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Glutathione Reductase and Lipoamide Dehydrogenase Have Opposite Stereospecificities for α-Lipoic Acid Enantiomers
Abstract
The reduction of exogenous alpha-lipoic acid to dihydrolipoate by mammalian cells and tissues confers additional antioxidant protection to the cell. Both (R+) and (S-) isomers of alpha-lipoic acid were analyzed as substrates with glutathione reductase from several sources and with mammalian lipoamide dehydrogenase. Mammalian glutathione reductase catalyzed faster reduction of (S)-lipoic acid (1.4-2.4-fold greater activity) than of (R)-lipoic acid, whereas lipoamide dehydrogenase had a very marked preference for (R)-lipoic acid (18-fold greater activity) over (S)-lipoic acid. Mammalian glutathione reductase showed better affinity for (S)-lipoic acid substrate; Km values were 3.5 mM for (S)-lipoic acid and and 7 mM for (R)-lipoic acid. Glutathione reductase from yeast reduced lipoic acid less efficiently than the mammalian enymes, had a Km for both stereoisomers of about 10 mM, and showed little stereospecificity. Although (S)-lipoic acid is not formed in nature, these findings indicate that exogenous (S)-lipoic acid may have a useful role as an antioxidant for mammalian systems.
... Alpha LA can increase GSH form it GSSG. It is said that existing glutathione is essential to prove the alpha LA antioxidant effects (23)(24)(25). It should be noted that alpha LA may have a good effect on glutathione cycle and maintain cellular glutathione by impacting on glutathione reductase (25). ...
... It is said that existing glutathione is essential to prove the alpha LA antioxidant effects (23)(24)(25). It should be noted that alpha LA may have a good effect on glutathione cycle and maintain cellular glutathione by impacting on glutathione reductase (25). DN is known as a common manifestation in advanced stages of diabetes (26). ...
Background: Myeloperoxidase (MPO) is involved in the initiation, progression, and complications of atherosclerosis in diabetic patients. Objectives: In the current study, the impact of alpha-lipoic acid (LA), a natural antioxidant and a cofactor in the enzyme complexes on MPO, catalase (CAT) and glutathione peroxidase (GPx) activity, glutathione (GSH) and malondialdehyde (MDA) level, histopathology of kidney and expression of antioxidant enzymes, superoxide dismutase (SOD), GPx and CAT which are involved in the detoxification of reactive oxygen species (ROS), was evaluated in alloxan-induced diabetic rats.
Materials and Methods: In this study, 30 male Rattus norvegicus rats randomly divided into three groups; control (C), non-treated diabetic (NTD), and LA-treated diabetics (LATD) was induced by alloxan monohydrate (100mg/kg; subcutaneous [SC]). Then treatment was performed with alphaLA (100 mg/kg intraperitoneal (i.p) daily to 6 weeks). Blood sample of animals collected to measure levels of MPO, CAT and GPx activity GSH and MDA. Kidney paraffin sections were prepared to estimate histological studies and to measure quantitative gene expression SOD, GPX and CAT in kidney.
Results: Induction of diabetes led to a significant increase in MPO and MDA, reduced GSH level and GPx and CAT activities (P < 0.05). However, treatment with alpha-LA led to a significant elevation in GPx, CAT and GSH levels with a reduction in MPO activities and MDA levels (P < 0.05). Furthermore, the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis results showed increased expressions of GPx, CAT and SOD enzyme in the treatment group compared with the diabetic control group. Histopathological lesions such as increased glomerular volume and lymphocyte infiltration were attenuated in the alpha-LA treated group.
Conclusions: Our findings indicated that alpha-LA supplementation is effective in preventing complications induced by oxidative stress and atherosclerosis in diabetic rats.
... Ovariectomy markedly decreases estrogen level and 17β-E 2 receptor activity in the different structures of the brain. 30,31 In this connection, a low dose of 17β-E 2 may play a trigger role in activation of 17β-E 2 receptors at the hypoestrogenic syndrome. Thus, we used a low dose of 17β-E 2 in our present study. ...
... 30,49 Projections from serotonergic neurons of the raphe nuclei terminate on GABAergic hippocampal interneurons increasing or decreasing their activity via serotonergic receptors. 31,50 In our study of cholecalciferol effects in hippocampus may be useful to study the respective contribution direct of serotonin and/or indirect of GABA in the treatment of CNS diseases. DA and NE are the most abundant excitatory monoamine neurotransmitters, widely distributed in the mammalian brain, including the hippocampus. ...
Vitamin D can be one of the candidate substances that are used as additional supplementation in the treatment of anxiety-related disorders in women with estrogen imbalance.
The aim of the present study was to examine the effects of chronic cholecalciferol administration (1.0, 2.5 or 5.0 mg/kg/day, s.c.) on the anxiety-like behavior and monoamines levels in the rat hippocampus following ovariectomy in female rats. Cholecalciferol was given to ovariectomized (OVX) rats and OVX rats treated with 17β-estradiol (17β-E
Cholecalciferol in high doses alone or in combination with 17β-E
Our results indicate that cholecalciferol in high doses has a marked anxiolytic-like effect due to an increase in the monoamines levels in the experimental rat model of estrogen deficiency.
... Moreover, S(À)-LA acts either as a poor substrate or as an inhibitor of R(þ)-LA in its interaction with 2-oxoacid dehydrogenase multienzyme complexes. However, both free LA enantiomers are reduced intracellularly to their respective reduced forms, albeit by different enzymatic interactions: R(þ)-LA by dihydrolipoamide dehydrogenase (the E3 enzyme in the PDH complex) and S(À)-LA by glutathione reductase (Pick et al. 1995;Haramaki et al. 1997). Consequently, R(þ)-LA=R(þ)-DHLA and S(À)-LA=S(À)-DHLA act as redox couples and free-radical scavengers in cells (Suzuki et al. 1991;Hermann et al. 1996). ...
... It appears that the amide bond linking LA to the bulky amphiphilic carrier was relatively resistant to amidase-induced hydrolysis in cells due to steric hindrance. Therefore, mitochondrial or cytoplasmic enzymatic reduction of LA to DHLA by dihydrolipoamide dehydrogenase or glutathione reductase was slowed (Pick et al. 1995;Haramaki et al. 1997). Consequently, a limited generation of functional LA=DHLA redox couple in cells may explain the marginal antioxidative effects of this derivative. ...
... Once in the cells, LA can undergo β-oxidation (Teichert et al., 2003;Shay et al., 2009) in mitochondria or reductive reactions to produce DHLA, which are important for the recycling of other antioxidants as stated in Section 1. At least three enzymes are known to reduce LA: cytosolic thioredoxin reductase, glutathione reductase and mitochondrial lipoamide dehydrogenase (Pick et al., 1995;Biewenga et al., 1996;Bustamante et al., 1998) (Fig. 2). In the reductive pathways, some important differences exist. ...
... As the C 6 is a chiral carbon, LA can occur as an R or S enantiomer. A higher activity of lipoamide dehydrogenase has been verified for the natural R-enantiomer, and a preference for the S-enantiomer has been reported for glutathione reductase (Pick et al., 1995;Biewenga et al., 1996). May et al. (2007) reported a lack of specificity of thioredoxin reductase from human red blood erythrocytes between R and S enantiomers. ...
Lipoic acid (LA) is a disulfide-containing compound derived from octanoic acid that is synthesized in mitochondria. This molecule acts as a co-factor for mitochondrial enzymes that catalyze oxidative decarboxylation reactions. Several antioxidant properties of LA enable it to be considered as an “ideal antioxidant”, having diverse benefits that allow it to deal with environmental or biological stress. Some of the effects induced by LA in aquatic organisms render it suitable for use in aquaculture. However, it is necessary to determine the appropriate dose(s) to be used with different species and even organs to maximize the beneficial antioxidant and detoxifying effects and to minimize the pro-oxidant toxic effects. This review analyzes and compiles existing data from aquatic organisms in which both benefits and drawbacks of LA have been described.
... ALA is a cofactor for mitochondrial α-keto-dehydrogenase complexes and participates in S-O transfer reactions that are naturally occurring cofactors that also possesslipid regulatory properties. ALA is reduced to DHLA in several tissues [15,48] and both ALA and DHLA have acted as antioxidants in relation to hydroxyl radicals and inhibit the oxidation of lipids and proteins [57]. Cell reduction of ALA to DHLA is performed by NAD (P) H-induced enzymes, thioredoxin reductase, lipoamide dehydrogenase and glutathione reductase. ...
