We show that mitochondrial function in the majority of hepatocytes isolated from old rats (24 mo)... more 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 O 2 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-=-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.
Mitochondrial decay has been postulated to be a significant underlying part of the aging process.... more Mitochondrial decay has been postulated to be a significant underlying part of the aging process. Decline in mitochondrial function may lead to cellular energy deficits, especially in times of greater energy demand, and compromise vital ATP-dependent cellular operations, including detoxification, repair systems, DNA replication, and osmotic balance. Mitochondrial decay may also lead to enhanced oxidant production and thus render the cell more prone to oxidative insult. In particular, the heart may be especially susceptible to mitochondrial dysfunction due to myocardial dependency on b-oxidation of fatty acids for energy and the postmitotic nature of cardiac myocytes, which would allow for greater accumulation of mitochondrial mutations and deletions. Thus, maintenance of mitochondrial function may be important to maintain overall myocardial function. Herein, we review the major age-related changes that occur to mitochondria in the aging heart and the evidence that two such supplements, acetyl-L-carnitine (ALCAR) and (R)-a-lipoic acid, may improve myocardial bioenergetics and lower the increased oxidative stress associated with aging. We and others have shown that feeding old rats ALCAR reverses the age-related decline in carnitine levels and improves mitochondrial boxidation in a number of tissues studied. However, ALCAR supplementation does not appear to reverse the age-related decline in cardiac antioxidant status and thus may not substantially alter indices of oxidative stress. Lipoic acid, a potent thiol antioxidant and mitochondrial metabolite, appears to increase low molecular weight antioxidant status and thereby decreases age-associated oxidative insult. Thus, ALCAR along with lipoic acid may be effective supplemental regimens to maintain myocardial function.
This study was performed to examine the effect of exogenous glutathione (GSH) or its precursor am... more 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). 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. 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. 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.
Studies were performed in rats that had been fasted 24 h, fed a glutathione (GSH)-free semisynthe... more Studies were performed in rats that had been fasted 24 h, fed a glutathione (GSH)-free semisynthetic diet (AIN-76), and fed the same diet supplemented with GSH. The results from the fasted rats and those fed GSH-free diet showed that the duodenum and jejunum contained 0.2-0.5 mumol of GSH/gram wet wt of luminal contents. The GSH contents of biliary juice was sufficient to maintain this amount of GSH in the intestinal lumen. Other analyses showed that cell sloughing, bacterial GSH content, and GSH secretion by epithelial cells of the jejunum were not sufficient to account for this content. GSH concentrations following consumption of a GSH-supplemented diet (5-50 mg/g AIN-76) showed a rapid increase in all regions of the small intestine and indicated that removal occurred primarily in the jejunum. However, the combined activities of brush-border gamma-glutamyltransferase and GSH uptake systems were not sufficient to remove all of the ingested GSH. Results from in situ vascular perfusi...
Plasma glutathione (GSH) concentration in rats increased from approximately 15 to 30 microM after... more Plasma glutathione (GSH) concentration in rats increased from approximately 15 to 30 microM after administration of GSH either as a liquid bolus (30 mumol) or mixed (2.5-50 mg/g) in AIN-76 semisynthetic diet. GSH concentration was maximal at 90-120 min after GSH administration and remained high for over 3 h. Administration of the amino acid precursors of GSH had little or no effect on plasma GSH values, indicating that GSH catabolism and resynthesis do not account for the increased GSH concentration seen. Inhibition of GSH synthesis and degradation by L-buthionine-[S,R]-sulfoximine and acivicin showed that the increased plasma GSH came mostly from absorption of intact GSH instead of from its metabolism. Plasma protein-bound GSH also increased after GSH administration, with a time course similar to that observed for free plasma GSH. Thus dietary GSH can be absorbed intact and results in a substantial increase in blood plasma GSH. This indicates that oral supplementation may be useful...
