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Research Notes: Alpha-lipoic AcidCarcinogenesis. 2007 Sep 24. alpha-lipoic acid is an antioxidant used in a number of conditions related to liver diseases. Herein, we investigated the effect of alpha-lipoic acid on the development of rat preneoplastic lesions generated by a model of hepatocarcinogenesis which has similarities in its histopathological sequence to human hepatocellular carcinoma development with cirrhosis. Initiation of hepatocytes was achieved by treatment with a single dose of diethylnitrosamine and promotion by feeding a choline-methionine deficient diet (CMD), with or without alpha-lipoic acid. Preneoplastic lesions were identified by their positivity to the placental form of glutathione S-transferase (GSTP) or to gamma-glutamyl transpeptidase (GGT). alpha-lipoic acid given to rats fed a CMD for 6 weeks dramatically increased the number of GSTP-positive foci as compared to rats fed a CMD diet alone (96/cm(2) vs 7/cm(2)), the mean foci area (0.033 mm(2) vs. 0.008 mm(2)) and the percentage of GSTP-positive liver tissue (3.01% vs. 0.07%). Essentially similar results were obtained after 10 weeks of treatment. Co-treatment with CMD + alpha-lipoic acid also resulted in the enhancement of fat accumulation, lipid peroxidation, and hepatocyte death; increased expression of tumour necrosis factor-alpha, cytochrome 2E1, cyclooxygenase-2, enhanced activation of JNK and STAT3, and chronic hepatocyte proliferation was also observed. No such effects were observed when alpha-lipoic acid was added to a choline-supplemented diet. In conclusion, administration of alpha-lipoic acid in conditions associated with hepatic damage aggravates liver injury and stimulates the development of preneoplastic lesions; the results also suggest caution in its use in the presence of chronic liver injury. Biogerontology. 2007 Jun. D-Galactose (D-gal) -induced aging models in Drosophila, houseflies, mice and rats have been widely used; however, the underlying mechanisms are poorly understood. To investigate the involvement of mitochondrial dysfunction of D-gal, mitochondrial function was examined in the brain and liver of C57BL/6J mice, subjected to a treatment of D-gal with or without a concomitant treatment with a mitochondrial nutrient, R-alpha-lipoic acid (LA). D-Gal treatment induced a significant decrease in succinate-linked respiratory control ratio (RCR) and ADP/O ratio in the liver and brain, and also a significant increase in the maximum velocity (Vmax) and substrate binding affinity (Km) of complex II in the liver. LA treatment to D-gal-injected animals restored mitochondrial RCR in both brain and liver, ADP/O and Km of complex II in the liver. These results suggest LA is effective in delaying D-gal toxicity by ameliorating mitochondrial dysfunction. Intern Med. 2007 Mar 1. Insulin Autoimmune Syndrome (IAS) is a rare disease characterized by hypoglycemia and autoantibodies to insulin without prior insulin administration. Here, we report a case of IAS associated with alpha lipoic acid (ALA). The patient is a 55-year-old man. He began to complain of hypoglycemic symptoms after taking ALA. He lost consciousness in the late postprandial period and blood glucose was found to be 27 mg/dl. A high insulin level and high titers of insulin antibodies were detected. His HLA genotype contains DRB1* 0406. As ALA comes to be used widely, the incidence of IAS due to ALA might increase. Diabetes Res Clin Pract. 2007 Mar. The insulin autoimmune syndrome is characterized as producing polyclonal or monoclonal anti-insulin autoantibodies in a patient with no previous history of exposure to exogenous insulin. The patient is 44-year-old Japanese woman and she had symptoms of hypoglycaemia without exposure to exogenous insulin. The patient was considered to have IAS because high titre of anti-insulin autoantibodies (96-98%: bound/total) were found in her serum. Her HLA DR beta1 DNA sequences analysis revealed that she has the DRB1(*)0406 and DRB1(*)0901. Our patient have been taken alpha lipoic acid (ALA) before onset. SH group compounds are known to play an important role in the pathogenesis of IAS, and ALA contains SH. From these data, we propose the possibility of the correlation between pathogenesis of IAS and ALA, and it will be important to pay attention for ALA as a cause of hypoglycemia in such cases. Muscle Nerve. 2007 Feb. Mitochondrial disorders share common cellular consequences: (1) decreased ATP production; (2) increased reliance on alternative anaerobic energy sources; and (3) increased production of reactive oxygen species. The purpose of the present study was to determine the effect of a combination therapy (creatine monohydrate, coenzyme Q(10), and lipoic acid to target the above-mentioned cellular consequences) on several outcome variables using a randomized, double-blind, placebo-controlled, crossover study design in patients with mitochondrial cytopathies. Three patients had mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), four had mitochondrial DNA deletions (three patients with chronic progressive external ophthalmoplegia and one with Kearns-Sayre syndrome), and nine had a variety of other mitochondrial diseases not falling into the two former groups. The combination therapy resulted in lower resting plasma lactate and urinary 8-isoprostanes, as well as attenuation of the decline in peak ankle dorsiflexion strength in all patient groups, whereas higher fat-free mass was observed only in the MELAS group. Together, these results suggest that combination therapies targeting multiple final common pathways of mitochondrial dysfunction favorably influence surrogate markers of cellular energy dysfunction. Future studies with larger sample sizes in relatively homogeneous groups will be required to determine whether such combination therapies influence function and quality of life. Muscle Nerve. 2006 Nov 1. Mitochondrial disorders share common cellular consequences: (1) decreased ATP production; (2) increased reliance on alternative anaerobic energy sources; and (3) increased production of reactive oxygen species. The purpose of the present study was to determine the effect of a combination therapy (creatine monohydrate, coenzyme Q(10), and lipoic acid to target the above-mentioned cellular consequences) on several outcome variables using a randomized, double-blind, placebo-controlled, crossover study design in patients with mitochondrial cytopathies. Three patients had mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), four had mitochondrial DNA deletions (three patients with chronic progressive external ophthalmoplegia and one with Kearns-Sayre syndrome), and nine had a variety of other mitochondrial diseases not falling into the two former groups. The combination therapy resulted in lower resting plasma lactate and urinary 8-isoprostanes, as well as attenuation of the decline in peak ankle dorsiflexion strength in all patient groups, whereas higher fat-free mass was observed only in the MELAS group. Together, these results suggest that combination therapies targeting multiple final common pathways of mitochondrial dysfunction favorably influence surrogate markers of cellular energy dysfunction. Future studies with larger sample sizes in relatively homogeneous groups will be required to determine whether such combination therapies influence function and quality of life. J Pharmacol Exp Ther. 2006 Sep 21. The antioxidant alpha-lipoic acid (LA) is a naturally-occurring compound that has been shown to possess promising anticancer activity due to its ability to preferentially induce apoptosis and inhibit proliferation of cancer cells relative to normal cells. However, the molecular mechanisms underlying the apoptotic effect of LA are not well understood. We report here that LA induced reactive oxygen species (ROS) generation and a concomitant increase in apoptosis of human lung epithelial cancer H460 cells. Inhibition of ROS generation by ROS scavengers or by overexpression of antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD) effectively inhibited LA-induced apoptosis, indicating the role of ROS, especially hydroperoxide and superoxide anion, in the apoptotic process. Apoptosis induced by LA was found to be mediated through the mitochondrial death pathway which requires caspase-9 activation. Inhibition of caspase activity by pan-caspase inhibitor (z-VAD-fmk) or caspase-9-specific inhibitor (z-LEHD-fmk) completely inhibited the apoptotic effect of LA. Likewise, the mitochondrial respiratory chain inhibitor rotenone potently inhibited the apoptotic and ROS inducing effects of LA, supporting the role of mitochondrial ROS in LA-induced cell death. LA induced downregulation of mitochondrial Bcl-2 protein through peroxide-dependent proteasomal degradation and overexpression of the Bcl-2 protein prevented the apoptotic effect of LA. Together, our findings indicate a novel pro-oxidant role of LA in apoptosis induction and its regulation by Bcl-2, which may be exploited for the treatment of cancer and related apoptosis disorders. Selections from the full text article: Introduction alpha-Lipoic acid (LA) is a naturally-occurring essential co-enzyme in mitochondrial multienzyme complexes catalyzing the oxidative decarboxylation of alpha-keto acids such as pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acid (Packer et al., 1995; Bilska and Wlodex, 2005). LA has been shown to combat oxidative stress by quenching a variety of intracellular reactive oxygen species (ROS) (Suzuki et al., 1991; Bilska and Wlodex, 2005). In addition to ROS scavenging, LA has also been shown to be involved in the recycling of other cellular antioxidants including vitamins C and E, and glutathione (Biewenga et al., 1997). LA has been demonstrated to be effective in preventing pathology in various experimental models in which ROS have been implicated, such as ischemia-reperfusion injury (Coombes et al., 2000), diabetes (Kocak and Karasu, 2002; Da Ros et al., 2005), diabetic neuropathy (Vincent et al., 2005), neurodegeneration (Pirlich et al., 2002), hypertension (de Champlain et al., 2004; Vasdev et al., 2005), radiation injury (Demir et al., 2005), and HIV activation (Patrick, 2000). On the other hand, LA has been reported to possess pro-oxidant activities (Dicter et al., 2002; Gorolska et al., 2003; Cakatay et al., 2005). For examples, LA dose dependently increases intramuscular ROS production and stimulates glucose uptake into adipocytes by increasing intracellular oxidant levels (Dicter et al., 2002). In cancer cells, ROS also play a crucial role in cell growth and apoptosis regulation. LA and its reduced form dihydrolipoic acid (DHLA) have been shown to inhibit proliferation and induce apoptosis of several cancer and transformed cell lines, while being less active toward normal non-transformed cells (Sen et al., 1999; Pack et al., 2001; Mark et al., 2003; Wenzel et al., 2005). [...] LA has been shown to act as a pro-oxidant (Dicter et al., 2002; Gorolska et al., 2003; Cakatay et al., 2005) as well as anti-oxidant (Suzuki et al., 1991; Bilska and Wlodex, 2005), depending on cell type and cellular oxidative status. The antioxidant role of LA is commonly associated with cells under oxidative stress and this action of LA has been attributed to its ability to regenerate other cellular antioxidants such as vitamin C and