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À professora Ana Lúcia dos Anjos Ferreira, em primeiro lugar, por me aceitar como sua orientada e participar ativamente de toda a execução deste trabalho. Mas, não poderia deixar de salientar que o papel de professora, pouco valor teria não fossem o carinho e a dedicação com que trata seus orientados. Isso a faz mais que uma orientadora tornando-a uma grande amiga. Obrigada pela oportunidade.
Ao Flávio (“Flavinho”) que, aos trancos e, sempre com muita paciência comigo, jamais deixou de me auxiliar na execução deste trabalho, mostrando muita compreensão especialmente nos momentos de maior nervosismo. Obrigada pela sua ajuda.
À professora Beatriz B. Matsubara pela fundamental cooperação na execução dos ecocardiogramas e na elucidação das dúvidas que foram surgindo durante este estudo.
Ao Professor Carlos Roberto Padovani pela valiosa orientação e pelo estudo estatístico.
À minha família, em especial à minha mãe. Gostaria muito de registrar minha gratidão e respeito por tudo que ela faz por mim até os dias de hoje e que sei que vai continuar fazendo por toda minha vida. Outro agradecimento especial vai para minha filha Isadora que, hoje, não pode ter ideia do que representa mais esta conquista. Um dia sei que vai saber que toda minha luta, empenho e carinho com esta tese passou a ter um significado maior depois que ela nasceu! Ao meu marido Alexandre pela companhia e ajuda constante com nossa filha nesta etapa final, toda minha gratidão.
A toda equipe do Laboratório Experimental de Clínica Médica por todo apoio e, em especial:
...à Sueli, deixo renovadas minhas desculpas e agradeço imensamente pela sua ajuda!
...à Camila pela ajuda com o HPLC, no início e, principalmente, na conclusão dos dados. Obrigada pela compreensão!
...ao Zé, sempre tão prestativo e carinhoso e que, em meio a tantas outras atribuições, sempre procurou me atender no que fosse possível. Valeu!!!
E como não poderia deixar de ser, aos ratinhos que foram
usados em meu trabalho, assim como tantos animais que são
sacrificados em nome da ciência. A vocês todo o meu respeito
e gratidão! Como veterinária, espero com este trabalho, poder
oferecer cada vez mais qualidade de vida àqueles que tanto
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Níveis de licopeno no tecido cardíaco (nmol/kg de tecido)
G1 G2 G3 G4
0,00 (0,00) A 64,93 (32,28) B 0,00 (0,00) A 0,00 (0,00) A
G5 G6 G7 G8
0,00 (0,00) A 86,23 (125,10) B 0,00 (0,00) A 0,00 (0,00) A
Níveis de α-tocoferol no tecido cardíaco (mmol/kg de tecido)
G1 G2 G3 G4
52,09 (17,62) A 62,03 (17,96) A 109,91 (18,43) B 114,86 (25,52) B
G5 G6 G7 G8
52,33 (19,66) A 49,86 (15,58) A 108,60 (34,49) B 106,64 (37,21) B
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G1 G2 G3 G4 0,00 (0,50) AB 0,00 (0,00) A 2,00 (0,50) C 1,00 (0,50) BC
G5 G6 G7 G8
1,00 (1,00) 1,00 (1,00) 2,00 (1,00) 1,00 (0,50)
Necrose Nuclear (NN)
G1 G2 G3 G4 0,00 (0,00) aA 0,00 (0,00) A 1,00 (0,50) B 1,50 (0,50) B
G5 G6 G7 G8
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Degeneração Miocítica (DM)
G1 G2 G3 G4 0,00 (0,00) aA 0,00 (0,00) A 2,00 (1,00) B 2,00 (0,50) B
G5 G6 G7 G8
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Desorganização Celular (DC)
G1 G2 G3 G4 0,00 (0,00) aA 0,00 (0,00) A 1,00 (0,50) B 1,50 (0,50) B
G5 G6 G7 G8
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G1 G2 G3 G4 54,50 (14,00) a 45,00 (10,00) a 45,50 (9,00) a 56,00 (16,00) a
G5 G6 G7 G8 315,00 (165,00) b 260,00 (189,50) b 193,00 (138,00) b 224,00 (96,50) b
LDL colesterol (mg/dL)
G1 G2 G3 G4 11,50 (4,00) aAB 9,00 (6,00) aA 11,50 (10,50) aAB 27,00 (6,00) B
G5 G6 G7 G8 88,50 (48,00) b 49,00 (45,50) b 63,50 (30,00) b 75,00 (70,50)
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G1 G2 G3 G4 G5 G6 G7 G8