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1998
Ascorbic acid recycling from dehydroascorbic acid and biosynthesis from gulono-1,4-lactone were u... more Ascorbic acid recycling from dehydroascorbic acid and biosynthesis from gulono-1,4-lactone were used as measures of cellular response capacity to increased oxidative stress induced by tert-butylhydroperoxide. The hepatic ascorbic acid concentration was 54% lower in cells from old rats when compared to cells isolated from young rats (P<0.0005). Freshly isolated hepatocytes from old rats exhibited a significantly decreased ascorbic acid recycling capacity in response to oxidative stress (P<0.005) compared to cells from young rats. Ascorbic acid synthesis in these cells from old animals was unaffected by various concentrations of tert-butylhydroperoxide, but amounted to only approximately half of the biosynthetic rate when compared to cells from young animals (P<0.001). Cells from young animals were not significantly affected by the tert-butylhydroperoxide treatments. The results demonstrate a declining ability with age to respond to increased oxidative stress. (R)-alpha-Lipoi...
Proceedings of the National Academy of Sciences of the United States of America, Jan 6, 2007
The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was recently shown ... more The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was recently shown to negatively regulate LPS-induced acute inflammatory responses. We previously observed that the metabolic thiol antioxidant alpha-lipoic acid (LA) inhibits LPS-induced expression of cellular adhesion molecules and adherence of monocytes to human aortic endothelial cells. Here we investigated the mechanism by which LA attenuates LPS-induced monocyte activation in vitro and acute inflammatory responses in vivo. Incubation of human monocytic THP-1 cells with LA induced phosphorylation of Akt in a time- and dose-dependent manner. In cells pretreated with LA followed by LPS, Akt phosphorylation was elevated initially and further increased during incubation with LPS. This LA-dependent increase in Akt phosphorylation was accompanied by inhibition of LPS-induced NF-kappaB DNA binding activity and up-regulation of TNFalpha and monocyte chemoattractant protein 1. Lipoic acid-dependent Akt phospho...
Oxidative mitochondrial decay is a major contributor to aging and neural degeneration. In old rat... more Oxidative mitochondrial decay is a major contributor to aging and neural degeneration. In old rats (vs. young rats) mitochondrial membrane potential, cardiolipin level, respiratory control ratio, and cellular O 2 uptake are lower; oxidants/O 2 , neuron RNA oxidation, and mutagenic aldehydes from lipid peroxidation are higher. Ambulatory activity and cognition declines with age. Feeding old rats for a few weeks with acetyl-L-carnitine (ALCAR), a mitochondrial metabolite, plus R-lipoic acid (LA), a mitochondrial coenzyme and antioxidant, restores mitochondrial function; lowers oxidants, neuronal RNA oxidation, and mutagenic aldehydes; and increases rat ambulatory activity and cognition (Skinner box/Morris water maze). The mechanism appears to be that with age increased oxidative damage to protein causes a deformation of structure of key enzymes, with a consequent lessening of affinity (K m) for the substrate. The loss with age of carnitine acetyl transferase binding affinity can be mimicked by reacting it with malondialdehyde (a lipid peroxidation product which increases with age). Feeding the substrate ALCAR with LA restores the velocity of the reaction, K m for ALCAR and CoA, and mitochondrial function. Heme biosynthesis is predominantly in the mitochondria. Interfering with heme synthesis causes specific loss of complex IV with consequent release of oxidants and neuronal degeneration. Iron deficiency (25% of menstruating women in the US ingest , 50% of the RDA) also causes release of oxidants and mitochondrial decay, presumably through lack of heme, accelerating brain aging. Vitamin B6 or zinc inadequacy (10% of Americans ingest , 50% of each RDA) should also cause a heme deficiency.