E, and glutathione (4). The pro-oxidant role of LA is generally observed under non-oxidative stress conditions, which is also supported by this study. In human colon cancer HT29 cells, LA was shown to act a pro-oxidant by increasing mitochondrial O2•- generation (Wenzel et al., 2005). The colon cancer cells were also shown to possess a lower antioxidant status and are more susceptible to LA-induced apoptosis as compared to normal non-transformed cells, thus providing a basis for the selective effect of LA on cancer cells. [...] The therapeutic potential of LA in cancer treatment has been demonstrated in several studies (Sen et al., 1999; Pack et al., 2001; Mark et al., 2003; Wenzel et al., 2005). Previously, chemotherapeutic agents such as doxorubicin, cisplatin, vincristine, and the alkaloid taxol have commonly been used as anti-tumor agents. However, at high concentrations these drugs are toxic to cells and cause adverse side-effects. In contrast, LA is an endogenous agent that has been widely used as a dietary supplement. It is known to increase cellular glutathione levels, regulate cellular redox balance and help protect against diabetic complications (Sen et al., 1997; Da Ros et al., 2005; Kocak and Karasu, 2002). Pharmacol Ther. 2006 Sep 19. Alzheimer's disease (AD) is a progressive neurodegenerative disorder that destroys patient memory and cognition, communication ability with the social environment and the ability to carry out daily activities. Despite extensive research into the pathogenesis of AD, a neuroprotective treatment - particularly for the early stages of disease - remains unavailable for clinical use. In this review, we advance the suggestion that lipoic acid (LA) may fulfil this therapeutic need. A naturally occurring precursor of an essential cofactor for mitochondrial enzymes, including pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (KGDH), LA has been shown to have a variety of properties which can interfere with pathogenic principles of AD. For example, LA increases acetylcholine (ACh) production by activation of choline acetyltransferase and increases glucose uptake, thus supplying more acetyl-CoA for the production of ACh. LA chelates redox-active transition metals, thus inhibiting the formation of hydroxyl radicals and also scavenges reactive oxygen species (ROS), thereby increasing the levels of reduced glutathione. Via the same mechanisms, downregulation redox-sensitive inflammatory processes is also achieved. Furthermore, LA can scavenge lipid peroxidation products such as hydroxynonenal and acrolein. The reduced form of LA, dihydrolipoic acid (DHLA), is the active compound responsible for most of these beneficial effects. R-alpha-LA can be applied instead of DHLA, as it is reduced by mitochondrial lipoamide dehydrogenase, a part of the PDH complex. In this review, the properties of LA are explored with particular emphasis on how this agent, particularly the R-alpha-enantiomer, may be effective to treat AD and related dementias. Diabetes Res Clin Pract. 2006 Sep 8. The insulin autoimmune syndrome is characterized as producing polyclonal or monoclonal anti-insulin autoantibodies in a patient with no previous history of exposure to exogenous insulin. The patient is 44-year-old Japanese woman and she had symptoms of hypoglycaemia without exposure to exogenous insulin. The patient was considered to have IAS because high titre of anti-insulin autoantibodies (96-98%: bound/total) were found in her serum. Her HLA DR beta1 DNA sequences analysis revealed that she has the DRB1(*)0406 and DRB1(*)0901. Our patient have been taken alpha lipoic acid (ALA) before onset. SH group compounds are known to play an important role in the pathogenesis of IAS, and ALA contains SH. From these data, we propose the possibility of the correlation between pathogenesis of IAS and ALA, and it will be important to pay attention for ALA as a cause of hypoglycemia in such cases. Life Sci. 2006 Sep 1. alpha-Lipoic acid (ALA) and its reduced form dihydrolipoic acid (DHLA) are powerful antioxidants both in hydrophilic and lipophylic environments with diverse pharmacological properties including anti-inflammatory activity. The mechanism of anti-inflammatory activity of ALA and DHALA is not known. The present study describes the interaction of ALA and DHALA with pro-inflammatory secretory PLA(2) enzymes from inflammatory fluids and snake venoms. In vitro enzymatic inhibition of sPLA(2) from Vipera russellii, Naja naja and partially purified sPLA(2) enzymes from human ascitic fluid (HAF), human pleural fluid (HPF) and normal human serum (HS) by ALA and DHLA was studied using (14)C-oleate labeled Escherichia coli as the substrate. Biophysical interaction of ALA with sPLA(2) was studied by fluorescent spectral analysis and circular dichroism studies. In vivo anti-inflammatory activity was checked using sPLA(2) induced mouse paw edema model. ALA but not DHLA inhibited purified sPLA(2) enzymes from V. russellii, N. naja and partially purified HAF, HPF and HS in a dose dependent manner. This data indicated that ALA is critical for inhibition. IC(50) value calculated for these enzymes ranges from 0.75 to 3.0 muM. The inhibition is independent of calcium and substrate concentration. Inflammatory sPLA(2) enzymes are more sensitive to inhibition by ALA than snake venom sPLA(2) enzymes. ALA quenched the fluorescence intensity of sPLA(2) enzyme in a dose dependent manner. Apparent shift in the far UV-CD spectra of sPLA(2) with ALA indicated change in its alpha-helical confirmation and these results suggest its direct interaction with the enzyme. ALA inhibits the sPLA(2) induced mouse paw edema in a dose dependent manner and confirms the sPLA(2) inhibitory activity in vivo also. These data suggest that ALA may act as an endogenous regulator of sPLA(2) enzyme activity and suppress inflammatory reactions. Basic Clin Pharmacol Toxicol. 2006 Sep. The effects of the antioxidant lipoic acid and its reduced form, dihydrolipoic acid (DHLA), were studied on the process of the erythrocytic toxicity of 4-aminophenol in human erythrocytes in vitro. 4-Aminophenol alone caused a stepwise increase in methaemoglobin formation, along with a commensurate decrease in total thiols. At 10 min., in the presence of lipoic acid alone and the thiol depletor 1-chloro-2,4-dinitrobenzene (CDNB) alone, 4-aminophenol-mediated methaemoglobin formation was significantly increased, whilst thiol levels were significantly reduced compared with the 4-aminophenol alone. At 10 min., with DHLA and CDNB alone, 4-aminophenol was associated with significantly increased methaemoglobin formation. However, thiol levels were not significantly different in the presence of DHLA compared with 4-aminophenol alone, although thiol levels were different compared with control (4-aminophenol alone) in the incubations with CDNB alone. At 15 min., only CDNB/4-aminophenol methaemoglobin formation differed from control, whilst thiol levels were significantly lower in the presence of CDNB alone compared with 4-aminophenol alone. Lipoic acid enhanced the toxicity of 4-aminophenol in terms of increased methaemoglobin formation coupled with increased thiol depletion, whilst DHLA showed increased 4-aminophenol-mediated methaemoglobin formation without thiol depletion. Lipoic acid, and to a lesser extent its reduced derivative DHLA, acted as a prooxidant in the presence of 4-aminophenol, enhancing the oxidative stress effects of the amine in human erythrocytes. [Note: 4-aminophenol (p-aminophenol) is used as a dye for textiles, hair, furs, and feathers, as a photographic developer (Rodinal), and is a chemical intermediate for pharmaceuticals and dyes. The lethal oral dose in humans is in the range of 50-500 mg/kg. 4-aminophenol induces methemoglobinemia in experimental animals, but it is not as potent as aniline. It is a potential sensitizer of the skin and respiratory tract.] Rheumatol Int. 2006 Aug 31. Objective: To test the ability of alpha-lipoic acid (LA) to attenuate the development of collagen-induced arthritis (CIA) in mice. Methods: Mice were divided into three groups and treated with intraperitoneal administration of LA (10 or 100 mg/kg) or placebo. Clinical, histologic, and biochemical parameters were assessed. Human synovial fibroblasts and peripheral blood mononuclear cells were cocultured in various concentrations of LA to evaluate the effects on osteoclastogenesis. Results: LA was associated with a dose-dependent reduction of CIA, as well as preventing bone erosion and destructive changes. Intracellular reactive oxygen species in lymphocytes obtained from inguinal lymph nodes, which was significantly higher in CIA than control mice, was significantly reduced in CIA by LA. The concentrations of TNF-alpha, IL-1beta, and IL-6 in the paws, and synovial NF-kappaB binding, all of which were markedly higher in CIA than control mice, were reduced by treatment with LA. In addition, LA inhibited the formation of human osteoclasts in vitro. Conclusion: Amelioration of joint disease by LA was associated with reduction in oxidative stress, as well as inhibition of inflammatory cytokine activation and NF-kappaB DNA binding activity. Moreover, LA inhibited bone destruction in vivo and osteoclastogenesis in vitro. Collectively, these results indicate that LA may be a new adjunctive therapy for rheumatoid arthritis. Neurochem Int. 2006 Aug 30. The antioxidant alpha-lipoic acid has been reported to prevent and reverse age-related impairments in learning and memory. However, it is unclear how alpha-lipoic acid improves cognitive function. In this study, the effect of alpha-lipoic acid on the release of endogenous glutamate from rat cerebrocortical nerve terminals (synaptosomes) was examined. We found that alpha-lipoic acid potently facilitated 4-aminopyridine (4AP)-evoked glutamate release, and this release facilitation results from an enhancement of vesicular exocytosis and not from an increase of non-vesicular release. Examination of the effect of alpha-lipoic acid on cytosolic [Ca(2+)] revealed that the facilitation of glutamate release was associated with an increase in voltage-dependent Ca(2+) influx. Consistent with this, alpha-lipoic acid-mediated facilitation of glutamate release was completely prevented in synaptosomes pretreated with a wide spectrum blocker of the N- and P/Q-type Ca(2+) channels, omega-conotoxin MVIIC. The facilitatory effect of alpha-lipoic acid on Ca(2+) influx was not due to an increase of synaptosomal excitability because alpha-lipoic acid did not alter the 4AP-evoked depolarization of the synaptosomal plasma membrane potential. In addition, both ionomycin and hypertonic sucrose-induced glutamate release were enhanced by alpha-lipoic acid. Furthermore, disruption of cytoskeleton organization with cytochalasin D occluded the facilitatory effect of alpha-lipoic acid on 4AP or ionomycin-evoked glutamate release. These results suggest that the antioxidant alpha-lipoic acid enhances the Ca(2+) entry through presynaptic N- and P/Q-type Ca(2+) channels as well as the vesicular release machinery to cause an increase in evoked glutamate release from rat cerebrocortical synaptosomes. Also, activation of PKA and PKC may underlie, at least in part, the alpha-lipoic acid-mediated facilitation of glutamate release observed here as alpha-lipoic acid-enhanced 4AP and ionomycin-evoked glutamate release were significantly attenuated by PKA and PKC inhibitors. This finding may provide some information regarding the mechanism of action of alpha-lipoic acid in the central nervous system (CNS). J Immunol. 