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Clinical Nutrition (2005) 24, 794–800

KEYWORDAging;MitochondElectron trchain;Tricarboxycycle;Carnitine;Lipoic acid

0261-5614/$ - sdoi:10.1016/j.c

�Correspondifax: 91 044 249

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ORIGINAL ARTICLE

Efficacy of levo carnitine and alpha lipoic acid inameliorating the decline in mitochondrial enzymesduring aging

S. Savitha�, K. Sivarajan, D. Haripriya, V. Kokilavani, C. Panneerselvam

Department of Medical Biochemistry, Dr. ALMPG. Institute of Basic Medical Science, University of Madras,Taramani, Chennai 600113, India

Received 17 February 2005; accepted 13 April 2005

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ria;ansport

lic acid

ee front matter & 2005lnu.2005.04.005

ng author. Tel.: 91 0442 6709.ess: sm_savitha@yahoo

SummaryBackground: Mitochondria are central to energy production and are therefore fullyintegrated into the rest of the cell’s physiological responses to stress. The age-related decline of capacity of each cell to manufacture energy (as ATP) is due to theprogressive loss of structural integrity of mitochondria. It is apparent that as thebody ages, the cells become less and less able to maintain threshold levels of cellularenergy production.Methods: In the present study we have evaluated the efficacy of carnitine, amitochondrial metabolite and lipoic acid, a potent antioxidant on the activities ofthe tri carboxylic acid (TCA) cycle enzymes like succinate dehydrogenase, malatedehydrogenase, a-ketoglutarate dehydrogenase, Isocitrate dehydrogenase andelectron transport complex I–IV in young and aged heart mitochondria.Result: We observed that there was an age-dependent decrement in the levels ofthe TCA cycle enzymes and electron transport chain complexes. Supplementation ofcarnitine (300mg/kg bw/day) and lipoic acid (100mg/kg bw/day) for 30 daysbrought the activities of these enzymes to almost near normal levels.Conclusion: These findings suggest that the combination of these drugs raises themitochondrial energy producing capabilities by reversing the age-associated declinein mitochondrial enzyme activities and thereby protecting mitochondria from aging.& 2005 Elsevier Ltd. All rights reserved.

Elsevier Ltd. All rights reserv

2492 5861;

.co.in (S. Savitha).

Introduction

Aging is a multifactorial phenomenon characterizedby time-dependent decline in physiological func-tion, which varies between different species.

ed.

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Efficacy of L- carnitine and DL a lipoic acid in ameliorating mitochondrial enzymes during aging 795

Several theories have been proposed to explain thisprocess. The free radical theory first proposed byHarman1 hypothesized that free radicals producedduring aerobic respiration cause cumulative oxida-tive damage to proteins, lipids and DNA resulting inaging and death. It also predicts that slowing therate of initiation of free radical reactions canmodulate the aging process.2–4 Many of thesignificant age-related changes are exhibited inpost-mitotic tissues such as brain, heart andskeletal muscle.

The heart is dependent on molecular oxygen andoxidative phosphorylation to provide high-energycompounds necessary for contraction, but thisexposes the myocardium to harmful reactive oxy-gen species that are generated continuously asnormal by-products of the mitochondrial electrontransport chain (ETC). The aging heart undergoessignificant functional and structural alterationsleading to atrophy and a compensatory hypertro-phy, followed by myocardial fibrosis.5 In addition,there is an age-related decline in the capacity towithstand stress, such as ischemia/reperfusion.6 Inits most severe form, cardiac decay results incongestive heart failure, one of the leading causesof death in people over the age of 65. Although themechanisms underlying cardiac decay are not clear,loss of mitochondrial function and a resultantincrease in oxidative stress has been proposed tobe one of the key factors in myocardial aging.7

Therefore, it seems very likely that age-relatedchanges at the mitochondrial level are important inthe decline of physiological function that accom-panies senescence.