Because standard culture media for human aortic endothelial cells (HAEC) do not contain vitamin C... more Because standard culture media for human aortic endothelial cells (HAEC) do not contain vitamin C, we hypothesized that HAEC may be under significant oxidative insult compared with the situation in vivo. To assess parameters of oxidative stress, intracellular vitamin C, glutathione (GSH), GSH/GSSG, and NAD(P)H/NAD(P)+ ratios, as well as oxidant appearance and oxidative damage, were measured in HAEC with or without vitamin C addition. The effect of vitamin C on eNOS activity was also determined. Results showed that HAEC without vitamin C treatment were essentially scorbutic. On addition of 100 mM vitamin C to the culture media, intracellular vitamin C levels increased and peaked at 6 h. A concomitant increase in the total GSH and the GSH/GSSG ratio was also observed; the NAD(P)H/NAD(P)+ ratio increased more slowly over the 24-h time course. Significantly lower (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05) oxidant appearance and steady-state oxidative damage were also observed following vitamin C repletion. Vitamin C treatment increased eNOS activity by 600%. Thus, HAEC are scorbutic under normal culture conditions and exhibit higher oxidative stress than vitamin C repleted cells. Vitamin C supplementation should be considered when using cultured cells, especially when experimental endpoints are related to cellular redox status and eNOS activity.
Beagle dogs between 7.6 and 8.8 years of age administered a twice daily supplement of ␣-lipoic ac... more Beagle dogs between 7.6 and 8.8 years of age administered a twice daily supplement of ␣-lipoic acid (LA) and acetyl-L-carnitine (ALC) over ϳ2 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 ageassociated cognitive decline by slowing the rate of mitochondrial decay and cellular aging.
a-Lipoic acid (LA) and its reduced form, dihydrolipoic acid (DHLA), have been suggested to chelat... more a-Lipoic acid (LA) and its reduced form, dihydrolipoic acid (DHLA), have been suggested to chelate transition metal ions and, hence, mitigate iron-and copper-mediated oxidative stress in biological systems. However, it remains unclear whether LA and DHLA chelate transition metal ions in a redox-inactive form, and whether they remove metal ions from the active site of enzymes. Therefore, we investigated the effects of LA and DHLA on iron-or copper-catalyzed oxidation of ascorbate, a sensitive assay for the redox activity of these metal ions. We found that DHLA, but not LA, significantly inhibited ascorbate oxidation mediated by Fe(III)-citrate, suggesting that reduced thiols are required for iron binding. DHLA also strongly inhibited Cu(II)(histidine) 2-mediated ascorbate oxidation in a concentration-dependent manner, with complete inhibition at a DHLA:Cu(II) molar ratio of 3:1. In contrast, no inhibition of copper-catalyzed ascorbate oxidation was observed with LA. To investigate whether LA and DHLA remove copper or iron from the active site of enzymes, Cu,Zn superoxide dismutase and the iron-containing enzyme aconitase were used. We found that neither LA nor DHLA, even at high, millimolar concentrations, altered the activity of these enzymes. Our results suggest that DHLA chelates and inactivates redox-active transition metal ions in small-molecular, biological complexes without affecting iron-or copperdependent enzyme activities.
Accumulation of divalent metal ions (e.g. iron and copper) has been proposed to contribute to hei... more 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.
Proceedings of the National Academy of Sciences, 1994
We argue for the critical role of oxidative damage in causing the mitochondrial dysfunction of ag... more We argue for the critical role of oxidative damage in causing the mitochondrial dysfunction of aging. Oxidants generated by mitochondria appear to be the major source of the oxidative lesions that accumulate with age. 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. Acetyl-L-carnitine, a high-energy mitochondrial substrate, appears to reverse many age-associated deficits in cellular function, in part by increasing cellular ATP production. Such 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.
Proceedings of the National Academy of Sciences, 1994
A transgenic mouse strain that expresses the hepatitis B virus (HBV) large envelope protein in th... more A transgenic mouse strain that expresses the hepatitis B virus (HBV) large envelope protein in the liver was used to determine the extent of oxidative DNA damage that occurs during chronic HBV infection. This mouse strain develops a chronic necroinflammatory liver disease that mimics the inflammation, cellular hyperplasia, and increased risk for cancer that is evident in human chronic active hepatitis. When perfused in situ with nitroblue tetrazolium, an indicator for superoxide formation, the liver of transgenic mice displayed intense formazan deposition in Kupffer cells, indicating oxygen radical production, and S-phase hepatocytes were commonly seen adjacent to the stained Kupffer cells. Similar changes were not observed in nontransgenic control livers. To determine whether these events were associated with oxidative DNA damage, genomic DNA from the livers of transgenic mice and nontransgenic controls was isolated and examined for 8-oxo-2'-deoxyguanosine, an oxidatively modif...