Dapsone (DDS) therapy can frequently lead to hematological side effects, such as methemoglobinemia and DNA damage. In this study, we aim to evaluate the protective effect of racemic alpha lipoic acid (ALA) and its enantiomers on methemoglobin induction. The pre- and post-treatment of erythrocytes with ALA, ALA isomers, or MB (methylene blue), and treatment with DDS-NOH (apsone hydroxylamine) was performed to assess the protective and inhibiting effect on methemoglobin (MetHb) formation. Methemoglobin percentage and DNA damage caused by dapsone and its metabolites were also determined by the comet assay. We also evaluated oxidative parameters such as SOD, GSH, TEAC (Trolox equivalent antioxidant capacity) and MDA (malondialdehyde). In pretreatment, ALA showed the best protector effect in 2.5 µg/mL of DDS-NOH. ALA (1000 µM) was able to inhibit the induced MetHb formation even at the highest concentrations of DDS-NOH. All ALA tested concentrations (100 and 1000 µM) were able to inhibit ROS and CAT activity, and induced increases in GSH production. ALA also showed an effect on DNA damage induced by DDS-NOH (2.5 µg/mL). Both isomers were able to inhibit MetHb formation and the S-ALA was able to elevate GSH levels by stimulating the production of this antioxidant. In post-treatment with the R-ALA, this enantiomer inhibited MetHb formation and increased GSH levels. The pretreatment with R-ALA or S-ALA prevented the increase in SOD and decrease in TEAC, while R-ALA decreased the levels of MDA; and this pretreatment with R-ALA or S-ALA showed the effect of ALA enantiomers on DNA damage. These data show that ALA can be used in future therapies in patients who use dapsone chronically, including leprosy patients.
... Lipoic acid has been proposed to be an antioxidant 41 , although evidence for direct action as an antioxidant is disputed 42 . Cellular antioxidant effects involving an enzymatic redox cycle will involve dihydrolipoamide dehydrogenase, which is more active for the naturally occurring R-(+) isomer although normally reduces lipoyl moieties 38,43 . However S-(−)-lipoic acid had very similar activity to R-(+)-lipoic acid ( Figure 3C,D). ...
... Lipoic acid has been proposed to be an antioxidant 41 , although evidence for direct action as an antioxidant is disputed 42 . Cellular antioxidant effects involving an enzymatic redox cycle will involve dihydrolipoamide dehydrogenase, which is more active for the naturally occurring R-(+) isomer although normally reduces lipoyl moieties 38,43 . However S-(−)-lipoic acid had very similar activity to R-(+)-lipoic acid ( Figure 3C,D). ...
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with few avenues for treatment. Many proteins implicated in ALS associate with stress granules, which are examples of liquid-like compartments formed by phase separation. Aberrant phase transition of stress granules has been implicated in disease, suggesting that modulation of phase transitions could be a possible therapeutic route. Here, we combine cell-based and protein-based screens to show that lipoamide, and its related compound lipoic acid, reduce the propensity of stress granule proteins to aggregate in vitro. More significantly, they also prevented aggregation of proteins over the life time of Caenorhabditis elegans. Observations that they prevent dieback of ALS patient-derived (FUS mutant) motor neuron axons in culture and recover motor defects in Drosophila melanogaster expressing FUS mutants suggest plausibility as effective therapeutics. Our results suggest that altering phase behaviour of stress granule proteins in the cytoplasm could be a novel route to treat ALS.
... Exogenously supplied lipoic acid (1.65 g/kg diet, 5 weeks) was absorbed, transported to tissues, and reduced to DHLA in adult hairless mice (Podda et al., 1994). Two enzymes systems were identified to reduce lipoic acid to DHLA (Pick et al., 1995). The mitochondrial dihydrolipoamide dehydrogenase is capable of reducing lipoic acid to DHLA in the expense of NADH. ...
The present study was undertaken to evaluate the potential protective effects
of lipoic acid against the toxicity induced by aluminium phosphide on biochemical
parameters as well as enzyme activities and thiobarbituric acid reactive substance
(TBARS) in plasma, liver, kidney, lung, testes and brain of rats. Twenty eight male
Wistar rats (each weighing 100-135g) were used. Animals were divided into 4 groups,
7 rats in each. The first group was used as control, the second group was treated with
lipoic acid (100 mg/k BW), the third group was treated with aluminium phosphide
(AlP, 2 mg/k BW), while the fourth group was administrated to lipoic acid and
aluminium phosphide as combination. The doses of lipoic acid and aluminium
phosphide were given orally daily for 30 days. At the end of the experimental period
body weight of rats were recorded. Then animals were sacrificed. Blood samples were
collected and kidney, liver, lung, heart, brain, spleen, testes and epididymis were
obtained and the tested parameters were carried out.
Treatment with lipoic acid alone did not cause significant changes in body weight
or relative weight of tested organs. While, treatment with aluminium phosphide lead
to significant decrease (P<0.05) in body weight and the relative weight of testes and
epididymes. While, treatment with aluminium phosphide caused significant (P<0.05)
increase in the relative weight of spleen. The concentrations of plasma total protein
showed insignificant increase due to treatment with lipoic acid alone, while treatment
with aluminium phosphide caused significant (P<0.05) decrease in plasma total
protein and the protein content of liver and kidney. Treatment with lipoic acid
significantly (P<0.05) decreased the concentration of plasma glucose, and
insignificant decrease in the concentration of urea, creatinine and bilirubin. On the
other hand, treatment with aluminium phosphide significantly increased (P<0.05)
bilirubin, urea and creatinine. Aluminium phosphide induced lipid peroxidations
which play an important role in the nephrotoxicity. The activities of plasma aspartate
aminotransaminase (AST), alanine aminotransaminase (ALT) and acid phosphatase
(AcP) did not change due to treatment with lipoic acid alone, while insignificant
decrease in the activity of alkaline phosphatase (AP) occurred. On the other hand,
aluminium phosphide significantly (p<0.05) increased plasma AST, ALT, AP and
AcP activities compared to control. Also, it caused significant (p<0.05) increase in the
activities of AST, ALT and AcP in liver. Aluminium phosphide induced lipid
peroxidations which play an important role in the hepatotoxicity. Treatment with
lipoic acid alone did not cause any significant changes on the activities of antioxidant
enzymes (glutathione S-Transferase, GST; super oxide dismutase, SOD; and catalase,
CAT) and did not cause any significant changes in the concentrations of thiobarbituric
acid-reactive substances (TBARS) in plasma and liver, while caused insignificant
decrease in the concentration of TBARS in kidney. On the other hand, treatment
with aluminium phosphide significantly (P<0.05) increased the activity of SOD and
decreased the activity of GST and CAT in plasma, liver and kidney. While,
significantly (P<0.05) increased the concentration of plasma, liver and kidney
TBARS and significantly decreased (P<0.05) the concentration of reduced glutathione
(GSH) in liver.
The observed phosphine induced oxidative damage in rats may be related to
oxyradicals, nitric oxide, peroxynitrite, or the combination of them. The mechanism
of phosphine induced lipid peroxidation could involve reactive oxygen species (ROS)
generated from inhibition of cellular respiration, or a direct reaction between
phosphine and H2O2. While, the structure of lipoic acid is suitable for modulating the
lipid peroxidation by ability to chelate transition metals thus inhibiting formation of
hydroxyl radical, capacity to scavenge reactive oxygen species, capacity to regenerate
endogenous antioxidants such as vitamin C, vitamin E, and GSH and ability to repair
oxidatively damaged protein. Administration of lipoic acid in combination with
aluminium phosphide was able to minimize and alleviate the hazardous effect of
aluminium phosphide on most of the measured parameters.
... Normally the dietary supplements made from ALA contain a mixture of both R-and S-enantiomers. This is the best choice because the two stereoisomers are absorbed and metabolized differently depending on the site of uptake, which implicates the involvement of different enzymes 36 . ...