2006 Aug 15. Reactive oxygen species (ROS) play an important role in various events underlying multiple sclerosis (MS) pathology. In the initial phase of lesion formation, ROS are known to mediate the transendothelial migration of monocytes and induce a dysfunction of the blood-brain barrier (BBB). In this study, we describe the beneficial effect of the antioxidant alpha-lipoic acid (LA) on these phenomena. In vivo, LA dose-dependently prevented the development of clinical signs in a rat model for MS, acute experimental allergic encephalomyelitis (EAE). Clinical improvement was coupled to a decrease in leukocyte infiltration into the CNS, in particular monocytes. Monocytes isolated from the circulation of LA-treated rats revealed a reduced migratory capacity to cross a monolayer of rat brain endothelial cells in vitro compared with monocytes isolated from untreated EAE controls. Using live cell imaging techniques, we visualized and quantitatively assessed that ROS are produced within minutes upon the interaction of monocytes with brain endothelium. Monocyte adhesion to an in vitro model of the BBB subsequently induced enhanced permeability, which could be inhibited by LA. Moreover, administration of exogenous ROS to brain endothelial cells induced cytoskeletal rearrangements, which was inhibited by LA. In conclusion, we show that LA has a protective effect on EAE development not only by affecting the migratory capacity of monocytes, but also by stabilization of the BBB, making LA an attractive therapeutic agent for the treatment of MS. Eur J Pharmacol. 2006 Aug 14. Chem Biol Interact. 2006 Aug 12. Background: Glutathione plays crucial roles in antioxidant defence and glutathione deficiency contributes to oxidative stress and may therefore play a key role in the pathogenesis of many diseases. The objectives of the present study were to evaluate the effects on glutathione turnover of thiol and non-thiol antioxidants in human cell cultures and if any of the antioxidant had a short-term cellular effect against different levels of hydrogen peroxide. Methods: We have investigated the effect on the total glutathione amount in HeLa and hepatoma cell cultures of thiol antioxidants in comparison with non-thiol antioxidants, such as a copper chelator, Vitamin C, and a flavonoid. Furthermore, we have investigated the short-term (within 24h) interaction of the different antioxidants with hydrogen peroxide. Results and conclusions: Lipoic acid and quercetin (Quer) were the two antioxidants that showed the highest stimulation of glutathione synthesis in cell cultures as judged by the total glutathione amount. However, no antioxidant protected against hydrogen peroxide present in concentrations that lowered cell protein. This finding may be attributed to the fact that it is necessary to incubate cell cultures with antioxidants or small doses of oxidants for a period before the cultures are exposed to hydrogen peroxide in order to enhance the antioxidant defence. The presence of Quer and Vitamin C lowered cell protein and total glutathione even in cell cultures containing hydrogen peroxide in concentrations that did not lower cell protein. This finding might be attributed to pro-oxidant properties and formation of excess reactive oxygen species in the presence of Quer and Vitamin C. Regul Toxicol Pharmacol. 2006 Aug 10. The safety of the antioxidant alpha-lipoic acid (racemic form) (ALA), also called thioctic acid (CAS RN 1077-28-7) was assessed in acute and subchronic toxicity studies as well as in in vitro and in vivo mutagenicity/genotoxicity studies. ALA was not acutely toxic to rats (LD(50)>2000mg/kg bw, OECD method 425). Administration of 31.6 or 61.9mg ALA/kg bw/day for 4 weeks to male/female Wistar rats did not show any adverse effects. Specifically, there was no significant difference between control and treated animals at 31.6 or 61.9mg ALA/kg bw with regard to body weight gain, feed consumption, animal behaviour, or haematological and clinical chemistry parameters. Only the high-dose of 121mg ALA/kg bw was associated with slight alterations in liver enzymes as well as histopathological effects on the liver and mammary gland. ALA did not possess any mutagenic activity in the Ames assays conducted with various bacterial strains of Salmonella typhimurium. Moreover, there was no evidence of genotoxic activity in a mouse micronucleus assay. The results of these studies support the safety of ALA. The no-observed-adverse-effect level (NOAEL) is considered to be 61.9mg/kg bw/day. Cell Biochem Funct. 2006 Aug 7. We have previously shown that one of the potential mediators of the deleterious effects of high glucose on extracellular matrix protein (ECM) expression in renal mesangial cells is its metabolic flux through the hexosamine biosynthesis pathway (HBP). Here, we investigate further whether the hexosamines induce oxidative stress, cell-cycle arrest and ECM expression using SV-40-transformed rat mesangial (MES) cells and whether the anti-oxidant alpha-lipoic acid will reverse some of these effects. Culturing renal MES cells with high glucose (HG, 25 mM) or glucosamine (GlcN, 1.5 mM) for 48 h stimulates laminin gamma1 subunit expression significantly approximately 1.5 +/- 0.2- and 1.9 +/- 0.3-fold, respectively, when compared to low glucose (LG, 5 mM). Similarly, HG and GlcN increase the level of G0/G1 cell-cycle progression factor cyclin D1 significantly approximately 1.7 +/- 0.2- and 1.4 +/- 0.04-fold, respectively, versus LG (p < 0.01 for both). Azaserine, an inhibitor of glutamine:fruc-6-PO(4) amidotransferase (GFAT) in the HBP, blocks the HG-induced expression of laminin gamma1 and cyclin D1, but not GlcN's effect because it exerts its metabolic function distal to GFAT. HG and GlcN also elevate reactive oxygen species (ROS) generation, pro-apoptotic caspase-3 activity, and lead to mesangial cell death as revealed by TUNEL and Live/Dead assays. FACS analysis of cell-cycle progression shows that the cells are arrested at G1 phase; however, they undergo cell growth and hypertrophy as the RNA/DNA ratio is significantly (p < 0.05) increased in HG or GlcN-treated cells relative to LG. The anti-oxidant alpha-lipoic acid (150 microM) reverses ROS generation and mesangial cell death induced by HG and GlcN. Alpha-lipoic acid also reduces HG and GlcN-induced laminin gamma1 and cyclin D1 expression in MES cells. In addition, induction of diabetes in rats by streptozotocin (STZ) increases both laminin gamma1 and cyclin D1 expression in the renal cortex and treatment of the diabetic rats with alpha-lipoic acid (400 mg kg(-1) body weight) reduces the level of both proteins significantly (p < 0.05) when compared to untreated diabetic rats. These results support the hypothesis that the hexosamine pathway mediates mesangial cell oxidative stress, ECM expression and apoptosis. Anti-oxidant alpha-lipoic acid reverses the effects of high glucose, hexosamine and diabetes on oxidative stress and ECM expression in mesangial cells and rat kidney. Regul Toxicol Pharmacol. 2006 Aug 7. alpha-Lipoic acid (ALA) (CAS RN 1077-28-7), also referred to as thioctic acid, has been demonstrated to exhibit strong anti-oxidant properties. In order to test the long-term toxicity of ALA, groups of 40-50 male and female, 5-6-week-old, Sprague-Dawley rats were subjected to oral administration of 20, 60, or 180mg/kg body weight (bw)/day ALA for 24 months. There was no significant difference between control animals and treated animals at 20 or 60mg/kg bw/day with respect to body weight gain, food consumption, behavioural effects, haematological and clinical chemistry parameters, and gross and histopathological findings. In all treatment groups, mortality was slightly lower as compared to the control. The absolute weights of the heart (high-dose males), thymus (high-dose males), and left adrenal (mid-dose males), liver (high-dose females), and lungs (high-dose females) were decreased in comparison to controls. These changes were of no toxicological significance. The only notable finding in rats of both sexes dosed at 180mg/kg bw/day was a reduction in food intake relative to the controls and a concomitant decrease in body weight. This decrease in body weight led to significant differences between the control and high-dose rats with respect to the absolute weights of certain organs. However, no gross or histopathological changes were associated with these findings. The no-observed-adverse-effect level (NOAEL) is considered to be 60mg/kg bw/day. J Neurochem. 2006 Aug. The work presented here verifies the hypothesis that RS-alpha-lipoic acid may exert its cholinoprotective and cholinotrophic activities through the maintenance of appropriate levels of acetyl-CoA in mitochondrial and cytoplasmic compartments of cholinergic neurons. Sodium nitroprusside (SNP) and amyloid-beta decreased pyruvate dehydrogenase, choline acetyltransferase activities, acetyl-CoA content in mitochondria and cytoplasm, as well as increased fraction of non-viable, trypan blue positive cells in cultured differentiated cholinergic SN56 neuroblastoma cells. Lipoic acid totally reversed toxin-evoked suppression of choline acetyltrasferase and pyruvate dehydrogenase activities, as well as mitochondrial and cytoplasmic acetyl-CoA levels, and partially attenuated increase of cell mortality. Significant negative correlations were found between enzyme activities, acetyl-CoA levels and cell mortality in different neurotoxic and neuroprotective conditions employed here. The level of cytoplamic acetyl-CoA correlated with mitochondrial acetyl-CoA, whereas choline acetyltransferase activity followed shifts in cytoplasmic acetyl-CoA. Thus, we conclude that, in cholinergic neurons, particular elements of the pyruvate-acetyl-CoA-acetylcholine pathway form a functional unit responding uniformly to neurotoxic and neuroprotectory conditions. J Diabetes Complications. 2006 Jul-Aug. The pathophysiology of diabetes includes oxidative stress and impaired heat shock protein (HSP) expression. We studied the effects of alpha-lipoic acid (LA) supplementation for 8 weeks and acute exercise on HSP60 expression and the oxidative stress marker 4-hydroxynonenal adducts (4-HNE) in streptozotocin-induced diabetic (SID) and nondiabetic control rats. Diabetes was associated with decreased HSP60 in the heart and increased levels of HSP60 and 4-HNE in the liver. LA increased HSP60 in the liver of control and diabetic rats and decreased 4-HNE in the liver and heart. Acute exercise increased liver 4-HNE, which was offset by LA. In conclusion, diabetes induced oxidative stress and impaired myocardial HSP60 expression, while LA partially offsets these alterations in a tissue-specific manner. J Gerontol A Biol Sci Med Sci. 2006 Jul. The aging process is characterized by a general decline in physiological functions that affects many tissues and increases the risk of death. In the present investigation using various substrates, the respiration rate was observed in young, middle-aged, and aged rats upon administration of carnitine (300 mg/kg body weight) and lipoic acid (100 mg/kg body weight). We observed that the rate of respiration, both State 3 and respiratory control ratio, decreased significantly in aged rats after using various substrates (except succinate). An increase in the State 4 respiration was observed in aged rats when beta-hydroxybutyrate as well as pyruvate and malate were used as substrates, whereas no change in the adenosine diphosphate/oxygen ratio ratio was observed. These changes were brought to normal levels upon cosupplementation of carnitine and lipoic acid. Thus, this study provides evidence for the role of carnitine and lipoic acid in alleviating the age-related decline in mitochondrial respiratory activity. Diabetologia. 