Relation between lipoic acid and cell redox status in wheat grown in excess copper

Cell redox status and lipoic acid contents were analysed in wheat (Triticum durum Desf. cv. Creso) plants treated with 150 μM Cu to elucidate the role of the antioxidant lipoic acid against oxidative stress. In comparison with shoots, roots suffered a higher oxidative stress showing a decrease in NADPH contents and an oxidation of glutathione and ascorbate. Shoots did not evidence a clear oxidative damage since Cu was translocated in small amounts. Lipoic acid as reduced (DHLA) or oxidised (LA) form was present in both leaves and roots of wheat. Analysis of the cell sap showed that this antioxidant was present also as free form. The analyses showed that stroma contained significant amounts of free LA and that, after acidic hydrolysis, higher amounts of LA and DHLA were released. However, lipoic acid was undetectable in both thylakoids and microsomal membranes. Cu treatment did not determine changes in the contents of total LA and DHLA in roots, they being likely involved in Cu chelation. In contrast, in leaves after 48 h of treatment the metal induced an increase in DHLA, which could in part explain the reduction in the oxidised glutathione levels. In leaves free lipoic acid was more prone to be oxidised compared to the bound form, and the reduced form disappeared in both leaves and roots after Cu treatment.     

Interactions of lipoic acid radical cations with vitamins C and E analogue and hydroxycinnamic acid derivatives

As a powerful natural antioxidant, lipoic acid exerts significant antioxidant activities in vivo and in vitro by deactivation of reactive oxygen and nitrogen species. In this study we present a novel synergistic interaction of lipoic acid with other endogenous or exogenous antioxidants. Antioxidants vitamins C and E analogue (Trolox C) and hydroxycinnamic acid derivatives were found to recycle lipoic acid by donating electrons to lipoic acid radical cations, thereby increasing the antioxidant capacity of lipoic acid in vivo and in vitro. The rate constant of the electron transfer is in the order 10(9)dm(3)mol(-1)s(-1), close to the diffusion-controlled limit, and transfer quantum yield is above 95%.     

Lipoic acid lessens Th1-mediated inflammation in lipopolysaccharide-induced uveitis reducing selectively Th1 lymphocytes-related cytokines release

Abstract

Inflammation results in the production of free radicals. We evaluated the anti-inflammatory and antioxidant capacity of lipoic acid in an experimental uveitis model upon a subcutaneous injection of endotoxin into Lewis rats. The role of oxidative stress in the endotoxin-induced uveitis model is well-known. Besides, the Th1 response classically performs a central part in the immunopathological process of experimental autoimmune uveitis. Exogenous sources of lipoic acid have been shown to exhibit antioxidant and anti-inflammatory properties. Our results show that lipoic acid treatment plays a preventive role in endotoxin-induced oxidative stress at 24 h post-administration and reduced Th1 lymphocytes-related cytokines by approximately 50–60%. Simultaneously, lipoic acid treatment caused a significant reduction in uveal histopathological grading and in the protein concentration in aqueous humors, but not in cellular infiltration.     

Chromatographic analysis of lipoic acid and related compounds

The analysis of lipoic acid and related compounds, such as its reduced form dihydrolipoic acid, its amide form lipoamide and other analogues, in biological and food samples is important in biochemistry, nutritional and clinical chemistry. This review summarizes the chromatographic methods for the determination of lipoic acid and related compounds, and their applications to various samples such as bacteria, tissues, drugs and food. Gas chromatographic methods with flame ionization detection and flame photometric detection are commonly used for the quantification of lipoic acid present as its protein-bound form, after acid or base hydrolysis of these samples. High-performance liquid chromatographic methods with ultraviolet, fluorescence and electrochemical detection are mainly used for the determination of free lipoic acid and related compounds, such as dihydrolipoic acid, lipoamide and other analogues. Moreover, gas chromatography–mass spectrometry and capillary electrophoresis methods are also developed.     

