Lipoic acid significantly restores, in rats, the age-related decline in vasomotion.

BACKGROUND AND PURPOSE: The age-related decline in vasorelaxation is largely due to ceramide-induced induction of phosphatase 2A (PP2A), which limits nitric oxide synthase (eNOS) phosphorylation at stimulatory sites. We hypothesized that ceramide accumulation was from an age-related loss of endothelial glutathione (GSH) and subsequent activation of neutral sphingomyelinase (nSMase), an enzyme whose activity increases when GSH is limited. EXPERIMENTAL APPROACH: Old (30-32 mo) F344xBN rats were given (R)-alpha-lipoic acid (LA), an agent known to induce GSH synthesis. Vasorelaxation was measured in aortic rings; GSH and ceramide levels, activity of nSMase and eNOS phosphorylation (by Western blot) was measured in aortic endothelial cells, isolated from the same aortas. KEY RESULTS: In old animals, endothelium-dependent relaxation in aortic rings was decreased, GSH levels and its redox state in aortic endothelia were over 30% lower and nSMase activity and endothelial ceramide levels were three-fold increased, relative to young (2-4 mo) rats. LA treatment of old animals improved relaxation in aortic rings, reversed the changes in endothelial GSH, in nSMase activities and in ceramide levels. Similar effects on GSH levels and nSMase activity in old rats were also induced by treatment with GSH monoethylester. Activation (by phosphorylation) of eNOS was decreased by about 50% in old rats and this age-related decrease was partially reversed by LA treatment. CONCLUSIONS AND IMPLICATIONS: Decreased endothelial GSH was partly responsible for the age-related loss of vascular endothelial function and LA might be therapeutically evaluated to treat endothelial dysfunction.

Acetyl-L-carnitine and alpha-lipoic acid supplementation of aged beagle dogs improves learning in two landmark discrimination tests.

Beagle dogs between 7.6 and 8.8 years of age administered a twice daily supplement of alpha-lipoic acid (LA) and acetyl-L-carnitine (ALC) over approximately 2 months made significantly fewer errors in reaching the learning criterion on two landmark discrimination tasks compared to controls administered a methylcellulose placebo. Testing started after a 5 day wash-in. The dogs were also tested on a variable delay version of a previously acquired spatial memory task; results were not significant. The improved performance on the landmark task of dogs supplemented with LA + ALC provides evidence of the effectiveness of this supplement in improving discrimination and allocentric spatial learning. We suggest that long-term maintenance on LA and ALC may be effective in attenuating age-associated cognitive decline by slowing the rate of mitochondrial decay and cellular aging.

Mitochondrial decay in aging.

Several mitochondrial functions decline with age. The contributing factors include, the intrinsic rate of proton leakage across the inner mitochondrial membrane (a correlate of oxidant formation), decreased membrane fluidity, and decreased levels and function of cardiolipin, which supports the function of many of the proteins of the inner mitochondrial membrane. Oxidants generated by mitochondria appear to be the major source of the oxidative lesions that accumulate with age. Evidence supports the suggestion that age-associated accumulation of mitochondrial deficits due to oxidative damage is likely to be a major contributor to cellular, tissue, and organismal aging.

(R)-alpha-lipoic acid reverses the age-associated increase in susceptibility of hepatocytes to tert-butylhydroperoxide both in vitro and in vivo.

Hepatocytes were isolated from young (3-5 months) and old (24-28 months) rats and incubated with various concentrations of tert-butylhydroperoxide (t-BuOOH). The t-BuOOH concentration that killed 50% of cells (LC50) in 2 hr declined nearly two-fold from 721 +/- 32 microM in cells from young rats to 391 +/- 31 microM in cells from old rats. This increased sensitivity of hepatocytes from old rats may be due, in part, to changes in glutathione (GSH) levels, because total cellular and mitochondrial GSH were 37.7% and 58.3% lower, respectively, compared to cells from young rats. Cells from old animals were incubated with either (R)- or (S)-lipoic acid (100 microM) for 30 min prior to the addition of 300 microM t-BuOOH. The physiologically relevant (R)-form, a coenzyme in mitochondria, as opposed to the (S)-form significantly protected hepatocytes against t-BuOOH toxicity. Dietary supplementation of (R)-lipoic acid [0.5% (wt/wt)] for 2 weeks also completely reversed the age-related decline in hepatocellular GSH levels and the increased vulnerability to t-BuOOH as well. An identical supplemental diet fed to young rats did not enhance the resistance to t-BuOOH, indicating that antioxidant protection was already optimal in young rats. Thus, this study shows that cells from old animals are more susceptible to oxidant insult and (R)-lipoic acid, after reduction to an antioxidant in the mitochondria, effectively reverses this age-related increase in oxidant vulnerability.