The mitochondrial ETC plays an important role inenergy production in aerobic organisms. Electronsfrom nicotinamide adenine dinucleotide (NADH)and flavin adenine dinucleotide (FADH2), producedduring the citric acid cycle, flow down the ETC andare coupled to the establishment of a protongradient that is utilized in the production of ATP.The final electron acceptor is molecular oxygen,which through four-electron reduction is convertedto H2O. The ETC is located in the inner membraneof the mitochondria and consists of five complexes.They are: NADH dehydrogenase (Complex I), Succi-nate dehydrogenase (Complex II), cytochrome bc1complex (Complex III), cytochrome c oxidase(Complex IV) and ATP synthase (Complex V).However, as mitochondria including the respiratorychain become more and more damaged, higheramounts of free radicals are generated. This kind ofa vicious cycle finally leads to non-functionalmitochondria.

Carnitine is a natural substance that acts as acarrier of fatty acids across the inner mitochondrial

membrane for subsequent b-oxidation. L-carnitineand its esters are endogenously synthesized in manand also found in diet.8 Carnitines are essentialcofactors of several enzymes necessary for thetransformation of long chain fatty acids. Thus, wechose to administer carnitine to aged rats toimprove the mitochondria-mediated bioenergetics.But despite the benefits of carnitine treatment,there are also potential adverse effects. Whilecarnitine supplementation reversed many of thealtered characteristics evident in mitochondrialmetabolism with age, the rate of oxidant produc-tion was higher in carnitine-supplemented rats.9

We further observed that the age-related in-crease in oxidant production and oxidative damagecan be reversed by co-supplementation with a-lipoic acid, a disulfide compound found naturally inplants and animals.10 The disulfide form of lipoicacid is reduced in mitochondria by specific dehy-drogenase and its supplementation would thustarget an antioxidant to the mitochondria, themajor site of free radical production.11 Supple-mentation with LA may also boost mitochondrialfunction because it is a co-factor for pyruvate anda-ketoglutarate dehydrogenase12,13 and as such,may be a useful dietary supplement in its own rightto increase overall mitochondrial metabolism. Inthe present study, we have observed the changes inthe activities of mitochondrial enzymes in aged ratsand the effect of co-supplementation of carnitineand lipoic acid on these enzymes.

Materials and methods

Source of chemicals

L-Carnitine and DL-a-lipoic acid were purchasedfrom Sigma chemical company (St. Louis, MO,USA). All other chemicals were of reagent gradeand were obtained from Glaxo Laboratories, CDHdivision, Mumbai, India and Sarabhai. M. Chemi-cals, Baroda, India.

AnimalsMale albino rats of Wistar strain approximately 3–4-months-old (young) and above 24-months-old(aged) were used in this study. They were healthyanimals procured from The King’s Institute ofPreventive Medicine, Chennai. The animals werehoused in large spacious cages and were given foodand water ad libitum. The animal room was wellventilated and had 12 h light/dark cycles through-out the experimental period. The animals weremaintained on a commercial rat feed which

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contained 5% fat, 21% protein, 55% nitrogen-freeextract and 4% fiber (wt/wt) with adequate mineraland vitamin contents.

Grouping of animals

The animals were divided into six groups. Eachgroup consisted of six animals.

Group I: Control young rats,Group II: Young rats administered L-carnitine andDL-a-lipoic acid,Group III: Control aged rats,Group VI: Aged rats administered carnitinealone,Group V: Aged rats administered a-lipoic acidalone,Group VI: Aged rats administered L-carnitine andDL-a-lipoic acid,

L-Carnitine (300mg/kg bw/day) was dissolved in0.89% physiological saline and DL-a-lipoic acid(100mg/kg bw/day) was dissolved in 0.5% of KOHin physiological saline and administered via intra-gastric canula for 30 days. Control animals receivedphysiological saline alone. On completion of theexperimental period, animals were killed by cervi-cal decapitation. Heart was excised immediatelyand immersed in physiological saline and used forfurther analysis.

