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.     

Regulation of the activity of choline acetyl transferase by lipoic acid

The observations reported in this article demonstrate that lipoic acid strongly influences the activity of a purified preparation of choline acetyl transferase. The reduced form, dihydrolipoic acid, is a powerful activator of the enzyme while lipoic acid itself has an inhibitory effect and counteracts the stimulatory effect of dihydrolipoic acid. It is proposed that dihydrolipoic acid serves an essential function in the action of this enzyme and that the ratio of reduced to oxidized lipoic acid in the cell may play an important role in the regulation of the activity of the enzyme. The implications of these findings for cell function and acetyl choline formation are discussed.Affiliation     

Analysis of lipoic acid in biological samples by gas chromatography with flame photometric detection

A selective and sensitive gas chromatographic method for the analysis of lipoic acid in biological samples has been developed. After base hydrolysis of the sample, the liberated lipoic acid was converted into its S,S-diethoxycarbonyl methyl ester derivative and measured by gas chromatography using a DB-210 capillary column and a flame photometric detector. The calibration curve was linear in the range 20–500 ng, and the detection limit was ca. 50 pg injected. The best hydrolysis conditions for the biological samples were obtained by using 2 M potassium hydroxide containing 4% bovine serum albumin at 110°C for 3 h. Using this method, lipoic acid in the hydrolysate could be selectively determined without any interference from matrix substances. Analytical results for the determination of lipoic acid in the mouse tissue and bacterial cell samples are presented.     

Lipoic acid and diabetes II: Mode of action of lipoic acid

Intraperitoneal administration of lipoic acid (10 mg/100 g) does not effect changes in serum insulin levels in normal and alloxan diabetic rats, while normalising increased serum pyruvate, and impaired liver pyruvic dehydrogenase characteristic of the diabetic state. Dihydrolipoic acid has been shown to participate in activation of fatty acids with equal facility as coenzyme A. Fatty acyl dihydrolipoic acid however is sparsely thiolyzed to yield acetyl dihydrolipoic acid. Also acetyl dihydrolipoic acid does not activate pyruvate carboxylase unlike acetyl coenzyme A. The reduced thiolysis of Β-keto fatty acyl dihydrolipoic acid esters and the lack of activation of pyruvic carboxylase by acetyl dihydrolipoic acid could account for the antiketotic and antigluconeogenic effects of lipoic acid     

Reaction of peroxyacetyl nitrate with cysteine,cystine, methionine,lipoic acid,papain, and lysozyme

Peroxyacetyl nitrate was reactive with small molecular-weight sulfur-containing compounds The order of susceptibility was cysteine > reduced lipoic acid = reduced lipoamide > oxidized lipoic acid > oxidized lipoamide > methionine ? cystine. From thiols the predominant product was disulfide. In the early stages of oxidation methionine yielded methionine sulfoxide. Products of oxidation of oxidized lipoic acid and lipoamide were the respective sulfoxides. Cystine was resistant to oxidation, yielding cysteic acid when oxidation took place.Papain was readily inactivated by peroxyacetyl nitrate while lysozyme was resistant. The small amount of inactivation of lysozyme was correlated with methionine oxidation. Papain inactivation was correlated with thiol oxidation and could be reversed by thiol compounds. The oxidation product was judged to be a dimer by methods for determining molecular weight.     

Simultaneous determination of lipoic acid (LA) and dihydrolipoic acid (DHLA) in human plasma using high-performance liquid chromatography coupled with electrochemical detection