OBJECTIVE: Alpha-lipoic acid is a natural molecule, which directly or by means of its reduced form, dihydrolipoic acid, exerts antioxidant, anti-inflammatory and immunomodulatory activities, very helpful also in preventing miscarriage and preterm delivery. Used as dietary supplement alpha-lipoic acid was demonstrated to be safe for living organisms even when administered at high doses. However, no study was made so far to verify the safety of its continuous administration on a substantial number of pregnant women. The present investigation was performed to answer this issue.
PATIENTS AND METHODS: An observational retrospective study was carried out analyzing 610 expectant mothers. They had been treated daily by oral route with 600 mg alpha-lipoic acid, for at least 7 weeks during gestation. The primary outcome was to verify alpha-lipoic acid safety in the mother and infant. Maternal safety was assessed by monitoring for adverse reactions, physical and clinical examination, including a morbidity assessment. Laboratory and clinical examinations were performed monthly. Neonatal safety was assessed by the evaluation of birth weight, gestational age, Apgar scores, neonatal death with the related cause of death. Data collected from the Birth Registry of Campania Region were used as control.
RESULTS: This study provided a very clear and reassuring picture about the safety of alpha-lipoic acid oral treatment during pregnancy. No adverse effect was noticed in mothers or newborns. The two sets of monitored data, from treated and controls, were completely superimposable or, in some cases, better in alpha-lipoic acid group.
CONCLUSIONS: Our results open a reassuring scenario regarding the administration of alpha-lipoic acid during pregnancy.
... These disturbances cause organelle damage, changes in gene expression followed by altered cellular responses which ultimately results into aging glucose uptake in L6 myotubes [14], as well as to enhance insulin-stimulated glucose uptake in obese Zucker rats [32]. On the other hand, the S-enantiomer exerts a slightly increased affinity for glutathione reductase [57], thus the two forms of LA differ in their ability to exercise the various biological activities. ...
Background:
The molecular nature of lipoic acid (LA) clarifies its capability of taking part to a variety of biochemical reactions where redox state is meaningful. The pivotal action of LA is the antioxidant activity due to its ability to scavenge and inactivate free radicals. Furthermore, LA has been shown to chelate toxic metals both directly and indirectly by its capability to enhance intracellular glutathione (GSH) levels. This last property is due to its ability to interact with GSH and recycle endogenous GSH. LA exhibits significant antioxidant activity protecting against oxidative damage in several diseases, including neurodegenerative disorders. Interestingly, LA is unique among natural antioxidants for its capability to satisfy a lot of requirements, making it a potentially highly effective therapeutic agent for many conditions related with oxidative damage. In particular, there are evidences showing that LA has therapeutic activity in lowering glucose levels in diabetic conditions. Similarly, LA supplementation has multiple beneficial effects on the regression of the mitochondrial function and on oxidative stress associated with several diseases and aging.
Aim:
The aim of the present review is to describe the molecular mechanisms underlying the beneficial effects of LA under various experimental conditions and disease and how to exploit such effect for clinical purposes.
Conclusion:
LA has pleiotropic effects in different pathways related with several diseases, its use as a potential therapeutic agent is very promising.
... LA is generated by the contribution of two sulfur atoms to the octanoyl group from the octanoic acid in the mitochondria, and it is located in mammalian cells that have great mitochondrial numbers and activity such as liver, heart, and testis [11]. This molecule is converted to dihydrolipoic acid (DHLA) by the mitochondrial dihydrolipoamide dehydrogenase and cytosolic glutathione reductase enzyme systems [12]. DHLA can act as a powerful reductant agent that can interact with the oxidized glutathione and other oxidized antioxidants such as vitamin E and vitamin C [13]. ...
Background:
We have investigated the effects of α-lipoic acid (LA), a powerful antioxidant, on the fatty acid (FA) profiles, aluminum accumulation, antioxidant activity and some minerals such as zinc, copper and iron against aluminum chloride (AlCl3)-induced oxidative stress in rat liver.
Methods:
Twenty-eight male Wistar rats were divided into four groups as control, LA, AlCl3 and LA+AlCl3. For 30 days, LA was intraperitoneally administrated (50 mg/kg) and AlCl3 was given via orogastric gavage (1600 ppm) every other day.
Results:
AlCl3-treated animals exhibited higher hepatic malondialdehyde concentration and lower glutathione peroxidase and catalase activity, whereas these alterations were restored by the LA supplementation. Total saturated FA of the AlCl3-treated group was higher than the LA supplementation groups. Moreover, total unsaturated FA level of the LA+AlCl3 group was higher than the AlCl3-treated group. Hepatic zinc level of the AlCl3-treated group was lower than the control group, whereas it was higher in the LA and the LA+AlCl3 groups. Hepatic copper levels did not significantly change in the experimental groups. Iron level was lower in the LA and LA+AlCl3 groups compared with the AlCl3-treated group. Moreover, the liver Al concentration was found to be lower in the LA and LA+AlCl3 groups compared to the AlCl3 group.
Conclusions:
These results indicate that AlCl3 treatment can induce oxidative stress in the liver. LA supplementation has a beneficial effect on the AlCl3-induced alterations such as high lipid peroxidation, Al accumulation, FA profile ratios and mineral concentrations.
... Then, 17β-E 2 was injected subcutaneously at a dose of 5.0 µg/rat. Ovariectomy markedly decreases estrogen level and 17β-E 2 receptor activity in the different structures of the brain [26,27]. In this connection, a low dose of 17β-E 2 may play a trigger role in the activation of 17β-E 2 receptors in hypoestrogenic syndrome (20). ...
The present preclinical study was created to determine the therapeutic effects of vitamin D hormone treatment as an adjunctive therapy alone or in a combination with low dose of 17β-estradiol (17β-E2) on anxiety-like behavior in female rats with long-term absence of estrogen. Accordingly, the aim of the current study was to examine the effects of chronic cholecalciferol administration (1.0, 2.5 or 5.0 mg/kg subcutaneously, SC, once daily, for 14 days) on the anxiety-like state after long-term ovariectomy in female rats. Twelve weeks postovariectomy, cholecalciferol was administered to ovariectomized (OVX) rats and OVX rats treated with 17β-E2 (0.5 µg/rat SC, once daily, for 14 days). Anxiety-like behavior was assessed in the elevated plus maze (EPM) and the light/dark test (LDT), and locomotor and grooming activities were tested in the open field test (OFT). Cholecalciferol at two doses of 1.0 and 2.5 mg/kg alone or in combination with 17β-E2 produced anxiolytic-like effects in OVX rats as evidenced in the EPM and the LDT, as well as increased grooming activity in the OFT. Our results indicate that cholecalciferol, at two doses of 1.0 and 2.5 mg/kg, has a profound anxiolytic-like effects in the experimental rat model of long-term estrogen deficiency.
... The disulfide bonds of -LA can potentially perturb the cellular redox environment by oxidizing protein thiols. The thiolane ring of -LA can be reduced to vicinal thiolslike dihydrolipoic acid (DHLA) in cells through NADH-and NADPH-dependent pathways such as mitochondrial electron transport chain, thioredoxin and thioredoxin reductase, lipoamide dehydrogenase and oxidized glu-tathione (GSSG) reductases [14][15][16]. -LAis rapidly converted to DHLA in mitochondria by an NADH-dependent reaction with lipoamide dehydrogenase, whereas in mitochondria-deficient cells, -LA can be reduced to DHLA via an NADPH-dependent glutathione and thioredoxin reductases [17]. Consequently, -LA serves as a cofactor for mitochondrial bioenergetic enzymes, and is a pharmacological antioxidant that can be generated by the body and absorbed from the diet via synthetic R-enantionmer (naturally occurring form) and the S-enantiomer. ...
Reactive oxygen species and reactive nitrogen species promote endothelial dysfunction in old age and contribute to the development of cardiovascular diseases such as atherosclerosis, diabetes, and hypertension. alpha-lipoic acid was identified as a catalytic agent for oxidative decarboxylation of pyruvate and alpha-ketoglutarate in 1951, and it has been studied intensively by chemists, biologists, and clinicians who have been interested in its role in energetic metabolism and protection from reactive oxygen species-induced mitochondrial dysfunction. Consequently, many biological effects of alpha-lipoic acid supplementation can be attributed to the potent antioxidant properties of alpha-lipoic acid and dihydro alpha-lipoic acid. The reducing environments inside the cell help to protect from oxidative damage and the reduction-oxidation status of alpha-lipoic acid is dependent upon the degree to which the cellular components are found in the oxidized state. Although healthy young humans can synthesize enough alpha-lipoic acid to scavenge reactive oxygen species and enhance endogenous antioxidants like glutathione and vitamins C and E, the level of alpha-lipoic acid significantly declines with age and this may lead to endothelial dysfunction. Furthermore, many studies have reported alpha-lipoic acid can regulate the transcription of genes associated with anti-oxidant and anti-inflammatory pathways. In this review, we will discuss recent clinical studies that have investigated the beneficial effects of alpha-lipoic acid on endothelial dysfunction and propose possible mechanisms involved.