Proceedings of the National Academy of Sciences, 2007
The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was recently shown ... more The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was recently shown to negatively regulate LPS-induced acute inflammatory responses. We previously observed that the metabolic thiol antioxidant α-lipoic acid (LA) inhibits LPS-induced expression of cellular adhesion molecules and adherence of monocytes to human aortic endothelial cells. Here we investigated the mechanism by which LA attenuates LPS-induced monocyte activation in vitro and acute inflammatory responses in vivo . Incubation of human monocytic THP-1 cells with LA induced phosphorylation of Akt in a time- and dose-dependent manner. In cells pretreated with LA followed by LPS, Akt phosphorylation was elevated initially and further increased during incubation with LPS. This LA-dependent increase in Akt phosphorylation was accompanied by inhibition of LPS-induced NF-κB DNA binding activity and up-regulation of TNFα and monocyte chemoattractant protein 1. Lipoic acid-dependent Akt phosphorylation an...
The use of antioxidants is now often used as a pharmacological adjunct to limit infertility. Inde... more The use of antioxidants is now often used as a pharmacological adjunct to limit infertility. Indeed, the lay public rightly perceives oxidative stress and, thus, antioxidant treatment as important modulators of infertility. While the direct effects of antioxidant treatment on the quality of semen and oocytes are still under investigation, a significant body of evidence points to loss of vascular tone as a root-cause of erectile dysfunction and, possibly, alterations to female reproduction. In this article, we will critically review the often neglected link between vascular dysfunction and infertility. A particular emphasis will be on the potential use of antioxidants to increase fertility and promote conception.
Diet, along with genetic and environmental factors, is considered a major aspect affecting longev... more Diet, along with genetic and environmental factors, is considered a major aspect affecting longevity as well as vascular disease outcome. Yet, inadequate nutritional intake is rampant among the elderly, affecting nearly 44% of otherwise healthy, community-dwellers in developed countries. Thus, malnutrition, both in quali-and quantitative terms and especially as related to micronutrient intake, may exacerbate intrinsic cardiovascular maladaptation associated with aging, affecting vascular disease outcomes as well as longevity. Conversely, there is accumulating evidence that diets enriched in micronutrients, including vitamins, polyphenols, and essential fatty acids, maintain cellular antioxidant status and stress response enzymes, which otherwise decrease with age. Thus, adequate intakes of micronutrients, either consequent to a correct diet or through supplementation, might afford the elderly protection from cardiovascular diseases. In this article we review the known effects of micronutrients on the aging heart and we propose strategies for dietary improvements.
We assayed levels of lipid peroxidation, protein carbonyl formation, glutamine synthetase (GS) ac... more We assayed levels of lipid peroxidation, protein carbonyl formation, glutamine synthetase (GS) activity and both oxidized and reduced glutathione to study the link between oxidative damage, aging and b-amyloid (Ab) in the canine brain. The aged canine brain, a model of human brain aging, naturally develops extensive diffuse deposits of human-type Ab. Ab was measured in immunostained prefrontal cortex from 19 beagle dogs (4-15 years). Increased malondialdehyde (MDA), which indicates increased lipid peroxidation, was observed in the prefrontal cortex and serum but not in cerebrospinal fluid (CSF). Oxidative damage to proteins (carbonyl formation) also increased in brain. An age-dependent decline in GS activity, an enzyme vulnerable to oxidative damage, and in the level of glutathione (GSH) was observed in the prefrontal cortex. MDA level in serum correlated with MDA accumulation in the prefrontal cortex. Although 11/19 animals exhibited Ab, the extent of deposition did not correlate with any of the oxidative damage measures, suggesting that each form of neuropathology accumulates in parallel with age. This evidence of widespread oxidative damage and Ab deposition is further justification for using the canine model for studying human brain aging and neurodegenerative diseases.
