Propionyl-l-carnitine prevents the progression of cisplatin-induced cardiomyopathy in a carnitine-depleted rat model

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Pharmacological Research xxx (2006) xxx–xxx

Propionyl-l-carnitine prevents the progression of cisplatin-inducedcardiomyopathy in a carnitine-depleted rat model

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Abdulhakeem A. Al-Majed, Mohamed M. Sayed-Ahmed∗, Abdulaziz A. Al-Yahya,Abdulaziz M. Aleisa, Salim S. Al-Rejaie, Othman A. Al-Shabanah

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Department of Pharmacology, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia7

Accepted 16 December 2005

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Abstract9

This study has been initiated to investigate whether endogenous carnitine depletion and/or carnitine deficiency is a risk factor during developmentof cisplatin (CDDP)-induced cardiomyopathy and if so, whether carnitine supplementation by propionyl-l-carnitine (PLC) could offer protectionagainst this toxicity. To achieve the ultimate goal of this study, a total of 60 adult male Wistar albino rats were divided into six groups. The first threegroups were injected intraperitoneally with normal saline, PLC (500 mg kg−1), andd-carnitine (500 mg kg−1) respectively, for 10 successive days.T ived ts werei hydrogenase( SH),t atic increasei in cardiact sei firmed theb sued and shouldb revents thep

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he 4th, 5th, and 6th groups were injected intraperitoneally with the same doses of normal saline, PLC andd-carnitine, respectively, for 5 successays before and after a single dose of CDDP (7 mg kg−1). On day 6 after CDDP treatment, animals were sacrificed, serum as well as hear

solated and analyzed. CDDP resulted in a significant increase in serum creatine phosphokinase isoenzyme (CK-MB) and lactate deLDH), thiobarbituric acid reactive substances (TBARS) and total nitrate/nitrite (NO(x)) and a significant decrease in reduced glutathione (Gotal carnitine, and adenosine triphosphate (ATP) content in cardiac tissues. In the carnitine-depleted rat model, CDDP induced dramn serum cardiomyopathy enzymatic indices, CK-MB and LDH, as well as progressive reduction in total carnitine and ATP contentissue. Interestingly, PLC supplementation resulted in a complete reversal of the increase in cardiac enzymes, TBARS and NOx, and the decrean total carnitine, GSH and ATP, induced by CDDP, to the control values. Moreover, histopathological examination of cardiac tissues coniochemical data, where PLC prevents CDDP-induced cardiac degenerative changes whiled-carnitine aggravated CDDP-induced cardiac tisamage. In conclusion, data from this study suggest for the first time that carnitine deficiency and oxidative stress are risk factorse viewed as mechanisms during development of CDDP-related cardiomyopathy and that carnitine supplementation, using PLC, progression of CDDP-induced cardiotoxicity.2005 Published by Elsevier Ltd.

eywords: Cisplatin; Carnitine deficiency; Cardiomyopathy;d-carnitine; Proionyl-l-carnitine

. Introduction

Cisplatin, cis-diamminedichloroplatinum II (CDDP), is annorganic platinum compound with a broad-spectrum antineo-lastic activity against various types of animal and human

umours[1]. Unfortunately, the optimal usefulness of CDDP asn important anticancer drug is usually limited secondary to itsose related nephrotoxicity[2,3]. It is well known that CDDP-

nduced nephrotoxicity is the most important dose-limiting fac-or in cancer chemotherapy[2,3].

∗ Corresponding author. Tel.: +966 506065734; fax: +966 1 467 7200.E-mail address: [email protected] (M.M. Sayed-Ahmed).

Earlier studies have reported that CDDP therapy is usassociated with cardiotoxicity[4,5]. Cardiac events, reportedmany case reports, may include electrocardiographic chaarrythmias, myocarditis, cardiomyopathy and congestivefailure [6–8]. CDDP-induced cardiotoxicity was reported todue to its disposition in the sinoatrial-node area, which mayto bradycardia[9]. Combinations of CDDP with other anticandrugs as methotrexate, 5-fluorouracil, bleomycin and doxbicin are associated with lethal cardiomyopathy[10–13].