... 13 GSHreductase is a cytosolic enzyme which uses NADPH as a cofactor. The mammalian GSH-reductase converts the Senantiomer 2.4 times faster than the R-enantiomer, 14 and thus has an opposite stereoselectivity compared to LipDH. ...
For the antioxidant effect of lipoic acid, reduction to dihydrolipoic acid is considered to be important. Dihydrolipoamide dehydrogenase (LipHD) preferentially reduces R-lipoic acid and in a subsequent reaction, the R-dihydrolipoic acid formed may non-enzymatically reduce S-lipoic acid. Using circular dichroism (CD) spectroscopy, the second order rate constant of the latter reaction was determined (k2 = 15 M−1 sec−1). In vitro, it was found that S-lipoic acid is reduced by LipDH using R-lipoic acid as a catalyst. The non-enzymatic dithiol-disulfide reaction leads to synergistic reduction of the enantiomers which can explain the higher antioxidant activity of racemic lipoic acid found in vivo (Maitra et al. Biochem. Biophys. Res. Commun. 221:422–429, 1996) in comparison to the enantiomers. Lipoic acid is therapeutically active in several diseases via antioxidant activity. These findings suggest that racemic lipoic acid can be therapeutically more active than the separate enantiomers. Chirality 9:362–366, 1997. © 1997 Wiley-Liss, Inc.
... Exogenously supplied LA (1.65 g/kg diet, 5 weeks) was absorbed, transported to tissues, and reduced to DHLA in adult hairless mice (Podda et al., 1994). Two enzyme systems were identified to reduce LA to DHLA (Pick et al., 1995). The mitochondrial dihydrolipoamide dehydrogenase is capable of reducing LA to DHLA at the expense of NADH. ...
... Both (+)-and (−)-thioctic acid are reduced intracellularly via two enzymatic pathways. (+)-thioctic acid is reduced by dihydrolipoamide dehydrogenase (the E3 enzyme in the PDH complex), whereas (−)-thioctic acid is reduced by glutathione reductase [18,19]. ...
Oxidative stress is an imbalance between the production of free radicals and antioxidant defense mechanisms, potentially leading to tissue damage. Oxidative stress has a key role in the development of cerebrovascular and/or neurodegenerative diseases. This phenomenon is mainly mediated by an enhanced superoxide production by the vascular endothelium with its consequent dysfunction. Thioctic, also known as alpha-lipoic acid (1,2-dithiolane-3-pentanoic acid), is a naturally occurring antioxidant that neutralizes free radicals in the fatty and watery regions of cells. Both the reduced and oxidized forms of the compound possess antioxidant ability. Thioctic acid has two optical isomers designated as (+)- and (-)-thioctic acid. Naturally occurring thioctic acid is the (+)-thioctic acid form, but the synthetic compound largely used in the market for stability reasons is a mixture of (+)- and (-)-thioctic acid. The present study was designed to compare the antioxidant activity of the two enantiomers versus the racemic form of thioctic acid on hydrogen peroxide-induced apoptosis in a rat pheochromocytoma PC12 cell line. Cell viability was evaluated by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and free oxygen radical species (ROS) production was assessed by flow cytometry. Antioxidant activity of the two enantiomers and the racemic form of thioctic acid was also evaluated in spontaneously hypertensive rats (SHR) used as an in vivo model of increased oxidative stress. A 3-h exposure of PC12 cells to hydrogen peroxide (H(2)O(2)) significantly decreased cell viability and increased levels of intracellular ROS production. Pre-treatment with racemic thioctic acid or (+)-enantiomer significantly inhibited H(2)O(2)-induced decrease in cell viability from the concentration of 50 μmol/L and 20 μmol/L, respectively. Racemic thioctic acid and (+)-salt decreased levels of intracellular ROS, which were unaffected by (-)-thioctic acid. In the brain of SHR, the occurrence of astrogliosis and neuronal damage, with a decreased expression of neurofilament 200 kDa were observed. Treatment of SHR for 30 days with (+)-thioctic acid reduced the size of astrocytes and increased the neurofilament immunoreaction. The above findings could contribute to clarify the role played by thioctic acid in central nervous system injury related to oxidative stress. The more pronounced effect of (+)-thioctic acid observed in this study may have practical therapeutic implications worthy of being investigated in further preclinical and clinical studies.
... The enzyme show a marked preference for the naturally occurring R-enantiomer of lipoate [10]. Lipoate is also a substrate for the NADPHdependent enzyme glutathione reductase [20]. Although lipoate is recognized by glutathione reductase as a substrate for reduction, the rate of reduction to DHLA is much slower than that of the natural substrate glutathione disulfide. ...
Lipoic acid (LA) is globally known and its supplements are widely used. Despite its importance for the organism it is not considered a vitamin any more. The multiple metabolic forms and the differences in kinetics (absorption, distribution and excretion), as well as the actions of its enantiomers are analysed in the present article together with its biosynthetic path. The proteins involved in the transfer, biotransformation and activity of LA are mentioned. Furthermore, the safety and the toxicological profile of the compound are commented, together with its stability issues. Mechanisms of lipoic acid intervention in the human body are analysed considering the antioxidant and non-antioxidant characteristics of the compound. The chelating properties, the regenerative ability of other antioxidants, the co-enzyme activity and the signal transduction by the implication in various pathways will be discussed in order to be elucidated the pleiotropic effects of LA. Finally, lipoic acid integrating analogues are mentioned under the scope of the multiple pharmacological actions they acquire towards degenerative conditions.
Graphic abstract
Malaria is still one of the most important global infectious diseases. Emergence of drug resistance and a shortage of new efficient antimalarials continue to hamper a malaria eradication agenda. Malaria parasites are highly sensitive to changes in the redox environment. Understanding the mechanisms regulating parasite redox could contribute to the design of new drugs. Malaria parasites have a complex network of redox regulatory systems housed in their cytosol, in their mitochondrion and in their plastid (apicoplast). While the roles of enzymes of the thioredoxin and glutathione pathways in parasite survival have been explored, the antioxidant role of α-lipoic acid (LA) produced in the apicoplast has not been tested. To take a first step in teasing a putative role of LA in redox regulation, we analysed a mutant Plasmodium falciparum (3D7 strain) lacking the apicoplast lipoic acid protein ligase B (lipB) known to be depleted of LA. Our results showed a change in expression of redox regulators in the apicoplast and the cytosol. We further detected a change in parasite central carbon metabolism, with lipB deletion resulting in changes to glycolysis and tricarboxylic acid cycle activity. Further, in another Plasmodium cell line (NF54), deletion of lipB impacted development in the mosquito, preventing the detection of infectious sporozoite stages. While it is not clear at this point if the observed phenotypes are linked, these findings flag LA biosynthesis as an important subject for further study in the context of redox regulation in asexual stages, and point to LipB as a potential target for the development of new transmission drugs.
The major focus of this study was to prepare poly(lactic-co-glycolic) acid (PLGA) microspheres containing atorvastatin calcium (ATR) in combination with alpha-lipoic acid (ALA). PLGA microspheres will maintain dual-release for providing neuroprotective effects for peripheral nerve injury. For this purpose, microspheres were prepared by spray dryer with different drug:polymer ratios. Microsphere formulations were evaluated for particle size distribution, preparation and encapsulation efficiency, surface morphology, in-vitro release and dose dependent cytotoxicity test with L-929 and B-35 cells. TGA, DSC and FTIR analysis were performed for the investigation of physicochemical properties of the PLGA microspheres. Encapsulation efficiencies were calculated as >70% for ALA and >62% for ATR. FTIR results indicated that there were no interaction between the polymer and the active ingredients. A novel analytical method has been developed and fully validated, which would allow for quantification of ATR and ALA simultaneously. Release profiles showed that ALA is released within the first 17 h and ATR release lasted for 17 days. Finally, results showed that there was no any toxicity associated with ALA and ATR containing PLGA formulations on both B-35 and L-929 cells. It was concluded that PLGA formulations with dual effects are promising systems for the treatment of peripheral nerve injury.
