John K. Lodge

α-Lipoic Acid in Liver Metabolism and Disease

R-alpha-Lipoic acid is found naturally occurring as a prosthetic group in alpha-keto acid dehydrogenase complexes of the mitochondria, and as such plays a fundamental role in metabolism. Although this has been known for decades, only recently has free supplemented alpha-lipoic acid been found to affect cellular metabolic processes in vitro, as it has the ability to alter the redox status of cells and interact with thiols and other antioxidants. Therefore, it appears that this compound has important therapeutic potential in conditions where oxidative stress is involved. Early case studies with alpha-lipoic acid were performed with little knowledge of the action of alpha-lipoic acid at a cellular level, but with the rationale that because the naturally occurring protein bound form of alpha-lipoic acid has a pivotal role in metabolism, that supplementation may have some beneficial effect. Such studies sought to evaluate the effect of supplemented alpha-lipoic acid, using low doses, on lipid or carbohydrate metabolism, but little or no effect was observed. A common response in these trials was an increase in glucose uptake, but increased plasma levels of pyruvate and lactate were also observed, suggesting that an inhibitory effect on the pyruvate dehydrogenase complex was occurring. During the same period, alpha-lipoic acid was also used as a therapeutic agent in a number of conditions relating to liver disease, including alcohol-induced damage, mushroom poisoning, metal intoxification, and CCl4 poisoning. Alpha-Lipoic acid supplementation was successful in the treatment for these conditions in many cases. Experimental studies and clinical trials in the last 5 years using high doses of alpha-lipoic acid (600 mg in humans) have provided new and consistent evidence for the therapeutic role of antioxidant alpha-lipoic acid in the treatment of insulin resistance and diabetic polyneuropathy. This new insight should encourage clinicians to use alpha-lipoic acid in diseases affecting liver in which oxidative stress is involved.

Antioxidants modulate acute solar ultraviolet radiation-induced NF-kappa-B activation in a human keratinocyte cell line

Exposure of the human skin to ultraviolet radiation (UVR) leads to depletion of cutaneous antioxidants, regulation of gene expression and ultimately to the development of skin diseases. Although exogenous supplementation of antioxidants prevents UVR-induced photooxidative damage, their effects on components of cell signalling pathways leading to gene expression has not been clearly established. In the present study, the effects of the antioxidants alpha-lipoic acid, N-acetyl-L-cysteine (NAC) and the flavonoid extract silymarin were investigated for their ability to modulate the activation of the transcription factors nuclear factor kappa B (NF-kappaB) and activator protein-1 (AP-1) in HaCaT keratinocytes after exposure to a solar UV simulator. The activation of NF-kappaB and AP-1 showed a similar temporal pattern: activation was detected 2 h after UV exposure and maintained for up to 8 h. To determine the capacity of activated NF-kappaB to stimulate transcription, NF-kappaB-dependent gene expression was measured using a reporter gene assay. The effects of the antioxidants on NF-kappaB and AP-1 activation were evaluated 3 h after exposure. While a high concentration of NAC could achieve a complete inhibition, low concentrations of alpha-lipoic acid and silymarin were shown to significantly inhibit NF-kappaB activation. In contrast, AP-1 activation was only partially inhibited by NAC, and not at all by alpha-lipoic acid or silymarin. These results indicate that antioxidants such as alpha-lipoic acid and silymarin can efficiently modulate the cellular response to UVR through their selective action on NF-kappaB activation.

Thiol Chelation of Cu2 By Dihydrolipoic Acid Prevents Human Low Density Lipoprotein Peroxidation

Mono-thiols can act either as pro- or anti-oxidants during metal-catalyzed low density lipoprotein (LDL) peroxidation, however investigation of the role of vicinal thiols has been neglected. Therefore dihydrolipoic acid (DHLA), a vicinal dithiol, and lipoic acid, its oxidized form, were used to investigate Cu2+-mediated LDL peroxidation. We demonstrate here that DHLA inhibited Cu2+-dependent LDL peroxidation by chelating copper. DHLA (0-20 microM) increased lag-times of conjugated diene formation in LDL (100 microg/ml) oxidized with 5 microM Cu2+ in a concentration dependent manner, and this effect was saturated after 5 microM DHLA; enough to chelate all of the added Cu2+. In a similar fashion DHLA prevented LDL-mediated reduction of Cu2+ to Cu+. Lipoic acid had no effect in these systems. DHLA alone also reduced Cu2+, however this was inhibited when DHLA was in excess of the copper concentration. Hence there is complex formation between the two species. Copper:DHLA complex formation was further investigated and found to be dependent upon pH and the presence of oxygen. At low pH (<6), or in the absence of oxygen, the complex is stable, presumably due to vicinal thiol chelation. As the pH is increased, the carboxylate group also participates in copper chelation, this results in a less stable complex which is susceptible to oxidation, and copper is eventually released. Electron spin resonance studies demonstrate the formation of hydroxyl, but not superoxide, radicals during Cu2+-catalyzed DHLA oxidation. Thus in our LDL experiments at physiological pH, DHLA is able to either reductively inactivate Cu2+ when Cu2+ is in excess, or effectively chelate Cu2+ when DHLA is in excess. The Cu2+:DHLA complex eventually undergoes copper-catalyzed oxidation, copper is released and LDL peroxidation proceeds. DHLA, thus, has both pro- and antioxidant properties depending upon the ratio of Cu2+:DHLA and the pH. These results provide an additional mechanism of thiol-mediated formation of radicals and metal chelation.

