Study -- (R)-a-Lipoic acid-supplemented old rats have improved mitochondrial fun

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(The FASEB Journal. 1999;13:411-418.)

© 1999 _FASEB_ (http://www.fasebj.org/misc/terms.shtml)

RESEARCH COMMUNICATION

®-α-Lipoic acid-supplemented old rats have improved mitochondrial

function, decreased oxidative damage, and increased metabolic rate

TORY M. HAGEN_1 _

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RMAT= & author1=Hagen,+T & author2=Ames,+B & titleabstract=L

ipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#FN1#FN1) ,

RUSSELL T. INGERSOLL, JENS LYKKESFELDT_2 _

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RMAT= & author1=H

agen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX\

=0 & resource

type=HWCIT#FN2#FN2) , JIANKANG LIU, CAROL M. WEHR, VLADIMIR VINARSKY,

JAMES C. BARTHOLOMEWa and BRUCE N. AMES_ 3 _

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RMAT= & author1=Hagen,+T & author2=

Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcet

ype=HWCIT#FN1#FN1)

Department of Molecular and Cell Biology, University of California at

Berkeley, Berkeley, California 94720, USA; and

a Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

ABSTRACT

A diet supplemented with ®-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 ®-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. ®-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

®-lipoic acid. The increase of oxidants with age, as measured by the

fluorescence produced on oxidizing 2',7'-dichlorofluorescin, was significantly

lowered in ®-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 ®-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 ®-lipoic acid supplementation. Thus, ®-lipoic acid

supplementation improves indices of metabolic activity as well as lowers

oxidative stress and damage evident in aging.—Hagen, T. M., Ingersoll, R. T.,

Lykkesfeldt, J., Liu, J., Wehr, C. M., Vinarsky, V., Bartholomew, J. C., Ames,

B. N. ®-α-Lipoic acid-supplemented old rats have improved mitochondrial

function, decreased oxidative damage, and increased metabolic rate.

INTRODUCTION

AGING IS A MULTIFACTORIAL PROCESS that leads to loss of function and the

inability to adequately respond to external stress. Mitochondrial

dysfunction appears to contribute to some of the loss of function accompanying

aging

_1_

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RMAT= & author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Rev

erses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B1#B1) , _2_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & auth

or1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & F

IRSTINDEX=0 & resourcetype=HWCIT#B2#B2) ) . Mitochondria from aged tissue

use oxygen inefficiently, which impairs ATP synthesis and results in increased

oxidant production _3_

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RMAT= & author1=Hagen,+T & author2=Ames,+B & titleabst

ract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B3#B3)

, _4_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+R

everses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B4#B4) ) . The high flux

of oxidants not only damages mitochondria, but other important cell

biomolecules as well. Antioxidant defenses also decline with age _5_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & aut

hor1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 &

FIRSTINDEX=0 & resourcetype=HWCIT#B5#B5) , _6_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & auth

or2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resou

rcetype=HWCIT#B6#B6) ) , making mitochondria even more susceptible to

oxidative injury _(1)_

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RMAT= & author1=Hagen,+T & author2=Ames,+B & titleabstract=L

ipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B1#B1) .

The resultant mitochondrial decay may eventually cause inadequate energy

production and/or the loss of calcium homeostasis. Such changes could result in

unwarranted cellular apoptosis and also lead to the general metabolic

decline evident in aging.

Lipoic acid is a disulfide compound found naturally in mitochondria as the

coenzyme for pyruvate dehydrogenase and α-ketoglutarate dehydrogenase. It

has been used as therapy for many diseases associated with impaired energy

utilization, such as type II diabetes _(7)_

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RMAT= & author1=Hagen,+T & autho

r2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resour

cetype=HWCIT#B7#B7) and diabetic polyneuropathies _8_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Ha

gen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTIN

DEX=0 & resourcetype=HWCIT#B8#B8) , _9_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ame

s,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype

=HWCIT#B9#B9) ) . Dietary supplementation also increases unbound lipoic

acid, which can act as a potent antioxidant and ameliorate oxidative stress

both in vitro and in vivo _10_

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RMAT= & author1=Hagen,+T & author2=Ames,+B & tit

leabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B

10#B10) -_15_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoi

c+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B15#B15) ) . To

a degree, aging results in the same type(s) of metabolic impairment and

increased oxidative stress as shown in these conditions.