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G1 G2 G3 G4 G5 G6 G7 G8
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G1 G2 G3 G4 G5 G6 G7 G8
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G1 G2 G3 G4 G5 G6 G7 G8
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G1 G2 G3 G4 G5 G6 G7 G8
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G1 G2 G3 G4 G5 G6 G7 G8
E A 0 10 20 30 40 50 60 70
G1 G2 G3 G4 G5 G6 G7 G8
A ¨d 0 10 20 30 40 50 60
G1 G2 G3 G4 G5 G6 G7 G8
Dd FE 74 76 78 80 82 84 86 88 90 92
G1 G2 G3 G4 G5 G6 G7 G8
FE FC 0 50 100 150 200 250 300 350
G1 G2 G3 G4 G5 G6 G7 G8
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G1 G2 G3 G4 G5 G6 G7 G8
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Colesterol total (mg / dl)
G1 G2 G3 G4 54,50 (14,00) a 45,00 (10,00) a 45,50 (9,00) a 56,00 (16,00) a
G5 G6 G7 G8 315,00 (165,00) b 260,00 (189,50) b 193,00 (138,00) b 224,00 (96,50) b
HDL colesterol (mg / dl)
G1 G2 G3 G4 32,50 (9,50) a 31,00 (14,00) a 27,50 (9,00) a 31,00 (9,00) a
G5 G6 G7 G8 126,50 (60,00) b 112,00 (59,00) b 91,00 (75,50) b 108,00 (84,50) b
Triglicérides (mg / dl)
G1 G2 G3 G4 74,50 (39,50) a 79,00 (53,50) a 111,00 (63,50) 75,50 (48,50) a
G5 G6 G7 G8 400,00 (832,00) b 779,00 (738,50) b 199,50 (422,50) 254,00(986,00) b
LDL colesterol (mg / dl)
G1 G2 G3 G4 11,50 (4,00) aAB 9,00 (6,00) aA 11,50 (10,50) aAB 27,00 (6,00) B
G5 G6 G7 G8 88,50 (48,00) b 49,00 (45,50) b 63,50 (30,00) b 75,00 (70,50)
J < ! ) ! ! #
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Artigo Original
Alpha-tocopherol-induced cardiotoxicity in rats: an echocardiographic and
histological assessment
Maria Carolina Munhoz Oliveira Nascimento1, Beatriz Bojikian Matsubara1, Luiz Shiguero
Matsubara1, Camila Renata Correa1, Elenize Jamas Pereira1, Priscila Lucelia Moreira1, Caio
Henrique Paganini Burini2, Kyung-Jin Yeum3, Ana Lucia Anjos Ferreira1
1
Department of Internal Medicine, Botucatu School of Medicine, UNESP, São Paulo State
University, CEP: 18618-970, Botucatu, SP, Brazil;
2
Department of Veterinary Pathology, School of Veterinary Medicine, UNESP, São Paulo
State University, CEP: 18618-000, Botucatu, SP, Brazil.
3
United States Department of Agriculture, Human Nutrition Research Center on Aging at
Tufts University, Boston, MA, 02111, USA;
Address correspondence to: Maria Carolina M.O. Nascimento; Dept. Clinica Medica;
Faculdade Medicina de Botucatu; UNESP; CEP: 18618-970; Botucatu; SP; Brazil; phone
+55-14-3882.2969; FAX: +55-14-3882.2238; carolinamon@terra.com.br
Abbreviations
A, late peak transmitral flow velocity;
E, early peak transmitral flow velocity;
E / A, left ventricle diastolic function index;
FS, left ventricle fractional shortening or left ventricle diameter variation percentage [(LVD
– LVS) / LVD] x 100;
EF, ejection fraction (EF) (LVD3 – LVS3) / LVD3;
Ao, aortic diameter;
HR, heart rate;
LA, left atrium antero-posterior systolic diameter during ventricle systole;
LV, left ventricle;
LVD, left ventricle end-dimension measured at maximal dimension;
LVS, left ventricle end-systolic dimension;
PWT, left ventricle posterior wall thickness measured at maximal dimension;
PWT/LVD ratio, free wall relative thickness was calculated by the ratio;
Introduction
Ascribed in 1922 as an essential lipid-soluble factor for maintaining normal
reproduction, vitamin E is currently recognized as an important dietary nutrient for all
animals (1). Vitamin E is a term used to describe a family of tocopherols (α, β, γ and δ -
tocopherols and α, β, γ and δ - tocotrienols) of which α-tocopherol is the most important
and abundant molecule (2). Bile production and secretion are essential to the absorption of
α-tocopherol which is carried mainly by low-density lipoprotein LDL fraction
(LDL-cholesterol) (3).
It has been shown that vitamin E may attenuate several events and diseases.