Copper-induced LDL peroxidation investigated by 1H-NMR spectroscopy

Oxidatively modified LDL (oLDL) is thought to play a key role in the pathogenesis of atherosclerosis. We have studied Cu(2+)-induced peroxidation reactions of LDL and have elucidated the sequence of events which subsequently occur within LDL particles by 1H-NMR spectroscopy. Studies of chloroform/methanol extracts show that LDL arachidonate is oxidised by Cu2+ at a higher rate and to a greater extent than linoleate, giving isomeric hydroperoxides with predominantly trans,trans double-bonds, whilst only cis,trans isomers were detected as intrinsic hydroperoxides in control LDL samples. These intrinsic hydroperoxides were not degraded during peroxidation, suggesting that they are not involved in the initiation of Cu(2+)-induced peroxidation. Aldehydes arising from the decomposition of hydroperoxides were also detected, as well as saturated fatty acids which were released into the external aqueous medium. Decomposition pathways of the two major isomeric hydroperoxides are discussed. Cu(2+)-induced oxidation of LDL cholesterol appears to occur only after hydroperoxide breakdown, with esterified cholesterol being oxidised to a greater extent than free cholesterol. Phospholipid hydrolysis appeared to parallel the peroxidation of arachidonic acid, and the released lysophosphatidylcholine may become associated with apoB. These results suggest that hydroperoxide breakdown (probably in phospholipids) may be a key event in the peroxidation process, leading to the oxidation of cholesterol and propagation into the core of LDL.

α-Lipoic Acid

There is increasing evidence that thiols play a role in various biological processes. This arises from their ability to undergo redox reactions; thus, they can act as efficient electron donators or acceptors. α-Lipoic acid is a dithiol-containing compound that plays an essential role in mitochondrial dehydrogenase reactions, but it has recently gained considerable interest as an antioxidant. Further investigations have shown lipoate to be an effective redox modulator of cell signaling and gene transcription. The various effects of α-lipoic acid at a cellular level are discussed here, highlighting the remarkable therapeutic potential for lipoate in a variety of disorders where oxidative stress is a factor.

Skeletal muscle and liver lipoyllysine content in response to exercise, training and dietary α‐lipoic acid supplementation

In human cells, α‐lipoic acid (LA) is present in a bound lipoyllysine form in mitochondrial proteins that play a central role in oxidative metabolism. The possible effects of oral LA supplementation, a single bout of strenuous exercise and endurance exercise training on the lipoyllysine content in skeletal muscle and liver tissues of rat were examined. Incorporation of lipoyl moiety to tissue protein was not increased by enhanced abundance of LA in the diet. Endurance exercise training markedly increased lipoyllysine content in the liver at rest. A bout of exhaustive exercise also increased hepatic lipoyllysine content. A significant interaction of exhaustive exercise and training to increase tissue lipoyllysine content was evident. In vastus lateralis skeletal muscle, training did not influence tissue lipoyllysine content. A single bout of exhaustive exercise, however, clearly increased the level of lipoyllysine ill the muscle. Comparison of tissue lipoyllysine data with that of free or loosely‐bound LA results showed a clear lack of association between the two apparently related parameters. Tightly protein‐bound lipoyllysine pool in tissues appeared to be independent of the loosely‐bound or free LA status in the tissue.

9 – Natural Sources of Lipoic Acid in Plant and Animal Tissues

Publisher Summary Naturally occurring lipoic acid is known to play a fundamental role in metabolism, serving as a cofactor in enzyme complexes, which function at strategic points in carbohydrate metabolism, citric-acid cycle, and amino-acid catabolism. In the last decade, experimental evidence has increased suggesting free lipoic acid to be a powerful antioxidant and redox regulator, and, as such, a potentially useful therapeutic tool. It is important that the distribution and content of lipoic acid in plants and animals be ascertained. Therefore, a method for measuring the naturally occurring protein-bound form of lipoic acid (lipoyllysine) has been developed, and this method has been used to measure the lipoyllysine content of various plant and animal tissues. In lipoate-containing enzymes (α-keto acid dehydrogenases), lipoic acid is bound covalently to a lysine residue. This is an important consideration for determination as the release of lipoic acid is crucial. Previous methods have tried to overcome this problem via hydrolysis with strong acid/base; however, a larger amount of lipoic acid is lost by such methods, and recoveries of only 50% have been reported, and no more than 70% have been found. To overcome this problem, proteolytic hydrolysis was introduced to liberate lipoic acid. This also has the advantage of liberating the actual protein-bound form (lipoyllysine). Lipoic acid from animal tissues has been determined previously, but with methods of detection such as refractive index, ultraviolet, and GC mass spectroscopy. The former methods are inadequate as tissue hydrolysates contain a large amount of contaminants, which absorb around 330 nm, and GC methods require prior derivatization. To overcome these problems, an electrochemical detection system was introduced.

Cu2+ -induced low density lipoprotein peroxidation is dependent on the initial O2 concentration: an O2 consumption study.

Atherosclerotic plaques form in the arterial intima, where low density lipoprotein (LDL) is thought to be oxidatively modified at sites which may contain catalytic amounts of copper in the presence of low O2 tension. We have investigated O2 consumption during LDL peroxidation induced by Cu2+ ions in vitro and found two phases: a lag phase followed by a phase of rapid O2 consumption. The length of the lag phase was dependent on Cu2+ and on initial O2 concentrations; increasing either decreased the lag time; however, LDL concentration had no effect. LDL-induced Cu2+ reduction, however, was not affected by low initial O2 concentrations, suggesting that O2 is not required for LDL-mediated reduction of Cu2+. Following the lag phase O2 consumption was dependent upon LDL or initial O2 concentrations; Cu2+ concentrations had little effect, suggesting that the propagation phase is more dependent on the presence of LDL lipids and O2 as substrates for the reaction. In summary, LDL peroxidation takes place in the presence of Cu2+ at low O2 tension; however, the reaction is dependent upon initial O2 concentrations; increases shorten the lag phase and accelerate O2 consumption.