Though its ability to improve energy metabolism _(16)_

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RMAT= & author1=Ha

gen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTIN

DEX=0 & resourcetype=HWCIT#B16#B16) and lower oxidative stress _11_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT

= & author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & search

id=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B11#B11) -_15_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,

+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=

0 & resourcetype=HWCIT#B15#B15) ) for certain disease states has been

described, it is not known whether lipoic acid supplementation may also reverse

energy-linked metabolic deficits or reduce the increased oxidative stress

seen in aging. The purpose of this study was twofold: to 1) determine whether

®-lipoic acid supplementation increased cellular and general metabolic

activity in old rats, and 2) examine whether this supplementation affected

hepatocellular antioxidant status, oxidant production, and oxidative damage.

We show that supplementing old rats with 0.5% (w/w) lipoic acid for 2 wk

partially reverses the age-associated loss of mitochondrial function, an

increase in oxidative stress, and the damage and decline in general metabolic

activity.

MATERIALS AND METHODS

The following chemicals were used: EGTA (ethylene glycol-bis (ß-aminoethyl

ether) N,N, N',N'-tetraacetic acid), 2,4-dinitrofluorobenzene, Trypan

blue, heparin (sodium salt), rhodamine 123 (R123),4 glutathione, reduced form

(GSH), and dithiothreitol (Sigma, St. Louis, Mo.); 2',7'-dichlorofluoroscin

diacetate (DCFH) (Molecular Probes, Eugene, Oreg.); collagenase (type D)

(Boehringer Mannheim, Indianapolis, Ind.); L-ascorbic acid, and

meta-phosphoric acid (Fluka, Ronkonkoma, N.Y.). All other reagents were reagent

grade or

better. Double-distilled/deionized water was used throughout.

Animals

Rats (Fisher 344, virgin male, outbred albino), both young (3–5 months;

Simonsen, Gilroy, Calif.) and old (24–26 months; National Institute of Aging

animal colonies), were acclimatized in the Berkeley animal facilities for

at least 1 wk prior to experimentation. The AIN-93M standard diet or one

supplemented with 0.5% (w/w) ®-lipoic acid, and water ad libitum was given

throughout.

Cell isolation

Liver tissue was dispersed into single cells by collagenase perfusion

_(17)_

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RMAT= & author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Rever

ses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B17#B17) . Cell number was

assessed using a hemocytometer, and viability (typically greater than 90%

in both age groups) was determined by Trypan blue exclusion.

Mitochondrial membrane potential

The average mitochondrial membrane potential in intact hepatocytes was

measured by flow cytometry using R123 as the fluorescent probe _(4)_

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RMAT= &

author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid

=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B4#B4) . Hepatocytes (2.0x106 cells)

were incubated with R123 (0.01 mg/ml) for 30 min at 37°C, then subjected to

flow cytometry using an instrument constructed according to the design of

Steinkamp et al. _(18)_

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ct=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B18#B18)

. Nonspecific light scatter was subtracted and cells showing a particular

fluorescence were quantified.

Oxygen consumption studies

Hepatocellular oxygen consumption was analyzed using a YSI 5300 oxygen

electrode and monitor (Yellow Springs, Ohio).

DCFH measurement

Formation of oxidants in cells were determined by assaying the

fluorescence of 2',7'-dichlorofluorescein, the oxidation product of DCFH _(19)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMA

T= & author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searc

hid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B19#B19) . Quadruplicate samples

were routinely analyzed. Fluorescence was monitored using a Cytofluor 2350

fluorescent measurement system (Millipore, Bedford, Mass.) using standard

fluorescein filters and Cytocalc software. Oxygen consumption was measured and

data were expressed as the fluorescence per µM O2 consumed/106 cells.

