Fatty acids increase the circulating levels of oxidative stress factors in mice with diet-induced obesity via redox changes of albumin

Plasma concentrations of free fatty acids are increased in metabolic syndrome, and the increased fatty acids may cause cellular damage via the induction of oxidative stress. The present study was designed to determine whether the increase in fatty acids can modify the free sulfhydryl group in position 34 of albumin (Cys34) and enhance the redox-cycling activity of the copper-albumin complex in high-fat diet-induced obese mice. The mice were fed with commercial normal diet or high-fat diet and water ad libitum for 3 months. The high-fat diet-fed mice developed obesity, hyperlipemia, and hyperglycemia. The plasma fatty acid/albumin ratio also significantly increased in high-fat diet-fed mice. The increased fatty acid/albumin ratio was associated with conformational changes in albumin and the oxidation of sulfhydryl groups. Moreover, an ascorbic acid radical, an index of redox-cycling activity of the copper-albumin complex, was detected only in the plasma from obese mice, whereas the plasma concentrations of ascorbic acid were not altered. Plasma thiobarbituric acid reactive substances were significantly increased in the high-fat diet group. These results indicate that the increased plasma fatty acids in the high-fat diet group resulted in the activated redox cycling of the copper-albumin complex and excessive lipid peroxidation.     

In vitro interaction between homocysteine and copper ions: Potential redox implications

Homocysteine (Hcys) has been implicated in various oxidative stress-related disorders. The presence of a thiol on its structure allows Hcys to exert a double-edge redox action. Depending on whether Cu2+ ions occur concomitantly, Hcys can either promote or prevent free radical generation and its consequences. We have addressed in vitro the interaction between Hcys and Cu2+ ions, in terms of the consequences that such interaction may have on the free radical scavenging properties of Hcys and on the redox state and redox activity of the metal. To this end, we investigated the free radical-scavenging, O2(*-)-generating, and ascorbate-oxidizing properties of the interacting species by assessing the bleaching of ABTS*+ radicals, the reduction of O2(*-)-dependent cytochrome c, and the copper-dependent oxidation of ascorbate, respectively. In addition, electron paramagnetic resonance and Cu(I)-bathocuproine formation were applied to assess the formation of paramagnetic complexes and the metal redox state. Upon a brief incubation, the Hcys/Cu2+ interaction led to a decrease in the free radical-scavenging properties of Hcys, and to a comparable loss of the thiol density. Both effects were partial and were not modified by increasing the incubation time, despite the presence of Cu2+ excess. Depending on the molar Hcys:Cu2+ ratio, the interaction resulted in the formation of mixtures that appear to contain time-stable and ascorbate-reducible Cu(II) complexes (for ratios up to 2:1), and ascorbate- and oxygen-redox-inactive Cu(I) complexes (for ratios up to 4:1). Increasing the interaction ratio beyond 4:1 was associated with the sudden appearance of an O2(*-)-generating activity. The data indicate that depending on the molar ratio of interaction, Hcys and Cu2+ react to form copper complexes that can promote either antioxidant or pro-oxidant actions. We speculate that the redox activity arising from a large molar Hcys excess may partially underlie the association between hyper-homocysteinemia and a greater risk of developing oxidative-related cardiovascular diseases.     

Lipid peroxyl radical intermediates in the peroxidation of polyunsaturated fatty acids by lipoxygenase. Direct electron spin resonance investigations

