Alpha Lipoic Acid

Reverse Oxidative Stress in the Heart with Alpha Lipoic Acid



Jung H. Suh, Eric T. Shigeno, Jason D. Morrow, Brian Cox, Alma E. Rocha, Balz Frei, and Tory M. Hagen



Linus Pauling Institute and Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, USA; and Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA



Abstract: Oxidative stress has been implicated as a causal factor in the aging process of the heart and other tissues. To determine the extent of age-related myocardial oxidative stress, oxidant production, antioxidant status, and oxidative DNA damage were measured in hearts of young (2 months) and old (28 months) male Fischer 344 rats. Cardiac myocytes isolated from old rats showed a nearly threefold increase in the rate of oxidant production compared to young rats, as measured by the rates of 2,7-dichlorofluorescin diacetate oxidation. Determination of myocardial antioxidant status revealed a significant twofold decline in the levels of ascorbic acid (P 5 0.03), but not a-tocopherol. A significant age-related increase (P 5 0.05) in steadystate levels of oxidative DNA damage was observed, as monitored by 8-oxo-2*-deoxyguanosine levels. To investigate whether dietary supplementation with (R)-a-lipoic acid (LA) was effective at reducing oxidative stress, young and old rats were fed an AIN-93M diet with or without 0.2% (w/w) LA for 2 wk before death. Cardiac myocytes from old, LA-supplemented rats exhibited a markedly lower rate of oxidant production that was no longer significantly different from that in cells from unsupplemented, young rats. Lipoic acid supplementation also restored myocardial ascorbic acid levels and reduced oxidative DNA damage. Our data indicate that the aging rat heart is under increased mitochondrial-induced oxidative stress, which is significantly attenuated by lipoic acid supplementation. Suh, J. H., Shigeno, E. T., Morrow, J. D., Cox, B., Rocha, A. E., Frei, B., Hagen, T. M. Oxidative stress in the aging rat heart is reversed by dietary supplementation with (R)-a-lipoic acid. FASEB J. 15, 700-706 (2001)



Key Words: aging z cardiac myocytes z oxidative stress z lipoic acid



Aging is associated with an increased incidence of cardiac arrhythmias and diastolic and systolic dysfunction, which may ultimately lead to heart failure. Heart failure alone is the leading cause of hospitalization, permanent disability, and death in persons over the age of 65 [1] in the U.S. Because of the enormous suffering and health care burden that cardiac dysfunction causes, much effort has gone into understanding the mechanisms leading to age-related myocardial decline. It has been difficult, however, to separate the effects of aging per se from those of age-associated diseases (atherosclerosis, diabetes, hypertension) on cardiac performance. Thus, the relative contribution of to myocardial dysfunction is not well defined.



Even though the mechanisms leading to alterations in cardiac performance are not well understood, there is reason to suspect increased oxidative stress to significantly contribute to myocardial dysfunction with age. It is generally agreed that isolated mitochondrial preparations from old compared to young hearts produce more reactive oxygen species (ROS), reflecting an age-related decline in coupling of electron transport to ATP production. These changes in mitochondria may lead to the reported increase in superoxide and hydrogen peroxide production in mitochondria prepared from old vs. young rats [2,4]. Thus, it is conceivable that dietary interventions with antioxidants, which could augment endogenous antioxidant compounds to either prevent the formation or quench the higher levels of oxidants, could provide an effective means to improve or maintain myocardial function with age.



(R)-a-lipoic acid (LA) is a thiol compound found naturally in plants and animals [4]. Lipoamide dehydrogenases, found only in mitochondria, reduce free LA to dihydrolipoic acid, which is a potent antioxidant. Thus, LA supplementation may increase cellular and mitochondrial antioxidant status, thereby effectively attenuating any putative increase in oxidative stress with age [5].



Aside from acting as a potent antioxidant in its own right, LA increases or maintains levels of other low molecular weight antioxidants such as ubiquinone, glutathione (GSH), and ascorbic acid. LA may exert these effects by sparing or reducing, ubiquinone [6], GSH, and vitamin C [7] or, in the case of GSH, by increasing the cellular uptake of cysteine [8], which is the rate-limiting substrate for GSH biosynthesis.



The purpose of the present study was to 1) examine the age-related changes to myocardial oxidant production, low molecular weight antioxidant status, and indices of oxidative damage, and 2) determine whether dietary supplementation of lipoic acid could improve those indices of oxidative stress. Overall, our results show that the aging rat myocardium exhibits increased oxidant production, significantly lower ascorbic acid levels, and a marked increase in steady-state levels of oxidative DNA damage. LA supplementation significantly reverses the age-related decline in myocardial ascorbic acid content, and lowers the rate of oxidant production and the steady-state levels of oxidative DNA damage. Our results thus indicate that dietary supplementation with lipoic acid may be an effective means tolower increased myocardial oxidative stress with age.