GSH analysis

Reduced GSH was measured by high-performance liquid chromatography (HPLC)

as described by et al. _(20)_

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& titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWC

IT#B20#B20) . Briefly, cells were mixed with 5-sulfosalicylic acid [7.5%

(w/v), final concentration] and the samples were spun for 1 min at 13,000

RPM in a microcentrifuge to remove denatured debris. An aliquot of the

supernatant was added to 100 µl of 1M Trizma Base buffer (pH 8), followed by

addition of 100 µl of 40 mM fresh aqueous iodoacetic acid (4 µmol). The

reaction mixture was brought to pH 8 with NaHCO3 and dinitrophenyl derivatives

were made by addition of 500 µl of 2,4-dinitrofluorobenzene [1.5% (v/v) in

absolute ethanol] and 100 to 200 µl of K2CO3. The resultant derivatives were

separated on a 10 µm Ultrasphere-amine column (4.6 mmx25 cm) using a Waters

HPLC system and solvents, as described _(20)_

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RMAT= & author1=Hagen,+T & au

thor2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & res

ourcetype=HWCIT#B20#B20) . GSH was quantified relative to standards.

Ascorbic acid analysis

Total ascorbic acid quantification was performed after reduction with

dithiothreitol, as described _(21)_

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RMAT= & author1=Hagen,+T & author2=Ames,+B & ti

tleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#

B21#B21) . The samples were placed in a chilled (2°C) auto sampler for

analysis. The system used for separation was reversed-phase HPLC

(Hewlett-Packard, Mountain View, Calif.) with coulometric detection (ESA Inc.,

Bedford,

Mass.). The peak area corresponding to ascorbic acid was integrated using

HP ChemStation software (Hewlett-Packard).

Malondialdehyde analysis

Lipid peroxidation was assayed using a recently developed sensitive and

specific gas chromatography-mass spectrometry method for malondialdehyde

(MDA) _22_

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RMAT= & author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+R

everses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B22#B22) , _23_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT=

& author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchi

d=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B23#B23) ) . Briefly, the hepatocytes

were lysed with phosphate-buffered saline containing 2.8 mM butylated

hydroxytoluene and 1% sodium dodecyl sulfate, pH 7.4. The protein-bound MDA was

hydrolyzed with H2SO4. MDA was converted into a stable derivative, using

pentafluorophenyl hydrazine at room temperature, and the derivative was

detected with a Hewlett Packard 5890 Series II gas chromatograph interfaced to

a

5989 mass spectrometry system equipped with a J & W Scientific DBWAX

capillary column (15 mx0.25 mm i. d., 0.25 µm film thickness) in the negative

chemical ionization mode. The results were indexed with protein, which was

measured with a modified Lowry method.

Ambulatory activity tests

Each night rats were moved from group housing to individual cages (48 cm

lx25 wx20 h) at least 4 h prior to the quantification of ambulatory

parameters. The room was on a 12 h light/dark cycle (lights on 6 AM to 6 PM).

At 8

PM, a very low light illuminated the test subjects for video tracking.

Quantification began at 9 PM and continued for 4 h. One hour later the low

light was turned off and the room remained in total darkness until 6 AM, when

the standard light/dark cycle began. A video signal from a camera suspended

directly above the individual cages was fed directly into a Videomex-V

(Columbus Instruments, Columbus, Ohio) computer system running the Multiple

Objects Multiple Zones software. The system quantified ambulatory activity

parameters and was calibrated to report distance traveled in centimeters. In

addition to total distance traveled, the time each subject spent in

ambulatory (locomotor), stereotypic (grooming), and resting (nonmovement)

activity

was recorded by an IBM computer. No additional modifications (such as fur

dying) were needed to continuously track the subjects. At 9 AM animals were

removed from individual housing and returned to group housing. Results are

shown as the mean centimeters traveled per hour ±SEM.

The ambulatory activity of each rat was recorded before lipoic acid

supplementation and for two consecutive nights. After lipoic acid

supplementation

and for two consecutive nights, the same spontaneous locomotor parameters

were determined. With this design, each rat acted as its own control. After

measurement of ambulatory activity, some lipoic acid-supplemented animals

were placed on an AIN-93M diet for three additional weeks and activity was

again measured.

Statistical analysis

Statistical significance was determined using the paired Student's t test

or one way analysis of variance. Results are expressed as the mean ±SEM. A

P value of less than 0.05 was considered significant.

