic acid and F2-isoprostane levels in human plasma samples after extracorporeal photoimmunotherapy.

To investigate the involvement of reactive oxygen species in extracorporeal photoimmunotherapy (photopheresis), we have introduced two highly sensitive and specific techniques for the detection and quantitative measurement of oxygenated nonenzymatically formed arachidonic acid isomers [mono-hydroxyeicosatetraenoic acids (HETEs) and F2-isoprostanes] by gas chromatography-mass spectrometry/negative ion chemical ionization (GC-MS/NICI) in plasma samples of patients suffering from cutaneous T-cell lymphoma and progressive systemic scleroderma II. The analysis of HETEs involved hydrogenation, solid phase extraction on a C18 cartridge, formation of pentafluorobenzyl bromide and trimethylsilyl ether derivatives. In the case of F2-isoprostanes, the analytical procedure was similar to that of HETEs except that the hydrogenation step was omitted. In the plasma of healthy volunteers picomole amounts of 2-, 5-, 8-12-, 15-HETEs, 8-iso-PGF(2alpha) and 9alpha,11alpha-PGF(2alpha) were quantified by using 12-hydroxy-heptadecatrienoic acid and PGF(2alpha)-d4 as internal standards of HETEs and isoprostanes, respectively. Analysis of plasma samples obtained from patients before and after extracorporeal photoimmunotherapy revealed characteristic increases in both, HETE and isoprostane levels. The enhancement of indicators of lipid peroxidation is in correspondence with a moderate loss of alpha-tocopherol, the most important lipid-soluble antioxidant in human plasma. Thus, our data confirm the involvement of lipid peroxidation in extracorporeal photoimmunotherapy.

Oxidative stress in cardio renal anemia syndrome: correlations and therapeutic possibilities.

Cardiovascular injury has been shown to be the most critical factor affecting quality of life and mortality in patients suffering from chronic renal failure. Oxidative stress has been thought to be an important risk factor for cardiovascular disorders. As oxidative stress parameters with high cardiovascular risk factor 4-hydroxynonenal and other aldehydic lipid peroxidation products, F2-isoprostanes, homocysteine, and cholesterol oxidation products were measured in chronic renal failure patients. 4-Hydroxynonenal and some cholesterol oxidation products correlated well with the degree of renal anemia. F2-isoprostane levels were related to inflammation, whereas homocysteine was increased due to malnutrition. Further, cholesterol oxidation products correlated well with the consumption of lipophilic antioxidants such as alpha-tocopherol. There was an almost linear correlation between the left ventricular mass index and 4-hydroxynonenal. Both parameters furthermore showed an inverse relationship to hemoglobin concentration. The correction of renal anemia by means of erythropoietin therapy led to an efficient strengthening of the antioxidative defence system. The improvement of the antioxidative capacity is of complex nature comprising both enzymatic pathways and low molecular antioxidants. The correction of renal anemia with its well documented reduction of the cardiovascular risk can be regarded as an antioxidative therapy, demonstrating the clinical efficiency of antioxidative protection in patients with chronic renal failure.

Oxidative stress in chronic renal failure as a cardiovascular risk factor.

Myocardial injury has been shown to be the most critical factor influencing quality of life and mortality in patients with chronic renal failure. Oxidative stress has been postulated to be an important risk factor for cardiovascular disorders. One reason for oxidative stress in patients with renal failure is the underlying disease itself. Renal toxicity, ischemia/reperfusion and immunological disorders of the kidney result in an elevated formation of reactive oxygen species active in the pathogenesis of kidney disease. However, treatment procedures were also shown to induce oxidative stress. Increased formation of free radicals leads to an accelerated lipid peroxidation (LPO). Furthermore, secondary aldehydic LPO products, e.g. malondialdehyde (MDA) and 4-hydroxynonenal (HNE), are formed which were shown to deplete antioxidants, inhibit protein syntheses, mitochondrial respiration, and enzyme functions. F2-isoprostanes, also metabolites of polyunsaturated fatty acids, represent an additional in vivo marker of oxidative stress. Both isoprostanes and aldehydic LPO products can be removed by hemodialysis, however, this suggests only in part their binding to other molecules which cause tissue damage. Protein carbonyls are end-products of such interventions. Oxysterols, another form of free-radical initiated oxidation products, were shown to initiate atherosclerosis and plaque formation increasing dramatically the risk of coronary heart disease. Today there is no doubt that the correction of the oxidant/antioxidant imbalance in patients with chronic renal failure is an important approach for the reduction of the risk of those patients to develop cardiovascular disorders. The complete correction of renal anemia represents an effective means of strengthening antioxidant capacity and, therefore, of reducting cardiovascular risk potential.