We show that mitochondrial function in the majority of hepatocytes isolated from old rats (24 mo)... more 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 O 2 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-=-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.
Mitochondrial decay has been postulated to be a significant underlying part of the aging process.... more Mitochondrial decay has been postulated to be a significant underlying part of the aging process. Decline in mitochondrial function may lead to cellular energy deficits, especially in times of greater energy demand, and compromise vital ATP-dependent cellular operations, including detoxification, repair systems, DNA replication, and osmotic balance. Mitochondrial decay may also lead to enhanced oxidant production and thus render the cell more prone to oxidative insult. In particular, the heart may be especially susceptible to mitochondrial dysfunction due to myocardial dependency on b-oxidation of fatty acids for energy and the postmitotic nature of cardiac myocytes, which would allow for greater accumulation of mitochondrial mutations and deletions. Thus, maintenance of mitochondrial function may be important to maintain overall myocardial function. Herein, we review the major age-related changes that occur to mitochondria in the aging heart and the evidence that two such supplements, acetyl-L-carnitine (ALCAR) and (R)-a-lipoic acid, may improve myocardial bioenergetics and lower the increased oxidative stress associated with aging. We and others have shown that feeding old rats ALCAR reverses the age-related decline in carnitine levels and improves mitochondrial boxidation in a number of tissues studied. However, ALCAR supplementation does not appear to reverse the age-related decline in cardiac antioxidant status and thus may not substantially alter indices of oxidative stress. Lipoic acid, a potent thiol antioxidant and mitochondrial metabolite, appears to increase low molecular weight antioxidant status and thereby decreases age-associated oxidative insult. Thus, ALCAR along with lipoic acid may be effective supplemental regimens to maintain myocardial function.
This study was performed to examine the effect of exogenous glutathione (GSH) or its precursor am... more 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). 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. 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. 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.
Studies were performed in rats that had been fasted 24 h, fed a glutathione (GSH)-free semisynthe... more Studies were performed in rats that had been fasted 24 h, fed a glutathione (GSH)-free semisynthetic diet (AIN-76), and fed the same diet supplemented with GSH. The results from the fasted rats and those fed GSH-free diet showed that the duodenum and jejunum contained 0.2-0.5 mumol of GSH/gram wet wt of luminal contents. The GSH contents of biliary juice was sufficient to maintain this amount of GSH in the intestinal lumen. Other analyses showed that cell sloughing, bacterial GSH content, and GSH secretion by epithelial cells of the jejunum were not sufficient to account for this content. GSH concentrations following consumption of a GSH-supplemented diet (5-50 mg/g AIN-76) showed a rapid increase in all regions of the small intestine and indicated that removal occurred primarily in the jejunum. However, the combined activities of brush-border gamma-glutamyltransferase and GSH uptake systems were not sufficient to remove all of the ingested GSH. Results from in situ vascular perfusi...
Plasma glutathione (GSH) concentration in rats increased from approximately 15 to 30 microM after... more Plasma glutathione (GSH) concentration in rats increased from approximately 15 to 30 microM after administration of GSH either as a liquid bolus (30 mumol) or mixed (2.5-50 mg/g) in AIN-76 semisynthetic diet. GSH concentration was maximal at 90-120 min after GSH administration and remained high for over 3 h. Administration of the amino acid precursors of GSH had little or no effect on plasma GSH values, indicating that GSH catabolism and resynthesis do not account for the increased GSH concentration seen. Inhibition of GSH synthesis and degradation by L-buthionine-[S,R]-sulfoximine and acivicin showed that the increased plasma GSH came mostly from absorption of intact GSH instead of from its metabolism. Plasma protein-bound GSH also increased after GSH administration, with a time course similar to that observed for free plasma GSH. Thus dietary GSH can be absorbed intact and results in a substantial increase in blood plasma GSH. This indicates that oral supplementation may be useful...