RESULTS

Effect of lipoic acid supplementation on metabolic activity

Hepatocellular oxygen consumption was monitored to assess age-related

changes in mitochondrial activity and whether ®-lipoic acid supplementation

affected cellular metabolic rate. Oxygen consumption in hepatocytes isolated

from old rats was only 59% of that of hepatocytes from young animals

(_Table 1_

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RMAT= & author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Re

verses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#T1#T1) ). This

significant decline (P<0.03) was completely reversed after a 2 wk feeding

regimen

of ®-lipoic acid _(Table 1)_

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RMAT= & author1=Hagen,+T & author2=Ames,+B & titl

eabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#T1

#T1) . ®-Lipoic acid supplementation had no significant effect on

oxygen consumption in cells isolated from young rats.

Table 1. Lipoic acid supplementation increases hepatocellular oxygen

consumption in old ratsa

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O2 Consumption (µmol O2/min per 107 cells)

Unsupplemented

®-Lipoic acid supplemented

Young

480 ± 60 (5)

500 ± 48 (8)

Old

281 ± 27 (5)

533 ± 28 (8)

a Numbers are expressed as the mean ± SEM. N values are indicated in

parentheses.

The mitochondrial membrane potential in hepatocytes was measured using

R123 fluorescence in order to test whether the lipoic acid-induced increase in

O2 consumption in hepatocytes from old rats was attributable to enhanced

mitochondrial function. The average mitochondrial membrane potential in the

majority of hepatocytes from old rats has previously been shown to be

approximately 40% that of hepatocytes from young rats, a significant loss

(P<0.02; N=8) of the driving force for ATP production _(4)_

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RMAT= & author1=Hage

n,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDE

X=0 & resourcetype=HWCIT#B4#B4) . ®-Lipoic acid supplementation caused

the mitochondrial membrane potential to increase by 50.0% ±7.9 (N=4) over

that of unsupplemented old rats, a marked improvement (P<0.03), but still

significantly lower (P<0.04) when compared to cells from young untreated rats.

Thus, ®-lipoic acid supplementation partially improves mitochondrial

function in old rats and may alleviate some loss of metabolic activity

associated with aging.

To determine whether ®-lipoic acid improved metabolic activity on a

physiological basis, we quantified ambulatory activity in rats with and without

lipoic acid treatment. Ambulatory activity declined almost threefold with

age (_Fig. 1_

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RMAT= & author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic

+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#F1#F1) ). This

significant decline was partially reversed by ®-lipoic acid

supplementation, which increased ambulatory activity by twofold over untreated

old

animals (P<0.0005). Activity in treated old rats compared to untreated young

rats was lower, but not significantly so (P<0.06). Feeding ®-lipoic acid to

young rats also increased ambulatory activity, but this increase was not

significant.

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Figure 1. ®-Lipoic acid supplementation increases ambulatory activity

in old rats. Ambulatory activity was measured as distance traveled in young

and old rats, fed with or without 0.5% (w/w) lipoic acid. Each bar

represents the mean distance traveled (cm/h) ±SEM from 8 h of quantification

as

described in Materials and Methods. Distance was determined from the same

young (n=5) and old (n=5) rats before and after ®-lipoic acid treatment.

Comparing old animals before and after lipoic acid supplementation: P =

0.0005. Comparing lipoic acid supplemented to unsupplemented young rats: P =

0.06.

To confirm the effect of ®-lipoic acid on metabolic activity, a

three-staged feeding regimen using the same aged rats was designed. Ambulatory

activity was monitored 1) after feeding an AIN-93M diet for 2 wk, 2) after

feeding ®-lipoic acid supplemented AIN-93M diet for 2 wk, and 3) after

feeding an AIN-93M diet (without lipoic acid) for three additional weeks. The

results of this experiment showed that during the lipoic acid supplementation

period, ambulatory activity again was significantly higher (P<0.03; _Fig.