Results: To determine the effects of aging on the general metabolic rate, O2 consumption in freshly isolated cardiac myocytes from young and old rats was determined. O2 consumption in cells from young rats was 3.18 6 0.53 mmol O2/min per 106 cells. In contrast, O2 consumption in cardiac myocytes from old rats was 1.74 6 0.13 mmol O2/min per 106 cells, a 45% decline compared to young rats. These results suggest an age-associated decrease in the basal metabolic rate of the cardiac myocytes.



To examine how the decline in mitochondrial function affected cellular oxidant production, the rates of DCFH oxidation in freshly isolated cardiac myocytes from old and young rats were determined. In cardiac myocytes isolated from old rats, total cellular oxidant production was 125% higher than in myocytes from young rats. This increase in oxidant production was even more pronounced when normalized to the rates of oxygen consumption. Thus, the rate of DCFH fluorescence per mmol O2 consumed was threefold higher in cardiac myocytes from old rats compared to young rats. These results strongly suggest that, with age, cardiac myocytes are under increased oxidative stress, possibly due to a decline in mitochondrial function. To investigate the effects of this age-associated increase in oxidative stress on cellular antioxidant capacities, ascorbate and a-tocopherol levels in the freshly isolated hearts from old and young rats were measured. Consistent with the data on oxidant production, we observed a 57% decrease in the tissue ascorbate concentration of old rats (1.4060.87 nmol/mg protein) compared to young rats (3.0861.10 pmol/mg tissue (P50.02). In contrast, no age-related decrease in vitamin E levels was observed (0.9760.29 and 1.1660.62 nmol/mg protein in hearts from young and old). This result suggests that there is a differential consumption of water-soluble vs. lipid-soluble antioxidants in vivo due to aging.



To further explore this notion of different levels of oxidative stress with aging in the aqueous and lipid compartments, we examined oxidative damage to DNA, as assessed by the levels of 8-oxo-dG, and lipids, as assessed by levels of F2-isoprostanes. Consistent with the pattern of antioxidant depletion with age, we found a twofold increase in oxidative DNA damage in the heart of old rats (4.0064.80-oxo-dG/105 dG bases) compared to young rats (2.0160.25 8-oxo-dG/105 dG bases (P50.002). In contrast, the levels of F2-isoprostanes in hearts from old rats (3.7460.41 ng/g wet tissue) were not significantly different from those in young rats (3.4760.02 ng/g wet tissue). These results suggest that in the heart, macromolecules in an aqueous environment may be more susceptible to damage due to age-associated increase in oxidative stress than membrane lipids.



In light of the above evidence of increased oxidative stress in the aging heart, we examined whether supplementation with (R)-a-lipoic acid can reverse the decline in mitochondrial function and reduce oxidative stress. To this end, Fischer 344 rats of varying ages (young: 2 months; old: 24-28 months) were fed an AIN-93M diet with or without 0.2% (w/w) (R)-a-lipoic acid for 2wk. Results show a significant decrease in the cellular oxidant production in the old LA-supplemented vs. old unsupplemented animals. In agreement withthese data, we also found a significant twofold improvement in ascorbate levels in the hearts from old LA treated vs. untreated animals (2.9060.36 vs 1.462.9nmol/mg protein, respectively; P50.03). In fact, cardiac ascorbate levels in LA-fed old rats were not different from those in young rats (2.9060.72 nmol/mg protein). However, a similar increase in tissue ascorbate levels was not achieved by LA feeding to young rats. Furthermore, there was no effect of LA treatment on the levels of a-tocopherol.


To investigate whether the decreased ROS production and increased ascorbate levels in LA-treated old rats translated into a decrease in oxidative damage to DNA and lipids, 8-oxo-dG and F2-isoprostane levels were measured. Indeed, we found a 30% decrease in cardiac 8-oxo-dG levels in the supplemented old rats (2.8460.39 8-oxo-dG/105 dG bases) compared to their age-matched controls (4.0060.40 8-oxo-dG/105 dG bases (P50.05). In the young animals, LA supplementation did not alter the steady-state levels of 8-oxo-dG in the heart. As expected, treatment with LA had no effect on the steady-state levels of F2-isoprostanes in young or old rats. Thus, by decreasing the rate of oxidant production and increasing the level of antioxidant protection in the aqueous phase, LA treatment caused a marked reversal in steady-state levels of 8-oxo-dG in old rats to levels found in the young rats.



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