Previous studies indicated that reduced androgen levels may contribute to both physical and cognitive disorders in men, including Alzheimer's disease. New drug candidates for Alzheimer's disease in patients with androgen deficiency should ideally be able to act not only on multiple brain targets but also to correct impaired endocrine functions in hypogonadal men with Alzheimer's disease. Ropren(®) is one such candidate for the treatment of Alzheimer's disease in men with an imbalance of androgens. Accordingly, the aim of the current study was to examine the effects of long-term Ropren(®) administration (8.6mg/kg, orally, once daily, for 28 days) on the anxiety-like behavior and monoamines levels in the rat hippocampus using a β-amyloid (25-35) rat model of Alzheimer's disease following gonadectomy. Ropren(®) was administered to the gonadectomized (GDX) rats and GDX rats treated with testosterone propionate (TP, 0.5mg/kg, subcutaneous, once daily, for 28 days). Anxiety-like behavior was assessed in the elevated plus maze (EPM) and the light-dark test (LDT), locomotor and grooming activities were assessed in the open field test (OFT). Ropren(®) alone or in combination with TP-induced anxiolytic effects as evidenced in the EPM and in the LDT and increased locomotor activity in the OFT. Additionally, it was observed that dopamine (DA) and serotonin (5-HT) levels increased while 5-hydroxyindoleacetic acid (5-HIAA)/5-HT ratio in the hippocampus decreased. Our results indicate that Ropren(®) has a marked anxiolytic-like action due to an increase in the monoamines levels in the experimental rat model of Alzheimer's disease with altered levels of androgens.
Purpose:
The purpose of the experiments described here was to determine the effects of lipoic acid (LA)-dependent disulfide reduction on mouse lens elasticity, to synthesize the choline ester of LA (LACE), and to characterize the effects of topical ocular doses of LACE on mouse lens elasticity.
Methods:
Eight-month-old mouse lenses (C57BL/6J) were incubated for 12 hours in medium supplemented with selected levels (0-500 μM) of LA. Lens elasticity was measured using the coverslip method. After the elasticity measurements, P-SH and PSSP levels were determined in homogenates by differential alkylation before and after alkylation. Choline ester of LA was synthesized and characterized by mass spectrometry and HPLC. Eight-month-old C57BL/6J mice were treated with 2.5 μL of a formulation of 5% LACE three times per day at 8-hour intervals in the right eye (OD) for 5 weeks. After the final treatment, lenses were removed and placed in a cuvette containing buffer. Elasticity was determined with a computer-controlled instrument that provided Z-stage upward movements in 1-μm increments with concomitant force measurements with a Harvard Apparatus F10 isometric force transducer. The elasticity of lenses from 8-week-old C57BL/6J mice was determined for comparison.
Results:
Lipoic acid treatment led to a concentration-dependent decrease in lens protein disulfides concurrent with an increase in lens elasticity. The structure and purity of newly synthesized LACE was confirmed. Aqueous humor concentrations of LA were higher in eyes of mice following topical ocular treatment with LACE than in mice following topical ocular treatment with LA. The lenses of the treated eyes of the old mice were more elastic than the lenses of untreated eyes (i.e., the relative force required for similar Z displacements was higher in the lenses of untreated eyes). In most instances, the lenses of the treated eyes were even more elastic than the lenses of the 8-week-old mice.
Conclusions:
As the elasticity of the human lens decreases with age, humans lose the ability to accommodate. The results, briefly described in this abstract, suggest a topical ocular treatment to increase lens elasticity through reduction of disulfides to restore accommodative amplitude.
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.
In this review the structure and function of the lipoamide dehydrogenase component of the keto acid dehydrogenase complexes is discussed. Three structural models are available and in recent years many new genes have been sequenced and expressed. Unsolved is the role that many non-complex-bound lipoamide dehydrogenases play in metabolism and the physiological function of free lipoic acid. Central in catalysis is the problem of stabilization of the 2-electron reduced enzyme. Mutagenesis studies and the use of a modified flavin cofactor revealed the various factors involved in this stabilization in which also the core component of the complex has a function.
Powders of α-lipoic acid (ALA)/cyclodextrin (CD) complexes containing 9-24% (w/w) ALA were prepared to improve the gastrointestinal absorption of ALA. ALA and the ALA/CD complexes, corresponding to 30 mg/kg of body weight of ALA in rats and 600 mg of ALA in humans, were orally administered under fasting conditions. In rats, the area under the ALA plasma concentration/time-course curve from 0 to 3 h (AUC 0-3 h) for the ALA/G2-β-CD® complex was larger than that for ALA alone. In humans, after administration of 3 ALA/CD complexes with γ-CD, G2-β-CD® and Isoeleat®P, the ALA plasma concentration value 0.5 h after administration of the ALA/G2-β-CD® complex was significantly higher (p < 0.05) than that for ALA alone, and the AUC 0-3 h value for the ALA/G2-β-CD® complex was 1.4 times larger than that for ALA alone, although the difference was not significant. We suggest that the water-soluble ALA/G2-β-CD® complex powder can enhance ALA absorption in rats and humans.
α-Lipoic acid, also known as thioctic acid, 1,2-dithiolane-3-pentanoic acid, 1,2-dithiolane-3-valeric, or 6,8-thioctic acid, is a naturally occurring potent antioxidant. It is present as lipoyllysine in various natural sources. In the plant material studied, the lipoyllysine content was the highest in spinach (3.15 μg/g dry weight; 92.51 μg/mg protein). When expressed as weight per dry weight of lyophilized vegetables, the abundance of naturally existing lipoate in spinach is over three- and five-fold higher than that in broccoli and tomato, respectively. A lower concentration of lipoyllysine is also detected in garden pea, brussel sprouts, and rice bran. However, lipoyllysine concentration has been found to be below detection limits in acetone powders of banana, orange peel, soybean, and horseradish. Lipoic acid is also an integral component of the mammalian cell. It is present in trace amounts as lipoamide in at least five proteins, where it is covalently linked to a lysyl residue. Four of these proteins are found in α-keto acid dehydrogenase complexes: pyruvate dehydrogenase complex, branched chain keto acid dehydrogenase complex, and α-ketoglutarate dehydrogenase complex. Three lipoamide-containing proteins are present in the E2 enzyme, dihydrolipoyl acyltransferase, which is different in each of the complexes and specific for the substrate of the complex. One lipoyl residue is found in protein X, which is the same in each complex. The fifth lipoamide residue is present in the glycine cleavage system.
Considering the current obesity epidemic in the United States (>100 million adults are overweight or obese), the prevalence of hypertriglyceridemia is likely to grow beyond present statistics of ∼30% of the population. Conventional therapies for managing hypertriglyceridemia include lifestyle modifications such as diet and exercise, pharmacological approaches, and nutritional supplements. It is critically important to identify new strategies that would be safe and effective in lowering hypertriglyceridemia. α-Lipoic acid (LA) is a naturally occurring enzyme cofactor found in the human body in small quantities. A growing body of evidence indicates a role of LA in ameliorating metabolic dysfunction and lipid anomalies primarily in animals. Limited human studies suggest LA is most efficacious in situations where blood triglycerides are markedly elevated. LA is commercially available as dietary supplements and is clinically shown to be safe and effective against diabetic polyneuropathies. LA is described as a potent biological antioxidant, a detoxification agent, and a diabetes medicine. Given its strong safety record, LA may be a useful nutraceutical, either alone or in combination with other lipid-lowering strategies, when treating severe hypertriglyceridemia and diabetic dyslipidemia. This review examines the current evidence regarding the use of LA as a means of normalizing blood triglycerides. Also presented are the leading mechanisms of action of LA on triglyceride metabolism.