2_

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RMAT= & author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Revers

es & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#F2#F2) ). Removal of

®-lipoic acid from the diet reversed this improvement _(Fig. 2)_

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RMAT= & author

1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIR

STINDEX=0 & resourcetype=HWCIT#F2#F2) . Control experiments where the

AIN-93M was given to old rats throughout the study, but otherwise treated

similarly to the experimental group, showed no change in ambulatory activity

(data not shown). Thus, ®-lipoic acid significantly increases overall

physiological activity among old rats.

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Figure 2. Removal of ®-lipoic acid from the diet reverses the

improvement in ambulatory activity. Ambulatory activity (distance traveled in

cm/h)

was measured in old rats after 2 wk on the AIN-93M diet (`Pre'), after 2 wk

of the ®-lipoic acid supplemented diet (`+LA'), and finally 3 wk after

removal of ®-lipoic acid from the diet (`off diet'). ®-Lipoic acid

increased ambulatory activity (P<0.03) whereas its removal resulted in a

complete reversal of the observed improvement. Control experiments where

AIN-93M

was given throughout resulted in no change in ambulatory activity (data not

shown).

Effect of lipoic acid supplementation on oxidant stress

®-Lipoic acid acts as a cofactor in several mitochondrial enzyme

complexes, but is also a powerful antioxidant and increases levels of other

endogenous antioxidants when given as a supplement. The effect of ®-lipoic

acid

supplementation on antioxidant status, oxidant production, and levels of

oxidative damage in hepatocytes from old rats was examined.

Hepatocellular GSH and ascorbic acid concentrations were measured to

determine whether these low molecular weight antioxidants declined with age.

Both GSH and ascorbic acid levels were significantly lower (P<0.05) in

hepatocytes from old compared to young rats, with declines of 23% and 50%,

respectively (_Fig. 3A, B_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames,+B & titleabstract=

Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#F3 F3#F3

F3) ). Supplementation of ®-lipoic acid for 2 wk prior to cell isolation

restored the level of antioxidants to that of young animals. In both young

and old rats, hepatocellular GSH levels were significantly higher vs. their

corresponding controls (P<0.03; _Fig. 3A_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=

Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcet

ype=HWCIT#F3#F3) ); GSH levels were more than twofold higher in old rats

than in unsupplemented animals. Lipoic acid supplementation also restored

the cellular ascorbic acid levels to that of young rats (_Fig. 3B_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= &

author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid

=1 & FIRSTINDEX=0 & resourcetype=HWCIT#F3#F3) ). Thus, ®-lipoic acid

reverses the age-associated decline in endogenous low molecular weight

antioxidants, and therefore may lower the increased risk for oxidative damage

that

occurs during aging.

_http://www.fasebj.org/content/13/2/411/F3.expansion.html_

(http://www.fasebj.org/content/13/2/411/F3.expansion.html)

Figure 3. Hepatocellular glutathione (GSH) and ascorbate levels increase

after ®-lipoic acid supplementation. GSH and ascorbate levels in

hepatocytes from rats either on control diet (AIN-93M) or a diet supplemented

with

®-lipoic acid were measured as described. A) GSH levels in young and old

rats fed either with or without 0.5% (w/w) ®-lipoic acid. ®-Lipoic acid

supplementation significantly (P<0.03) increased GSH levels in both young

and old rat hepatocytes. Hepatocellular ascorbate levels in young and

old rats supplemented with or without 0.5% (w/w) lipoic acid. Lipoic acid

treatment reversed the age-associated decline in cellular ascorbate levels.

Results are the mean ±SEM for at least 5 experiments.

We previously showed that hepatocytes from old rats have a higher rate of

oxidant production per oxygen consumed, as measured by the fluorescence

formed on oxidizing DCFH _(4)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames,+B & titlea

bstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B4#B

4) . To determine whether dietary ®-lipoic acid could lower the

increased rate of oxidant production seen in aging, we measured the

fluorescence

in supplemented animals and their corresponding controls. Cells from old

rats had significantly higher oxidant production (P<0.005), nearly twofold

more than that in young rats (_Fig. 4_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames,

+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=H

WCIT#F4#F4) ). In contrast, oxidant production was lowered in cells from

lipoic acid-treated old rats to a level not significantly different from

those of untreated young rats _(Fig. 4)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ame

s,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype

=HWCIT#F4#F4) .