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1998
Ascorbic acid recycling from dehydroascorbic acid and biosynthesis from gulono-1,4-lactone were u... more Ascorbic acid recycling from dehydroascorbic acid and biosynthesis from gulono-1,4-lactone were used as measures of cellular response capacity to increased oxidative stress induced by tert-butylhydroperoxide. The hepatic ascorbic acid concentration was 54% lower in cells from old rats when compared to cells isolated from young rats (P<0.0005). Freshly isolated hepatocytes from old rats exhibited a significantly decreased ascorbic acid recycling capacity in response to oxidative stress (P<0.005) compared to cells from young rats. Ascorbic acid synthesis in these cells from old animals was unaffected by various concentrations of tert-butylhydroperoxide, but amounted to only approximately half of the biosynthetic rate when compared to cells from young animals (P<0.001). Cells from young animals were not significantly affected by the tert-butylhydroperoxide treatments. The results demonstrate a declining ability with age to respond to increased oxidative stress. (R)-alpha-Lipoi...
Proceedings of the National Academy of Sciences of the United States of America, Jan 6, 2007
The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was recently shown ... more The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was recently shown to negatively regulate LPS-induced acute inflammatory responses. We previously observed that the metabolic thiol antioxidant alpha-lipoic acid (LA) inhibits LPS-induced expression of cellular adhesion molecules and adherence of monocytes to human aortic endothelial cells. Here we investigated the mechanism by which LA attenuates LPS-induced monocyte activation in vitro and acute inflammatory responses in vivo. Incubation of human monocytic THP-1 cells with LA induced phosphorylation of Akt in a time- and dose-dependent manner. In cells pretreated with LA followed by LPS, Akt phosphorylation was elevated initially and further increased during incubation with LPS. This LA-dependent increase in Akt phosphorylation was accompanied by inhibition of LPS-induced NF-kappaB DNA binding activity and up-regulation of TNFalpha and monocyte chemoattractant protein 1. Lipoic acid-dependent Akt phospho...
Oxidative mitochondrial decay is a major contributor to aging and neural degeneration. In old rat... more Oxidative mitochondrial decay is a major contributor to aging and neural degeneration. In old rats (vs. young rats) mitochondrial membrane potential, cardiolipin level, respiratory control ratio, and cellular O 2 uptake are lower; oxidants/O 2 , neuron RNA oxidation, and mutagenic aldehydes from lipid peroxidation are higher. Ambulatory activity and cognition declines with age. Feeding old rats for a few weeks with acetyl-L-carnitine (ALCAR), a mitochondrial metabolite, plus R-lipoic acid (LA), a mitochondrial coenzyme and antioxidant, restores mitochondrial function; lowers oxidants, neuronal RNA oxidation, and mutagenic aldehydes; and increases rat ambulatory activity and cognition (Skinner box/Morris water maze). The mechanism appears to be that with age increased oxidative damage to protein causes a deformation of structure of key enzymes, with a consequent lessening of affinity (K m) for the substrate. The loss with age of carnitine acetyl transferase binding affinity can be mimicked by reacting it with malondialdehyde (a lipid peroxidation product which increases with age). Feeding the substrate ALCAR with LA restores the velocity of the reaction, K m for ALCAR and CoA, and mitochondrial function. Heme biosynthesis is predominantly in the mitochondria. Interfering with heme synthesis causes specific loss of complex IV with consequent release of oxidants and neuronal degeneration. Iron deficiency (25% of menstruating women in the US ingest , 50% of the RDA) also causes release of oxidants and mitochondrial decay, presumably through lack of heme, accelerating brain aging. Vitamin B6 or zinc inadequacy (10% of Americans ingest , 50% of each RDA) should also cause a heme deficiency.