Lipid peroxyl radicals resulting from the peroxidation of polyunsaturated fatty acids by soybean lipoxygenase were directly detected by the method of rapid mixing, continuous-flow electron spin resonance spectroscopy. When air-saturated borate buffer (pH 9.0) containing linoleic acid or arachidonate acid was mixed with lipoxygenase, fatty acid-derived peroxyl free radicals were readily detected; these radicals have a characteristic g-value of 2.014. An organic free radical (g = 2.004) was also detected; this may be the carbon-centered fatty acid free radical that is the precursor of the peroxyl free radical. The ESR spectrum of this species was not resolved, so the identification of this free radical was not possible. Fatty acids without at least two double bonds (e.g. stearic acid and oleic acid) did not give the corresponding peroxyl free radicals, suggesting that the formation of bisallylic carbon-centered radicals precedes peroxyl radical formation. The 3.8-G doublet feature of the fatty acid peroxyl spectrum was proven (by selective deuteration) to be a hyperfine coupling due to a gamma-hydrogen that originated as a vinylic hydrogen of arachidonate. Arachidonate peroxyl radical formation was shown to be dependent on the substrate, active lipoxygenase, and molecular oxygen. Antioxidants are known to protect polyunsaturated fatty acids from peroxidation by scavenging peroxyl radicals and thus breaking the free radical chain reaction. Therefore, the peroxyl signal intensity from micellar arachidonate solutions was monitored as a function of the antioxidant concentration. The reaction of the peroxyl free radical with Trolox C was shown to be 10 times slower than that with vitamin E. The vitamin E and Trolox C phenoxyl radicals that resulted from scavenging the peroxyl radical were also detected.     

Three histidine residues of amyloid-beta peptide control the redox activity of copper and iron

Zinc, iron and copper are concentrated in senile plaques of Alzheimer disease. Copper and iron catalyze the Fenton-Haber-Weiss reaction, which likely contributes to oxidative stress in neuronal cells. In this study, we found that ascorbate oxidase activity and the intensity of ascorbate radicals measured using ESR spectroscopy, generated by free Cu(II), was decreased in the presence of amyloid-beta (Abeta), the major component of senile plaques. Specifically, the ascorbate oxidase activity was strongly inhibited (85% decrease) in the presence of Abeta1-16 or Abeta1-42, whereas it was only slightly inhibited in the presence of Abeta1-12 or Abeta25-35 (<20% inhibition). Ascorbate-dependent hydroxyl radical generation by free Cu(II) decreased in the presence of Abeta in the identical order of Abeta1-42, Abeta1-16 > Abeta1-12 and was abolished in the presence of 2-fold molar excess glycylhystidyllysine (GHK). Ascorbate oxidase activity and ascorbate-dependent hydroxyl radical generation by free Fe(III) were inhibited by Abeta1-42, Abeta1-16, and Abeta1-12. Although Cu(II)-Abeta shows a significant SOD-like activity, the rate constant for the reaction of superoxide with Cu(II)-Abeta was much slower than that with SOD. Overall, our results suggest that His6, His13, and His14 residues of Abeta1-42 control the redox activity of transition metals present in senile plaques.     

The ascorbate-driven reduction of extracellular ascorbate free radical by the erythrocyte is an electrogenic process

Erythrocytes can reduce extracellular ascorbate free radicals by a plasma membrane redox system using intracellular ascorbate as an electron donor. In order to test whether the redox system has electrogenic properties, we studied the effect of ascorbate free radical reduction on the membrane potential of the cells using the fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide. It was found that the erythrocyte membrane depolarized when ascorbate free radicals were reduced. Also, the activity of the redox system proved to be susceptible to changes in the membrane potential. Hyperpolarized cells could reduce ascorbate free radical at a higher rate than depolarized cells. These results show that the ascorbate-driven reduction of extracellular ascorbate free radicals is an electrogenic process, indicating that vectorial electron transport is involved in the reduction of extracellular ascorbate free radical.     

Isolation of esterified fatty acids bound to serum albumin purified from human plasma and characterised by MALDI mass spectrometry.