_http://www.fasebj.org/content/13/2/411/F4.expansion.html_

(http://www.fasebj.org/content/13/2/411/F4.expansion.html)

Figure 4. ®-Lipoic acid treatment lowers the age-associated increase in

oxidant production per µM O2 used as measured by the fluorescence formed on

oxidizing DCFH. Hepatocytes from young and old rats that had been

supplemented with or without ®-lipoic acid were incubated with DCFH, as

described, and the increase in mean fluorescence was monitored to determine the

rate

of oxidant production. Results show that cells isolated from old untreated

rats had a significantly higher rate of oxidant appearance. Lipoic acid

treatment reversed this increase. Results are the mean ±SEM for 7

experiments.

To gauge whether the lipoic acid-induced decline in oxidant production

translated into lower levels of oxidative damage, MDA was measured as an

indicator of cellular lipid peroxidation _22_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=

Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcet

ype=HWCIT#B22#B22) , _23_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMA

T= & author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=\

1 & FIRSTINDEX=0 & resourcetype=HWCIT#B23#B

23) ) . Hepatocytes from unsupplemented old rats had fivefold more MDA

than the cells from young rats (P<0.01; _Fig. 5_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & aut

hor2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & reso

urcetype=HWCIT#F5#F5) ). ®-Lipoic acid supplementation reduced MDA

levels markedly in old rats (P<0.01) _(Fig. 5)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author

2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourc

etype=HWCIT#F5#F5) . Although this decline in lipid peroxidation was

substantial, the levels observed in the lipoic acid-supplemented old rats were

still significantly higher than those found in cells from unsupplemented

young rats (P=0.05). MDA levels were higher in lipoic acid-supplemented young

animals, but this increase was not significant. Thus, ®-lipoic acid

supplementation markedly lowers oxidant production and the attendant increase

in oxidative damage associated with aging.

_http://www.fasebj.org/content/13/2/411/F5.expansion.html_

(http://www.fasebj.org/content/13/2/411/F5.expansion.html)

Figure 5. ®-Lipoic acid supplementation lowers the age-related increase

in hepatocellular lipid peroxidation. Lipid peroxidation, as measured by

hepatocellular MDA levels, was measured in cells isolated from rats fed with

or without ®-lipoic acid. The MDA levels increased significantly with age

(P<0.01). 2 wk of ®-lipoic acid supplementation significantly lowered

MDA levels in old rats (P<0.01). Similar supplementation to young rats

resulted in no significant change in MDA levels (P=0.05). Results are expressed

as

the mean ±SEM for 5 experiments.

*DISCUSSION

The ®-form of lipoic acid used in this study is the naturally occurring

enantiomer in mammalian cells _(24)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames

,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=

HWCIT#B24#B24) . Only the ®-form is used by mitochondrial α-keto acid

dehydrogenases and specifically reduced to dihydrolipoic acid, a powerful

antioxidant, via mitochondrial lipoamide dehydrogenase. There is evidence

that ®-lipoic acid supplementation may be more potent than either the

racemic mixture (the form sold commercially as α-lipoic acid) or

(S)-enantiomer,

and thus a more relevant supplement for this study. Addition of ®-lipoic

acid increases ATP synthesis and aortic blood flow during reoxygenation

after hypoxia in a working heart model _(25)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author

2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourc

etype=HWCIT#B25#B25) . The (S)-enantiomer had no effect on ATP synthesis

and improved blood flow at only 10-fold the effective dose of ®-lipoic

acid. Packer and colleagues _(26)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames,+B & t

itleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT

#B26#B26) also showed that ®-lipoic acid significantly reduced

buthionine-S,R-sulfoximine-induced cataract formation, but (S)-lipoic acid had

little effect at the same concentration. ®-Lipoic acid increased glucose

uptake and the number of glucose transporters in muscle tissue much more

effectively than (S)-lipoic acid _(27)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames,+B

& titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWC

IT#B27#B27) . The ®-enantiomer more effectively chelated copper and

prevented copper-induced lipid peroxidation _(28)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & a

uthor2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & re

sourcetype=HWCIT#B28#B28) .