Because standard culture media for human aortic endothelial cells (HAEC) do not contain vitamin C... more Because standard culture media for human aortic endothelial cells (HAEC) do not contain vitamin C, we hypothesized that HAEC may be under significant oxidative insult compared with the situation in vivo. To assess parameters of oxidative stress, intracellular vitamin C, glutathione (GSH), GSH/GSSG, and NAD(P)H/NAD(P)+ ratios, as well as oxidant appearance and oxidative damage, were measured in HAEC with or without vitamin C addition. The effect of vitamin C on eNOS activity was also determined. Results showed that HAEC without vitamin C treatment were essentially scorbutic. On addition of 100 mM vitamin C to the culture media, intracellular vitamin C levels increased and peaked at 6 h. A concomitant increase in the total GSH and the GSH/GSSG ratio was also observed; the NAD(P)H/NAD(P)+ ratio increased more slowly over the 24-h time course. Significantly lower (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05) oxidant appearance and steady-state oxidative damage were also observed following vitamin C repletion. Vitamin C treatment increased eNOS activity by 600%. Thus, HAEC are scorbutic under normal culture conditions and exhibit higher oxidative stress than vitamin C repleted cells. Vitamin C supplementation should be considered when using cultured cells, especially when experimental endpoints are related to cellular redox status and eNOS activity.
Beagle dogs between 7.6 and 8.8 years of age administered a twice daily supplement of ␣-lipoic ac... more Beagle dogs between 7.6 and 8.8 years of age administered a twice daily supplement of ␣-lipoic acid (LA) and acetyl-L-carnitine (ALC) over ϳ2 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 ageassociated cognitive decline by slowing the rate of mitochondrial decay and cellular aging.
a-Lipoic acid (LA) and its reduced form, dihydrolipoic acid (DHLA), have been suggested to chelat... more a-Lipoic acid (LA) and its reduced form, dihydrolipoic acid (DHLA), have been suggested to chelate transition metal ions and, hence, mitigate iron-and copper-mediated oxidative stress in biological systems. However, it remains unclear whether LA and DHLA chelate transition metal ions in a redox-inactive form, and whether they remove metal ions from the active site of enzymes. Therefore, we investigated the effects of LA and DHLA on iron-or copper-catalyzed oxidation of ascorbate, a sensitive assay for the redox activity of these metal ions. We found that DHLA, but not LA, significantly inhibited ascorbate oxidation mediated by Fe(III)-citrate, suggesting that reduced thiols are required for iron binding. DHLA also strongly inhibited Cu(II)(histidine) 2-mediated ascorbate oxidation in a concentration-dependent manner, with complete inhibition at a DHLA:Cu(II) molar ratio of 3:1. In contrast, no inhibition of copper-catalyzed ascorbate oxidation was observed with LA. To investigate whether LA and DHLA remove copper or iron from the active site of enzymes, Cu,Zn superoxide dismutase and the iron-containing enzyme aconitase were used. We found that neither LA nor DHLA, even at high, millimolar concentrations, altered the activity of these enzymes. Our results suggest that DHLA chelates and inactivates redox-active transition metal ions in small-molecular, biological complexes without affecting iron-or copperdependent enzyme activities.
Accumulation of divalent metal ions (e.g. iron and copper) has been proposed to contribute to hei... more 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.
Proceedings of the National Academy of Sciences, 1994
We argue for the critical role of oxidative damage in causing the mitochondrial dysfunction of ag... more We argue for the critical role of oxidative damage in causing the mitochondrial dysfunction of aging. Oxidants generated by mitochondria appear to be the major source of the oxidative lesions that accumulate with age. 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. Acetyl-L-carnitine, a high-energy mitochondrial substrate, appears to reverse many age-associated deficits in cellular function, in part by increasing cellular ATP production. Such 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.
Proceedings of the National Academy of Sciences, 1994
A transgenic mouse strain that expresses the hepatitis B virus (HBV) large envelope protein in th... more A transgenic mouse strain that expresses the hepatitis B virus (HBV) large envelope protein in the liver was used to determine the extent of oxidative DNA damage that occurs during chronic HBV infection. This mouse strain develops a chronic necroinflammatory liver disease that mimics the inflammation, cellular hyperplasia, and increased risk for cancer that is evident in human chronic active hepatitis. When perfused in situ with nitroblue tetrazolium, an indicator for superoxide formation, the liver of transgenic mice displayed intense formazan deposition in Kupffer cells, indicating oxygen radical production, and S-phase hepatocytes were commonly seen adjacent to the stained Kupffer cells. Similar changes were not observed in nontransgenic control livers. To determine whether these events were associated with oxidative DNA damage, genomic DNA from the livers of transgenic mice and nontransgenic controls was isolated and examined for 8-oxo-2'-deoxyguanosine, an oxidatively modif...