Differential oxidative injury in extrapancreatic tissues during experimental pancreatitis: modification of lung proteins by 4-hydroxynonenal.

Oxidative stress is considered to be a pathogenic factor for multisystem organ failure during acute pancreatitis. Infusion of 3% and 5% sodium taurocholate into the pancreatic duct of rats resulted in a 24-hr lethality of 8% and 82%, respectively. Kidney tissue showed a long-lasting significant elevation of malondialdehyde (lipid peroxidation). Only small amounts of this aldehyde were formed in the liver. In the lung malondialdehyde was increased during the first 6 hr after pancreatitis induction. Malondialdehyde levels were not different for pancreatitis initiated by 3% or 5% taurocholate. Protein-bound carbonyls (protein oxidation) in the tissues were not significantly changed at any time point. However, after infusion of 5% taurocholate, lung proteins were oxidatively modified by the product of lipid peroxidation, 4-hydroxynonenal. Another parameter characteristic for pancreatitis with high lethality was the high number of neutrophils in the lungs. We conclude that oxidative stress is important for the injury of extrapancreatic tissues during pancreatitis, but survival is determined by the degree of systemic inflammation.

A sensitive gas chromatography-mass spectrometry assay reveals increased levels of monohydroxyeicosatetraenoic acid isomers in human plasma after extracorporeal photoimmunotherapy and under in vitro ultraviolet A exposure.

Extracorporeal photoimmunotherapy (photopheresis) is a highly effective therapy in the treatment of various disorders. Although extracorporeal photoimmunotherapy has been successfully used for more than 10 y, its mechanism of action is still unclear. The formation of reactive oxygen species have been implicated in extracorporeal photoimmunotherapy, but malonyl dialdehyde as a marker of systemic lipid peroxidation did not increase significantly during treatment. To investigate further the involvement of reactive oxygen species in extracorporeal photoimmunotherapy, we have introduced a highly sensitive negative ion gas chromatography-mass spectrometry based method for quantitating oxygenated arachidonic acid isomers (hydroxyeicosatetraenoic acids) in plasma samples of patients treated with extracorporeal photoimmunotherapy. In the plasma of healthy volunteers pmole amounts of 2-, 3-, 5-, 8-12-, and 15-hydroxyeicosatetraenoic acid were detected and we observed a dose-dependent augmentation in these metabolites when the blood was irradiated with increasing doses of ultraviolet A in the presence of the photosensitizer 8-methoxypsoralen. Analysis of plasma samples obtained from patients before and after extracorporeal photoimmunotherapy revealed a characteristic increase in total hydroxyeicosatetraenoic acid levels, particularly of 5-hydroxyeicosatetraenoic acid which contributed 80% to the sum of all hydroxyeicosatetraenoic acid isomers. Chiral phase high-performance liquid chromatography indicated almost equal amounts of 5S- and 5R-hydroxyeicosatetraenoic acid suggesting that the majority of lipid peroxidation products are formed via nonenzymatic oxidation reactions.

Comparison of protein oxidation and aldehyde formation during oxidative stress in isolated mitochondria.