This review continues a general presentation of the principles of stereochemistry with special emphasis on the biomedicinal sciences. Here, we discuss and illustrate the phenomenon of substrate stereoselectivity in biochemistry (endogenous metabolism) and principally in xenobiochemistry or drug metabolism. The review begins with an overview of the stereoselective processes occurring in the biomedicinal sciences. The general rule is for distinct stereoisomers, be they enantiomers or diastereoisomers, to elicit different pharmacological responses (Part 5), to a lesser extent be transported with different efficacies (Part 5), and to be metabolized at different rates (this Part). In other words, biological environments discriminate between stereoisomers both when acting on them and when being acted upon by them. The concept of substrate stereoselectivity describes this phenomenon in endogenous biochemistry and xenobiotic metabolism, as discussed and illustrated in the present Part. The sister concept of product stereoselectivity will be presented in Part 8.
This review continues a general presentation of the principles of stereochemistry with special emphasis on the biomedicinal sciences. Here, we discuss and illustrate the phenomenon of substrate stereoselectivity in biochemistry (endogenous metabolism) and principally in xenobiochemistry or drug metabolism. The review begins with an overview of the stereoselective processes occurring in the biomedicinal sciences. The general rule is for distinct stereoisomers, be they enantiomers or diastereoisomers, to elicit different pharmacological responses (Part 5), to a lesser extent be transported with different efficacies (Part 5), and to be metabolized at different rates (this Part). In other words, biological environments discriminate between stereoisomers both when acting on them and when being acted upon by them. The concept of substrate stereoselectivity describes this phenomenon in endogenous biochemistry and xenobiotic metabolism, as discussed and illustrated in the present Part. The sister concept of product stereoselectivity will be presented in Part 8.
Reactive oxygen species and reactive nitrogen species promote endothelial dysfunction in old age and contribute to the development of cardiovascular diseases such as atherosclerosis, diabetes, and hypertension. α-lipoic acid was identified as a catalytic agent for oxidative decarboxylation of pyruvate and α-ketoglutarate in 1951, and it has been studied intensively by chemists, biologists, and clinicians who have been interested in its role in energetic metabolism and protection from reactive oxygen species-induced mitochondrial dysfunction. Consequently, many biological effects of α-lipoic acid supplementation can be attributed to the potent antioxidant properties of α-lipoic acid and dihydro α-lipoic acid. The reducing environments inside the cell help to protect from oxidative damage and the reduction-oxidation status of α-lipoic acid is dependent upon the degree to which the cellular components are found in the oxidized state. Although healthy young humans can synthesize enough α-lipoic acid to scavenge reactive oxygen species and enhance endogenous antioxidants like glutathione and vitamins C and E, the level of α-lipoic acid significantly declines with age and this may lead to endothelial dysfunction. Furthermore, many studies have reported α-lipoic acid can regulate the transcription of genes associated with anti-oxidant and anti-inflammatory pathways. In this review, we will discuss recent clinical studies that have investigated the beneficial effects of α-lipoic acid on endothelial dysfunction and propose possible mechanisms involved.
Reactive oxygen species (ROS) are known mediators of intracellular signaling cascades. Excessive production of ROS may, however, lead to oxidative stress, loss of cell function, and ultimately apoptosis or necrosis. A balance between oxidant and antioxidant intracellular systems is hence vital for cell function, regulation, and adaptation to diverse growth conditions. Thioredoxin reductase (TrxR) in conjunction with thioredoxin (Trx) is a ubiquitous oxidoreductase system with antioxidant and redox regulatory roles. In mammals, extracellular forms of Trx also have cytokine-like effects. Mammalian TrxR has a highly reactive active site selenocysteine residue resulting in a profound reductive capacity, reducing several substrates in addition to Trx. Due to the reactivity of TrxR, the enzyme is inhibited by many clinically used electrophilic compounds including nitrosoureas, aurothioglucose, platinum compounds, and retinoic acid derivatives. The properties of TrxR in combination with the functions of Trx position this system at the core of cellular thiol redox control and antioxidant defense. In this review, we focus on the reactions of the Trx system with ROS molecules and different cellular antioxidant enzymes. We summarize the TrxR-catalyzed regeneration of several antioxidant compounds, including ascorbic acid (vitamin C), selenium-containing substances, lipoic acid, and ubiquinone (Q10). We also discuss the general cellular effects of TrxR inhibition. Dinitrohalobenzenes constitute a unique class of immunostimulatory TrxR inhibitors and we consider the immunomodulatory effects of dinitrohalobenzene compounds in view of their reactions with the Trx system.
The therapeutic potential of α-lipoic acid (thioctic acid) was evaluated with respect to its influence on cellular reducing equivalent homeostasis. The requirement of NADH and NADPH as cofactors in the cellular reduction of α-lipoic acid to dihydrolipoate has been reported in various cells and tissues. However, there is no direct evidence describing the influence of such reduction of α-lipoate on the levels of cellular reducing equivalents and homeostasis of the NAD(P)HNAD(P) ratio. Treatment of the human Wurzburg T-cell line with 0.5 mM α-lipoate for 24 hr resulted in a 30% decrease in cellular NADH levels. α-Lipoate treatment also decreased cellular NADPH, but this effect was relatively less and slower compared with that of NADH. A concentration-dependent increase in glucose uptake was observed in Wurzburg cells treated with α-lipoate. Parallel decreases (30%) in cellular NADHNAD+ and in lactate/pyruvate ratios were observed in α-lipoate-treated cells. Such a decrease in the NADHNAD+ ratio following treatment with α-lipoate may have direct implications in diabetes, ischemia-reperfusion injury, and other pathologies where reductive (high NADHNAD+ ratio) and oxidant (excess reactive oxygen species) imbalances are considered as major factors contributing to metabolic disorders. Under conditions of reductive stress, α-lipoate decreases high NADH levels in the cell by utilizing it as a co-factor for its own reduction process, whereas in oxidative stress both α-lipoate and its reduced form, dihydrolipoate, may protect by direct scavenging of free radicals and recycling other antioxidants from their oxidized forms.
Ageing is characterized by a failure to maintain homeostasis under conditions of physiological stress, with an increasing susceptibility to disease and death. The accumulation of errors committed by faulty biochemical reactions over a vast period generates the cumulative effect observed during ageing. The most notable among the effects of ageing are the age-related disorders where free radicals are the major cause. When the level of free radicals increases because of diet, lifestyle, environment or other influences, it results in subsequent reduction of antioxidants. Reduced glutathione is one of the most fascinating molecules virtually present in all animal cells in often quite higher concentrations. An essential mechanism that accounts for most of the metabolic and cell regulatory properties of glutathione is the thiol disulfide exchange equilibria. We evaluated the age-associated alterations in glutathione dependent enzymes, glutathione and hydroxyl radicals in young and aged rats with respect to lipoate supplementation. In aged rats, activities of glutathione peroxidase, glutathione reductase, glutathione-S-transferase and glucose-6-phosphate dehydrogenase and the level of glutathione were low, whereas the level of hydroxyl radical was higher than in the young ones. Administration of dl-α-lipoic acid, a thiol antioxidant intraperitoneally to the aged rats, led to a time-dependent reduction in hydroxyl radicals and elevation in the activities/level of glutathione systems. Hence it can be suggested that lipoate, a dithiol prevents the oxidation of reduced glutathione and protects its related enzymes from peroxidative damage.
Methylglyoxal (MG), a reactive dicarbonyl compound, is a metabolic byproduct of glycolysis often found at high levels in blood from diabetic patients. The effect of lipoic acid on MG-induced oxidative stress was investigated using LLC-PK1 renal tubular epithelial cells, which are susceptible to oxidative stress. MG (500 gm) treatment induced LLC-PK1 cell death to nearly 50% compared with non-treated control cells, but lipoic acid significantly inhibited the MG-induced cytotoxicity in a concentration-dependent manner. In addition, lipoic acid treatment dose-dependently reduced the intracellular reactive oxygen species level increased by 500 mu M MG. The nitric oxide level was also increased by 500 mu m MG treatment, but it was significantly inhibited by lipoic acid. Furthermore, lipoic acid treatment at 50 mu M inhibited the nuclear translocation of nuclear factor-kappa B induced by MG treatment in LLC-PK1 cells. These findings indicate that lipoic acid has potential as a therapeutic agent against the development of diabetic complications related to MG-induced oxidative stress in diabetes.