Human serum albumin (HSA) is the most abundant protein in plasma. It is known to transport drugs as well as endogenous ligands, like free fatty acids (FFA). A mass spectrometry based method was applied to analyze the albumin bound lipid ligands. HSA was isolated from a human plasma pool by cold ethanol fractionation and ion exchange chromatography. HSA was defatted using a solvent extraction method to release the copurified lipids bound to the protein. The extracts were then analyzed by matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Using this method, phospholipids and acylglycerols were detected. The phospholipids were identified to be lyso-phosphatidylcholine (lyso-PC) with distribution of different fatty acids (palmitic, stearic, oleic, and linoleic acids). An abundant species in the HSA lipid extract was found to be a diacylglycerol, composed of two linoleic and/or oleic acid chains. The identified motifs reflect structures that are known to be present in plasma. The binding of lysophospholipids has already been described but it is the first ever-reported evidence of native diacylglycerol ligands bound to HSA. Besides the native ligands from plasma a triacylglycerol was detected that has been added during the albumin preparation steps.     

The effect of a free radical scavenger and platelet-activating factor antagonist on FFA accumulation in post-ischemic canine brain

The effects of the platelet-activating factor antagonist BN 50739 and a free radical scavenger dimethyl sulfoxide on the accumulation of free fatty acids in post-ischemic canine brain are reported. Following 14 min of complete normothermic ischemia and 60 min of reperfusion, the total brain FFAs were approximately 150% higher than in the control group (p<0.05). Perfusion with the platelet-activating factor antagonist BN50739 in its diluent dimethyl sulfoxide during 60 min of post-ischemic reoxygenation resulted in a 61.8% (p<0.01) reduction in the total brain free fatty acid accumulation. Palmitic, stearic, oleic, linoleic, and arachidonic acids decreased by 53.8%, 63.5%, 69.0%, 47.4%, and 57.2%, respectively. Although dimethyl sulfoxide alone caused stearic and arachidonic acids to return to the normal concentration range, BN 50739 had a significant influence on recovery of palmitic, oleic, and linoleic acids and was previously shown to provide significant therapeutic protection against damage to brain mitochondria following an ischemic episode. Because free fatty acid accumulation is one of the early phenomena in cerebral ischemia, this study provides evidence to support the hypothesis that both platelet-activating factor and free radicals are involved in initiating cerebral ischemic injury.     

Inhibition of type 1 and type 2 5alpha-reductase activity by free fatty acids,active ingredients of Permixon

In different cell systems, the lipido-sterolic extract of Serenoa repens (LSESr, Permixon inhibits both type 1 and type 2 5alpha-reductase activity (5alphaR1 and 5alphaR2). LSESr is mainly constituted of fatty acids (90+/-5%) essentially as free fatty acids (80%). Among these free fatty acids, the main components are oleic and lauric acids which represent 65% and linoleic and myristic acids 15%.To evaluate the inhibitory effect of the different components of LSESr on 5alphaR1 or 5alphaR2 activity, the corresponding type 1 and type 2 human genes have been cloned and expressed in the baculovirus-directed insect cell expression system Sf9. The cells were incubated at pH 5.5 (5alphaR2) and pH 7.4 (5alphaR1) with 1 or 3nM testosterone in presence or absence of various concentrations of LSESr or of its different components. Dihydrotestosterone formation was measured with an automatic system combining HPLC and an on-line radiodetector.The inhibition of 5alphaR1 and 5alphaR2 activity was only observed with free fatty acids: esterified fatty acids, alcohols as well as sterols assayed were inactive. A specificity of the fatty acids in 5alphaR1 or 5alphaR2 inhibition has been found. Long unsaturated chains (oleic and linolenic) were active (IC(50)=4+/-2 and 13+/-3 microg/ml, respectively) on 5alphaR1 but to a much lesser extent (IC(50)>100 and 35+/-21 microg/ml, respectively) on 5alphaR2. Palmitic and stearic acids were inactive on the two isoforms. Lauric acid was active on 5alphaR1 (IC(50)=17+/-3 microg/ml) and 5alphaR2 (IC(50)=19+/-9 microg/ml). The inhibitory activity of myristic acid was evaluated on 5alphaR2 only and found active on this isoform (IC(50)=4+/-2 microg/ml).The dual inhibitory activity of LSESr on 5alpha-reductase type 1 and type 2 can be attributed to its high content in free fatty acids.     