Oxidative stress is known to cause oxidative protein modification and the generation of reactive aldehydes derived from lipid peroxidation. Extent and kinetics of both processes were investigated during oxidative damage of isolated rat liver mitochondria treated with iron/ascorbate. The monofunctional aldehydes 4-hydroxynonenal (4-HNE), n-hexanal, n-pentanal, n-nonanal, n-heptanal, 2-octenal, 4-hydroxydecenal as well as thiobarbituric acid reactive substances (TBARS) were detected. The kinetics of aldehyde generation showed a lag-phase preceding an exponential increase. In contrast, oxidative protein modification, assessed as 2,4-dinitrophenylhydrazine (DNPH) reactive protein-bound carbonyls, continuously increased without detectable lag-phase. Western blot analysis confirmed these findings but did not allow the identification of individual proteins preferentially oxidized. Protein modification by 4-HNE, determined by immunoblotting, was in parallel to the formation of this aldehyde determined by HPLC. These results suggest that protein oxidation occurs during the time of functional decline of mitochondria, i.e. in the lag-phase of lipid peroxidation. This protein modification seems not to be caused by 4-HNE.

Role of endogenous and exogenous antioxidants in the defence against functional damage and lipid peroxidation in rat liver mitochondria.

Mitochondria are cellular organelles where the generation of reactive oxygen species may be high. They are, however, effectively protected by their high capacities of antioxidative systems, as enzymes and either water or lipid soluble low molecular weight antioxidants. These antioxidative defence systems can be effectively regenerated after or during an oxidative stress as long as the mitochondria are in an energized state. Energization of mitochondria mainly depends on the availability of suitable respiratory substrates which can provide hydrogen for the reduction of either the glutathione- or alpha-tocopherol-system, since GSH is regenerated by glutathione reductase with the substrate NADPH and the alpha-tocopheroxyl-radical likely by reduced coenzyme Q. It was shown that mitochondria do not undergo damages as long as they can keep a high energy state. The delicate balance between prooxidative/antioxidative activities can be shifted towards oxidation, if experimentally prooxidants were added. After exhaustion of the antioxidative defence systems damages of mitochondrial functions become expressed followed by membrane injuries along with the oxidation and degradation of mitochondrial lipids and proteins leading finally to the total degradation of the mitochondria. Extramitochondrial antioxidants may assist the mitochondrial antioxidative defence systems in a complex way, whereby particularly ascorbic acid can act both as prooxidant and as antioxidant.

Short-term impairment of energy production in isolated rat liver mitochondria by hypoxia/reoxygenation: involvement of oxidative protein modification.

The aim of the present study was to elucidate the role of mitochondria in liver impairment after ischaemia/reperfusion. It is commonly assumed that mitochondria are in part responsible for tissue damage by impaired oxidative phosphorylation as a consequence of the attack of radicals generated within the mitochondria. The principal support for this hypothesis was found by exposing isolated mitochondria to temporary hypoxia in combination with alterations of substrate supply. Rat liver mitochondria treated in this way responded with impaired ADP-stimulated respiration after reoxygenation, which decreased with time of hypoxia and reoxygenation. The decline of the activity of the NADH-cytochrome c-oxidoreductase complex found under these conditions is likely to cause the drop in active respiration. No changes in the content of respiratory chain complexes, determined by Blue Native PAGE, could be demonstrated. However, oxidative modifications of mitochondrial proteins, indicated by carbonyl formation, were found. Likewise, products of lipid peroxidation, such as lipid peroxides and malondialdehyde, were formed. Mitochondria were still able to build up a transmembrane potential and did not show drastic changes in membrane conductivity after hypoxia/reoxygenation stress. The presence of water-soluble antioxidants exhibited a beneficial effect, diminishing the decline of active respiration after 5 min of hypoxia and 10 min of reoxygenation. These observations strongly suggest that mitochondria play a pathogenic role in ischaemia/reperfusion injury, which is at least in part mediated by an oxygen-derived free-radical-linked mechanism.

Identification of short-chain oxidized phosphatidylcholine in human plasma.

