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Purpose: To determine whether (R)-alpha-lipoic acid (LA) protects cultured human fetal retinal pigment epithelial (hfRPE) cells against oxidative injury and identify the pathways that may mediate protection., Methods: Cultured hfRPE cells were pretreated with various concentrations of LA for 14 to 16 hours followed by treatment with a chemical oxidant, tert-butylhydroperoxide (t-BuOOH; 0.8 mM, 3 hours). Reactive oxygen species (ROS) production and cell viability were measured using H(2)DCF and MTT assays, respectively. RPE cells were evaluated with fluorescent dyes (SYTOX Orange and SYTO Green; Molecular Probes, Eugene, OR), which differentiate between live and dead cells. Apoptosis was visualized by using the TUNEL assay. Changes in mitochondrial membrane potential were detected by JC-1 dye. Intracellular levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured by HPLC. Regulation of gamma-glutamylcysteine ligase (GCL), the rate-controlling enzyme of GSH production, was assayed by RT-PCR., Results: Pretreatment of hfRPE cells with LA, 0.2 mM and 0.5 mM, significantly reduced the levels of t-BuOOH-induced intracellular ROS, by 23% and 49%, respectively. LA (0.5 mM) prevented oxidant-induced cell death and apoptosis and also increased the viability of oxidant-treated hfRPE cells from 38% to 90% of control. LA upregulated the mRNA expression of GCL, and was protective against t-BuOOH-induced decreases in both mitochondrial membrane potential and intracellular levels of GSH and GSH/GSSG., Conclusions: The present study suggests that the protective effect of LA involves multiple pathways and that LA could be effective against age-associated increase in oxidative stress and mitochondrial dysfunction in RPE cells.

Accumulation of divalent metal ions (e.g. iron and copper) has been proposed to contribute to heightened oxidative stress evident in aging and neurodegenerative disorders. To understand the extent of iron accumulation and its effect on antioxidant status, we monitored iron content in the cerebral cortex of F344 rats by inductively coupled plasma atomic emission spectrometry (ICP-AES) and found that the cerebral iron levels in 24-28-month-old rats were increased by 80% (p<0.01) relative to 3-month-old rats. Iron accumulation correlated with a decline in glutathione (GSH) and the GSH/GSSG ratio, indicating that iron accumulation altered antioxidant capacity and thiol redox state in aged animals. Because (R)-alpha-Lipoic acid (LA) is a potent chelator of divalent metal ions in vitro and also regenerates other antioxidants, we monitored whether feeding LA (0.2% [w/w]; 2 weeks) could lower cortical iron and improve antioxidant status. Results show that cerebral iron levels in old LA-fed animals were lower when compared to controls and were similar to levels seen in young rats. Antioxidant status and thiol redox state also improved markedly in old LA-fed rats versus controls. These results thus show that LA supplementation may be a means to modulate the age-related accumulation of cortical iron content, thereby lowering oxidative stress associated with aging.

Graft-versus-host disease (GVHD) is a common complication of allogeneic bone marrow transplantation (BMT). Upregulation of inflammatory cytokines precedes the clinical presentation of GVHD and predicts its severity. In this report, thiol/redox metabolomics was used to identify metabolic perturbations associated with early preclinical (Day+4) and clinical (Day+10) stages of GVHD by comparing effects in Syngeneic (Syn; major histocompatibility complex- identical) and allogeneic transplant recipients (Allo BMT) in experimental models. While most metabolic changes were similar in both groups, plasma glutathione (GSH) was significantly decreased, and GSH disulfide (GSSG) was increased after allogeneic compared to syngeneic recipient and non-transplant controls. The early oxidation of the plasma GSH/GSSG redox couple was also observed irrespective of radiation conditioning treatment and was accompanied by significant rise in hepatic protein oxidative damage and ROS generation. Despite a significant rise in oxidative stress, compensatory increase in hepatic GSH synthesis was absent following Allo BMT. Early shifts in hepatic oxidative stress and plasma GSH loss preceded a statistically significant rise in TNF-α. To identify metabolomic biomarkers of hepatic GVHD injury, plasma metabolite concentrations analyzed at Day+10 were correlated with hepatic organ injury. GSSG (oxidized GSH) and β-alanine, were positively correlated, and plasma GSH cysteinylglycine, and branched chain amino acids were inversely correlated with hepatic injury. Although changes in plasma concentrations of cysteine, cystathionine (GSH precursors) and cysteinylglycine (a GSH catabolite) were not significant by univariate analysis, principal component analysis (PCA) indicated that accumulation of these metabolites after Allo BMT contributed significantly to early GVHD in contrast to Syn BMT. In conclusion, thiol/redox metabolomic profiling implicates that early dysregulation of host hepatic GSH metabolism and oxidative stress in sub-clinical GVHD before elevated TNF-α levels is associated with GVHD pathogenesis. Future studies will probe the mechanisms for these changes and examine the potential of antioxidant intervention strategies to modulate GVHD.