Isolation of mitochondria

Heart mitochondria were isolated by the method ofTakasawa et al.14 The heart tissue was put into ice-cold 50mM Tris-HCl (pH 7.4) containing 0.25Msucrose and homogenized. The homogenates werecentrifuged at 700g for 20min and then thesupernatant obtained were centrifuged at 14,000gfor 15min. The pellets were washed with 10mMTris-HCl (pH 7.8) containing 0.25M sucrose andfinally resuspended in the same buffer. Mitochon-drial protein was estimated by the method of Lowryet al.15 The purity of the mitochondria was checkedusing specific marker enzyme, succinate dehydro-genase.

Assay of Tri carboxylic acid (TCA) cycleenzymes

The activity of Isocitrate dehydrogenase wasassayed by the method of King.16 The activity ofa-ketoglutarate dehydrogenase was assayed by themethod of Reed and Mukherjee17 according to thecolorimetric determination of ferrocyanide pro-

duced by the decarboxylation of a-ketoglutaratewith ferricyanide as electron acceptor. The activityof succinate dehydrogenase was assayed by themethod of Slater and Bonner,18 in which the rate ofreduction of potassium ferricyanide was measuredin the presence of potassium cyanide. The activityof malate dehydrogenase was assayed by themethod of Ochoa.19

Assay of ETC complexes

Complex I activity was measured by following thedecrease in the absorbance due to the oxidation ofNADH at 340 nm and Complex II activity wasmeasured by following the reduction of 2,6-dichlorophenol indophenol at 600 nm according tothe method of Birch-Maching et al.20 The reductionof cytochorme c Fe3+ by decyl ubiquinol was usedfor measuring the activity of Complex III accordingto the method of Krahenbuhl et al.21 and ComplexIV activity was measured by following the oxidationof cytochrome c Fe2+.22

Statistical analysis

Values were expressed as mean7SD for six rats ineach group. Statistical significance of changes indifferent groups was evaluated by one-way analysisof variance followed by a post-hoc test using acomputerized SPSS package. Differences wereconsidered statistically significant at P valueso0.05.

Result

Table 1 depicts the TCA cycle enzymes in heartmitochondria of control and treated rats. In thepresent study, an age-dependent decrementðPo0:05Þ in the activities of TCA cycle enzymeshas been observed. The decrease being 32% forsuccinate dehydrogenase, 33% for malate andisocitrate dehydrogenase, and 35% for a-ketoglul-tarate dehydrogenase. Administration of bothcarnitine and lipoic acid for 30 days restored theactivities of these enzymes. The increase wasapproximately 1.4-fold for succinate dehydrogen-ase, 1.5-fold for malate dehydrogenase, 1.4-foldfor isocitrate dehydrogenase and 1.5-fold for a-ketoglutarate dehydrogenase in aged rats treatedwith carnitine and lipoic acid. No significant changewas observed in young rats treated with carnitineand lipoic acid.

Aging has caused a lowering of not only the TCAcycle enzymes but also the ETC complexes. Figures

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Table

1Effect

ofca

rnitinean

dlipoicac

idon

theTCAcycleen

zymes

ofhe

artmitoc

hond

riain

youn

gan

dag

edrat.