CDDP is a well-known renal tubular toxin, leadingincreased excretion of a number of vital endogenous substincludingl-carnitine[14,15]. Under normal physiological coditions,l-carnitine is highly conserved since 90% of the filtel-carnitine is reabsorbed at the proximal tubular level[16]. Ithas been reported that CDDP inhibited carnitine reabsor

043-6618/$ – see front matter © 2005 Published by Elsevier Ltd.oi:10.1016/j.phrs.2005.12.005

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2 A.A. Al-Majed et al. / Pharmacological Research xxx (2006) xxx–xxx

with the consequent increase in urinary excretion of carnitine53

that represents an early marker of CDDP-induced nephrotoxic-54

ity [15]. Recent study in our laboratory have demonstrated the55

progression of CDDP-induced nephrotoxicity under condition56

of carnitine deficiency and that carnitine supplementation could57

protect against this toxicity[17].58

However, the effects of CDDP on the cardiac function under59

condition of carnitine depletion and the role of carnitine in60

CDDP-related cardiomyopathy are not studied yet. Therefore,61

the present study have selected the natural short chain deriva-62

tive of l-carnitine, Propionyl-l-carnitine (PLC), which proved63

efficacy in treatment of many forms of cardiomyopathies and64

have several therapeutic advantages overl-carnitine or acetyl-65

carnitine. First, PLC has a higher transport rate into cardiac66

myocytes thanl-carnitine, thus increasing myocardial carni-67

tine content and substrates oxidation rates[18–20]. Second,68

PLC has higher affinity for muscular carnitine transferase, thus69

PLC is highly specific to cardiac and skeletal muscles[18,21].70

Third, PLC stimulates with a better efficiency of the krebs71

cycle by providing it with a very easily usable substrate, pro-72

pionate, which is rapidly transformed into succinate without73

energy consumption (anaplerotic pathway)[18,20]. Further-74

more, PLC suppresses the formation of hydroxyl radicals, thus75

acting as a free radical scavenger[22]. Finally, due to the par-76

ticular structure of the molecule with a long lateral tail, PLC77

has a specific pharmacological action independently of its effect78

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store, KSU, KSA. PLC andd-carnitine (products of Sigma-Tau105

Pharmaceuticals, Pomezia, Italy) were kindly supplied by Dr.106

Menotti Calvani, Sigma-Tau, Pomezia, Italy. All other chemi-107

cals used were of the highest analytical grade. 108

2.3. Carnitine-depleted model 109

Experimental animal models of carnitine deficiency were110

developed by Paulson and Shug[24], Whitmer[25]; and Tsoko 111

et al.[26]. In the current study, carnitine deficiency was induced112

in rats by daily intraperitoneal (i.p.) injection ofd-carnitine, 113

the inactive isomer, at dose level of 500 mg kg−1 for 10 suc- 114

cessive days according to Sayed-Ahmed et al.[17]. Depletion 115

of l-carnitine byd-carnitine occurs via an exchange of thed- 116

andl-isomers across the cell membrane where the intracellu-117

lar l-carnitine was shown to exchange with the extracellular118

d-carnitine. Moreover,d-carnitine possesses an inhibitory effect119

upon carnitine transferase enzymes and competitive inhibitory120

effect uponl-carnitine uptake[24–26]. 121

2.4. Experimental design 122

A total of 60 adult male Wistar albino rats were used and123

divided at random into 6 groups of 10 animals each. The first124

three groups were injected with normal saline (0.5 ml 200 gm−1125

body weight, i.p.), PLC (500 mg kg−1, i.p.), andd-carnitine 126

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n metabolism resulting in peripheral dilatation and posnotropism[23].

Up to date, in the literature, we could not find any stnvestigating the effects of CDDP on the myocardium unondition of endogenous carnitine depletion and the rolel-arnitine supplementation in CDDP-induced cardiomyopaherefore, this study has been initiated to investigate whndogenous carnitine depletion and/or carnitine deficiencisk factor and should be viewed as a mechanism during dpment of CDDP-induced cardiomyopathy and if so, whearnitine supplementation by PLC could offer protection aghis toxicity.

. Materials and methods

.1. Animals

Adult male Wistar albino rats, weighing 230–250 g, wbtained from the Animal Care Center, College of Pharming Saud University, Riyadh, Saudi Arabia and were hou

n metabolic cages under controlled environmental condi25◦C and a 12 h light/dark cycle). Animals had free accesulverized standard rat pellet food and tap water unless oise indicated. The protocol of this study has been approveesearch Ethics Committee of College of Pharmacy, Kingniversity (KSU), Riyadh, Kingdom Saudi Arabia (KSA).