We did not measure hepatic tissue concentrations of ®-lipoic acid or

dihydrolipoic acid after oral supplementation. However, the characteristics of

its uptake and tissue distribution in the rat have previously been

examined, although not on an age-related basis. Lipoic acid is rapidly absorbed

in

the gastrointestinal tract but is subject to considerable presystemic

elimination _29_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+

Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B29#B29) , _30_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTF

ORMAT= & author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & s

earchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B30#B30) ) . Between 27 to 34% of

orally administered lipoic acid is available for tissue uptake, and the

liver is one of the major organs of clearance _(31)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+

T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0

& resourcetype=HWCIT#B31#B31) . Studies where radiolabeled lipoic acid was

infused into rats revealed that the liver has a high capacity for both

uptake and accumulation of lipoic acid _(32)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2

=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resource

type=HWCIT#B32#B32) . Thus, dietary supplementation of lipoic acid would

be expected to elevate hepatocellular lipoic acid concentrations

considerably in both young and old rats, though its release from the liver may

also

be rapid.

A pharmacological dose of ®-lipoic acid was given to maximize the

possibility of observing whether it could affect metabolic activity and lower

the

increased oxidative stress evident in old rats. Even though the

supplemental dose given was relatively high, it was considerably lower than the

reported LD50 concentration for ®- or (R,S)-lipoic acid for old rats _(24)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTF

ORMAT= & author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & s

earchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B24#B24) . Rats fed the lipoic

acid-supplemented diet for 2 wk exhibited no adverse side effects other than

a small amount of weight loss, which we attribute to increased general

metabolic activity. We are currently determining whether lower levels of

®-lipoic acid in the diet would be equally effective in partially restoring

metabolic function and decreasing oxidative stress in old rats.

We demonstrate that lipoic acid supplementation of old rats markedly

improves the average mitochondrial membrane potential and restores the cellular

oxygen consumption _(Table 1)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames,+B & tit

leabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#T

1#T1) in hepatocytes to that of young rats. Rats on this feeding regimen

were significantly more active, which further shows that ®-lipoic acid

acts physiologically to increase general metabolic activity. While the

underlying causes for this increased energy metabolism were not explored, it is

plausible that lipoic acid improves mitochondrial function through a number

of mechanisms. Administration of lipoic acid stimulates insulin-dependent

and independent glucose uptake into cells _(33)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & au

thor2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & res

ourcetype=HWCIT#B33#B33) and also enhances nonoxidative and oxidative

glucose metabolism. Reduced ®-lipoic acid has also been shown to increase

ATP synthase activity _(16)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames,+B & titleab

stract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B16#B

16) , which in combination with increased glucose utilization would be

expected to enhance overall cellular metabolism. Finally, as a potent

antioxidant, dihydrolipoic acid may also maintain critical thiol groups in a

reduced state and allow mitochondrial protein carriers to function more

effectively _(16)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Ac

id+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B16#B16) .

We also show that feeding ®-lipoic acid significantly attenuates the

age-related increase in hepatocellular oxidant production as well as lipid

peroxidation. This reduction in oxidative stress may be directly attributable

to increased unbound dihydrolipoic acid or indirectly due to higher levels

of other antioxidants. Lipoic acid raises GSH values by increasing cysteine

availability _(12)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames,+B & titleabstract

=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B12#B12)

, which is the rate-limiting factor in its biosynthesis. Lipoic acid

decreases levels of GSH protein-mixed disulfides _(34)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T

& author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 &

resourcetype=HWCIT#B34#B34) . Lipoic acid also causes faster ascorbic

acid recycling _(13)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames,+B & titleabstract=L

ipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B13#B13) .

This may be important because ascorbic acid recycling in times of oxidative

insult is markedly impaired in cells from old rats _(14)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1

=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRS

TINDEX=0 & resourcetype=HWCIT#B14#B14) and ®-lipoic acid supplementation

reverses this decline _(14)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames,+B & title

abstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B14

#B14) . Thus, feeding lipoic acid generally improves cellular antioxidant

status, which declines with age.