Parameters

Youn

gco

ntrol

Youn

gco

mbination

Age

dco

ntrol

Age

d+c

arnitine

Age

d+lipoicac

idAge

dco

mbination

Succinatedeh

ydroge

nase

39.837

4.71

4174.69

27.177

2.93�

33.177

3.54

y,z

3273.46

y,z

38.257

3.09

y

Malatedeh

ydroge

nase

21.137

2.37

21.837

2.54

14.087

1.43�

17.127

1.70

y,z

16.677

1.99

y,z

20.087

2.50

y

Isoc

itrate

deh

ydroge

nase

13.017

1.41

13.267

1.59

8.70

71.08�

10.447

0.16

y,z

10.277

1.18

y,z

12.027

1.41

y

a-ke

toglutaratedeh

ydroge

nase

8.92

71.07

9.44

71.16

5.75

70.71�

7.27

70.81

y,z

7.02

70.80

y,z

8.58

71.02

y

Values

areex

pressed

asmea

n7SD

forsixrats

inea

chgrou

p.Su

ccinatedeh

ydroge

nase,micromoles

ofsuccinateox

idized

per

min

per

mgprotein;malatedeh

ydroge

nase,na

nomoles

ofNADH

oxidized

per

min

per

mgprotein,isoc

itrate

deh

ydroge

nase,na

nomoles

ofa-ke

toglutarateform

edper

min

per

mgprotein;a-ke

toglutaratedeh

ydroge

nase,micromoles

ofpotassium

ferroc

yanideliberated

per

min

per

mgprotein.

�Differenc

esco

mpared

withyo

ungco

ntrolrats.

yDifferenc

esco

mpared

withag

edco

ntrolrats.

zDifferenc

esco

mpared

withag

edrats

trea

tedwithca

rnitinean

dlipo

icac

id.

Efficacy of L- carnitine and DL a lipoic acid in ameliorating mitochondrial enzymes during aging 797

1–4 present the activities of the ETC complexes.Observation of the aged rats has shown a decreasein the activities of these enzymes ðPo0:05Þ, thedecrease being 1.6-fold for Complex I, 1.5-fold forComplex II, 1.6-fold for Complex III and 1.5-fold forComplex IV in Group III when compared with Group I

Figure 1 Effect of carnitine and lipoic acid on Complex Iactivity in young and aged rat mitochondria. (a)Differences compared with young control rats, (b)Differences compared with aged control rats, (c) Differ-ences compared with aged rats treated with carnitineand lipoic acid. —Group I: Young rats administeredwith vehicle alone, —Group II: Young rats administeredwith L-carnitine and DL-a-lipoic acid, —Group III: Agedrats administered with vehicle alone, —Group IV: Agedrats administered with L-carnitine alone, —Group V:Aged rats administered with DL-a-lipoic acid alone,

—Group VI: Aged rats administered with L-carnitineand DL-a-lipoic acid.

Figure 2 Effect of carnitine and lipoic acid on Complex IIactivity in young and aged rat mitochondria. (a)Differences compared with young control rats, (b)Differences compared with aged control rats, (c) Differ-ences compared with aged rats treated with carnitineand lipoic acid. —Group I: Young rats administeredwith vehicle alone, —Group II: Young rats administeredwith L-carnitine and DL-a-lipoic acid, —Group III: Agedrats administered with vehicle alone, —Group IV: Agedrats administered with L-carnitine alone, —Group V:Aged rats administered with DL-a-lipoic acid alone,

—Group VI: Aged rats administered with L-carnitineand DL-a-lipoic acid.

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Figure 3 Effect of carnitine and lipoic acid on Complex IIIactivity in young and aged rat mitochondria. (a)Differences compared with young control rats, (b)Differences compared with aged control rats, (c) Differ-ences compared with aged rats treated with carnitineand lipoic acid. —Group I: Young rats administeredwith vehicle alone, —Group II: Young rats administeredwith L-carnitine and DL-a-lipoic acid, —Group III: Agedrats administered with vehicle alone, —Group IV: Agedrats administered with L-carnitine alone, —Group V:Aged rats administered with DL-a-lipoic acid alone,

—Group VI: Aged rats administered with L-carnitineand DL-a-lipoic acid.