Oxidized phospholipids have been recognized as potentially important compounds that carry biological activities similar to the platelet-activating factor, but their presence in biological tissue has not been firmly established. We developed a novel technique for the quantitative analysis of phospholipids with oxidized acyl chains. The method involves 1) lipid extraction, 2) chromatographic enrichment of phospholipids with short acyl chains, 3) derivatization with 9-(chloromethyl)anthracene, 4) solid-phase extraction of the derivatives, and 5) reversed-phase HPLC with fluorescence detection. The technique was capable of measuring dicarboxylate-containing phosphatidylcholines (PCs) at the picomole level. The method was suited to monitor the generation of oxidized phospholipids from 1-palmitoyl-2-arachidonoyl-PC in the presence of Fe21/ascorbate. The new procedure was used to isolate lipids from human plasma that were identified as anthracene derivatives of short-chain oxidized PC on the basis of chromatographic enzymatic, and spectroscopic evidence. The plasma concentration, determined with an internal standard (1-palmitoyl-2-suberoyl-PC), was 0.6 +/- 0.2 microM (n = 11). The analytical method did not produce oxidation antifacts in significant amount. We concluded that human blood contains oxidatively fragmented PC in submicromolar concentration.

Electrophoretic evidence for the impairment of complexes of the respiratory chain during iron/ascorbate induced peroxidation in isolated rat liver mitochondria.

The impairment of the complexes of the respiratory chain was studied in isolated rat liver mitochondria under the conditions of an iron/ascorbate-mediated oxidative stress. Using blue native electrophoresis technique the NADH-ubiquinone oxidoreductase, ubiquinol-cytochrome-c oxidoreductase, cytochrome oxidase and ATP-synthetase were separated from mitochondrial samples at different stages of peroxidation and quantified by densitometry. In the second dimension the protein complexes were separated into their individual subunits by Tricine/SDS-electrophoresis. In relation to the time course of lipid peroxidation protein losses were moderate in the exponential phase and enhanced towards plateau phase of TBARS formation, when the intensity of staining for the native complexes became reduced by 84%, 69%, 63% and 24% for complexes I, III, V and IV, respectively, and a high molecular aggregation band as a putative marker of oxidative stress was formed. The decline of overall staining by 23%, a decrease in trichloroacetic acid precipitable protein and the formation of acid soluble primary amines suggest the occurrence of fragmentation or degradation processes. Apparently, the impairment of the respiratory chain complexes during peroxidation was not reflected in altered electrophoretic mobilities or specific losses of protein subunits of these innermitochondrial membrane components.

Morphological changes of isolated rat liver mitochondria during Fe2+/ascorbate-induced peroxidation and the effect of thioctacid.

Fe2+/ascorbate-induced peroxidation of isolated rat liver mitochondria leads to initial volume changes and, ultimately, to severe damage characterized by gross swelling and loss of cristae and matrix material. Only the last phase is associated with significant production of malondialdehyde. The shrinkage of mitochondria during the onset of peroxidation matches changes observed in mitochondria of aging animals. Thioctacid (alpha-lipoic acid) prevents this initial shrinkage. However, its main effect in the system studied here is inhibition of active respiration.

Studies of lipid peroxidation in isolated rat heart mitochondria.

Peroxidation in isolated, functionally intact rat heart mitochondria was induced by iron/ascorbate or ADP-iron/NAD(P)H. Compared to liver mitochondria, MDA formation was very low and lipohydroperoxides not detected. The NADPH-mediated peroxidation which generally resulted in somewhat higher MDA levels was accompanied by an increasing inhibition of ADP-stimulated respiration. The active respiration was sensitively inhibited at very early stages of MDA formation, whereas in the same period the CAT-insensitive respiration exhibited almost no response at all. It was demonstrated that the decrease in active respiration correlated with the time for half-maximum MDA formation. No considerable degradation of major mitochondrial phospholipids was observed during two hours of incubation. It was not until after complete inhibition of respiration and onset of enhanced MDA formation that cardiolipin, phosphatidylethanolamine and, later, phosphatidylcholine were diminished.

Injury of mitochondrial respiration and membrane potential during iron/ascorbate-induced peroxidation.