Liposomal Drug with Carnosine and Lipoic Acid: Preparation,Antioxidant and Antiplatelet Properties

The conditions for producing phosphatidylcholine liposomes containing lipoic acid and carnosine together were determined. The obtained liposomes are 180–250-nm spherical particles with an efficiency of lipoic acid inclusion of 50–70% (for carnosine, 17–33%). Based on the model of the oxidation of phosphatidylcholine by hydrogen peroxide, an antioxidant effect of carnosine, lipoic acid or lipoic acid with carnosine together was demonstrated; it consisted in inhibition of lipid peroxidation process, which was manifested in a decrease in the formation of lipid peroxidation products that react with thiobarbituric acid. It was established that lipoic acid (5 mM) and carnosine (0.1–10 mM) in liposomes exhibit an antioxidant effect. At the same time, it was demonstrated that the content of the appropriate lipid peroxidation products in liposomes with antioxidants (lipoic acid + carnosine) was 15 times lower than in control liposomes (without antioxidants). The effect of the obtained liposomal drugs on the platelet aggregation induced by arachidonic acid was evaluated. It was found that the liposomal drug containing lipoic acid (1.5 mM) and carnosine (2.1 mM) inhibited platelet aggregation by 50–55% relative to the control (platelets and arachidonic acid), while liposomes without antioxidants and water-soluble forms of carnosine and lipoic acid had almost no effect on platelet aggregation caused by arachidonic acid.

     

Do spin traps also act as classical chain-breaking antioxidants? A quantitative kinetic study of phenyl tert-butylnitrone (PBN) in solution and in liposomes

Free radical spin traps such as phenyl tert-butylnitrone (PBN) are often reported to provide protection of the central nervous system of animal models against free radical damage, and the effects are attributed to its "antioxidant activity." The effects of PBN and p-CH(3)O-PBN were compared with known antioxidants, alpha-tocopherol and 2,2,5,7,8-pentamethyl-6-hydroxychroman (PMHC), in quantitative kinetic studies of lipid peroxidation thermally initiated under controlled conditions. Results obtained on the spin traps in organic solvents and in dilinoleoyl phosphatidylcholine (DLPC) bilayers indicated that the spin traps do not act as peroxyl radical trapping antioxidants but rather act only as moderate "retarders" of oxygen uptake at relatively high concentration. At low oxygen partial pressures, e.g., 14 torr, which better reflect oxygen partial pressures in biological systems, PBN provides a more significant reduction in oxygen uptake (up to 50%) by DLPC bilayers but still did not act as a typical antioxidant. However, at low partial pressures, PBN does act cooperatively with PMHC. It is suggested that its role in biological fluids and tissues may be to extend the suppressed oxidation by natural antioxidants expected to be present. The combination of antioxidant/spin trap, alpha-(3, 5-di-tert-butyl-4-hydroxyphenyl)-N-tert-butylnitrone did not exhibit any enhanced antioxidant efficiency compared with the related hindered phenol, 2,6-di-tert-butyl-4-methoxyphenol.     

Induction of neurite outgrowth in PC12 cells by alpha -phenyl-N-tert-butylnitron through activation of protein kinase C and the Ras-extracellular signal-regulated kinase pathway.

The spin trap alpha-phenyl-N-tert-butylnitron (PBN) is widely used for studies of the biological effects of free radicals. We previously reported the protective effects of PBN against ischemia-reperfusion injury in gerbil hippocampus by its activation of extracellular signal-regulated kinase (ERK) and suppression of both stress-activated protein kinase and p38 mitogen-activated protein kinase. In the present study, we found that PBN induced neurite outgrowth accompanied by ERK activation in PC12 cells in a dose-dependent manner. The induction of neurite outgrowth was inhibited significantly not only by transient transfection of PC12 cells with dominant negative Ras, but also by treatment with mitogen-activated protein kinase/ERK kinase inhibitor PD98059. The activation of receptor tyrosine kinase TrkA was not involved in PBN-induced neurite outgrowth. A protein kinase C (PKC) inhibitor, GF109203X, was found to inhibit neurite outgrowth. The activation of PKCepsilon was observed after PBN stimulation. PBN-induced neurite outgrowth and ERK activation were counteracted by the thiol-based antioxidant N-acetylcysteine. From these results, it was concluded that PBN induced neurite outgrowth in PC12 cells through activation of the Ras-ERK pathway and PKC.     