R(+)-alpha-lipoic acid is a natural occurring compound that acts as an essential cofactor for certain dehydrogenase complexes. The redox couple alpha-lipoic acid/dihydrolipoic acid possesses potent antioxidant activity. Exogenous racemic alpha-lipoic acid orally administered for the symptomatic treatment of diabetic polyneuropathy is readily and nearly completely absorbed, with a limited absolute bioavailability of about 30% caused by high hepatic extraction. Although the pharmacokinetics of the parent drug have been well characterized in humans, relatively little is known regarding the excretion of alpha-lipoic acid and the pharmacokinetics of any metabolites in humans. In the present study, plasma concentration-time courses, urinary excreted amounts, and pharmacokinetic parameters of alpha-lipoic acid metabolites were evaluated in 9 healthy volunteers after multiple once-daily oral administration of 600 mg racemic alpha-lipoic acid. The primary metabolic pathways of alpha-lipoic acid in man, S-methylation and β-oxidation, were quantitatively confirmed by an HPLC-electrochemical assay newly established prior to the beginning of this study. Major circulating metabolites were the S-methylated β-oxidation products 4,6-bismethylthio-hexanoic acid and 2,4-bismethylthio-butanoic acid, whereas its conjugated forms accounted for the major portion excreted in urine. There was no statistically significant difference in the pharmacokinetic parameters Cmax, AUC, and tmaxbetween day 1 and day 4. Despite the prolonged half-lives of the major metabolites compared to the parent drug, no evidence of accumulation was found. Mean values of 12.4% of the administered dose were recovered in the urine after 24 hours as the sum of alpha-lipoic acid and its metabolites. The results of the present study revealed that urinary excretion of alpha-lipoic acid and five of its main metabolites does not play a significant role in the elimination of alpha-lipoic acid. Therefore, biliary excretion, further electrochemically inactive degradation products, and complete utilization of alpha-lipoic acid as a primary substrate in the endogenous metabolism should be considered.
Healthy aging and disease prevention by antioxidant nutrition is increasingly a subject of academic and public health interest
and research. Antioxidants play an important role in maintaining physiological redox status of cellular constituents against
free radicals. Antioxidants may change their redox state, be targeted for destruction, regulate oxidative processes involved
in signal transduction, affect gene expression and pathways of cell proliferation, as well as differentiation and death. This
chapter will provide an overview of the antioxidant defense system with special relevance to the brain and aging.
The aim of this study was to investigate the effect of the mitochondrial cofactor α-lipoic acid [R (+) LA] or its lipoamide analogue, 2-(N,N-dimethylamine) ethylamido lipoate [R (+) LA-plus], on nitric oxide (NO) production in RAW 264.7 macrophages. NO production from RAW 264.7 cells stimulated with 10 μg/mL of lipopolysaccharide and 50 U/mL of interferon-γ was measured directly by electron spin resonance using spin-trapping techniques. R (+) LA or R (+) LA-plus was found to inhibit NO production at pharmacologically relevant concentrations. However, in a cell-free chemical system, neither R (+) LA nor R (+) LA-plus was able to directly scavenge NO. Furthermore, in the presence of 2.5 or 25 mM glucose, the inhibitory effects of R (+) LA and R (+) LA-plus on NO production were decreased markedly, while they showed more potent inhibitory effects in the presence of 2 μM rotenone or 5 μg/mL of antimycin A, inhibitors of mitochondrial complex I and complex III, respectively. Glucose, rotenone, or antimycin A alone resulted in an increase of NO production. These results suggest that NO production in macrophages can be regulated by glucose and mitochondrial respiration, and that modulation of NO production by lipoic acid or lipoamide analogues in inflammatory situations is attributed not to their radical scavenging activity but to their redox properties.
In cellular, tissue, and organismal systems, exogenously supplied α-lipoic acid (thioctic acid) has a variety of significant effects, including direct radical scavenging, redox modulation of cell metabolism, and potential to inhibit oxidatively-induced injury. Because reduction of lipoate to dihydrolipoate is a crucial step in many of these processes, we investigated mechanisms of its reduction. The mitochondrial NADH-dependent dihydrolipoamide dehydrogenase exhibits a marked preference for R(+)-lipoate, whereas NADPH-dependent glutathione reductase shows slightly greater activity toward the S(−)-lipoate stereoisomer. Rat liver mitochondria also reduced exogenous lipoic acid. The rate of reduction was stimulated by substrates which increased the NADH content of the mitochondria, and was inhibited by methoxyindole-2-carboxylic acid, a dihydrolipoamide dehydrogenase inhibitor. In rat liver cytosol, NADPH-dependent reduction was greater than NADH, and lipoate reduction was inhibited by glutathione disulfide. In rat heart, kidney, and brain whole cell-soluble fractions, NADH contributed more to reduction (70–90%) than NADPH, whereas with liver, NADH and NADPH were about equally active. An intact organ, the isolated perfused rat heart, reduced R-lipoate six to eight times more rapidly than S-lipoate, consistent with high mitochondrial dihydrolipoamide dehydrogenase activity and results with isolated cardiac mitochondria. On the other hand, erythrocytes, which lack mitochondria, somewhat more actively reduced S- than R-lipoate. These results demonstrate differing stereospecific reduction by intact cells and tissues. Thus, mechanisms of reduction of α-lipoate are highly tissue-specific and effects of exogenously supplied α-lipoate are determined by tissue glutathione reductase and dihydrolipoamide dehydrogenase activity. Copyright © 1996 Elsevier Science Inc.
To examine the stereospecific effects of lipoic compounds on pyruvate metabolism, the effects of R-lipoic acid (R-LA), S-lipoic acid (S-LA) and 1,2-diselenolane-3-pentanoic acid (Se-LA) on the activities of the mammalian pyruvate dehydrogenase complex (PDC) and its catalytic components were investigated. Both S-LA and R-LA markedly inhibited PDC activity; whereas Se-LA displayed inhibition only at higher concentrations. Examination of the effects on the individual catalytic components indicated that Se-LA inhibited the pyruvate dehydrogenase component; whereas R-LA and S-LA inhibited the dihydrolipoamide acetyltransferase component. The three lipoic compounds lowered dihydrolipoamide dehydrogrenase (E3) activity in the forward reaction by about 30 to 45%. The kinetic data of E3 showed that both R-LA and Se-LA are used as substrates by E3 for the reverse reaction. Decarboxylation of [1-14C]pyruvate via PDC by cultured HepG2 cells was not affected by R-LA, but moderately decreased with S-LA and Se-LA. These findings indicate that (i) purified PDC and its catalytic components are affected by lipoic compounds based on their stereoselectivity; and (ii) the oxidation of pyruvate by intact HepG2 cells is not inhibited by R-LA. The later finding with the intact cells is in support of therapeutic role of R-LA as an antioxidant.
Lipoic acid has been reported recently to be an effective antioxidant in biological systems. It may act in vivo through reduction to its dithiol form, dihydrolipoic acid. Using a dual Hg/Au electrode, and HPLC with electrochemical detection, a method was developed which allowed simultaneous measurement of lipoic acid and dihydrolipoic acid, at nanomolar levels. (RS)-α-Lipoic acid was added to human cells in tissue culture (Jurkat T-lymphocytes and primary neonatal diploid fibroblasts). Lipoic acid was converted rapidly by the cells to dihydrolipoic acid, which accumulated in the cell pellet. Monitored over a 2-hr interval, dihydrolipoic acid was released, and several-fold more dihydrolipoic acid could be found in the medium than in the pellet.