A fast, simple, and a reliable high-performance liquid chromatography linked with electrochemical detector (HPLC-ECD) method for the assessment of lipoic acid (LA) and dihydrolipoic acid (DHLA) in plasma was developed using naproxen sodium as an internal standard (IS) and validated according to standard guidelines. Extraction of both analytes and IS from plasma (250 μl) was carried out with a single step liquid-liquid extraction applying dichloromethane. The separated organic layer was dried under stream of nitrogen at 40°C and the residue was reconstituted with the mobile phase. Complete separation of both compounds and IS at 30°C on Discovery HS C18 RP column (250 mm × 4.6 mm, 5 μm) was achieved in 9 min using acetonitrile: 0.05 M phosphate buffer (pH 2.4 adjusted with phosphoric acid) (52:48, v/v) as a mobile phase pumped at flow rate of 1.5 ml min(-1) using electrochemical detector in DC mode at the detector potential of 1.0 V. The limit of detection and limit of quantification for lipoic acid were 500 pg/ml and 3 ng/ml, and for dihydrolipoic acid were 3 ng/ml and 10 ng/ml, respectively. The absolute recoveries of lipoic acid and dihydrolipoic acid determined on three nominal concentrations were in the range of 93.40-97.06, and 93.00-97.10, respectively. Similarly coefficient of variations (% CV) for both intra-day and inter-day were between 0.829 and 3.097% for lipoic acid and between 1.620 and 5.681% for dihydrolipoic acid, respectively. This validated method was applied for the analysis of lipoic acid/dihydrolipoic acid in the plasma of human volunteers and will be used for the quantification of these compounds in patients with oxidative stress induced pathologies.     

Targeting lipoic acid to mitochondria: synthesis and characterization of a triphenylphosphonium-conjugated alpha-lipoyl derivative

Lipoic acid (LA) is a widely used antioxidant that protects mitochondria from oxidative damage in vivo. Much of this protection is thought to be due to the reduction of LA to dihydrolipoic acid (LAH(2)). This reduction is catalyzed in vivo by thioredoxin, thioredoxin reductase (TrxR), and lipoamide dehydrogenase. We hypothesized that specifically targeting LA to mitochondria, the site of most cellular reactive oxygen species production, would make it a more effective antioxidant. To do this, we made a novel molecule, MitoLipoic acid, by attaching lipoic acid to the lipophilic triphenylphosphonium cation. MitoL was accumulated rapidly within mitochondria several-hundred fold driven by the membrane potential. MitoL was reduced to the active antioxidant dihydroMitoLipoic acid by thioredoxin and by lipoamide dehydrogenase but not by TrxR. In isolated mitochondria or cells MitoL was only slightly reduced (5-10%), while, in contrast, LA was extensively reduced. This difference was largely due to the reaction of LA with TrxR, which did not occur for MitoL. Furthermore, in cells MitoL was quantitatively converted to an S-methylated product. As a consequence of its lack of reduction, MitoL was not protective for mitochondria or cells against a range of oxidative stresses. These results suggest that the protective action of LA in vivo may require its reduction to LAH(2) and that this reduction is largely mediated by TrxR.     

Nitric oxide and dihydrolipoic acid modulate the activity of caspase 3 in HepG2 cells

Herein, we report that dihydrolipoic acid and lipoic acid (LA) plus lipoamide dehydrogenase and NADH denitrosate S-nitrosocaspase 3 (CASP-SNO). In HepG2 cells, S-nitroso-l-cysteine ethyl ester (SNCEE) impeded the activity of caspase 3 (CASP-SH), while a subsequent incubation of the cells in SNCEE-free medium resulted in endogenous denitrosation and reactivation of CASP-SH. The latter process was inhibited in thioredoxin reductase-deficient HepG2 cells, in which, however, LA markedly reactivated CASP-SH. The data obtained are discussed with focus on low molecular mass dithiols that mimic the activity of thioredoxin in reactions of protein S-denitrosation.     

Lipoic acid and diabetes: Effect of dihydrolipoic acid administration in diabetic rats and rabbits

Relative α-lipoic acid content of diabetic livers was considerably less than that of normal livers as determined by gas chromatography. It was not possible to detect any dihydrolipoic acid in the livers. Biochemical abnormalities such as hyperglycaemia, ketonemia, reduction in liver glycogen and impaired incorporation of [2-¹⁴C] -acetate into fatty acids in alloxan diabetic rats were brought to near normal levels by the oral or intraperitoneal administration of dihydrolipoic acid. The effect of α-lipoic acid was comparable to that of dihydrolipoic acid in reducing the blood sugar levels of diabetic rabbits during a glucose tolerance test. The results suggest that the mode of action of lipoic acid was through stimulation of pyruvate dehydrogenase.     