Effects of lipoic acid on citrate content, aconitate hydratase activity, and oxidative status during myocardial ischemia in rats

The effects of lipoic acid on intensity of free radical reactions, citrate content, and aconitate hydratase during myocardial ischemia have been investigated. Treatment with lipoic acid normalized biochemiluminescence parameters and citrate level, which were increased in the myocardial pathology. Treatment with lipoic acid also increased specific activity of aconitate hydratase, which was decreased in myocardium and blood of animals with myocardial ischemia. Administration of lipoic acid decreased DNA fragmentation observed during myocardial ischemia. The data suggest that lipoic acid can be effectively used as a cardioprotector preventing the development of free radical oxidation during myocardial ischemia.     

Neuroprotective role of lipoic acid against acute toxicity of N-acetylaspartic acid

N-Acetylaspartic acid (NAA) accumulates in Canavan disease, a severe inherited neurometabolic disorder clinically characterized by mental retardation, hypotonia, macrocephaly, and seizures. The mechanisms of brain damage in this disease remain poorly understood. Recent studies developed by our research group showed that NAA induces oxidative stress in vitro and in vivo in cerebral cortex of rats. Lipoic acid is considered as an efficient antioxidant which can easily cross the blood–brain barrier. Considering the absence of specific treatment to Canavan disease, this study evaluates the possible prevention of the oxidative stress promoted by NAA in vivo by the antioxidant lipoic acid to preliminarily evaluate lipoic acid efficacy against pro-oxidative effects of NAA. Fourteen-day-old Wistar rats received an acute administration of 0.6 mmol NAA/g body weight with or without lipoic acid (40 mg/kg body weight). Catalase (CAT), glutathione peroxidase (GPx), and glucose 6-phosphate dehydrogenase activities, hydrogen peroxide content, thiobarbituric acid-reactive substances (TBA-RS), spontaneous chemiluminescence, protein carbonyl content, total antioxidant potential, and DNA–protein cross-links were assayed in the cerebral cortex of rats. CAT, GPx activities, and total antioxidant potential were significantly reduced, while hydrogen peroxide content, TBA-RS, spontaneous chemiluminescence, and protein carbonyl content were significantly enhanced by acute administration of NAA. Those effects were all prevented by lipoic acid pretreatment. Our results clearly show that lipoic acid may protect against the oxidative stress promoted by NAA. This could represent a new therapeutic approach to the patients affected by Canavan disease.     

Effect of DL-alpha-lipoic acid on the status of lipid peroxidation and antioxidants in mitochondria of aged rats

The life span of a species is thought to be determined by the rate of mitochondrial damage which in turn is inflicted by free radicals in the mitochondria during the course of normal metabolism. The level of lipid peroxidation and antioxidants were measured in liver and kidney mitochondria of young and aged rats before and after DL-alpha-lipoic acid supplementation. In both liver and kidney, mitochondrial lipid peroxidation increased with age and a decrease in the enzymatic and non-enzymatic antioxidants were observed. DL-alpha-lipoic acid treated aged rats showed a decrease in the level of lipid peroxides and an increase in the antioxidant status. Our results conclude that supplementation of lipoic acid restores the depleted mitochondrial antioxidant status and suggest that it could be an effective therapeutic agent in treatment of age-associated disorders where free radicals are the major causative factor.     