2006 Jul. Aims/Hypothesis: The antioxidant compound alpha-lipoic acid (alpha-LA) possesses antidiabetic and anti-obesity properties. In the hypothalamus, alpha-LA suppresses appetite and prevents obesity by inhibiting AMP-activated protein kinase (AMPK). Given the therapeutic potential of alpha-LA for the treatment of type 2 diabetes and obesity, and the importance of AMPK in beta cells, we examined the effect of alpha-LA on pancreatic beta cell function. Materials and Methods: Isolated rat islets and MIN6 beta cells were treated acutely (15-90 min) or chronically (18-24 h) with alpha-LA or the known AMPK-activating compounds 5'-amino-imidazole-4-carboxamide ribonucleoside (AICAR) and metformin. Insulin secretion, the AMPK-signalling pathway, mitochondrial function and cell growth were assessed. Results: Acute or chronic treatment of islets and MIN6 cells with alpha-LA led to dose-dependent rises in phosphorylation of the AMPK alpha-subunit and acetyl CoA carboxylase. Chronic exposure to alpha-LA, AICAR or metformin caused a reduction in insulin secretion. alpha-LA inhibited the p70 s6 kinase translational control pathway, and inhibited MIN6 growth in a manner similar to rapamycin. Unlike AICAR and metformin, alpha-LA also acutely inhibited insulin secretion. Examination of the effect of alpha-LA on mitochondrial function showed that acute treatment with this compound elevated reactive oxygen species (ROS) production and enhanced mitochondrial depolarisation induced by Ca(2+). Conclusions/Interpretation: This study is the first to demonstrate that alpha-LA directly affects beta cell function. The chronic effects of alpha-LA include AMPK activation and reductions in insulin secretion and content, and cell growth. Acutely, alpha-LA also inhibits insulin secretion, an effect probably involving the ROS-induced impairment of mitochondrial function. Pol Merkuriusz Lek. 2006 Jun. The aim of this study was to investigate the effect of lipoic acid (LA) on lipid peroxidation, hydrogen peroxide concentration (H2O2), sulphydryl group (-SH) contents and total capacity protein a few hours after administration lipopolysaccharide (LPS)-induced oxidative stress in rats. Materials and methods: Male albino Wistar rats were used in this study. The animals were divided into four groups, each group consisting of ten male animals. Control group received physiological saline alone i.v. The second group was given LA (60 mg/kg b.w., i.v.). The third second group received LPS (30 mg/kg b.w., i.v.). The fourth group received LPS (30 mg/kg b.w., i.v.) and after 0.5h received LA (60 mg/kg b.w., i.v.). In the lung homogenates were measured thiobarbituric acid reactants (TBARS), hydrogen peroxide concentration, -SH groups and protein contents. Results: The administration of LA after to LPS-induced oxidative stress caused a significant decrease in TBARS and H2O2 concentrations (appropriate 3.65 +/- 0.2 microM and 0.014 +/- 0.008 microM) compared with the group treated with LPS (appropriate 6.030 +/- 0.16 microM and 0.189 +/- 0.03 microM). Treatment of LPS-injected rats with LA caused increase in -SH groups and protein concentration (p < 0.05). Conclusion: The early administration of LA [caused] a significant decrease [in] symptoms of oxidative stress-induced LPS, [including a] decrease in lipid peroxidation process [and] H2O2 concentration and [an] increase in -SH groups and protein contents in rat's lung homogenates. Pharmacol Rep. 2006 May-Jun. Lipopolysaccharide (LPS), called endotoxin, is a major component of Gram-negative bacteria cell wall. LPS stimulates the synthesis and release of several metabolites from mammalian phagocytes which leads to fulminant systemic inflammation (endotoxic shock). Among LPS-induced metabolites, reactive oxygen species are considered to play crucial role in the pathogenesis of endotoxic shock via oxidative stress generation. In this study, the effect of early administration of antioxidant alpha-lipoic acid (LA) on plasma lipid peroxidation and total antioxidant blood capacity was evaluated in endotoxic shock in rats. Lipid peroxidation was measured as plasma thiobarbituric acid reactive substances (TBARS) levels, while total blood antioxidant capacity was assessed as ferric reducing ability of plasma (FRAP). The endotoxic shock was induced by administration of LPS (Escherichia coli 026:B6, 30 mg/kg, iv) in anesthetized rats. Then, 30 min later, animals were treated intravenously (iv) with LA at 60 mg/kg. After 5 h observation animals were killed and blood from heart was taken for TBARS and FRAP measurements. LPS injected to saline-pretreated animals resulted in development of oxidative stress indicated by significant increases in plasma TBARS and significant decrease in total antioxidant capacity of plasma. Conversely, LA injected to saline pretreated animals caused an increase in FRAP values and the decrease in TBARS levels. The administration of LA0.5 h after LPS challenge resulted in an increase in FRAP values and decrease in plasma lipid peroxidation as compared to LPS group. Moreover, the levels of TBARS and FRAP in LPS + LAgroup were similar to those observed in LA group. In conclusion, our present study demonstrates that early treatment with LA significantly protects against endotoxin-induced oxidative stress in rats. Clin Appl Thromb Hemost. 2006 Apr. Vitamin E and alpha-lipoic acid are potent nutritional antioxidants, and when used together, their antioxidant capabilities are improved as alpha-lipoic acid recycles vitamin E. Supplementation of vitamin E has been shown to prolong platelet aggregation but the effects of vitamin E and alpha-lipoic acid supplementation on bleeding tendency have yet to be reported. Young, male rats consumed either control diet (n=5) or vitamin E and alpha-lipoic acid-supplemented diet (n=5) for 14 weeks. Activated partial thromboplastin time (APTT) and prothrombin time (PT) were measured as markers of intrinsic and extrinsic coagulation pathways respectively in addition to lipid peroxidation (malondialdehyde). Supplementation significantly prolonged APTT (23.8+/-1.5 vs 31.4+/-1.2s, p<0.05) compared to the control diet; however, there was no significant difference in PT (27.8+/-1.5 vs 26.6+/-0.9s, p>0.05). While vitamin E was increased (p<0.05), there was no significant difference in plasma levels of malondialdehyde (p>0.05). Dietary supplementation of vitamin E and alpha-lipoic acid increases bleeding tendency via inhibition of the intrinsic coagulation pathway with no change in markers of lipid peroxidation. Such supplementation could benefit patients with cardiovascular disease who exhibit elevated levels of coagulation and oxidative stress. Pharmazie. 2005 Oct. The present study investigates the antiglycating effect of alpha-lipoic acid (LA) in high fructose-fed rats in vivo and its potential to inhibit the process of glycation in vitro. In addition, the effect of LA on glucose utilisation in rat diaphragm was also studied. Rats fed a high fructose diet (60% total calories) were administered with 35 mg/kg b.w, lipoic acid (LA) intraperitoneally for 20 days. The effects of LA on plasma glucose, fructosamine, protein glycation and glycated haemoglobin in high fructose rats and on in vitro glycation were studied. In vitro utilization of glucose was carried out in normal rat diaphragm in the presence and absence of insulin in which LA was used as an additive. The contents of glucose, glycated protein, glycated haemoglobin and fructosamine were significantly lowered on LA administration to high fructose-fed rats. LA prevented in vitro glycation and the accumulation of advanced glycation end products. Further LA enhanced glucose utilization in the rat diaphragm. This effect was additive to that of insulin and did not interfere with the action of insulin. The findings provide evidence for the therapeutic utility of lipoic acid in diabetes and its complications. Biochem Biophys Res Commun. 2005 Jul 8. Triglyceride accumulation in skeletal muscle contributes to insulin resistance in obesity. We recently showed that alpha-lipoic acid (ALA) reduces body weight and prevents the development of diabetes in diabetes-prone obese rats by reducing triglyceride accumulation in non-adipose tissues. AMP-activated protein kinase (AMPK) is a major regulator of cellular energy metabolism. We examined whether ALA lowers triglyceride accumulation in skeletal muscle by activating AMPK. Alpha2-AMPK activity was decreased in obese rats compared to control rats. Administration of ALA to obese rats increased insulin-stimulated glucose disposal in whole body and in skeletal muscle. ALA also increased fatty acid oxidation and activated AMPK in skeletal muscle. Adenovirus-mediated administration of dominant negative AMPK into skeletal muscle prevented the ALA-induced increases in fatty acid oxidation and insulin-stimulated glucose uptake. These results suggest that ALA-induced improvement of insulin sensitivity is mediated by activation of AMPK and reduced triglyceride accumulation in skeletal muscle. Free Radic Res. 2004 Oct. The four pyruvate dehydrogenase kinase (PDK) and two pyruvate dehydrogenase phosphatase (PDP) isoenzymes that are present in mammalian tissues regulate activity of the pyruvate dehydrogenase complex (PDC) by phosphorylation/dephosphorylation of its pyruvate dehydrogenase (E1) component. The effect of lipoic acids on the activity of PDKs and PDPs was investigated in purified proteins system. R-lipoic acid, S-lipoic acid and R-dihydrolipoic acid did not significantly affect activities of PDPs and at the same time inhibited PDKs to different extents (PDK1>PDK4 approximately PDK2>PDK3 for R-LA). Since lipoic acids inhibited PDKs activity both when reconstituted in PDC and in the presence of E1 alone, dissociation of PDK from the lipoyl domains of dihydrolipoamide acetyltransferase in the presence of lipoic acids is not a likely explanation for inhibition. The activity of PDK1 towards phosphorylation sites 1, 2 and 3 of E1 was decreased to the same extent in the presence of R-lipoic acid, thus excluding protection of the E1 active site by lipoic acid from phosphorylation. R-lipoic acid inhibited autophosphorylation of PDK2 indicating that it exerted its effect on PDKs directly. Inhibition of PDK1 by R-lipoic acid was not altered by ADP but was decreased in the presence of pyruvate which itself inhibits PDKs. An inhibitory effect of lipoic acid on PDKs would result in less phosphorylation of E1 and hence increased PDC activity. This finding provides a possible mechanism for a glucose (and lactate) lowering effect of R-lipoic acid in diabetic subjects. Curr Med Chem. 2004 May. alpha-Lipoic acid (LA), a naturally occurring dithiol compound, has long been known as an essential cofactor for mitochondrial bioenergetic enzymes. Aside from its enzymatic role, in vitro and in vivo studies suggest that LA also acts as a powerful micronutrient with diverse pharmacologic and antioxidant properties. Pharmacologically, LA improves glycemic control, polyneuropathies associated with diabetes mellitus, and effectively mitigates toxicities associated with heavy metal poisoning. As an antioxidant, LA directly terminates free radicals, chelates transition metal ions (e.g. iron and copper), increases cytosolic glutathione and vitamin C levels and prevents toxicities associated with their loss. These diverse actions suggest that LA acts by multiple mechanisms both physiologically and pharmacologically, many of which are only now being explored. Herein, we review the known biochemical properties of LA with particular reference to how LA may be an effective agent to ameliorate certain pathophysiologies of many chronic diseases. Mol Genet Metab. 