Dihydrolipoamide dehydrogenase (E3) is the common component of the three α-ketoacid dehydrogenase complexes oxidizing pyruvate, α-ketoglutarate, and the branched-chain α-ketoacids. E3 also participates in the glycine cleavage system. E3 belongs to the enzyme family called pyridine nucleotide-disulfide oxidoreductases, catalyzing the electron transfer between pyridine nucleotides and disulfide compounds. This review summarizes the information available for E3 from a variety of species, from a halophilic archaebacterium which has E3 but no α-ketoacid dehydrogenase complexes, to mammalian species. Evidence is reviewed for the existance of two E3 isozymes (one for pyruvate dehydrogenase complex and α-ketoglutarate dehydrogenase complex and the other for branched-chain α-ketoacid dehydrogenase complex) in Pseudomonas species and for possible mammalian isozymes of E3, one associated with the three α-ketoacid dehydrogenase complexes and one for the glycine cleavage system. The comparison of the complete amino acid sequences of E3 from Escherichia coli, yeast, pig, and human shows considerable homologies of certain amino acid residues or short stretches of sequences, especially in the specific catalytic and structural domains. Similar homology is found with the limited available amino acid sequence information on E3 from several other species. Sequence comparison is also presented for other member flavoproteins [e.g., glutathione reductase and mercury(II) reductase] of the pyridine nucleotide-disulfide oxidoreductase family. Based on the known tertiary structure of human glutathione reductase it may be possible to predict the domain structures of E3. Additionally, the sequence information may help to better understand a divergent evolutionary relationship among these flavoproteins in different species.
dl-[1,6-14C]Lipoic acid was administered by intraperitoneal injection to rats at the level of 0.5 mg/100 g body weight. Approximately 56% of the radioactivity was recovered in the urine. When acidified and extracted with benzene, 92% of the radioactivity remained in the aqueous phase. Gel-filtration and paper chromatography were used to identify three of the compounds in the benzene extract as lipoic, bisnorlipoic and tetranorlipoic acids. In addition, a keto compound appears to be present. The aqueous phase contained several radioactive components separable by ion-exchange and paper chromatographies. Two of these compounds were identified as lipoate and β-hydroxybisnorlipoate. No evidence for oxidation of the dithiolane ring of lipoic acid was observed. dl-[7,8-14C]Lipoic acid was administered to rats under the same conditions. The urine contained 81% of the radioactivity, 72% of which remained in the aqueous phase and 28% was extracted into benzene. In contrast to over 30% of the label from dl-(1,6-14C] lipoate being expired as 14CO2, a negligible amount of 14CO2 was produced by rats injected with dl-[7,8-14C]lipoate. The catabolites identified were the same as those found using the 1,6-labeled lipoate. Another dithiolane-intact compound was also isolated. It appears that the rat, similar to Pseudomonas putida LP, metabolizes lipoate mainly via β-oxidation of the valeric acid side chain.
The hypothesis that dihydrolipoamide dehydrogenases (E3s) have tertiary structures very similar to that of human glutathione reductase (GR) was tested in detail by three separate criteria: (1) by analyzing each putative secondary structural element for conservation of appropriate polar/nonpolar regions, (2) by detailed comparison of putative active site residues in E3s with their authentic counterparts in human GR, and (3) by comparison of residues at the putative dimeric interface of the E3s with the authentic residues in GR. All three criteria are satisfied in a convincing way for the 7 E3s that were considered, supporting the conclusion that the structural scaffolding and the overall tertiary structure (which determines the location of functional sites and residues) are remarkably similar for the E3s and for GR. These analyses together with the crystal structures of human erythrocyte GR formed the basis for construction of a molecular model for human E3. The cofactor FAD and the substrates NAD and lipoic acid were also included in the model. Unexpectedly, the surface residues in the cleft that holds the lipoamide were found to be highly charged and predominantly acidic, allowing us to predict that the region around the lipoamide in the subunit should be basic in nature. The molecular model can be tested by site-directed mutagenesis of residues predicted to be in the dihydrolipoamide acetyltransferase subunit binding cleft.
Thioctic (lipoic) acid is used as a therapeutic agent in a variety of diseases in which enhanced free radical peroxidation of membrane phospholipids has been shown to be a characteristic feature. It was suggested that the antioxidant properties of thioctic acid and its reduced form, dihydrolipoic acid, are at least in part responsible for the therapeutic potential. The reported results on the antioxidant efficiency of thioctic and dihydrolipoic acids obtained in oxidation models with complex multicomponent initiation systems are controversial. In the present work we used relatively simple oxidation systems to study the antioxidant effects of dihydrolipoic and thioctic acids based on their interactions with: (1) peroxyl radicals which are essential for the initiation of lipid peroxidation, (2) chromanoxyl radicals of vitamin E, and (3) ascorbyl radicals of vitamin C, the two major lipid- and water-soluble antioxidants, respectively. We demonstrated that: (1) dihydrolipoic acid (but not thioctic acid) was an efficient direct scavenger of peroxyl radicals generated in the aqueous phase by the water-soluble azoinitiator 2,2'-azobis(2-amidinopropane)-dihydrochloride, and in liposomes or in microsomal membranes by the lipid-soluble azoinitiator 2,2'-azobis(2,4-dimethylvaleronitrile); (2) both dihydrolipoic acid and thioctic acid did not interact directly with chromanoxyl radicals of vitamin E (or its synthetic homologues) generated in liposomes or in the membranes by three different ways: UV-irradiation, peroxyl radicals of 2,2'-azobis(2,4-dimethylvaleronitrile), or peroxyl radicals of linolenic acid formed by the lipoxygenase-catalyzed oxidation; and (3) dihydrolipoic acid (but not thioctic acid) reduced ascorbyl radicals (and dehydroascorbate) generated in the course of ascorbate oxidation by chromanoxyl radicals. This interaction resulted in ascorbate-mediated dihydrolipoic acid-dependent reduction of the vitamin E chromanoxyl radicals, i.e. vitamin E recycling. We conclude that dihydrolipoic acid may act as a strong direct chain-breaking antioxidant and may enhance the antioxidant potency of other antioxidants (ascorbate and vitamin E) in both the aqueous and the hydrophobic membraneous phases.
Acquired immunodeficiency syndrome (AIDS) results from infection with a human immunodeficiency virus (HIV). The long terminal repeat (LTR) region of HIV proviral DNA contains binding sites for nuclear factor kappa B (NF-kappa B), and this transcriptional activator appears to regulate HIV activation. Recent findings suggest an involvement of reactive oxygen species (ROS) in signal transduction pathways leading to NF-kappa B activation. The present study was based on reports that antioxidants which eliminate ROS should block the activation of NF-kappa B and subsequently HIV transcription, and thus antioxidants can be used as therapeutic agents for AIDS. Incubation of Jurkat T cells (1 x 10(6) cells/ml) with a natural thiol antioxidant, alpha-lipoic acid, prior to the stimulation of cells was found to inhibit NF-kappa B activation induced by tumor necrosis factor-alpha (25 ng/ml) or by phorbol 12-myristate 13-acetate (50 ng/ml). The inhibitory action of alpha-lipoic acid was found to be very potent as only 4 mM was needed for a complete inhibition, whereas 20 mM was required for N-acetylcysteine. These results indicate that alpha-lipoic acid may be effective in AIDS therapeutics.
alpha-Lipoic acid, an essential cofactor in mitochondrial dehydrogenases, has recently been shown to be a potent antioxidant in vitro, as well as being capable of regenerating vitamin E in vitro. In this study, using a new animal model for rapid vitamin E deficiency in adult animals and a new technique for tissue extraction of oxidized and reduced alpha-lipoic acid, we examined the antioxidant action of alpha-lipoic acid in vivo. Vitamin E-deficient adult hairless mice displayed obvious symptoms of deficiency within five weeks, but if the diet was supplemented with alpha-lipoic acid the animals were completely protected. At five weeks on a vitamin E-deficient diet animals exhibited similar decreases in tissue vitamin E levels, whether supplemented or unsupplemented with alpha-lipoic acid: vitamin E levels in liver, kidney, heart, and skin decreased 70 to 85%; levels in brain decreased only 25%. These data show that there was no effect of alpha-lipoic acid supplementation on vitamin E tissue concentrations, arguing against a role for alpha-lipoic acid in regenerating vitamin E in vivo.
Effects of dietary vitamin E supplementation in rats were studied to determine whether or not they have a higher tolerance against cardiac ischemia-reperfusion injury using the working or Langendorff heart systems. Also, dihydrolipoic acid, recently reported to have potent antioxidant properties and accelerate vitamin E recycling of membrane in vitro, was perfused into the heart model systems to investigate its in vivo relationship with vitamin E. Tissue vitamin E content was increased by vitamin E feeding, but heart preparations did not show any improved functional recovery. Control hearts perfused with dihydrolipoic acid also did not show any improvement. However, a synergistic response is observed with the combination of dihydrolipoic acid perfusion and high dietary vitamin E using both perfusion systems in improvement of cardiac recovery. These results indicate that a high concentration of myocardial vitamin E does not increase tolerance to ischemia-reperfusion injury by itself, but, the combination of exogenous dihydrolipoic acid and high endogenous vitamin E can produce synergistic protective effects on recovery from ischemia during reperfusion.