Identification of iodotyrosines and some iodothyronines as dimethylaminoazobenzenethiohydantoin derivatives. Application to the sequencing of iodoamino acid containing peptides

A method has been developed for the gas chromatographic analysis of lipoic acid in biological samples. The lipoic acid is released from the samples by acid hydrolysis in the presence of the internal standards 1,2-dithiolane-3-butyric acid and/or 1,2-dithiolane-3-caproic acid. After hydrolysis, the lipoic acid and the internal standards are extracted from the hydrolysate and converted into the S,S-dibenzylmethyl esters. Gas chromatographic analysis of this mixture completely separates each of the homolog derivatives from the lipoic acid derivative and allows for the quantitation of the lipoic acid in the sample. Samples containing more than ～50 ng of lipoic acid can be easily assayed. Results are presented that show that the lipoic acid content of Escherichia coli depends on the carbon source used for its growth.     

Lipoic acid: a unique antioxidant in the detoxification of activated oxygen species

Lipoic acid (1,2-dithiolane-pentanoic acid) is a dithiol which is effective in affording protection against oxidative stress by virtue of its two sulphydryl moieties. It is present in all kinds of eukaryotic and prokaryotic cells. As lipoamide, it functions as a cofactor in the multienzyme complexes that catalyse the oxidative decarboxylation of α-keto acids such as pyruvate, α-ketoglutarate, and branched-chain α-keto acids. The complete enzyme pathway responsible for the de novo synthesis of lipoic acid has not yet been elucidated. Octanoic acid appears to be the precursor for the eight-carbon fatty acid chain, and cysteine the source of sulfur. Lipoic acid is unique, among antioxidants, because it retains powerful antioxidant properties in both its reduced (dihydrolipoic acid) and oxidised (lipoic acid) forms. Both lipoic and dihydrolipoic acids have metal-chelating ability and quench activated oxygen species either in the cytosol or in the hydrophobic domains. Dihydrolipoic acid has more antioxidant properties than lipoic acid, and it plays an important role in the recycling of other oxidised radical scavengers such as glutathione, ascorbate and tocopherol. However, dihydrolipoic acid can also exert pro-oxidant properties both by its iron-reducing ability and by its ability to generate sulfur-containing radicals that can damage proteins. There are few quantitative data on lipoic acid contents in vegetables. It has been found in asparagus, wheat and potatoes, and recently, the presence of both lipoic and dihydrolipoic acids in roots, leaves and in the stroma of wheat has been demonstrated.     

Effectiveness of 4-hydroxy phenyl N-tert-butylnitrone (4-OHPBN) alone and in combination with other antioxidant drugs in the treatment of acute acoustic trauma in chinchilla

Acute acoustic trauma (AAT) results in oxidative stress to the cochlea through overproduction of cellular reactive oxygen, nitrogen, and other free radical species appearing from 1 h to 10 days after noise exposure. It has been shown that N-acetyl-L-cysteine (NAC), a glutathione prodrug, and acetyl-L-carnitine (ALCAR), a mitochondrial biogenesis agent, are effective in reducing noise-induced hearing loss. Phenyl N-tert-butylnitrone (PBN), a nitrone-based free radical trap, appears to suppress oxidative stress in a variety of disorders and several biological models. In this study, we tested whether 4-hydroxy PBN (4-OHPBN), a major metabolite of PBN, administered 4 h after noise exposure is effective in treating noise-induced hearing loss and whether a combination of antioxidant drugs (4-OHPBN plus NAC and 4-OHPBN plus NAC plus ALCAR) provides greater efficacy in attenuating AAT since each agent addresses different injury mechanisms. Chinchilla were exposed to a 105 dB octave-band noise centered at 4 kHz for 6 h. 4-OHPBN and combinations of antioxidant drugs were intraperitoneally administered beginning 4 h after noise exposure. Hearing threshold shifts in auditory brainstem responses and missing outer hair cell counts were obtained. 4-OHPBN reduced threshold shifts in a dose-dependent manner while both drug combinations showed greater effects. These results demonstrate that 4-OHPBN and combinations of antioxidants can effectively treat acute acoustic trauma and drug combinations may increase the effectiveness of treatment and decrease the required individual medication dose.     