.2. Materials

Cisplatin (cisplatyl 50 mg, Laboratoire Roger Bellon, Franas purchased from King Khalid University Hospital d

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500 mg kg−1, i.p.) respectively, for 10 successive days.th, 5th, and 6th groups were injected with the same df normal saline, PLC andd-carnitine, respectively for 5 suessive days before a single dose of CDDP (7 mg kg−1, i.p.)nd continued for 5 successive days after CDDP treatmenay 6 after CDDP administration, animals were anestheith ether, and blood samples were obtained by heart punerum was separated for measurement of lactate dehydrog

LDH), creatine phosphokinase iso-enzyme (CK-MB) andarnitine. Animals were then sacrificed by decapitationxposure to ether in a dessicator kept in a well-functioningnd heart was quickly excised, washed with saline, blottedpiece of filter paper and homogenized, in normal salin

% perchloric acid as indicated in the procedures of meaent of each parameter, using a Branson sonifier (250, Vcientific, Danbury, Conn., USA). Heart specimens from eroup were removed to be examined histopathologically,ere fixed in 10% neutral buffered formalin, sectioned at 3�mnd stained with Hematoxylin and Eosin (H&E) stain for liicroscopic examination to evaluate the following parame

1) muscle fiber damage; (2) cytoplasmic damage; (3) hehage. The marking system used was according to the follocale: (A) no degenerative change: 0+; (B) mild degenerhange: 1+; (C) moderate degenerative change: 2+; (D) segenerative change: 3+.

.5. Assessment of cardiac enzymes

Serum activities of LDH and CK-MB were determinccording to the methods of Buhl and Jackson[27] and Wund Bowers[28], respectively.

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A.A. Al-Majed et al. / Pharmacological Research xxx (2006) xxx–xxx 3

2.6. Determination of reduced glutathione and lipid155

peroxidation in cardiac tissues156

The tissue levels of the acid soluble thiols, mainly GSH,157

were assayed spectrophotometrically at 412 nm, according to158

the method of Ellman[29], using a Shimadzu (Tokyo, Japan)159

spectrophotometer. The contents of GSH were expressed as160

�mol g−1 wet tissue. The degree of lipid peroxidation in cardiac161

tissues was determined by measuring thiobarbituric acid reactive162

substances (TBARS) in the supernatant tissue from homogenate163

[30]. The homogenates were centrifuged at 3500 rpm and super-164

natant was collected and used for the estimation of TBARS. The165

absorbance was measured spectrophotometrically at 532 nm and166

the concentrations were expressed as nmol TBARS g−1 wet tis-167

sue.168

2.7. Determination of total nitrate/nitrite (NO(x))169

concentrations in cardiac tissues170

Total nitrate/nitrite (NO(x)) was measured as stable end prod-171

uct, nitrite, according to the method of Miranda et al.[31]. The172

assay is based on the reduction of nitrate by vanidium trichloride173

combined with detection by the acidic griess reaction. The diazo-174

tization of sulfanilic acid with nitrite at acidic pH and subsequent175

coupling with N-(10 naphthyl)-ethylenediamine produced an176

intensely colored product that is measured spectrophotometri-177

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using ODS-Hypersil, 150× 4.6 mm i.d., 5�m column (Supelco 205

SA, Gland, Switzerland) and 75 mM ammonium dihydrogen206

phosphate as mobile phase. The peak elution was followed at207

254 nm. 208

2.10. Statistical analysis 209

Differences between obtained values (mean± S.E.M., 210

n = 10) were carried out by one way analysis of variance211

(ANOVA) followed by the Tukey–Kramer multiple compari- 212

son test. Ap-value of 0.05 or less was taken as a criterion for a213

statistically significant difference. 214

3. Results 215

Fig. 1shows the effects of CDDP on serum cardiac enzymes,216

LDH (A) and CK-MB (B), in PLC-supplemented and carnitine-217

Fig. 1. Effect of CDDP, PLC,d-carnitine and their combination on serum car-diomyopathy enzymatic indices, LDH (A) and CK-MB (B) in rats. Data arepresented as mean± S.E.M. (n = 10). (*) and (#) indicate significant changefrom control and CDDP, respectively, atp < 0.05 using ANOVA followed byTukey–Kramer as a post ANOVA test.

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ally at 540 nm. The levels of NOx were expressed as�mol g−1

et tissue.