Figure 4 Effect of carnitine and lipoic acid on ComplexIV activity in young and aged rat mitochondria. (a)Differences compared with young control rats, (b)Differences compared with aged control rats, (c) Differ-ences compared with aged rats treated with carnitineand lipoic acid. —Group I: Young rats administeredwith vehicle alone, —Group II: Young rats administeredwith L-carnitine and DL-a-lipoic acid, —Group III: Agedrats administered with vehicle alone, —Group IV: Agedrats administered with L-carnitine alone, —Group V:Aged rats administered with DL-a-lipoic acid alone,

—Group VI: Aged rats administered with L-carnitineand DL-a-lipoic acid.

S. Savitha et al.798

rats. Supplementation of carnitine and lipoic acidreverted the activities of these complexes to nearnormal levels. The increase was 50% for Complex I,39% for Complex II, 54% for Complex III and 40% forComplex IV in Group VI rats. Administration ofcarnitine and lipoic acid did not bring any sig-nificant changes in the mitochondrial enzymes ofyoung rats.

Discussion

For many years it has been proposed that the life-long production and accumulation of free radicalsas by-product of oxidative metabolism is at thebasis of the aging process.23 The enzymes locatedin mitochondria catalyze the oxidation of a numberof substrates via the citric acid cycle yieldingreducing equivalents. These reducing equivalentsare channeled through the respiratory chain for thesynthesis of ATP by oxidative phosphorylation,which provides the energy needed for many cellularfunctions.

The result of the present study shows that thedecline in mitochondrial activity is an age-relatedprocess. The enzymes of the TCA cycle were seento decrease significantly in aged rats which is inagreement with earlier reports which show asimilar decrease in the mitochondrial enzymes inageing.24 This reduction in the activities of specificdehydrogenases could be because of increased freeradical production leading to inefficient electrontransport resulting in oxidative damage to themitochondria thereby compromising their abilityto meet cellular energy demands. The inhibition ofthese enzymes may affect mitochondrial substrateoxidation resulting in reduced rate of transfer ofreducing equivalents to molecular oxygen anddepletion of energy production.

Levels of myocardial carnitine, an amino acidnecessary for shuttling fatty acyl moieties from thecytosol into the mitochondria for b oxidation wasseen to decline significantly in the rat heart withage.25 A deficiency of L-carnitine interferes withthe transport of fatty acids into the mitochondriaproducing an accumulation of both free fatty acidsin the cytoplasm and acyl CoA within the mitochon-dria. This leads to a significant loss in b oxidationof fatty acids and suggests that mitochondriabecome deprived of their major source for ATPsynthesis.26

Treatment with carnitine and lipoic acid hasshown to improve the activities of these enzymes inaged rats to near normalcy. Previous studies in ourlaboratory have shown carnitine to increase mito-chondrial enzymes by shuttling fatty acids into themitochondria.27 Similarly, earlier reports haveshown that lipoic acid improved the activities ofpyruvate dehydrogenase and a-ketoglutarate dehy-drogenase by improving the co-factor availability.28

Lipoic acid has also shown to increase glucoseuptake in cells.29 This glucose through the processof glycolysis could also increase pyruvate avail-ability for TCA cycle. Lipoic has also shownto possess potent antioxidant properties againsthydroxyl, peroxyl and superoxide radicals.30

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Efficacy of L- carnitine and DL a lipoic acid in ameliorating mitochondrial enzymes during aging 799

Therefore, preventing the free radical-inducedenzyme inactivation could be another possiblemechanism for the increase in the activities ofthe enzymes upon treatment.

Apart from a minor contribution of anaerobicglycolysis, the ATP essential for cellular activity isalmost exclusively mitochondrial in origin. TheseATP productions depend upon the proper function-ing of the mitochondrial ETC and of the functionallycoupled proton motive ATP-synthase (ATPase). Thedecline in the ability of cells to produce energy inthe form of ATP is one popular explanation for theage-related decline in the physiological function ofan organism. Therefore, we have evaluated theeffect of age on these electron chain complexes.