2004 Apr. Marked progress has been made over the past 15 years in defining the specific biochemical defects and underlying molecular mechanisms of oxidative phosphorylation disorders, but limited information is currently available on the development and evaluation of effective treatment approaches. Metabolic therapies that have been reported to produce a positive effect include coenzyme Q(10) (ubiquinone), other antioxidants such as ascorbic acid and vitamin E, riboflavin, thiamine, niacin, vitamin K (phylloquinone and menadione), and carnitine. The goal of these therapies is to increase mitochondrial ATP production, and to slow or arrest the progression of clinical symptoms. In the present study, we demonstrate for the first time that there is a significant increase in ATP synthetic capacity in lymphocytes from patients undergoing cofactor treatment. We also examined in vitro cofactor supplementation in control lymphocytes in order to determine the effect of the individual components of the cofactor treatment on ATP synthesis. A dose-dependent increase in ATP synthesis with CoQ(10) incubation was demonstrated, which supports the proposal that CoQ(10) may have a beneficial effect in the treatment of oxidative phosphorylation (OXPHOS) disorders. J Am Diet Assoc. 2003 Aug. Mitochondrial disorders are degenerative diseases characterized by a decrease in the ability of mitochondria to supply cellular energy requirements. Substantial progress has been made in defining the specific biochemical defects and underlying molecular mechanisms, but limited information is available about the development and evaluation of effective treatment approaches. The goal of nutritional cofactor therapy is to increase mitochondrial adenosine 5'-triphosphate production and slow or arrest the progression of clinical symptoms. Accumulation of toxic metabolites and reduction of electron transfer activity have prompted the use of antioxidants, electron transfer mediators (which bypass the defective site), and enzyme cofactors. Metabolic therapies that have been reported to produce a positive effect include Coenzyme Q(10) (ubiquinone); other antioxidants such as ascorbic acid, vitamin E, and lipoic acid; riboflavin; thiamin; niacin; vitamin K (phylloquinone and menadione); creatine; and carnitine. A literature review of the use of these supplements in mitochondrial disorders is presented. Proc Natl Acad Sci USA. 2002 Feb 19. Mitochondrial-supported bioenergetics decline and oxidative stress increases during aging. To address whether the dietary addition of acetyl-l-carnitine [ALCAR, 1.5% (wt/vol) in the drinking water] and/or (R)-alpha-lipoic acid [LA, 0.5% (wt/wt) in the chow] improved these endpoints, young (2-4 mo) and old (24-28 mo) F344 rats were supplemented for up to 1 mo before death and hepatocyte isolation. ALCAR+LA partially reversed the age-related decline in average mitochondrial membrane potential and significantly increased (P = 0.02) hepatocellular O(2) consumption, indicating that mitochondrial-supported cellular metabolism was markedly improved by this feeding regimen. ALCAR+LA also increased ambulatory activity in both young and old rats; moreover, the improvement was significantly greater (P = 0.03) in old versus young animals and also greater when compared with old rats fed ALCAR or LA alone. To determine whether ALCAR+LA also affected indices of oxidative stress, ascorbic acid and markers of lipid peroxidation (malondialdehyde) were monitored. The hepatocellular ascorbate level markedly declined with age (P = 0.003) but was restored to the level seen in young rats when ALCAR+LA was given. The level of malondialdehyde, which was significantly higher (P = 0.0001) in old versus young rats, also declined after ALCAR+LA supplementation and was not significantly different from that of young unsupplemented rats. Feeding ALCAR in combination with LA increased metabolism and lowered oxidative stress more than either compound alone. Excerpts from the full text article: [...] Mitochondria are targets of their own oxidant by-products. The steady-state oxidative damage in mitochondria is high relative to other organelles, and the percentage of oxygen converted to superoxide increases with age (3, 4, 5, 6). This leads to a vicious cycle of increasing mitochondrial damage, which adversely affects cell function (7), and results in a loss of ATP-generating capacity, especially in times of greater energy demand, thereby compromising vital ATP-dependent reactions. Cellular processes affected by mitochondrial decay include detoxification, repair systems, DNA replication, osmotic balance, and higher-order >processes (7), such as cognitive function (7, 8, 9). [...] Several dietary supplements, including the mitochondrial cofactor and antioxidant lipoic acid (LA), increase endogenous antioxidants or mitochondrial bioenergetics (13, 14, 15). Feeding old rats acetyl-l-carnitine (ALCAR), a mitochondrial metabolite, reverses the age-related decline in tissue carnitine levels and improves mitochondrial fatty acid β-oxidation in the tissues studied (15, 16, 17, 18). ALCAR supplementation also reverses the age-related alterations in fatty acid profiles and loss in cardiolipin levels, an essential phospholipid required for mitochondrial substrate transport (15, 16, 17). We demonstrated that ALCAR supplementation reverses the age-associated decline in metabolic activity in rats, suggesting that ALCAR improves mitochondrial function and increases general metabolic activity (19, 20). ALCAR-induced improvement in metabolic parameters appear to be responsible for improving short-term memory deficits and cognitive function in elderly subjects given ALCAR (21, 22) and in old rats (9). This increased metabolic activity may come at a price, however, because supplementing rats with high levels of ALCAR lowered hepatocellular antioxidant status (19). This ALCAR-induced antioxidant loss was not seen, however, in other organs (T.M.H. and D. Heath, unpublished work) or when lower doses were given (23). We also showed that giving high [1.5% (wt/vol)], but not lower [0.5% (wt/vol)], supplemental doses of ALCAR to old rats increased mitochondrial oxidant flux, suggesting that while high ALCAR supplementation may increase electron flow through the electron transport chain, it also heightens formation of ROS as a consequence. We thus hypothesized that ALCAR supplemented with an antioxidant may have the salutary effect of increasing mitochondrial function and general metabolic activity without a concomitant increase in oxidative stress. We chose LA as a cosupplement for two reasons: (i) it is a naturally occurring cofactor for mitochondrial α-keto acid dehydrogenases (24), which may aid in cellular glucose-dependent ATP production (25); and (ii) in its reduced form, LA is a potent antioxidant and also increases intracellular ascorbate and glutathione concentrations (15, 26). Thus, LA and ALCAR may act together to reverse age-related metabolic decline and also reduce indices of oxidative stress. We show that the combined supplementation of ALCAR and LA (ALCAR+LA) reverses age-related metabolic decline, improves hepatocellular ascorbate levels, and lowers oxidant appearance and oxidative damage. Materials and Methods [...] ALCAR Supplementation. Old and young rats were given a 1.5% (wt/vol; pH adjusted to ≈6) solution of ALCAR in their drinking water and allowed to drink ad libitum for 1 mo before death and hepatocyte isolation. Both young and old rats typically drank ≈20 ml/rat per day (data not shown), which provided a daily ALCAR dose of ≈0.75 g/kg body wt per day for old rats and 1.2 g/kg body wt per day for young rats. LA Supplementation. Young and old rats were given LA [0.5% (wt/wt)] mixed into the AIN-93M chow (Dyets, Bethlehem, PA) for 2 weeks before death. Unsupplemented animals were fed Purina rodent chow and water ad libitum. The pellets were made into a mush and fed to some young and old rats for 2 weeks before cell isolation. Both young and old rats typically ate ≈15 g/rat per day (data not shown), which provides a daily LA dose of 0.12 g/kg body weight for young rats and 0.075 g/kg body weight for old rats. Results [...] We previously showed significantly lower average mitochondrial membrane potential (delta-Ψ) in the majority of hepatocytes from old rats compared with young rats, but a 1-mo feeding regimen of 1.5% (wt/vol) ALCAR reversed this decline in delta-Ψ (19). For the present study, we also found a marked age-related decline in this key parameter of mitochondrial function (Fig. 1). Relative to mean fluorescence characteristics seen in hepatocytes from young unsupplemented animals, the average delta-Ψ for hepatocytes from old rats was 53.8 ± 8.0% lower (n = 5), representing a significant loss (P = 0.02). Feeding ALCAR+LA to old rats markedly reversed this decline (Fig. 1). Old rats on the ALCAR+LA supplemented diet had an average delta-Ψ that was only 22.8 ± 6.0% lower relative to young unsupplemented rats. Thus, dietary supplementation with ALCAR+LA partially restored the loss of mitochondrial delta-Ψ although the improvement was not as great as previously observed with ALCAR alone (19). We previously showed in separate reports that ALCAR or LA supplementation increased hepatocellular and myocardial oxygen consumption, indicating that either compound was able to increase cellular metabolism (18, 19). Young and old rats were supplemented with or without ALCAR+LA before cell isolation, and this general parameter of metabolic rate was monitored by using an oxygen electrode. Hepatocellular oxygen consumption declined from 1.03 ± 0.17 (n = 5) to 0.54 ± 0.09 μmol/min per 106 cells (n = 5) in young versus old unsupplemented rats, a significant (P = 0.03) decline of 47.6% with age. These results are in agreement with our previous results and suggest that there is an age-related decline in hepatocellular metabolic rate. Oxygen consumption in hepatocytes from old rats treated with ALCAR+LA was 0.82 ± 0.07 μmol O2/min per 106 cells versus 0.95 ± 0.05 μmol/min per 106 in unsupplemented (n = 5) young rats (P = 0.02). Thus, feeding ALCAR+LA to old rats significantly reversed the age-related decline in hepatocellular oxygen consumption. Ambulatory Activity. To further explore whether ALCAR+LA generally improved metabolic rate on a whole animal basis, we studied ambulatory activity in animals fed with or without ALCAR+LA. Old rats exhibited a 3-fold decline in ambulatory activity in terms of overall movement and the