.8. Determination of total carnitine levels in serum andardiac tissues

Heart homogenate was prepared in ice-cold 6% perchcid and centrifuged at 8000× g for 10 min. The supernatant wsed for the estimation of free carnitine, whereas the remaellet was used for determination of long chain acyl ca

ine after hydrolysis in 1 M KOH at 65◦C for 1 h according tlhomida[32]. Carnitine was determined in serum and thebtained tissue samples using HPLC after precolumn deriva

ion with l-aminoanthracene as previously described by Lot al. [33]. The mobile phase was prepared by mixing 70f 0.1 M ammonium acetate pH 3.5 with 300 ml of acetonithromatographic separation was performed at a flow ra.3 ml min−1, using a Kromasil C18, 250× 4.6 mm i.d. 5�molumn (Saulentechnik Knayer, Berlin, Germany). The exion and emission wavelengths of the spectrofluorimeter48 and 418 nm, respectively.

.9. Determination of adenosine triphosphate in cardiacissues

Adenosine triphosphate was determined in heart tissuesPLC according to Botker et al.[34]. In brief, heart tissuas homogenized in ice-cold 6% perchloric acid, centrifut 1000 rpm for 15 min at 0.5◦C, and the supernatant fluid w

njected into HPLC after neutralization to pH 6–7. Chromraphic separation was performed at a flow rate of 1.2 ml m−1,

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4 A.A. Al-Majed et al. / Pharmacological Research xxx (2006) xxx–xxx

Fig. 2. Effect of CDDP, PLC,d-carnitine and their combination on the levels ofthiobarbituric acid reactive substances (A), reduced glutathione (B), and totanitrate/nitrite (C) in rat cardiac tissues. Data are presented as mean± S.E.M.(n = 10). (*) and (#) indicate significant change from control and CDDP, respec-tively, atp < 0.05 using ANOVA followed by Tukey–Kramer as a post ANOVAtest.

depleted rats. Six days after treatment, a single dose of CDDP218

(7 mg kg−1) resulted in a significant 35 and 51% increase in219

serum LDH and CK-MB, respectively, as compared to the con-220

trol group. Administration of either PLC ord-carnitine alone for 221

10 successive days showed non-significant change. Combined222

treatment with CDDP andd-carnitine resulted in a significant 223

24 and 100% increase in serum LDH and CK-MB, respectively,224

as compared to CDDP alone. Interestingly, administration of225

PLC in combination with CDDP resulted in a complete reversal226

of CDDP-induced increase in serum LDH and CK-MB to the227

control values. 228

Fig. 2shows the effects of CDDP, PLC,d-carnitine and their 229

combination on oxidative stress biomarkers namely TBARS (A),230

GSH (B), and NOx (C) in cardiac tissues. CDDP resulted in a231

significant 40% decrease in GSH and a significant 60 and 99%232

increase in TBARS and NO(x), respectively, as compared to233

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lig. 3. Effect of CDDP, PLC,d-carnitine and their combination on total carni-

ine levels in serum (A) and cardiac tissues (B) in rats. Data are presented asean± S.E.M. (n = 10). (*) and (#) indicate significant change from control andDDP, respectively, atp < 0.05 using ANOVA followed by Tukey–Kramer as aost ANOVA test.

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Fig. 4. Effect of CDDP, PLC,d-carnitine and their combination on adenosinetriphosphate level in rat cardiac tissues. Data are presented as mean± S.E.M.(n = 10). (*) and (#) indicate significant change from control and CDDP, respec-tively, atp < 0.05 using ANOVA followed by Tukey–Kramer as a post ANOVAtest.