First functional events during peroxidation in mitochondria consisted in a progressive inhibition of the phosphorylating and uncoupled respiration with succinate and glutamate/malate as substrates, whereas the resting state respiration during the same period was virtually not influenced. The membrane potential registered at a time with the respiration rates was capable of being built up for a relatively long time interval with only minor decreases, and broke down rather promptly when the active respiration was highly diminished. Inhibition of respiration proceeded mainly during the initiation phase of peroxidation. Lag phases of varied length, of malondialdehyde formation which were predominantly attributed to the iron/protein ratios correlated closely with different time intervals needed to attain maximal inhibition of respiration and decrease in glutathione. Hence, the lessening of respiration, drop of membrane potential and loss of the antioxidant, glutathione, represent early stages in the causal chain of events which precede the onset of intensive lipid peroxidation.

Impairment of the respiratory chain in the b-c1 region as early functional event during Fe2+/ascorbate induced peroxidation in rat liver mitochondria.

An attack at the level of the respiratory chain was established as the reason for the early inhibition of oxygen uptake during Fe2+/ascorbate induced peroxidation by following the activities of complexes I-IV separately. The close correlation between the decline of mitochondrial respiration and the activity of complex III strongly suggests that an impairment of the respiratory chain in the b-c1 region represents one of the first functional events in the causal sequence of peroxidative reactions preceding the phase of massive malondialdehyde production.

Relations between enzyme activities connected with energy metabolism and parameters of food energy utilization in young and adult rats. Part 2. Enzyme activities related to alpha-glycerophosphate shuttle in various tissues.

The possible significance of food composition connected with the alpha-glycerophosphate (alpha GP) shuttle, a putative metabolic pathway of energy dissipation, was investigated at the level of enzyme activities. Liver, adipose tissue, slow-twitch and fast-twitch muscle of weaned male Wistar rats fed ad libitum for seven and for forty weeks a normal-protein (NP), a low-protein (LP), and a high-fat (HF) diet were examined. No striking dietary influences on cytosalic (NAD-linked glycerophosphate dehydrogenase, glyceraldehyde-phosphate dehydrogenase) and mitochondrial (succinate dehydrogenase, cytochrome c oxidase) enzyme activities could be detected, but mitochondrial alpha-glycerophosphate dehydrogenase (m-GPDH) showed an about twofold increase of its activity in the liver of LP-fed animals after seven weeks. A relationship between the "gross efficiency of food energy utilization" and tissue m-GPDH levels could not be established in general. The proposed inducing effect of a LP diet on the magnitude of the GP shuttle observed in the liver of young and adult rats seems to be interconnected reciprocally with the degree of metabolic energy dissipation only under the conditions of growth. The calculated capacities of the alpha GP shuttle are compatible with the assumption of its function as an energy dissipating pathway which is restricted in its magnitude.

Phospholipid patterns and phospholipase A2 activities in gastric mucosa of rats developing carcinomas after surgically induced enterogastric reflux.

Phospholipid patterns of the gastric mucosa from rats after operations inducing enterogastric reflux showed characteristic differences in relation to control rats: diminution of parent phospholipids phosphatidylethanolamine and phosphatidylcholine concomitant with increases of the corresponding lysophospholipids, increase of the sphingomyelin portion, indications for lipid peroxidations possibly connected with small drops of acidic phospholipids, and an elevated phospholipase A2 activity. The observed phospholipid changes support the assumption of disturbed membrane functions caused by activation of lipolytic activities as one factor involved in progressive mucosal alterations mostly manifested as carcinomas of the stomach.

Involvement of periodic deacylation-acylation cycles of mitochondrial phospholipids during Sr2+-induced oscillatory ion transport in rat liver mitochondria.

Lysophosphatidylcholine and lysophosphatidylethanolamine levels were determined during Sr2+-induced oscillating ion fluxes in mitochondria prelabelled in vivo with 32Pi. Periodic fluctuations of both lyso compounds were established with an increase at the stage of simultaneously monitored K+ influx and a decrease at K+ efflux. The periodic activations and inactivations of phospholipase were found to be associated with periodic changes in the incorporation rates of labelled polyunsaturated fatty acids with an apparent phase difference of 180 degrees. Periodic deacylation-acylation cycles of phospholipids accompanying the periodic cycles of reversible ion accumulation and release are suggested to be involved in the trigger mechanism generating the permeability changes during oscillatory ion transport.