Betamethasone activation of CTP: Cholinephosphate cytidylyltransferase is mediated by fatty acids

The purpose of the present study was to determine the mechanisms by which glucocorticoids increase the activity of CTP: cholinephosphate cytidylyltransferase, a key enzyme required for the synthesis of surfactant phosphatidylcholine. Lung cytidylyltransferase exists as an inactive, light form low in lipids (L-form) and an active, heavy form high in lipid content (H-form). In vivo, fatty acids stimulate and aggregate the inactive L-form to the active H-form. In vivo, betamethasone increases the amount of H-form while decreasing the amount of L-form in fetal lung. There is also a coordinate increase in total free fatty acids in the H-form. In the present study, we used gas chromatography–mass spectrometry to measure the fatty acid species associated with the H-forms in fetal rat lung after the mothers were treated with betamethasone (1 mg/kg). In vivo, betamethasone increased the total amount of free fatty acids associated with the H-form by 62%. Further, the hormone selectively increased the mass of myristic and oleic acids in H-form by 52 and 82%, respectively. However, betamethasone produced the greatest increase in the amount of H-form linoleic acid, which increased fourfold relative to control. In vitro, each of the fatty acids increased L-form activity in a dose-dependent manner; however, linoleic acid was the most potent. Linoleic and oleic acids also effectively increased L-form aggregations. These observations suggest that in vivo glucocorticoids elevate the level of specific fatty acids which convert cytidylyltransferase to the active form. © 1995 Wiley-Liss, Inc.     

Redox regulation of copper-metallothionein

Copper (Cu) is an essential element whose localization within cells must be carefully controlled to avoid Cu-dependent redox cycling. Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotective effects during metal exposure and oxidative stress. The specific role of MTs, however, in modulating Cu-dependent redox cycling remains unresolved. Our studies utilized a chemically defined model system to study MT modulation of Cu-dependent redox cycling under reducing (Cu/ascorbate) and mild oxidizing (Cu/ascorbate + H2O2) conditions. In the presence of Cu and ascorbate, MT blocked Cu-dependent lipid oxidation and ascorbyl radical formation with a stoichiometry corresponding to Cu/MT ratios 相似文献   

A site-specific mechanism for free radical induced biological damage: the essential role of redox-active transition metals

The metal-mediated site-specific mechanism for free radical-induced biological damage is reviewed. According to this mechanism, cooper- or iron-binding sites on macromolecules serve as centers for repeated production of hydroxyl radicals that are generated via the Fenton reaction. The aberrations induced by superoxide, ascorbate, isouramil, and paraquat are summarized. An illustrative example is the enhancement of double-strand breaks by ascorbate/copper. Prevention of the site-specific free radical damage can be accomplished by using selective chelators for iron and copper, by displacing these redox-active metals with other redox-inactive metals such as zinc, by introducing high concentrations of hydroxyl radicals scavengers and spin trapping agents, and by applying protective enzymes that remove superoxide or hydrogen peroxide. Histidine is a special agent that can intervene in free radical reactions in variety of modes. In biological systems, there are traces of copper and iron that are at high enough levels to catalyze free-radical reactions, and account for such deleterious processes. In the human body Fe/Cu = 80/1 (w/w). Nevertheless, both (free) copper and iron are soluble enough, and the rate constants of their reduced forms with hydrogen peroxide are sufficiently high to suggest that they might be important mediators of free radical toxicity.     