the control group. Treatment with PLC for 10 successive days234

showed a significant 32% increase in GSH and a significant 52235

and 54% decrease in TBARS and NOx, respectively. Similarly,236

d-carnitine alone induced a significant 50% increase in GSH237

and a significant 50 and 72% decrease in TBARS and NOx, 238

respectively. Administration of either PLC ord-carnitine for 5 239

days before and after a single dose of CDDP, resulted in a com-240

plete reversal of CDDP-induced decrease in GSH and increase241

in TBARS and NOx levels in heart tissues to the control values.242

Fig. 3shows the effects of CDDP, PLC,d-carnitine and their 243

combination on total carnitine levels in serum (A) and cardiac244

tissues (B). Treatment with CDDP resulted in a significant 33%245

decrease in total carnitine level in heart tissues, whereasd- 246

carnitine resulted in a significant 61% decrease as compared to247

the control group. Combination ofd-carnitine with a single dose 248

of CDDP induced a significant 44, 67 and 78% decrease of total249

carnitine content in cardiac tissues compared to the results of250

d-carnitine, CDDP and control, respectively. Administration of251

PLC for 5 days before and after a single dose of CDDP resulted252

in a complete reversal of cisplatin-induced decrease in total car-253

nitine content in cardiac tissues to the control values. Worth254

mentioning is that none of these treatments showed any signifi-255

cant changes in total carnitine levels in serum. 256

The effects of CDDP on ATP level in cardiac tissues from257

carnitine-depleted and supplemented rats are shown inFig. 4. 258

CDDP resulted in a significant 18% decease of ATP level in259

cardiac tissues relative to the values of the control group. Admin-260

istration of d-carnitine for 10 successive days showed 35%261

decrease, whereas PLC resulted in non-significant change, as262

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ig. 5. A photomicrographs of the heart specimen stained with hematoxylin anbers and homogenous acidophilic cytoplasm. (B) Heart of rat treated with CDith blood. (C) Heart of rat treated with CDDP andd-carnitine showing extensive vundles indicate massive bleeding. (D) Heart of rat treated with CDDP and PL

n small restricted foci in the vicinity of normal muscle fibers.

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d eosin (H&E X 200). (A) Heart from control rat showing normal bundles of muscleDP showing vacuolated cytoplasm of many muscle cells, blood vessels are engorgedacuolation of the cytoplasm of cardiac muscle fibers, blood cells in-betweens theC in which the cardiac muscle fibers appear as control with minimal sign of toxicity

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6 A.A. Al-Majed et al. / Pharmacological Research xxx (2006) xxx–xxx

compared to the control group. In carnitine-depleted rats, CDDP263

resulted in a significant 50 and 38% decrease in ATP level as264

compared to the control and CDDP groups, respectively. Con-265

versely, administration of PLC for 5 days before and after a266

single dose of CDDP resulted in a complete reversal of CDDP-267

induced decrease in ATP level in cardiac tissues to the control268

values.269

Fig. 5shows the histopathological changes in cardiac tissues270

induced by CDDP in carnitine-depleted and supplemented rats.271

Sections from cardiac tissues of control rats showed bundles272

of normal muscle fibers and the cytoplasm appears homoge-273

nous acidophilic with central nucleus (Fig. 5A). On the other274

hand, animals treated with CDDP alone showed degenerative275

changes, vacuolated cytoplasm of many muscle cells and blood276

vessels are engorged with blood (score 2,Fig. 5B). This tissue277

injury was aggravated in cardiac sections of rats treated with278

CDDP plusd-carnitine, where they showed massive degenera-279

tive changes. The muscle fibers appeared markedly vacuolated in280

wide areas, beside the bleeding where the blood cells were scat-281

tered in-between bundles of cardiac muscle (score 3,Fig. 5C).282

Interestingly, heart specimens from rats treated with CDDP and283

PLC (Fig. 5D) revealed minimal degenerative changes in which284

the cardiac muscle fibers appear as control with minimal sign of285

toxicity in small restricted foci in the vicinity of normal muscle286

fibers.287

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tion of CDDP to carnitine-depleted rats produced a progressive318