Proceedings of the National Academy of Sciences, 2007
The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was recently shown ... more The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was recently shown to negatively regulate LPS-induced acute inflammatory responses. We previously observed that the metabolic thiol antioxidant α-lipoic acid (LA) inhibits LPS-induced expression of cellular adhesion molecules and adherence of monocytes to human aortic endothelial cells. Here we investigated the mechanism by which LA attenuates LPS-induced monocyte activation in vitro and acute inflammatory responses in vivo . Incubation of human monocytic THP-1 cells with LA induced phosphorylation of Akt in a time- and dose-dependent manner. In cells pretreated with LA followed by LPS, Akt phosphorylation was elevated initially and further increased during incubation with LPS. This LA-dependent increase in Akt phosphorylation was accompanied by inhibition of LPS-induced NF-κB DNA binding activity and up-regulation of TNFα and monocyte chemoattractant protein 1. Lipoic acid-dependent Akt phosphorylation an...
The use of antioxidants is now often used as a pharmacological adjunct to limit infertility. Inde... more The use of antioxidants is now often used as a pharmacological adjunct to limit infertility. Indeed, the lay public rightly perceives oxidative stress and, thus, antioxidant treatment as important modulators of infertility. While the direct effects of antioxidant treatment on the quality of semen and oocytes are still under investigation, a significant body of evidence points to loss of vascular tone as a root-cause of erectile dysfunction and, possibly, alterations to female reproduction. In this article, we will critically review the often neglected link between vascular dysfunction and infertility. A particular emphasis will be on the potential use of antioxidants to increase fertility and promote conception.
Diet, along with genetic and environmental factors, is considered a major aspect affecting longev... more Diet, along with genetic and environmental factors, is considered a major aspect affecting longevity as well as vascular disease outcome. Yet, inadequate nutritional intake is rampant among the elderly, affecting nearly 44% of otherwise healthy, community-dwellers in developed countries. Thus, malnutrition, both in quali-and quantitative terms and especially as related to micronutrient intake, may exacerbate intrinsic cardiovascular maladaptation associated with aging, affecting vascular disease outcomes as well as longevity. Conversely, there is accumulating evidence that diets enriched in micronutrients, including vitamins, polyphenols, and essential fatty acids, maintain cellular antioxidant status and stress response enzymes, which otherwise decrease with age. Thus, adequate intakes of micronutrients, either consequent to a correct diet or through supplementation, might afford the elderly protection from cardiovascular diseases. In this article we review the known effects of micronutrients on the aging heart and we propose strategies for dietary improvements.
We assayed levels of lipid peroxidation, protein carbonyl formation, glutamine synthetase (GS) ac... more We assayed levels of lipid peroxidation, protein carbonyl formation, glutamine synthetase (GS) activity and both oxidized and reduced glutathione to study the link between oxidative damage, aging and b-amyloid (Ab) in the canine brain. The aged canine brain, a model of human brain aging, naturally develops extensive diffuse deposits of human-type Ab. Ab was measured in immunostained prefrontal cortex from 19 beagle dogs (4-15 years). Increased malondialdehyde (MDA), which indicates increased lipid peroxidation, was observed in the prefrontal cortex and serum but not in cerebrospinal fluid (CSF). Oxidative damage to proteins (carbonyl formation) also increased in brain. An age-dependent decline in GS activity, an enzyme vulnerable to oxidative damage, and in the level of glutathione (GSH) was observed in the prefrontal cortex. MDA level in serum correlated with MDA accumulation in the prefrontal cortex. Although 11/19 animals exhibited Ab, the extent of deposition did not correlate with any of the oxidative damage measures, suggesting that each form of neuropathology accumulates in parallel with age. This evidence of widespread oxidative damage and Ab deposition is further justification for using the canine model for studying human brain aging and neurodegenerative diseases.
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Papers by Tory Hagen