Ascorbate removes key precursors to oxidative damage by cell-free haemoglobin in vitro and in vivo

Haemoglobin initiates free radical chemistry. In particular, the interactions of peroxides with the ferric (met) species of haemoglobin generate two strong oxidants: ferryl iron and a protein-bound free radical. We have studied the endogenous defences to this reactive chemistry in a rabbit model following 20% exchange transfusion with cell-free haemoglobin stabilized in tetrameric form [via cross-linking with bis-(3,5-dibromosalicyl)fumarate]. The transfusate contained 95% oxyhaemoglobin, 5% methaemoglobin and 25 microM free iron. EPR spectroscopy revealed that the free iron in the transfusate was rendered redox inactive by rapid binding to transferrin. Methaemoglobin was reduced to oxyhaemoglobin by a slower process (t(1/2) = 1 h). No globin-bound free radicals were detected in the plasma. These redox defences could be fully attributed to a novel multifunctional role of plasma ascorbate in removing key precursors of oxidative damage. Ascorbate is able to effectively reduce plasma methaemoglobin, ferryl haemoglobin and globin radicals. The ascorbyl free radicals formed are efficiently re-reduced by the erythrocyte membrane-bound reductase (which itself uses intra-erythrocyte ascorbate as an electron donor). As well as relating to the toxicity of haemoglobin-based oxygen carriers, these findings have implications for situations where haem proteins exist outside the protective cell environment, e.g. haemolytic anaemias, subarachnoid haemorrhage, rhabdomyolysis.     

Activation of thiol-dependent antioxidant activity of human serum albumin by alkaline pH is due to the B-like conformational change

Antioxidant activity of human serum albumin (HSA) increased steeply as the reaction mixture was shifted from neutral to alkaline pH. The antioxidant activity was also remarkably increased by Ca(2+) or a cationic detergent (cetyltrimethylammonium chloride). Carboxyl group modification of HSA resulted in about 40-fold increase of the antioxidant activity. The chemical modification study indicated that in addition to functional cysteine(s), cationic amino acid residues such as histidine, arginine and lysine appeared to involve in the antioxidant reaction. HSA also exhibited alkaline-pH dependent peroxidase activity to remove fatty acid hydroperoxide. At neutral pH, only two thiols of Cys-289 and free Cys-34 of HSA were modified by a thiol-specific modification reagent, 5-((((2-iodoacetyl)amino)ethy)amino)naphthalene-1-sulfonic acid (I14), regardless of the presence or absence of dithiothreitol (DTT), and the resultant antioxidant activity was not decreased, suggesting that Cys-289 and Cys-34 did not participate in the antioxidant reaction. At alkaline pH, I14 modified several additional HSA thiols in the presence, but did not in the absence of DTT. The antioxidant activity of the modified HSA was remarkably decreased to as much as 30% of the antioxidant activity given by the unmodified HSA in the absence of DTT. The HPLC pattern for tryptic peptides containing modified cysteine(s) derived from the I14-treated c-HSA (carboxyl group-modified HSA) at pH 7.0 with DTT was very similar to that of the I14-modified HSA at pH 8.0 with DTT. Taken together, these results suggest that activation of thiol-dependent antioxidant activity of HSA at alkaline pH is due to the conformational change favorable for the functional cysteine(s)-mediated catalysis.     

Transient Formation of Superoxide Radicals in Polyunsaturated Fatty Acid-Induced Brain Swelling

The involvement of superoxide free radicals and lipid peroxidation in brain swelling induced by free fatty acids has been studied in brain slices and homogenates. The polyunsaturated fatty acids linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4), and docosahexaenoic acid (22:6) caused brain swelling concomitant with increases in superoxide and membrane lipid peroxidation. Palmitic acid (16:0) and oleic acid (18:1) had no such effect. Furthermore, superoxide formation was stimulated by NADPH and scavenged by the addition of exogenous superoxide dismutase in cortical slice homogenates. These in vitro data support the hypothesis that both superoxide radicals and lipid peroxidation are involved in the mechanism of polyunsaturated fatty acid-induced brain edema.     