Nitric oxide and thioredoxin modulate the activity of caspase 9 in HepG2 cells

The interdependent and finely tuned balance between the well-established redox-based modification, S-nitrosylation, and its counteractive mechanism of S-nitrosothiol degradation, i.e., S-denitrosylation of biological protein or non-protein thiols defines the cellular fate in the context of redox homeostasis. S-nitrosylation of cysteine residues by S-nitrosoglutathione, S-nitroso-L-cysteine-like physiological and S-nitroso-L-cysteine ethyl ester-like synthetic NO donors inactivate Caspase-3, 8, and 9, thereby hindering their apoptotic activity. However, spontaneous restoration of their activity upon S-denitrosylation of S-nitrosocaspases into their reduced, free thiol active states, aided by the members of the ubiquitous cellular redoxin (thioredoxin/ thioredoxin reductase/ NADPH) and low molecular weight dithiol (lipoic acid/ lipoamide dehydrogenase/ dihydrolipoic acid/ NADPH) systems imply a direct relevance to their proteolytic activities and further downstream signaling cascades. Additionally, our previous and current findings offer crucial insight into the concept of redundancy between thioredoxin and lipoic acid systems, and the redox-modulated control of the apoptotic and proteolytic activity of caspases, triggering their cyto- and neurotoxic effects in response to nitro-oxidative stress. Thus, this might lay the foundation for the exogenous introduction of precise and efficient NO or related donor drug delivery systems that can directly participate in catering to the S-(de)-nitrosylation-mediated functional outcomes of the cysteinyl proteases in pathophysiological settings.     

Lipoic acid content of Escherichia coli and other microorganisms

A mutant strain of Escherichia coli K12 requiring lipoic acid, W1485 lip 2 (ATCC 25645), was used to develop a turbidimetric assay for lipoic acid and a polarographic assay based on the oxidation of pyruvate by suspensions of lipoic acid-deficient organisms. The turbidimetric assay was more sensitive with a working range equivalent to 0.2–2.0 ng of dl-α-lipoic acid compared with 5–50 ng for the polarographic method. The mutant responded equally to racemic mixtures of α-lipoic acid, β-lipoic acid and dihydrolipoic acid but gave little response to lipoamide, and other derivatives without prior hydrolysis; 8-methyllipoic acid was a competitive inhibitor of the response to lipoic acid. A high specificity of the mutant for the natural stereoisomer was indicated by the fact that (+)-α-lipoic acid had twice the activity of the racemic mixture. Escherichia coli K12 contained less than 0.05 ng of free (+)-α-lipoic acid per mg dry weight but, depending on the growth substrate, the equivalent of between 13 and 47 ng of (+)-α-lipoic acid per mg dry weight after acid extraction. There was a strong correlation between the lipoic acid content and the sum of the specific activities for the pyruvate and α-ketoglutarate dehydrogenase complexes. Experiments with washed suspensions of Escherichia coli showed only small increases in lipoic acid content (18%) when incubated with pyruvate, cysteine and methionine. When supplied with exogenous lipoic acid the mutant, W1485 lip 2, accumulated very little more than was demanded by its metabolism. The lipoic acid contents of several organisms were measured and correlated with their metabolism.     

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.     

Electrocatalytic reduction of lipoic acid and electroenzymatic reduction of NAD(P)(+) for integrated dehydrogenase biosensors

Biosensors for the oxidized substrates of NAD(P)(+)-specific dehydrogenases demand the reductive recycling of the coenzymes. So far, suitable catalysts for the corresponding two-electron transfer are not available. In the present paper, this transport has been realized by a combined electrocatalytical and electroenzymatic process. Lipoic acid has been reduced on graphite electrodes functionalized with Fe(II)-phthalocyanine in 95% yield at-1200 mV in phosphate buffer pH 7.0. With the electrocatalytically reduced product, dihydrolipoic acid, lipoamide dehydrogenase could reduce NAD(+) in 20% yield and thioredoxin reductase NADP(+) in 18.4% yield. So far, the combined electrocatalytic/electroenzymatic system has not yet been realized, mainly because at the potential needed for the lipoic acid reduction, a parallel one-electron reduction of NAD(P)(+) was observed, implying the dimerization of the coenzyme.     