NADH-ubiquinone oxidoreductase, also known asComplex I, is a multisubunit integral membranecomplex of the mitochondrial ETC which catalyzeselectron transfer from NADH to ubiquinone.Coupled to the transfer of electrons, protons arevectorially translocated across the mitochondrialinner membrane to establish an electrochemicalgradient used for the synthesis of ATP. We observeda decrease in the activities of Complex I in agedrats on comparison with young rats. Complex I isgenerally reported to be impaired with age incardiac mitochondria.31 This decrease in theactivity of Complex I could be ascribed to theshortage of formation of its substrate, NADPH, thereducing equivalent formed in the TCA cycle. Asignificant increase was observed in the activity ofthis enzyme in Group VI rats when compared withGroup III rats. This could be attributed to theimprovement in the activities of TCA cycle enzymesby carnitine and lipoic acid, which would providethe substrates for the ETC. Additionally carnitineand lipoic acid could have increased the levels ofNADPH by increasing the levels of GSH.32

Cytochrome bc1 complex (Complex III) is consid-ered to be crucial for the activity of the entirerespiratory chain and appears to be well coupled withSuccinate dehydrogenase (Complex II). In the presentstudy, we observed an age-related decrease in bothcomplexes II and III activities. A possibility is that thephospholipid membrane environment, surroundingthe protein complexes, may become relatively lessoptimal during aging. Age-related alterations in themitochondrial membrane fluidity have indeed beenreported,33 which can have a considerable impact onthe activity of the respiratory chain as well as thegeneration of proton gradient. Activities of Com-plexes II and III also have shown to decline in oldrats.34 Combined treatment with carnitine and lipoicacid has shown to increase the activities of bothComplexes II and III in aged rats. Carnitine and lipoicacid stimulates the repair of mitochondrial mem-

branes and returns functionality to mitochondria.Thus, the mitochondrial membranes that havechanged over time are restored to the integrity tothose of young animals upon treatment.

Cytochrome c oxidase is the terminal enzyme ofthe mitochondrial respiratory chain, catalyzing thereduction of molecular oxygen with electrons fromreduced cytochrome c and concomitantly conser-ving the reaction energy by pumping protons acrossthe inner mitochondrial membrane. The activity ofthis enzyme was also seen to decrease in aged rats.This is in line with earlier reports, which show adecline in the activity of Complex IV in ageing.35

Paradies et al.36 have observed increased cyto-chrome c oxidase in aged rats on supplementationof acetyl carnitine. Cardiolipin is a structuralphospholipid that complexes with, and structurallysupports, electron transport system enzymes espe-cially the cytochrome c oxidase. Acetyl carnitinehas shown to restore cellular cardiolipin fatty acidcomposition,36 which could indirectly improve thelevels of this complex.

Mitochondrial DNA (mtDNA) is vulnerable to theattacks by ROS and free radicals that are generatedby electron leak of the respiratory chain of mito-chondria.37 Mitochondrial genome encodes ND1-6and NDL4 for Complex I; cytochrome c for ComplexII; COI-III for Complex IV and ATPase 6 and 8 forComplex V. Several mtDNA deletions have beenidentified in various tissues of old humans.38,39 Thusit can be hypothesized that multiple mtDNA re-arrangements occurring in tissue from aged indivi-duals might correlate with the observed progressivedecrease in the activity of these complexes. Increas-ing the antioxidant status in aged rats by carnitineand lipoic acid can protect the mtDNA against thefree radical attack and maintain its integrity.

In conclusion, our observations suggest that agingresults in the selective depletion of TCA cycleenzymes and a marked decline in the ETC com-plexes. Co-supplementation of carnitine with lipoicacid improved the status of these enzymes, theeffect being more pronounced with the combinedtreatment than when treated alone. As a conse-quence the utilization of carnitine and lipoic acidcan lead to an improvement in the quality of livingduring the later stages of life by revitalizing themitochondria and reversing the declining level ofthe body’s energy metabolism.

Acknowledgement

The present study was supported by the financialassistance of Science City, Government of TamilNadu, India.

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