Regulation of γ-Aminobutyric Acid/Barbiturate Receptor-Gated Chloride Ion Flux in Brain Vesicles by Phospholipase A2: Possible Role of Oxygen Radicals

Preincubation of brain membranes with phospholipase A2 (PLA2) has been shown previously to affect the binding characteristics of various recognition sites associated with the gamma-aminobutyric acid (GABA) receptor complex. In the present study, we have investigated the effects of PLA2 (from Naja naja siamensis venom) on the functional activity of the GABA receptor/chloride ion channel. PLA2 (0.001-0.02 U/mg protein) preincubation decreased pentobarbital-induced 36Cl- efflux and muscimol-induced 36Cl- uptake in rat cerebral cortical synaptoneurosomes. The effect of PLA2 was prevented by EGTA and two nonselective PLA2 inhibitors, mepacrine and bromophenacyl bromide. The removal of free fatty acids by addition of bovine serum albumin both prevented and reversed the effect of PLA2. Products of the catalytic activity of PLA2, such as the unsaturated free fatty acids, arachidonic and oleic acids, mimicked the effect of PLA2. However, the saturated fatty acid, palmitic acid, and lysophosphatidyl choline had no effect on pentobarbital-induced 36Cl- efflux. Because unsaturated free fatty acids are highly susceptible to peroxidation by oxygen radicals, the role of oxygen radicals was investigated. Xanthine plus xanthine oxidase, a superoxide radical generating system, mimicked the effect of PLA2, whereas the superoxide radical scavenger, superoxide dismutase, diminished the effects of PLA2 and arachidonic acid on pentobarbital-induced 36Cl- efflux. Similarly, the effect of PLA2 was also inhibited by methanol (1 mM), a scavenger of the hydroxyl radical, and by catalase. These data indicate that exogenously added PLA2 induces alterations in membrane phospholipids, possibly promoting the generation of oxygen radicals and fatty acid peroxides which can ultimately modulate GABA/barbiturate receptor function in brain.     

Lipid peroxidation induced by the Cu,Zn-superoxide dismutase and hydrogen peroxide system.

Cu,Zn-superoxide dismutase (SOD) can catalyze hydroxyl radical generation using H2O2 as a substrate. Lipid peroxidation induced by the Cu,Zn-SOD and H2O2 system was investigated. When linoleic acids micelles or phosphatidylcholine liposomes were incubated with Cu,Zn-SOD and H2O2, lipid peroxidation was gradually increased in a time-dependent manner. The extent of lipid peroxidation was proportional to Cu,Zn-SOD and H2O2 concentrations. Hydroxyl radical scavengers and copper chelator inhibited lipid peroxidation induced by the Cu,Zn-SOD and H2O2 system. These results suggest that lipid peroxidation is mediated by the Cu,Zn-SOD and H2O2 system via the generation of hydroxyl radicals by a combination of the peroxidative reaction of Cu,Zn-SOD and the Fenton-like reaction of free copper released from oxidatively damaged SOD.     

Thermodynamic and spectroscopic study of Cu(II) and Ni(II) binding to bovine serum albumin

The thermodynamics of Cu(II) and Ni(II) binding to bovine serum albumin (BSA) have been studied by isothermal titration calorimetry (ITC). The Cu(II) binding affinity of the N-terminal protein site is quantitatively higher when the single free thiol, Cys-34, is reduced (mercaptalbumin), compared to when it is oxidized or derivatized with N-ethylmaleimide. This increased affinity is due predominantly to entropic factors. At higher pH (approximately 9), when the protein is in the basic (B) form, a second Cu(II) binds with high affinity to albumin with reduced Cys-34. The Cu(II) coordination has been characterized by UV-vis absorption, CD, and EPR spectroscopy, and the spectral data are consistent with thiolate coordination to a tetragonal Cu(II), indicating this is a type 2 copper site with thiolate ligation. Nickel(II) binding to the N-terminal site of BSA is also modulated by the redox/ligation state of Cys-34, with higher Ni(II) affinity for mercaptalbumin, the predominant circulating form of the protein.     