The role of lipoic acid residues in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

Two lipoic acid residues on each dihydrolipoamide acetyltransferase (E2) chain of the pyruvate dehydrogenase multienzyme complex of Escherichia coli were found to undergo oxidoreduction reactions with NAD+ catalysed by the lipoamide dehydrogenase component. It was observed that: (a) 2 mol of reagent/mol of E2 chain was incorporated when the complex was incubated with N-ethylmaleimide in the presence of acetyl-SCoA and NADH; (b) 4 mol of reagent/mol of E2 chain was incorporated when the complex was incubated with N-ethylmaleimide in the presence of NADH; (c) between 1 and 2 mol of acetyl groups/mol of E2 chain was incorporated when the complex was incubated with acetyl-SCoA plus NADH; (d) 2 mol of acetyl groups/mol of E2 chain was incorporated when the complex was incubated with pyruvate either before or after many catalytic turnovers through the overall reaction. There was no evidence to support the view that only half of the dihydrolipoic acid residues can be reoxidized by NAD+. However, chemical modification of lipoic acid residues with N-ethylmaleimide was shown to proceed faster than the accompanying loss of enzymic activity under all conditions tested, which indicates that not all the lipoyl groups are essential for activity. The most likely explanation for this result is an enzymic mechanism in which one lipoic acid residue can take over the function of another.     

R-alpha-lipoic acid action on cell redox status, the insulin receptor, and glucose uptake in 3T3-L1 adipocytes.

The insulin signaling pathway has been reported to mediate R-alpha-lipoic acid- (R-LA-)-stimulated glucose uptake into 3T3-L1 adipocytes and L6 myotubes. We investigated the role of the thiol antioxidant dihydrolipoic acid (DHLA) and intracellular glutathione (GSH) in R-LA-stimulated glucose transport and explored the hypothesis that R-LA could increase glucose uptake into 3T3-L1 adipocytes in an oxidant-mimetic manner. R-LA pretreatment of 3T3-L1 cells stimulated glucose transport at early time points (30 min - 6 h), whereas it inhibited glucose uptake at later time points. Analysis of the oxidized and reduced content of LA in cells and medium showed that >90% of lipoic acid present was in its oxidized form. Furthermore, all oxidized forms of LA (S-, R-, and racemic LA) stimulated glucose uptake, whereas the reduced form, dihydrolipoic acid, was ineffective. Intracellular GSH levels were not changed at the early time points (before 12 h), while longer preincubation (24 - 48 h) of cells with R-LA significantly increased intracellular GSH. Pretreatment of adipocytes with R-LA increased intracellular peroxide levels at early time points (30 min - 6 h), after which it was decreased (12 - 48 h). R-LA also increased tyrosine phosphorylation of immunoprecipitated insulin receptors from 3T3-L1 adipocytes. These results indicate that (i) 3T3-L1 adipocytes have a low capacity to reduce R-LA and the oxidized form of lipoic acid is responsible for stimulating glucose uptake, (ii) R-LA modulates glucose uptake by changing the intracellular redox status, and (iii) the insulin receptor is a potential cellular target for R-LA action.     

Lipoic acid stimulates cAMP production via G protein-coupled receptor-dependent and -independent mechanisms

Lipoic acid (LA) is a naturally occurring fatty acid that exhibits anti-oxidant and anti-inflammatory properties and is being pursued as a therapeutic for many diseases including multiple sclerosis, diabetic polyneuropathy and Alzheimer's disease. We previously reported on the novel finding that racemic LA (50:50 mixture of R-LA and S-LA) stimulates cAMP production, activates prostanoid EP2 and EP4 receptors and adenylyl cyclases (AC), and suppresses activation and cytotoxicity in NK cells. In this study, we present evidence that furthers our understanding of the mechanisms of action of LA. Using various LA derivatives, such as dihydrolipoic acid (DHLA), S,S-dimethyl lipoic acid (DMLA) and lipoamide (LPM), we discovered that only LA is capable of stimulating cAMP production in NK cells. Furthermore, there is no difference in cAMP production after stimulation with either R-LA, S-LA or racemic LA. Competition and synergistic studies indicate that LA may also activate AC independent of the EP2 and EP4 receptors. Pretreatment of PBMCs with KH7 (a specific peptide inhibitor of soluble AC) and the calcium inhibitor (Bapta) prior to LA treatment resulted in reduced cAMP levels, suggesting that soluble AC and calcium signaling mediate LA stimulation of cAMP production. In addition, pharmacological inhibitor studies demonstrate that LA also activates other G protein-coupled receptors, including histamine and adenosine but not the β-adrenergic receptors. These novel findings provide information to better understand the mechanisms of action of LA, which can help facilitate the use of LA as a therapeutic for various diseases.     