increase in the activities of LDH and CK-MB as well as massive319

bleeding and degenerative changes in cardiac tissues (Fig. 5C). 320

Data from this study revealed that CDDP significantly321

increased NOx and TBARS and decreased GSH in cardiac tis-322

sues, suggesting that oxidative stress induced by CDDP may323

play a role in CDDP-induced cardiac damage. It seems that the324

heart is the most susceptible organ to oxidative stress since car-325

diac tissues has very low level of antioxidant enzymes such as326

catalase and superoxide dismutase[37]. However, no previous 327

studies are available on the role of oxidative stress exerted by328

CDDP in cardiac-induced damage. Recent studies have reported329

that beside kidney, CDDP-induced oxidative stress damage to330

liver and lens tissues in rats secondary to the formation of both331

reactive oxygen and nitrogen species[38,39]. Moreover, Lee 332

et al. [40] reported that the degeneration of the auditory sys-333

tem of mice by CDDP was due to CDDP-induced increase in334

hydroxyl radical, a marker for lipid peroxidation, and nitroty-335

rosine, a marker for protein peroxidation. The contribution of336

NOx in CDDP-induced cytotoxicity and organs toxicity has been337

previously reported[41–43]. Nitric oxide is known to inhibit 338

DNA repair proteins, thereby inhibiting the ability of the cell to339

repair damaged DNA[42]. Previous studies have demonstrated340

that increased NOx concentrations after CDDP was a secondary341

event following the increase in the inducible nitric oxide syn-342

thase[43–45]. 343

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. Discussion

It has been reported that increased urinary excretionl-arnitine is an early marker in CDDP-induced nephrotox15]. Under such condition, CDDP may induce a seconarnitine deficiency that might enhance CDDP-induced organs toxicity. Recent study in our laboratory have demtrated the progression of CDDP-induced nephrotoxicity uondition of carnitine deficiency[17]. However, the effects oDDP on the heart under condition of carnitine depletion

he role of carnitine in CDDP-induced cardiotoxicity aretudied yet. This study has been initiated to investigate whndogenous carnitine depletion and/or carnitine deficiencisk factor during development of CDDP-induced cardiomyohy and if so, whether carnitine supplementation by PLC cffer protection against this toxicity.

Data presented here demonstrate that CDDP increasedardiotoxicity enzymatic indices (LDH and CK-MB) and cauevere histopathological lesions in cardiac tissues (Fig. 5B). Thisffect could be a secondary event following CDDP-inducederoxidation of cardiac membranes with the consequent inc

n the leakage of LDH and CK-MB from cardiac myocytncreased release of LDH by CDDP has been previously rep35]. Fascinatingly, PLC prevented the increase in LDH andB induced by CDDP, suggesting that PLC may have po

ial protective effect against CDDP-induced cardiac damhis effect could be due to membrane stabilization by thl-arnitine portion of PLC with the consequent decrease in ref cardiac enzymes. Indeed, the interaction ofl-carnitine witharcolemmal phospholipids and mitochondrial membraneeen previously reported[36]. On the other hand, administr

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In the current study, both PLC andd-carnitine prevented thncrease in TBARS and NOx and the decrease in GSH inducedDDP in cardiac tissues, suggesting that both compounds

and/or induce) antioxidant effect. Previous studies have detrated that both thed- andl-forms of carnitine and its shohain derivatives have similar non-enzymatic free radical snging activity[17,46,47]. Although, both PLC andd-carnitineave similar antioxidant activity, in our study, PLC preventedrogression of CDDP-induced cardiomyopathy andd-carnitineggravated this toxicity. Therefore, one can anticipate that o

ive stress plays an important role but not the only mechay which CDDP-induced its cardiotoxicity.

Data presented here showed that CDDP significaecreased total carnitine in cardiac tissues. It seems thaesults are unique since no available data about the effect oitine deficiency or carnitine supplementation on cardiac tisf rats treated with CDDP. Since it has been reported thatroximal tubules are severely damaged by CDDP[48], there-

ore, impaired endogenous synthesis and inhibition of tubeabsorption of carnitine is the most likely explanation foecrease after CDDP administration. It was reported thatent with CDDP is associated with a tenfold increase in r

arnitine excretion, most likely due to inhibition of carniteabsorption by the proximal tubule of the nephron[15,49].

Under our experimental conditions,d-carnitine significantlyecreased the level of carnitine in cardiac tissues whereas p

evels were not changed. Our results are in good agreeith those previously reported[24,25]. Although, d-carnitinelone decreased cardiac carnitine content more than CDDPDDP increased LDH and CK-MB. This argues against ca

ine deficiency as a risk factor in CDDP-induced cardiac dam

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A.A. Al-Majed et al. / Pharmacological Research xxx (2006) xxx–xxx 7