In vivo metabolism of labeled oleic and linoleic acids by the laying hen.

Radioactive oleic and linoleic acids, labeled with 3H in the chain and 14C in the carbonyl group, were administered to white leghorn laying hens. Mixtures fed in separate experiments included: (1) 3H- and 14C-labeled oleic acid, (2) 3H- and 14C-labeled linoleic acid and (3) [3H]oleic aicd and [14C] linoleic acid. The 3H/14C ratios of both the neutral lipid and phospholipid fractions from the egg yolk and of the isolated acids from these lipid fractions were compared to that in the administered mixture. Agreement in the 3H/14C ratios for the neutral lipid fraction from each of the feeding experiments indicated that neither the 3H- and 14C labeled acids nor the oleic or linoleic acids were distinguishable during synthesis of the neutral lipid. Analysis of the phospholipid fractions showed that when dual-labeled mixtures of oleic acid were administered, 3H/14C ratios were elevated and, therefore, there was selective elimination of the 14C label. When dual-labeled mixtures of linoleic acid were administered, the 3H/14C ratios were in agreement; and when the two acids were administered simultaneously as a dual-labeled mixture, there was selective incorporation of linoleic acid. These findings indicate separate metabolic pathways for synthesis of neutral lipid and phospholipid in egg yolk as expected, as well as preferential use of the essential fatty acid in the phospholipid by the hen.     

Free radical involvement in the generation of trans‐root potential

The identity of the naturally occurring compounds that accept electrons from plasma membrane-bound redox systems in vivo is obscure. We analysed the effect of ascorbate, oxygen, iron, as well as their free radical forms, and also the free radical-generating and -quenching systems on the trans-root electrical potential, which had previously been shown to be coupled to plasma membrane-bound redox systems. The material was the primary root of 8-day-old maize (Zea mays L.) seedlings. Trans-root electrical potential difference was measured across excised roots. Different ascorbate (ascorbate, dehydroascorbate and ascorbate free radical) and oxygen redox forms (superoxide and hydroxide radicals and hydrogen peroxide), as well as scavenging agents of oxygen species (superoxide dismutase, catalase, mannitol), and ferric and ferrous ions were added to the solution flowing around the root. Ascorbate free radical induced the greatest depolarization of the trans-root potential when compared to other ascorbate redox forms, which is consistent with its suggested role as a natural electron acceptor. Addition of xanthine oxidase, with or without xanthine, also produced depolarizing effects. The presence of SOD magnified this effect both with ascorbate free radical and xanthine oxidase. When ferric or ferrous chloride and ferric EDTA were applied to the bathing medium, only free ferric ion produced a very pronounced depolarization. The magnitude and kinetics of trans-root potential depolarization, induced by the ascorbate redox forms and systems for the generation and scavenging of oxygen species, argue in favour of the mutually competing electron transfer role of ascorbate free radicals and superoxide radicals in the extracellular space of the root. These results provide evidence that at least a part of the electrical potential difference occurring across plant roots arises from current flow from the symplast, via the plasma membrane-bound redox systems, to naturally occurring compounds in the apoplast, and that this transfer is achieved through the mediation of their free radical forms.     

Effect of phosphatidylcholine and free fatty acids on the activity of pancreatic lipase-colipase

Mixed micelles of bile salt and phospholipids inhibit the lipase-colipase-catalysed hydrolysis of triacylglycerols. Free fatty acids can reverse this inhibition and reactivate lipase-colipase. This reactivation is either due to the formation of a high-affinity complex between lipase and colipase induced by free fatty acids and/or to a change of the quality of the interface. Lauric acid, oleic acid and linoleic acid are the most potent reactivators, while short-chain free fatty acids have no effect and long-chain, saturated free fatty acids inhibit the lipase-colipase activity further. The physiological relevance of these results is evident as the glyceride emulsion reaching the duodenum already contains free fatty acids due to the activity of lingual lipase in the stomach.