α-Lipoic acid has been used as a therapeutic agent in humans, especially

for diabetes _7_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames,+B & titleabstract=Lip

oic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B7#B7) , _9_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULT

FORMAT= & author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses &

searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B9#B9) , _35_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=H

agen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTI

NDEX=0 & resourcetype=HWCIT#B35#B35) ) as well as certain toxicological and

pathological conditions of the liver _24_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=

Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcet

ype=HWCIT#B24#B24) , _36_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames,+B & titleab

stract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B36#B

36) , _37_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+A

cid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B37#B37) ) .

However, little is known about whether ®-lipoic acid may be an effective

anti-aging supplement or therapy for certain diseases in humans. Our present

findings using rats would suggest that ®-lipoic acid supplementation may be a

safe and effective means to improve general metabolic activity and increase

antioxidant status, affording increased protection against external

oxidative and xenobiotic insults with age.

Other critical metabolites that become limiting due to age-associated

metabolic changes may also be beneficial as dietary supplements. A number of

studies report that administration of acetyl-L-carnitine (ALCAR), a

derivative of carnitine involved in fatty acid transport into mitochondria,

enhanced

mitochondrial function in aged tissue _38_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & autho

r2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resour

cetype=HWCIT#B38#B38) -_42_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames,+B & title

abstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT#B42

#B42) ) . We previously found _(43)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames

,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=

HWCIT#B43#B43) that ALCAR fed to old rats restores decayed mitochondria

for cardiolipin content, membrane potential, and oxygen consumption and

restores ambulatory activity of the rats. However, ALCAR supplementation also

increased the rate of oxidant production, oxidative damage, and decreased

cellular antioxidant levels _(43)_

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=Ames,+B & t

itleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcetype=HWCIT

#B43#B43) . This indicated that ALCAR supplementation improved

mitochondrial electron flux but did not reverse the increased inefficiency of

electron transport. In a separate study (T. M. Hagen et al., unpublished

results),

we find that feeding ALCAR in combination with lipoic acid to old rats

effectively increases mitochondrial metabolism without an increase in

oxidative stress. Long-term feeding studies are warranted to determine whether

these observed changes in mitochondria will significantly diminish decline in

energy metabolism and the increased oxidative stress evident in aging.

ACKNOWLEDGMENTS

The authors would like to thank Dr. H. Tritschler, Asta Medica, for the

generous gift of ®-lipoic acid. We also express our thanks to Anitra Carr,

Balz Frei, Mark McCall, and Lester Packer for critical comments. This study

was supported by National Cancer Institute Outstanding Investigator Grant

CA39910 and NIEHS Center Grant ESO1896 (B.N.A.), a Sandoz Gerontological

Foundation Grant (T.M.H.), and a Danish Natural Science Research Council

Grant (SNF9502434) (J.L.).

FOOTNOTES

3 Correspondence: Department of Molecular and Cell Biology, 401 Barker

Hall, University of California at Berkeley, Berkeley, CA 94720, USA. E-mail:

_bnames@..._ (mailto:bnames@...)

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT=

& author1=Hagen,+T & author2=Ames,+B & titleabstract=Lipoic+Acid+Reverses & searchi

d=1 & FIRSTINDEX=0 & resourcetype=HWCIT#RFN1#RFN1)

1 Present address: Linus ing Institute, Oregon State University, 571

Weniger Hall, Corvallis, OR 97331, USA.

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=A

mes,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcety

pe=HWCIT#RFN1#RFN1)

2 Department of Pharmacology and Pathobiology, Royal Veterinary and

Agricultural University, Copenhagen, Denmark.

(http://www.fasebj.org/content/13/2/411.full?maxtoshow= & HITS=10 & hits=10 & RESULTFO\

RMAT= & author1=Hagen,+T & author2=A

mes,+B & titleabstract=Lipoic+Acid+Reverses & searchid=1 & FIRSTINDEX=0 & resourcety

pe=HWCIT#RFN2#RFN2)

4 Abbreviations: ALCAR, acetyl-L-carnitine; GSH: glutathione; HPLC,

high-performance liquid chromatography; DCFH: 2',7'-dichlorofluorescin

diacetate;

MDA: malondialdehyde; R123, rhodamine 123.

Received for publication August 3, 1998. Revision received October 15,

1998.

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