Determination of caffeine and its metabolites by micellar electrokinetic capillary electrophoresis

The determination of caffeine and its analogues is important for a wide variety of analyses and is performed in an assortment of matrices ranging from food to clinical samples. While reversed-phase HPLC has become the standard analysis protocol in most laboratories, capillary electrophoresis has the advantages of higher separation efficiency and shorter separation time. The micellar capillary electrophoresis (MECC) separation of caffeine and its metabolites, theobromine, paraxanthine, theophylline and 1,3,7-trimethyluric acid was investigated using sodium dodecyl sulphate (SDS) as the micellar phase. The effects of pH, micelle concentration, buffer concentration, ionic strength, buffer salts, applied voltage and injection time were studied to select the optimum conditions for the determination of caffeine and its four analogues in drugs, foods and body fluids. Caffeine and its three analogues were resolved within 120 s with detection limits less than 1 μg/ml. Samples could be analyzed utilizing direct injection with satisfactory resolution and reproducibility.     

Homogeneous enzyme immunoassay for lipoic acid based on the pyruvate dehydrogenase complex: a model for an assay using a conjugate with one ligand per subunit.

A homogeneous enzyme immunoassay for lipoic acid was developed by using an enzyme-ligand conjugate containing only one ligand per enzyme subunit. Theoretical studies have shown that the traditional use of multisubstituted enzyme-ligand conjugates has limited the detection limits and sensitivity obtainable with these assays. The use of conjugates with a smaller number of ligands should allow for improved assays. The pyruvate dehydrogenase complex was chosen for this study because each polypeptide chain of dihydrolipoyl transacetylase contains one lipoic acid as a covalently attached prosthetic group. Thus, the naturally occurring enzyme can be considered as an enzyme-lipoic acid conjugate. Anti-lipoic acid antibodies were developed in New Zealand White rabbits to be used as the analyte-specific binders. Association and binder dilution curves were prepared in order to optimize the reagent concentrations and the analytical conditions. Unexpected inhibition by free lipoic acid resulted in a biphasic dose-response curve with a detection limit of 5 x 10(-6) M lipoic acid. This technique has several advantages over previous electrochemical and chromatographic techniques for lipoic acid determination.     

Separation methods for camptothecin and related compounds

This paper reviews working procedures for the analytical determination of camptothecin and analogues. We give an overview of aspects such as the chemistry, structure–activity relationships, stability and mechanism of action of these antitumor compounds. The main body of the review describes separation techniques. Sample treatment and factors influencing high-performance liquid chromatography development are delineated. Published high-performance liquid chromatographic methods are summarized to demonstrate the variability and versatility of separation techniques and a critical evaluation of separation efficiency, detection sensitivity and specificity of these methods is reported.     

Determination of pilocarpine, isopilocarpine, pilocarpic acid and isopilocarpic acid in human plasma and urine by high-performance liquid chromatography with tandem mass spectrometric detection

A method is described for the determination of pilocarpine and its degradation products isopilocarpine, pilocarpic acid and isopilocarpic acid in human plasma and urine. The method is based on a simple sample preparation step – ultrafiltration for plasma and dilution for urine samples – followed by a reversed-phase liquid chromatographic separation of the analytes and detection by means of tandem mass spectrometry. Parameters affecting the performance of these steps are discussed. The high sensitivity and selectivity of the method allow low ng/ml concentrations to be determined for all compounds in plasma and undiluted urine, which enables the investigation of the metabolic fate and elimination of pilocarpine after oral administration to humans.