Design and synthesis of 1,2-dithiolane derivatives and evaluation of their neuroprotective activity

We designed and synthesized new analogues containing 1,2-dithiolane-3-alkyl and protected or free catechol moieties connected through heteroaromatic rings such as triazole, 1,2,4-oxadiazole, 1,3,4-oxadiazole, tetrazole or thiazole in order to explore the influence of the bioisosteric replacement of the amide group on the neuroprotective activity of the lipoic acid/dopamine conjugate. Evaluation of the activity of the new compounds, using glutamate-challenged hippocampal HT22 cells, showed that incorporation of heteroaromatic rings in the alkyl-1,2-dithiolane moieties in conjunction with another antioxidant, in this case catechol, may result in strong neuroprotective activity.     

Antioxidant DL-alpha lipoic acid as an attenuator of adriamycin induced hepatotoxicity in rat model

Protective efficacy of DL-alpha lipoic acid on adriamycin induced hepatotoxicity was evaluated in rats. Adriamycin toxicity, induced by a single injection (ip; 15 mg/kg body wt), was expressed by an elevation in alanine transaminase, aspartate transaminase, bilirubin levels in serum and alkaline phosphatase, lactate dehydrogenase, alanine transaminase, aspartate transaminase activity in hepatic tissue. Adriamycin produced significant increase in malondialdehyde levels indicating tissue lipid peroxidation and potentially inhibiting the activity of antioxidant, reduced glutathione and antioxidant enzymes, catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, glucose-6-phosphate dehydrogenase. The present results showed that pretreatment with lipoic acid [75 mg/kg body wt/day (ip), 24 h prior to administration of adriamycin] significantly restored various cellular activity suggesting the antioxidant potential of lipoic acid in ameliorating the hepatotoxicity induced by adriamycin.     

Interaction of 1-hydroxyethyl radical with antioxidant enzymes

There is considerable interest in the role of the 1-hydroxyethyl radical (HER) in the toxic effects of ethanol. The goal of this study was to evaluate the effects of HER on classical antioxidant enzymes. The interaction of acetaldehyde with hydroxylamine-o-sulfonic acid has been shown to produce 1, 1'-dihydroxyazoethane (DHAE); this compound appears to be highly unstable, and its decomposition leads to the generation of HER. Addition of DHAE into a solution of PBN led to the appearance of the typical EPR spectra of PBN/HER adduct. No PBN/HER spin adduct was detected when DHAE was incubated with 0.1 M PBN in the presence of GSH. In the absence of PBN, DHAE oxidized ascorbic acid to semidehydroascorbyl radical, presumably via an ascorbate-dependent one-electron reduction of HER back to ethanol. Catalase was progressively inactivated by exposure to DHAE-generated HER in a time and HER concentration-dependent manner. Ascorbic acid and PBN gave full protection to catalase against HER-dependent inactivation. The antioxidants 2-tert-butyl-4-methylphenol, propylgallate, and alpha-tocopherol-protected catalase against inactivation by 84, 88, and 39%, respectively. Other antioxidant enzymes were also sensitive to exposure to HER. Glutathione reductase, glutathione peroxidase, and superoxide dismutase were inactivated by 46, 36, and 39%, respectively, by HER. The results reported here plus previous results showing HER interacts with GSH, ascorbate, and alpha-tocopherol suggest that prolonged generation of HER in cells from animals chronically exposed to ethanol may lower the antioxidant defense status, thereby contributing to mechanisms by which ethanol produces a state of oxidative stress and produces toxicity.     