amount of time spent in movement (Table 1). The speed of old animals when in movement was not different from that shown by young animals, suggesting that the age-related decline in activity was not caused by pain or the inability to move, but rather it reflected a general loss of metabolic activity. Animals were then fed ALCAR+LA for 1 mo (in the case of LA 2 weeks) and again tested for ambulatory activity. Results show that ALCAR+LA significantly improved ambulatory activity in young and old animals. For the young animals, the amount of active time and the overall distance traveled increased by ≈31% when compared with their activity before ALCAR+LA supplementation. A much greater increase was observed in old rats. Ambulation and overall distance traveled more than doubled from 20 ± 2 s per movement and 177 ± 19 cm/h to 43 ± 3 s per movement and 376 ± 23 cm/h, respectively. This increase, although still not as good, on average, as young untreated rats, nevertheless represented a significant (P = 0.03) improvement versus that of old untreated animals. Thus, ALCAR+LA supplementation not only reverses the age-related decline in oxygen consumption, a cellular parameter of metabolic activity, but also increases ambulatory activity, a general physiological parameter of metabolic activity. Antioxidant Status/Oxidative Stress. We previously observed that feeding 1.5% (wt/vol) ALCAR alone to old rats, although markedly increasing metabolic activity through improved mitochondrial function, also resulted in heightened oxidant production and decreased low molecular weight antioxidant status. This finding was presumably caused by increased formation of ROS/reactive nitrogen species as by-products of heightened metabolic activity. To understand whether feeding ALCAR+LA could ameliorate this potential increase in oxidative stress, we measured ascorbic acid status, overall oxidant production, and markers of oxidative damage in freshly isolated hepatocytes taken from young and old rats fed with or without ALCAR+LA. Hepatocytes from old rats had significantly lower ascorbate levels as compared with young rats (7.29 ± 2.97 versus 3.38 ± 0.67; P = 0.003) (Fig. 2), suggesting that liver antioxidant status may be compromised with age. We observed, as previously, that ALCAR supplementation at 1.5% (wt/vol) resulted in a further and significant decline in ascorbate levels beyond the observed age-related loss in this key antioxidant. However, ALCAR+LA supplementation reversed the ALCAR-induced and age-related loss of ascorbate such that there was no longer a significant difference (P = 0.3) in hepatocellular ascorbate values between ALCAR+LA-treated old rats and that of untreated young animals (Fig. 2). To further investigate whether ALCAR+LA actually affected oxidative stress parameters in old rats, hepatocellular oxidant production was monitored by using 2′,7′-dichlorofluorescin oxidation. This cell permeant dye becomes fluorescent when it is oxidized. Thus, general oxidant production can be monitored in cells by measuring the rate of increased fluorescence over time. Cells isolated from young and old rats exhibited a marked difference in fluorescence appearance (Fig. 3). Oxidant production increased over 30.8% with age from 2,942.3 ± 99.3 to 3,835.22 ± 303.6 fluorescence units/min per μmol O2 consumed per 106 cells. This finding is in agreement with our previously published results (18) and is consistent with lower antioxidant status and heightened mitochondrial oxidant production during aging. Addition of ALCAR+LA to the diet of old rats caused a significant decline in appearance of oxidants to 2,801.79 ± 308.0 fluorescence units/min per μmol O2 consumed per 106 cells, which was not different from untreated or ALCAR+LA-fed young rats. Thus, the combination of ALCAR with LA not only reverses the age-related increase in oxidants, but also the additional oxidants induced by high doses of ALCAR. These results suggest that ALCAR+LA supplementation not only improves metabolic rate and physiological activity, but does so without causing a concomitant increase in oxidants. To further assess whether ALCAR+LA modulated age-related and ALCAR-induced oxidative stress, we also measured steady-state levels of MDA, a marker of lipid peroxidation (Fig. 4). Hepatocellular MDA levels in old untreated rats were more than 4-fold higher than the levels seen in young rats, a significant increase (P = 0.0001). Similar to results shown for oxidant production, we observed a small, but significant, increase in steady-state MDA levels in liver tissue from old rats fed ALCAR alone (Fig. 4); on average, a similar increase in young rats was not significant. These results again suggest that high ALCAR alone, although improving metabolism and cognitive function, also increased oxidative stress in the liver. When LA was given along with ALCAR, we observed that there was a significant decline in MDA levels (Fig. 4). Most importantly, hepatic MDA concentrations in old ALCAR+LA fed rats no longer statistically differed from those found in young untreated animals. Discussion We previously demonstrated that feeding old rats ALCAR markedly improves the average mitochondrial membrane potential, a key indicator of mitochondrial function, to a level no longer significantly different from that of young rats (18). This reversal of membrane potential appears to be caused, in part, by replenishment of carnitine, a betaine that shuttles fatty acids into the mitochondrion for β-oxidation. ALCAR administration also appears to reverse the age-related decline in cardiolipin levels. Cardiolipin is a key phospholipid cofactor for a number of mitochondrial substrate transporters as well as the protein complexes in the electron transport chain. Thus, age-related decline in cardiolipin could profoundly and adversely affect mitochondria. Our results, in combination with studies by Hagen, Paradies, Gadaleta, and others (15, 16), clearly demonstrate that ALCAR improves metabolic function in a number of tissues, most likely by improving substrate and electron flux through mitochondria. ALCAR does not, however, improve one aspect of mitochondrial decay in old rats, namely, the age-related increase in oxidants. Electron transfer through the mitochondrial electron transport chain becomes less efficient with age, which leads to increased oxidant leakage. ALCAR at the 1.5% level used in our initial experiments appears to increase electron flow through the electron transport chain, which further increases the appearance of ROS. In support of this concept, we observed higher oxidant appearance and lower hepatocellular antioxidant status after ALCAR supplementation (18). Feeding 1.5% ALCAR to old rats improved the age-related decline in metabolic rate, but increased oxidant appearance to a small, yet significant, degree. This ALCAR-induced increase in hepatocellular oxidative stress may be unique to the liver or caused by the relatively high levels of ALCAR used in this feeding study. In recent studies where old rats were fed 1.5% ALCAR, we did not observe any ALCAR-induced increased oxidative stress in the heart but saw a significant improvement in mitochondrial function and cellular metabolism (T.M.H., J. Suh, and D. Heath, unpublished results). In other studies using lower ALCAR doses [0.5% (wt/vol)], Liu et al.(22) noted no ALCAR-induced changes in parameters of oxidative stress in rat brain, yet found that this dose significantly improved cognitive function in aged animals (9). Thus, smaller doses of ALCAR may effectively improve metabolic function without higher oxidative stress. The rationale for the present study was to determine whether other mitochondrial metabolites fed along with ALCAR could improve metabolic parameters and lower the age-related increase in oxidative stress. We chose to cosupplement LA with ALCAR because LA is easily taken up into a variety of tissues and can be reduced to a powerful antioxidant, dihydrolipoic acid (23). Even though LA/dihydrolipoic acid is quickly removed from most cells, this compound also induces cystine/cysteine uptake and can thereby increase glutathione synthesis (25). LA supplementation maintains and actually reverses the age-related decline in hepatocellular and myocardial ascorbate and glutathione levels, even when cells were incubated with tert-butylhydroperoxide, a model alkyl peroxide (32, 33). Thus, LA may not only act synergistically with ALCAR to improve mitochondrial-supported bioenergetics but may also improve general antioxidant status, which declines with age. LA elicits other cell responses that may complement the actions of ALCAR on the cell. LA enhances glucose uptake by increasing glucose transporters at the surface of cells (24). It is also a cofactor for α-keto acid dehydrogenases found in the mitochondria, and its supplementation in the diet of aging animals may thus correct any age-associated decline in α-ketoglutarate dehydrogenase and pyruvate dehydrogenase caused by lost cofactors. Humphries and Szweda (34) showed that pyruvate dehydrogenase and α-ketoglutarate dehydrogenase can be modified by adduction with 4-hydroxy-nonenal (R. Moreau and T. M. Hagen, personal communication), rendering it unable to transfer acetyl groups. MDA and 4-hydroxy-nonenal also inactivate carnitine acyltransferase and decrease the binding affinity for substrates (35). Thus, LA may act synergistically with ALCAR to improve both fatty acid and glucose catabolism and energy production. Indeed, we have previously shown that LA alone also increases oxygen consumption and mitochondrial membrane potential, although not as effectively as ALCAR (14). Supplementing the diet of old rats with ALCAR+LA significantly improves many of the most frequently encountered age-related changes in mammals - namely loss of energy metabolism, increased oxidative stress, decreased physical activity, and as shown in ref. 9, impaired cognitive function. This affect on cognitive function has been previously observed for both ALCAR and LA (36), but to our knowledge, has not been observed for the combination of the two supplements. How ALCAR and LA affect short-term memory is not well understood, but may be caused by a number of factors, including increased neurotransmitter production, improved mitochondrial function, and/or calcium handling by the neuron (20, 21, 36). We have also recently found that LA alone significantly reduces the age-related accumulation of iron and copper in the brain (J. H. Suh, personal communication). Thus, the LA component may also increase neuro-cognitive function by potentially lowering iron and copper-induced oxidative stress. Presently, only short-term feeding regimens of ALCAR+LA have been given to aged animals. The present study suggests that long-term feeding experiments are warranted to monitor how effectively ALCAR+LA supplementation ameliorates oxidative stress, loss of metabolic function, and mild cognitive impairment seen in older animals... Proc Natl Acad Sci USA. 2002 Feb 19. Accumulation of oxidative damage to mitochondria, protein, and nucleic acid in the brain may lead to neuronal and cognitive dysfunction. The effects on cognitive function, brain mitochondrial structure, and biomarkers of oxidative damage were studied after feeding old rats two mitochondrial metabolites, acetyl-l-carnitine (ALCAR) [0.5% or 0.2% (wt/vol) in drinking water], and/or R-alpha-lipoic acid (LA) [0.2% or 0.1% (wt/wt) in diet]. Spatial memory was assessed by using the Morris water maze; temporal memory was tested by using the peak procedure (a time-discrimination procedure). Dietary supplementation with ALCAR and/or LA improved memory, the combination being the most effective for two different tests of spatial memory (P < 0.05; P < 0.01) and for temporal memory (P < 0.05). Immunohistochemical analysis showed that oxidative damage to nucleic acids (8-hydroxyguanosine and 8-hydroxy-2'-deoxyguanosine) increased with age in the hippocampus, a region important for memory. Oxidative damage to nucleic acids occurred predominantly in RNA. Dietary administration of ALCAR and/or LA significantly reduced the extent of oxidized RNA, the combination being the most effective. Electron microscopic studies in the hippocampus showed that ALCAR and/or LA reversed age-associated mitochondrial structural decay. These results suggest that feeding ALCAR and LA to old rats improves performance on memory tasks by lowering oxidative damage and improving mitochondrial function. Nutrition. 