Lipoic acid as a potential therapy for chronic diseases associated with oxidative stress.

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.

Protection of retinal pigment epithelium from oxidative injury by glutathione and precursors.

PURPOSE: This study was performed to examine the effect of exogenous glutathione (GSH) or its precursor amino acids on oxidative injury in cultured human retinal pigment epithelium (RPE). METHODS: Cultured human RPE cells were suspended in Krebs-Henseleit medium, and 150 microM t-butylhydroperoxide was added. Cell viability was assessed by 0.2% trypan blue exclusion 30, 60, and 120 minutes after the addition of GSH or its amino acid precursors. RESULTS: Added GSH provided protection at concentrations of 0.01 mM and higher. The amino acid precursors for GSH, glutamate, cysteine, and glycine also protected against injury, but this required at least 0.1 mM of each amino acid. Inhibition of intracellular GSH synthesis by inclusion of 1 mM buthionine sulfoximine eliminated the protection by added amino acids but did not alter the protection by added GSH. CONCLUSIONS: These results indicate that protection by the amino acid precursors is mediated through synthesis of GSH, and they also show that exogenous GSH can provide protection against oxidative injury.

Protection against paraquat-induced injury by exogenous GSH in pulmonary alveolar type II cells.

Exogenous GSH provided rat alveolar type II cells with significant protection against injury induced by paraquat. This protection was also observed in cells treated with acivicin to inhibit GSH degradation and buthionine sulfoximine to inhibit GSH synthesis. Exogenous GSH was transported into cells by a Na+-dependent system. Addition of inhibitors of this transport system, gamma-glutamyl-glutamate and probenecid, prevented the protection against injury afforded by GSH. Thus, the results indicate that alveolar type II cells can supplement endogenous synthesis of GSH with uptake of exogenous GSH to protect against paraquat-induced injury.

Mitochondrial decay in aging. Reversal through supplementation of acetyl-L-carnitine and N-tert-butyl-alpha-phenyl-nitrone.

We show that mitochondrial function in the majority of hepatocytes isolated from old rats (24 mo) is significantly impaired. Mitochondrial membrane potential, cardiolipin levels, respiratory control ratio, and overall cellular O2 consumption decline, and the level of oxidants increases. To examine whether dietary supplementation of micronutrients that may have become essential with age could reverse the decline in mitochondrial function, we supplemented the diet of old rats with 1% (w/v) acetyl-L-carnitine (ALCAR) in drinking water. ALCAR supplementation (1 month) resulted in significant increases in cellular respiration, mitochondrial membrane potential, and cardiolipin values. However, supplementation also increased the rate of oxidant production, indicating that the efficiency of mitochondrial electron transport had not improved. To counteract the potential increase in oxidative stress, animals were administered N-tert-butyl-alpha-phenyl-nitrone (30 mg/kg) (PBN) with or without ALCAR. Results showed that PBN significantly lowered oxidant production as measured by 2,7'-dichlorofluorescin diacetate (DCFH), even when ALCAR was coadministered to the animals. Thus, dietary supplementation with ALCAR, particularly in combination with PBN, improves mitochondrial function without a significant increase in oxidative stress.

Transepithelial transport of glutathione in vascularly perfused small intestine of rat.

We used an in situ closed-loop vascular perfusion of rat small intestine to see whether intact glutathione (GSH) was transported from the lumen into the mesenteric circulation. Results using [3H] GSH showed radiolabel appearance in the perfusate, and high-performance liquid chromatography analysis confirmed that the transported radiolabeled species was intact GSH. Transepithelial transport was Na+ dependent, where Na+-free solutions on the luminal side inhibited transport but in the vasculature stimulated transport. Transport was inhibited by the GSH analogues ophthalmic acid and gamma-glutamylglutamate when added to the lumen. Probenecid caused inhibition only when added on the vascular side, suggesting that the transport systems on the two poles of the cells may be different. In vivo studies with GSH administration via stomach gavage caused marked increases in plasma GSH concentrations. These results establish that transepithelial transport of intact GSH occurs in rat small intestine. This process may allow dietary or biliary derived GSH to be utilized by various cell types that can take up intact GSH from the plasma. Moreover, the results show that oral administration of GSH may be therapeutically useful.