Dietary intake modulates disease risk, but little is known how components within food mixtures affect pathophysiology. A low-calorie, high-fiber, fruit-based nutrient-dense bar of defined composition (e.g., vitamins and minerals, fruit polyphenolics, β-glucan, do-cosahexaenoic acid) appropriate for deconstruction and mechanistic studies is described and evaluated in a pilot trial. The bar was developed in collaboration with the U.S. Department of Agriculture. Changes in cardiovascular disease and diabetes risk biomarkers were measured after 2 wk twice-daily consumption of the bar, and compared against baseline controls in 25 healthy adults. Plasma HDLrcholesterol (HDL-c) increased 6.2% (P=0.001), due primarily to a 28% increase in large HDL (HDL-L; P<0.0001). Total plasma homocysteine (Hey) decreased 19% (P=0.017), and glutathione (GSH) increased 20% (P=0.011). The changes in HDL and Hey are in the direction associated with decreased risk of cardiovascular disease and cognitive decline; increased GSH reflects improved antioxidant defense. Changes in biomarkers linked to insulin resistance and inflammation were not observed. A defined food-based supplement can, within 2 wk, positively impact metabolic biomarkers linked to disease risk. These results lay the groundwork for mech-anistic/deconstruction experiments to identify critical bar components and putative synergistic combinations responsible for observed effects. [ABSTRACT FROM AUTHOR]

Accumulation of divalent metal ions (e.g. iron and copper) has been proposed to contribute to heightened oxidative stress evident in aging and neurodegenerative disorders. To understand the extent of iron accumulation and its effect on antioxidant status, we monitored iron content in the cerebral cortex of F344 rats by inductively coupled plasma atomic emission spectrometry (ICP-AES) and found that the cerebral iron levels in 24–28-month-old rats were increased by 80% (p<0.01) relative to 3-month-old rats. Iron accumulation correlated with a decline in glutathione (GSH) and the GSH/GSSG ratio, indicating that iron accumulation altered antioxidant capacity and thiol redox state in aged animals. Because (R)-α-Lipoic acid (LA) is a potent chelator of divalent metal ions in vitro and also regenerates other antioxidants, we monitored whether feeding LA (0.2% [w/w]; 2 weeks) could lower cortical iron and improve antioxidant status. Results show that cerebral iron levels in old LA-fed animals were lower when compared to controls and were similar to levels seen in young rats. Antioxidant status and thiol redox state also improved markedly in old LA-fed rats versus controls. These results thus show that LA supplementation may be a means to modulate the age-related accumulation of cortical iron content, thereby lowering oxidative stress associated with aging. [ABSTRACT FROM AUTHOR]

Age-related depletion of GSH levels and perturbations in its redox state may be especially deleterious to metabolically active tissues, such as the heart and brain. We examined the extent and the mechanisms underlying the potential age-related changes in cerebral and myocardial GSH status in young and old F344 rats and whether administration of (R)-α-lipoic acid (LA) can reverse these changes. Our results show that GSH/GSSG ratios in the aging heart and the brain declined by 58 and 66% relative to young controls, respectively (p<0.001). Despite a consistent loss in GSH redox status in both tissues, only cerebral GSH levels declined with age (p<0.01). To discern the potential mechanisms underlying this differential loss, the levels and the activities of γ-glutamylcysteine ligase (GCL) and cysteine availability were determined. There were no significant age-related changes in substrate or enzyme levels, or GCL activity when saturating amounts of substrates were provided. However, kinetic analysis of GCL in brains of old rats displayed a significant increase (p<0.05) in the apparent Km for cysteine (Km cys) vs. young rats (84.3 ± 25.4 vs. 179.0 ± 49.0; young and old, respectively), resulting in a 40% loss in apparent catalytic turnover of the enzyme. Thus, the age-related decline in total GSH appears to be mediated, in part, by a general decrement in GCL catalytic efficiency. Treating old rats with LA (40 mg/kg body wt; by i.p.) markedly increased tissue cysteine levels by 54% 12 h following treatment and subsequently restored the cerebral GSH levels. Moreover, LA improved the age-related changes in the tissue GSH/GSSG ratios in both heart and the brain. These results demonstrate that LA is an effective agent to restore both the age-associated decline in thiol redox ratio as well as increase cerebral GSH levels that otherwise decline with age. [Copyright &y& Elsevier]

Cardiac mitochondria are composed of two distinct subpopulations: one beneath the sarcolemma (subsarcolemmal mitochondria: SSM), and another along the myofilaments (interfibrillary mitochondria: IFM). Previous studies suggest a preferential loss of IFM function with age; however, the age-related changes in oxidative stress in these mitochondrial subpopulations have not been examined. To this end, the changes in mitochondrial antioxidant capacity, oxidant output, and oxidative damage to Complex IV in IFM and SSM from young and old rats were studied. Results show no apparent differences in any parameters examined between IFM and SSM from young rats. However, relative to young, only IFM from old rats had a significantly higher rate of oxidant production and a decline in mitochondrial ascorbate levels and GSH redox status. The age-related decline in mitochondrial antioxidant capacity in IFM was accompanied by a marked loss in glutaredoxin and GSSG reductase activities, suggesting a diminished reductive capacity in IFM with age. Moreover, the loss in Complex IV activity was limited to the IFM of old rats, which was accompanied by a 4-fold increase in 4-hydroxynonenal-modified Complex IV. Thus, mitochondrial decay is not uniform and further indicates that myofibrils may be uniquely under oxidative stress in the aging heart. [Copyright &y& Elsevier]