2001 Oct. Alpha-lipoic acid (LA) and its reduced form, dihydrolipoic acid, are powerful antioxidants. LA scavenges hydroxyl radicals, hypochlorous acid, peroxynitrite, and singlet oxygen. Dihydrolipoic acid also scavenges superoxide and peroxyl radicals and can regenerate thioredoxin, vitamin C, and glutathione, which in turn can recycle vitamin E. There are several possible sources of oxidative stress in diabetes including glycation reactions, decompartmentalization of transition metals, and a shift in the reduced-oxygen status of the diabetic cells. Diabetics have increased levels of lipid hydroperoxides, DNA adducts, and protein carbonyls. Available data strongly suggest that LA, because of its antioxidant properties, is particularly suited to the prevention and/or treatment of diabetic complications that arise from an overproduction of reactive oxygen and nitrogen species. In addition to its antioxidant properties, LA increases glucose uptake through recruitment of the glucose transporter-4 to plasma membranes, a mechanism that is shared with insulin-stimulated glucose uptake. Further, recent trials have demonstrated that LA improves glucose disposal in patients with type II diabetes. In experimental and clinical studies, LA markedly reduced the symptoms of diabetic pathologies, including cataract formation, vascular damage, and polyneuropathy. To develop a better understanding of the preventative and therapeutic potentials of LA, much of the current interest is focused on elucidating its molecular mechanisms in redox dependent gene expression. Diabetes Technol Ther. 2000 Autumn. Alpha-Lipoic acid (LA) is a disulfide compound that is produced in small quantities in cells, and functions naturally as a co-enzyme in the pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase mitochondrial enzyme complexes. In pharmacological doses, LA is a multifunctional antioxidant. LA has been used in Germany for over 30 years for the treatment of diabetes-induced neuropathy. In patients with type 2 diabetes, recent studies have reported that intravenous (i.v.) infusion of LA increases insulin-mediated glucose disposal, whereas oral administration of LA has only marginal effects. If the limitations of oral therapy can be overcome, LA could emerge as a safe and effective adjunctive antidiabetic agent with insulin sensitizing activity. Metabolism. 1999 Apr. Lipoic acid (LA) is a unique antioxidant that increases peripheral glucose utilization in diabetic patients. This study was conducted to investigate whether the inhibition of glucose production could be an additional mechanism for the action of LA. Intravenous (i.v.) LA injection (100 or 60 mg/kg body weight) to fasting nondiabetic or streptozotocin (STZ)-induced diabetic rats caused a rapid reduction in blood glucose with no effect on circulating insulin levels. In vivo conversion of fructose to glucose was not inhibited by LA, whereas the gluconeogenesis flux from alanine was completely prevented. Reduced liver pyruvate carboxylase (PC) activity in vivo is suggested by the finding that LA induced a decrease in liver coenzyme A (CoA) content (44% and 28% reduction in nondiabetic and diabetic rats, respectively, compared with vehicle-treated animals) and liver acetyl CoA content (80% and 67% reduction in nondiabetic and diabetic rats, respectively). A reduction in plasma free carnitine (42% and 22% in nondiabetic and diabetic rats, respectively) was observed in LA-treated animals, and acylcarnitine levels were increased twofold. This could be attributed to elevated levels of C16 and C18 acylcarnitine, without a detectable accumulation of lipoylcarnitine. Under such conditions, a significant increase in the plasma free fatty acid (FFA) concentration (204% in nondiabetic and 151% in diabetic animals) with no elevation in beta-hydroxybutyrate levels was noted. In conclusion, this study suggests that short-term administration of LA at high dosage to normal and diabetic rats causes an inhibition of gluconeogenesis secondary to an interference with hepatic fatty acid oxidation. This may render LA an antihyperglycemic agent for the treatment of diabetic subjects, who display glucose overproduction as a major metabolic abnormality. FASEB J. 1999 Feb. A diet supplemented with (R)-lipoic acid, a mitochondrial coenzyme, was fed to old rats to determine its efficacy in reversing the decline in metabolism seen with age. Young (3 to 5 months) and old (24 to 26 months) rats were fed an AIN-93M diet with or without (R)-lipoic acid (0.5% w/w) for 2 wk, killed, and their liver parenchymal cells were isolated. Hepatocytes from untreated old rats vs. young controls had significantly lower oxygen consumption (P<0. 03) and mitochondrial membrane potential. (R)-Lipoic acid supplementation reversed the age-related decline in O2 consumption and increased (P<0.03) mitochondrial membrane potential. Ambulatory activity, a measure of general metabolic activity, was almost threefold lower in untreated old rats vs. controls, but this decline was reversed (P<0.005) in old rats fed (R)-lipoic acid. The increase of oxidants with age, as measured by the fluorescence produced on oxidizing 2',7'-dichlorofluorescin, was significantly lowered in (R)-lipoic acid supplemented old rats (P<0.01). Malondialdehyde (MDA) levels, an indicator of lipid peroxidation, were increased fivefold with age in cells from unsupplemented rats. Feeding rats the (R)-lipoic acid diet reduced MDA levels markedly (P<0.01). Both glutathione and ascorbic acid levels declined in hepatocytes with age, but their loss was completely reversed with (R)-lipoic acid supplementation. Thus, (R)-lipoic acid supplementation improves indices of metabolic activity as well as lowers oxidative stress and damage evident in aging. Free Radic Biol Med. 1998 Apr. R-alpha-Lipoic acid is found naturally occurring as a prosthetic group in alpha-keto acid dehydrogenase complexes of the mitochondria, and as such plays a fundamental role in metabolism. Although this has been known for decades, only recently has free supplemented alpha-lipoic acid been found to affect cellular metabolic processes in vitro, as it has the ability to alter the redox status of cells and interact with thiols and other antioxidants. Therefore, it appears that this compound has important therapeutic potential in conditions where oxidative stress is involved. Early case studies with alpha-lipoic acid were performed with little knowledge of the action of alpha-lipoic acid at a cellular level, but with the rationale that because the naturally occurring protein bound form of alpha-lipoic acid has a pivotal role in metabolism, that supplementation may have some beneficial effect. Such studies sought to evaluate the effect of supplemented alpha-lipoic acid, using low doses, on lipid or carbohydrate metabolism, but little or no effect was observed. A common response in these trials was an increase in glucose uptake, but increased plasma levels of pyruvate and lactate were also observed, suggesting that an inhibitory effect on the pyruvate dehydrogenase complex was occurring. During the same period, alpha-lipoic acid was also used as a therapeutic agent in a number of conditions relating to liver disease, including alcohol-induced damage, mushroom poisoning, metal intoxification, and CCl4 poisoning. Alpha-Lipoic acid supplementation was successful in the treatment for these conditions in many cases. Experimental studies and clinical trials in the last 5 years using high doses of alpha-lipoic acid (600 mg in humans) have provided new and consistent evidence for the therapeutic role of antioxidant alpha-lipoic acid in the treatment of insulin resistance and diabetic polyneuropathy. This new insight should encourage clinicians to use alpha-lipoic acid in diseases affecting liver in which oxidative stress is involved. Gen Pharmacol. 1997 Sep. 1. Lipoic acid is an example of an existing drug whose therapeutic effect has been related to its antioxidant activity. 2. Antioxidant activity is a relative concept: it depends on the kind of oxidative stress and the kind of oxidizable substrate (e.g., DNA, lipid, protein). 3. In vitro, the final antioxidant activity of lipoic acid is determined by its concentration and by its antioxidant properties. Four antioxidant properties of lipoic acid have been studied: its metal chelating capacity, its ability to scavenge reactive oxygen species (ROS), its ability to regenerate endogenous antioxidants and its ability to repair oxidative damage. 4. Dihydrolipoic acid (DHLA), formed by reduction of lipoic acid, has more antioxidant properties than does lipoic acid. Both DHLA and lipoic acid have metal-chelating capacity and scavenge ROS, whereas only DHLA is able to regenerate endogenous antioxidants and to repair oxidative damage. 5. As a metal chelator, lipoic acid was shown to provide antioxidant activity by chelating Fe2+ and Cu2+; DHLA can do so by chelating Cd2+. 6. As scavengers of ROS, lipoic acid and DHLA display antioxidant activity in most experiments, whereas, in particular cases, pro-oxidant activity has been observed. However, lipoic acid can act as an antioxidant against the pro-oxidant activity produced by DHLA. 7. DHLA has the capacity to regenerate the endogenous antioxidants vitamin E, vitamin C and glutathione. 8. DHLA can provide peptide methionine sulfoxide reductase with reducing equivalents. This enhances the repair of oxidatively damaged proteins such as alpha-1 antiprotease. 9. Through the lipoamide dehydrogenase-dependent reduction of lipoic acid, the cell can draw on its NADH pool for antioxidant activity additionally to its NADPH pool, which is usually consumed during oxidative stress. 