A possible explanation for this is thatd-carnitine, via its anti-375

lipid peroxidation, causes stabilization of cardiac membranes376

and prevents the leakage of cardiac enzymes, whereas CDDP,377

via increasing lipid peroxidation, causes irreversible modifica-378

tion of membrane structures and functions with the consequent379

leakage of cardiac enzymes.380

The marked decrease in total carnitine level in the heart381

by CDDP in the carnitine-depleted rat model (Fig. 3B), sug-382

gests thatd-carnitine and CDDP depletesl-carnitine by differ-383

ent mechanisms.d-carnitine-induced carnitine deficiency was384

reported to be due to the inhibition of carnitine transferase385

enzyme, inhibition of carnitine transport and competitive inhi-386

bition of l-carnitine uptake or exchange with extracellulard-387

carnitine[24–26]. On the other hand, secondary carnitine defi-388

ciency induced by the nephrotoxic effects of CDDP may be389

attributed to the inhibition of carnitine reabsorbtion by the prox-390

imal tubules of the nephron. Since kidney is the major site391

for endogenous carnitine biosynthesis in rats, therefore, kidney392

damage induced by CDDP may lead to inhibition of carni-393

tine synthesis and consequently leading to carnitine deficiency394

[15,49]. Moreover, hyponatraemia induced by CDDP might hin-395

der the action of sodium-dependent carnitine transporter, which396

may worsen the condition. This marked decrease (78%) of car-397

nitine level in cardiac tissue after combined treatment of CDDP398

andd-carnitine was parallel to the marked increase in LDH and399

CK-MB and the massive degenerative changes in cardiac tis-400

s itine401

d thy.402

M403

C itine404

d vate405

c rdia406

c acid407

o408

a on o409

l ATP410

p411

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c reas415

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C PLC418

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c ours422

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models of CDDP and paclitaxel-induced neuropathy, recent432

studies have recommended ALC as a specific protective agent for433

chemotherapy-induced neuropathy without showing any inter-434

ference with the antitumour activity of the drugs[54,55]. Worth 435

mentioning is thatl-carnitine, ALC and PLC do not interfere436

with the antitumour activity of anticancer drugs[55–57]. 437

The progression of CDDP-induced cardiomyopathy438

observed in this study could be secondary to its major dose-439

limiting nephrotoxicity. This hypothesis is supported by the fact440

that secondary carnitine deficiency reported in patients with441

end-stage renal disease undergoing hemodialysis is associated442

with severe cardiovascular and neuromuscular disorders[58]. 443

Our results are pioneer to studies that will be performed444

with carnitine and its short chain derivatives to protect from445

CDDP-induced cardiomyopathy. In conclusion, data from this446

study suggest for the first time that: (1) endogenous carnitine447

depletion and/or deficiency is a risk factor and should be448

viewed as a mechanism during development of CDDP-related449

cardiomyopathy; (2) oxidative stress plays an important role in450

CDDP-induced cardiomyopathy; (3) PLC prevents the progres-451

sion of CDDP-induced cardiotoxicity. It would be worthwhile452

studying the effects of carnitine supplementation in CDDP-453

treated cancer patients, in the hope of reducing CDDP-induced454

nephrotoxicity, neurotoxicity, and cardiomyopathy. 455

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ues, which may point to the possible consideration of carneficiency as a risk factor in CDDP-induced cardiomyopaost probably,d-carnitine via its depletion ofl-carnitine, andDDP partly through oxidative stress and partly due to carnepletion produced such myocardial damage. This aggraardiomyopathy could be explained on the basis of myocaarnitine deficiency with subsequent impairment of fattyxidation and ATP production. It is well known thatl-carnitine isn essential cofactor for mitochondrial transport and oxidati

ong chain fatty acids which are the preferred substrates forroduction in normal, well-oxygenated adult myocardium[50].epletion of the heart from carnitine either by CDDP,d-carnitinend both would impair the beta-oxidation of long chain fcids with the consequent decrease in ATP production andontractile function. This was supported by the marked decf ATP levels in heart tissues observed in carnitine-depats, which renders the cardiac cells vulnerable to damagDDP. On the other hand, carnitine supplementation byrevented CDDP-induced decrease in ATP by replenishinyocardium with adequate carnitine for its energy produche animal model used in this study could reflect the clinourse of CDDP in cachectic patients with malignant tumhom serum and urinary carnitine profile are altered and dped hypocarnitinaemia after repeated cycles of chemothe

ncluding CDDP[15,51].In this study, the protective effect achieved by PLC aga

DDP-induced cardiomyopathy is in good agreementecent results from clinical pilot studies which have demtrated that acetyl-l-carnitine (ALC) seems to be an effective aell-tolerated agent for the treatment of CDDP and paclita

nduced peripheral neuropathy[52,53]. Moreover, in an anima

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cknowledgements

The present work was supported by operating grantesearch Center, College of Pharmacy, King Saud Unive

CPRC154).

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