In Vivo 31P NMR Spectroscopy Studies of Halothane Induced Porcine Stress Syndrome. No Effect of C-Phenyl N-Tertbutyl Nitrone (PBN)

Porcine stress syndrome (PSS) which is an example of malignant hyperthermia (MH) in swine has previously been attributed to oxidative stress primarily due to an inherited antioxidant abnormality in MH susceptible (MHS) animals. C-phenyl-N-tert-butyl nitrone (PBN), a free radical spin trap, was selected to investigate whether free radicals are involved in MH. If free radicals cause the MH stress attack, then PBN should alter the time required for the onset of the stress attack, or perhaps protect the animal from experiencing the stress attack. In vivo phosphorus-31 (³¹P) magnetic resonance spectroscopy (MRS) was used to monitor metabolism in three to four week old normal and MHS piglets administered halothane as the stress challenge. Malignant hyperthermia was not reproducibly induced by halothane anesthesia. For those animals which did develop MH a dramatic fall in the level of PCr and a rise in the level of Pi was detected by ³¹P MRS. Intravenous administration of PBN prior to halothane exposure had no effect on the number of animals experiencing the stress attack. PBN does not appear to prevent, delay or reverse the onset of halothane-induced MH in three to four week old MHS piglets. The primary events leading to the MH syndrome do not appear to be influenced by the intervention of the type of free radicals normally trapped by PBN.     

Interplay between lipoic acid and glutathione in the protection against microsomal lipid peroxidation

Reduced glutathione (GSH) delays microsomal lipid peroxidation via the reduction of vitamin E radicals, which is catalyzed by a free radical reductase (Haenen, G.R.M.M. et al. (1987) Arch. Biochem. Biophys. 259, 449-456). Lipoic acid exerts its therapeutic effect in pathologies in which free radicals are involved. We investigated the interplay between lipoic acid and glutathione in microsomal Fe2+ (10 microM)/ascorbate (0.2 mM)-induced lipid peroxidation. Neither reduced nor oxidized lipoic acid (0.5 mM) displayed protection against microsomal lipid peroxidation, measured as thiobarbituric acid-reactive material. Reduced lipoic acid even had a pro-oxidant activity, which is probably due to reduction of Fe3+. Notably, protection against lipid peroxidation was afforded by the combination of oxidized glutathione (GSSG) and reduced lipoic acid. It is shown that this effect can be ascribed completely to reduction of GSSG to GSH by reduced lipoic acid. This may provide a rationale for the therapeutic effectiveness of lipoic acid.     

Beneficial effects of alpha-lipoic acid and ascorbic acid on endothelium-dependent, nitric oxide-mediated vasodilation in diabetic patients: relation to parameters of oxidative stress.

The impairment of nitric oxide (NO)-mediated vasodilation in diabetes has been attributed to increased vascular oxidative stress. Lipoic acid has been shown to have substantial antioxidative properties. The aim of this study was to assess the effect of lipoic acid on NO-mediated vasodilation in diabetic patients in comparison with the well-recognized effect of ascorbic acid. Using venous occlusion plethysmography, we examined the effects of lipoic acid (0.2 mM) and ascorbic acid (1 and 10 mM) on forearm blood flow responses to acetylcholine, sodium nitroprusside and concomitant infusion of the NO-inhibitor, N(G)-monomethyl-L-arginine, in 39 diabetic patients and 11 control subjects. Plasma levels of antioxidants and parameters of lipid peroxidation were measured and correlated to endothelial function tests. Lipoic acid improved NO-mediated vasodilation in diabetic patients, but not in controls. NO-mediated vasodilation was improved by ascorbic acid at 10 mM, but not 1 mM. Improvements of endothelial function by ascorbic acid and lipoic acid were closely related. The beneficial effects of lipoic acid were positively related to plasma levels of malondialdehyde and inversely related to levels of ubiquinol-10. These findings support the concept that oxidative stress contributes to endothelial dysfunction and suggest a therapeutic potential of lipoic acid particularly in patients with imbalance between increased oxidative stress and depleted antioxidant defense.