10. Within drug-related antioxidant pharmacology, lipoic acid is a model compound that enhances understanding of the mode of action of antioxidants in drug therapy. J Nutr. 1997 Sep. In the past, lipoic acid has been administered to patients and test animals as therapy for diabetic neuropathy and various intoxications. Lipoic acid and the vitamin biotin have structural similarities. We sought to determine whether the chronic administration of lipoic acid affects the activities of biotin-dependent carboxylases. For 28 d, rats received daily intraperitoneal injections of one of the following: 1) a small dose of lipoic acid [4.3 micromol/( kg.d)]; 2) a large dose of lipoic acid [15.6 micromol/(kg.d)]; or 3) a large dose of lipoic acid plus biotin [15.6 and 2.0 micromol/(kg.d), respectively]. Another group received n-hexanoic acid [14.5 micromol/(kg.d)], which has structural similarities to lipoic acid and biotin and thus served as a control for the specificity of lipoic acid. A fifth group received phosphatidylcholine in saline injections and served as the vehicle control. The rat livers were assayed for the activities of acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, and beta-methylcrotonyl-CoA carboxylase. Urine was analyzed for lipoic acid; serum was analyzed for indicators of liver damage and metabolic aberrations. The mean activities of pyruvate carboxylase and beta-methylcrotonyl-CoA carboxylase were 28-36% lower in the lipoic acid-treated rats compared with vehicle controls (P < 0.05). Rats treated with lipoic acid plus biotin had normal carboxylase activities. Carboxylase activities in livers of n-hexanoic acid-treated rats were normal despite some evidence of liver injury. Propionyl-CoA carboxylase and acetyl-CoA carboxylase were not significantly affected by administration of lipoic acid. This study provides evidence consistent with the hypothesis that chronic administration of lipoic acid lowers the activities of pyruvate carboxylase and beta-methylcrotonyl-CoA carboxylase in vivo by competing with biotin. Free Radic Biol Med. 1997. Reactive oxygen species are thought to be involved in a number of types of acute and chronic pathologic conditions in the brain and neural tissue. The metabolic antioxidant alpha-lipoate (thioctic acid, 1, 2-dithiolane-3-pentanoic acid; 1, 2-dithiolane-3 valeric acid; and 6, 8-dithiooctanoic acid) is a low molecular weight substance that is absorbed from the diet and crosses the blood-brain barrier. alpha-Lipoate is taken up and reduced in cells and tissues to dihydrolipoate, which is also exported to the extracellular medium; hence, protection is afforded to both intracellular and extracellular environments. Both alpha-lipoate and especially dihydrolipoate have been shown to be potent antioxidants, to regenerate through redox cycling other antioxidants like vitamin C and vitamin E, and to raise intracellular glutathione levels. Thus, it would seem an ideal substance in the treatment of oxidative brain and neural disorders involving free radical processes. Examination of current research reveals protective effects of these compounds in cerebral ischemia-reperfusion, excitotoxic amino acid brain injury, mitochondrial dysfunction, diabetes and diabetic neuropathy, inborn errors of metabolism, and other causes of acute or chronic damage to brain or neural tissue. Very few neuropharmacological intervention strategies are currently available for the treatment of stroke and numerous other brain disorders involving free radical injury. We propose that the various metabolic antioxidant properties of alpha-lipoate relate to its possible therapeutic roles in a variety of brain and neuronal tissue pathologies: thiols are central to antioxidant defense in brain and other tissues. The most important thiol antioxidant, glutathione, cannot be directly administered, whereas alpha-lipoic acid can. In vitro, animal, and preliminary human studies indicate that alpha-lipoate may be effective in numerous neurodegenerative disorders. Biochem Pharmacol. 1995 Aug 25. Lipoic acid (alpha-lipoic acid, thioctic acid) is applied as a therapeutic agent in various diseases accompanied by polyneuropathia such as diabetes mellitus. The stereoselectivity and specificity of lipoic acid for the pyruvate dehydrogenase complex and its component enzymes from different sources has been studied. The dihydrolipoamide dehydrogenase component from pig heart has a clear preference for R-lipoic acid, a substrate which reacts 24 times faster than the S-enantiomer. Selectivity is more at the stage of the catalytic reaction than of binding. The Michaelis constants of both enantiomers are comparable (Km = 3.7 and 5.5 mM for R- and S-lipoic acid, respectively) and the S-enantiomer inhibits the R-lipoic acid dependent reaction with an inhibition constant similar to its Michaelis constant. When three lipoic acid homologues were tested, RS-1,2-dithiolane-3-caproic acid was one carbon atom longer than lipoic acid, while RS-bisnorlipoic acid and RS-tetranorlipoic acid were two and four carbon atoms shorter, respectively. All are poor substrates but bind to and inhibit the enzyme with an affinity similar to that of S-lipoic acid. No essential differences with respect to its reaction with lipoic acid enantiomers and homologues exist between free and complex-bound dihydrolipoamide dehydrogenase. Dihydrolipoamide dehydrogenase from human renal carcinoma has a higher Michaelis constant for R-lipoic acid (Km = 18 mM) and does not accept the S-enantiomer as a substrate. Both enantiomers of lipoic acid are inhibitors of the overall reaction of the bovine pyruvate dehydrogenase complex, but stimulate the respective enzyme complexes from rat as well as from Escherichia coli. The S-enantiomer is the stronger inhibitor, the R-enantiomer the better activator. The two enantiomers have no influence on the partial reaction of the bovine pyruvate dehydrogenase component, but do inhibit this enzyme component from rat kidney. The implications of these results are discussed. Free Radic Biol Med. 1995 Aug. alpha-Lipoic acid, which plays an essential role in mitochondrial dehydrogenase reactions, has recently gained considerable attention as an antioxidant. Lipoate, or its reduced form, dihydrolipoate, reacts with reactive oxygen species such as superoxide radicals, hydroxyl radicals, hypochlorous acid, peroxyl radicals, and singlet oxygen. It also protects membranes by interacting with vitamin C and glutathione, which may in turn recycle vitamin E. In addition to its antioxidant activities, dihydrolipoate may exert prooxidant actions through reduction of iron. alpha-Lipoic acid administration has been shown to be beneficial in a number of oxidative stress models such as ischemia-reperfusion injury, diabetes (both alpha-lipoic acid and dihydrolipoic acid exhibit hydrophobic binding to proteins such as albumin, which can prevent glycation reactions), cataract formation, HIV activation, neurodegeneration, and radiation injury. Furthermore, lipoate can function as a redox regulator of proteins such as myoglobin, prolactin, thioredoxin and NF-kappa B transcription factor. We review the properties of lipoate in terms of (1) reactions with reactive oxygen species; (2) interactions with other antioxidants; (3) beneficial effects in oxidative stress models or clinical conditions. Arzneimittelforschung. 1991 Dec. The influence of alpha-lipoic acid (LA, thioctic acid, CAS 62-46-4) on thyroid hormone metabolism and serum lipid-, protein- and glucose levels was investigated. In the first setup of experiments administration of LA together with thyroxine (T4) for 9 days suppressed the T4 induced increase of T3 generation by 56%. This suppression was similar to that affected by 6-propylthiouracil (54%). LA or T4 alone did not affect the cholesterol level, but together they led to a reduction. LA decreased the triglyceride level by 45%; the decrease induced by T4 or LA plus T4 was not significant. Total protein and albumin levels decreased by LA plus T4 treatment when compared to the LA control. The slight increase in glucose level by LA or T4 alone was not observed when they were administered together. In the second setup of experiments the administration of T4 for 22 days increased the serum T3 level 3-fold. When LA was combined with T4 and the treatment continued, the T3 production decreased by 22%. T4 reduced cholesterol level by 30%, and LA plus T4 further reduced it by 47%. The triglycerides were not affected. A moderate decrease in total protein was observed after treatment with T4 plus LA; T4 and LA plus T4 decreased the albumin level. The decrease in serum glucose by T4 recovers by LA treatment. These results demonstrate that LA interferes with the production of T3 from T4 when it is co-administered with T4. The elevated level of T3, after T4 administration, is reduced by treatment with LA. Pediatr Res. 1990 Jan. Lactic acidosis and accumulation of 3-hydroxybutyrate and other citric acid cycle intermediates were found in an infant with a lethal syndrome of metabolic acidosis and renal tubular acidosis. Nevertheless, the patient was relatively well for 4 mo of life. The activity of the pyruvate dehydrogenase complex, 2-oxoglutarate dehydrogenase, and branched-chain keto acid dehydrogenase were all reduced to levels 9 to 29% of control. In contrast, the activity of lipoamide dehydrogenase was normal. The conversion of 1-14C-leucine and 1-14C-valine to 14CO2 and of U-L-14C-valine to its major metabolic product 3-hydroxyisobutyric acid by fibroblasts derived from the patient was less than 5% of control. Cultivation of the patient's fibroblasts in medium enriched with lipoic acid markedly improved these in vitro conversions of leucine and valine. Eur J Pediatr. 1989 Apr. A 2-day-old girl developed a severe lactic acidosis with a normal lactate/pyruvate ratio and hyperammonaemia. Plasma arginine and citrulline levels were below the limit of detection. In muscle total pyruvate dehydrogenase complex (PDHC) and pyruvate decarboxylase (E1) activities were reduced to a fraction of lower control values. The acute neonatal period was bridged with peritoneal dialysis, dichloroacetate therapy, supplements of arginine and branched chain amino acids, a complete vitamin B complex and lipoic acid. Lactate homeostasis responded to pharmacological supplements of lipoic acid. At age 1 year the child was hypotonic, showed severe developmental retardation, optic atrophy and cranial dysmorphism. She died aged 1 year 8 months with signs of respiratory paralysis but with normal lactate levels under assisted breathing. Pathological findings at autopsy were suggestive of Leigh syndrome, interstitial pneumonia and extensive fatty infiltration of hepatocytes. Regression analysis of data from 187 plasma amino acid determinations from the patient over a period of 1 year 8 months revealed a persistent imbalance involving alanine, glutamic acid, glutamine, proline, citrulline and branched chain amino acids. Aspects of acute and long-term therapy in this patient and some implications of the imbalances in plasma amino acids are discussed. Biochem J. 1984 May 1. Hepatocytes obtained from starved rats were incubated in oxygenated Krebs bicarbonate buffer containing 2% defatted bovine serum albumin. DL-alpha-Lipoic (dithio-octanoic) acid (1.0 mM) caused striking reductions in hepatic glucose output in the presence of each of the following substrates: pyruvate, lactate, alanine, dihydroxyacetone, glycerol, and fructose. With lactate as substrate, 0.1-1.0 mM-lipoate caused a concentration-dependent inhibition of gluconeogenesis. With the same substrate, e.g. lactate, 0.25-2.0 mM-octanoate abolished the effect of lipoate in a dose-dependent manner. Additional experimental data are presented which support the concept that the antigluconeogenic effects of lipoic acid in liver can be attributed largely, if not entirely, to sequestration of intramitochondrial coenzyme. A, presumably as lipoyl-CoA, bisnorlipoyl-CoA, or tetranorlipoyl-CoA. |