Although its protective mechanism is not completely understood, the antioxidant effect
seems to be the most important (1, 2, 4). It is also essential for tumour inhibition, immune
response stimuli, membrane stability (5) and inhibiting the conversion of nitrites into
nitrosamines (2). Its protection has been examined specifically in cardiovascular diseases
(6-8) including hypertension (9) and angina pectoris (10). A protective effect on
myocardium infarction-induced-left ventricle dilatation has been identified when Vitamin E
is associated with ascorbate (11). Cardiovascular observational studies that examined
mortality (7)(12) and coronary artery event (13) risks showed their inverse association with
high intake of food rich in Vitamin E. On the other hand, there was no Vitamin E
supplementation efficacy on coronary disease in a double-bind study (10) or under
cardiotoxicity experimental models (14, 15).
The reported side effects of vitamin E supplementation have included
gastrointestinal disturbances, increase in prothrombin time, decrease in absorption of
promotion of mice skin tumour (17) at high doses. Few studies, however, have examined
the undesirable effect of vitamin E on cardiac diseases. They showed that vitamin E was
associated with a strong in vitro pro-oxidant action on low-density lipoprotein (18), with a
20% higher probability of developing congestive heart failure in humans with ventricular
dysfunction (19) and with augmented mortality risk in patients with coronary disease (20).
However, to our knowledge, the effect of Vitamin E supplementation on hearts free of
disease has not been studied. Factors that may determine the switch from antioxidant to
pro-oxidant behaviour of vitamin E can be associated, in part, with the vulnerability of organs
already compromised by a disease-induced heavy oxidative stress (21).
In light of these considerations, the present study was undertaken to evaluate the
effect of Į-tocopherol supplementation on the heart of animals without previous cardiac
disease. Additionally, we estimated Į-tocopherol uptake into the heart. A significant
cardiotoxicty followed by Į-tocopherol treatment was found for all parameter investigated.
Uptake of the supplement into the heart was confirmed by chromatography.
Material and Methods
Chemical Products. α-tocopherol (dl-α-tocopherol-acetate) was donated by Relthy Labs
(Indaiatuba, SP, Brazil). HPLC-grade methanol and water were obtained from J. T. Baker
Chemical (Philipsburg, NJ). Methyltert-butyl ether was purchased from Aldrich Chemical
(Milwaukee, WI). All HPLC solvents were passed through a 0.45-mm membrane filter and
degassed before use. The standard (DL-α-tocopherol) was stored at -70°C until use.
Pentobarbital sodium (Cristalia, Paulinia, SP, Brazil), ketamine chlorhydrate (Vetbrands,
Animals. Male Wistar rats (400 g) from CEMIB (Centro Multidisciplinar de Investigação
Biológica, UNICAMP, Campinas, SP, Brazil) were housed for seven weeks (wks) and kept
three per cage in plastic cages in the animal facility at the Experimental Laboratory of
Internal Medicine Department, Botucatu School of Medicine, UNESP where they
consumed water and commercial diet (22) ad libitum. Rats were randomly assigned either
to C (control, n = 11) or E (α-tocopherol, n = 11). α-tocopherol or corn oil was
administered daily by gavage every morning for the entire seven-wk period. The α
-tocopherol supplement mixed with corn oil [250 mg α-tocopherol /(Kg body wt)/day] was
given to E animals, while the C group received only corn oil. To avoid differences in the
energy provided, all groups received the same corn oil volume [(~0.5 mL/(Kg body wt) /
day]. One day before euthanasia all animals underwent a transthoracic Doppler
echocardiogram using light anaesthesia [ketamine [50mg/(kg body wt)] and xylazine
[1mg/(kg body wt)]. Rats fasted overnight before euthanasia under pentobarbital sodium IP
[50 mg/(kg body wt)] anaesthesia and necropsied immediately after death. After collection,
heart and plasma were stored at -80°C until α-tocopherol analyses. Tissues were fixed with
10% buffered formalin for 48 h for morphological analysis. The protocol used was in
accordance with the Ethical Principles for Animal Research adopted by the Brazilian
College of Animal Experimentation (COBEA) and was approved by the local Animal
Research Ethics Committee (Document # 445/2004).
Alpha-tocopherol preparation.The α-tocopherol-corn oil mixture was stirred for 20 min in
a water bath at 54°C before being fed to the animals. Each milliliter of solution contained
250 mg of α-tocopherol. The α-tocopherol was monitored at 294 nm and confirmed by
was checked by HPLC, while the α-tocopherol was stable in the α-tocopherol-corn oil
mixture for 9 weeks at 25°C.
The extraction of α-tocopherol was performed by adding 100 µL of α-tocopherol solution
to 900 µL of ethanol. To dissolve α-tocopherol, the solution was sonicated three times at
4°C (1 minute) and also vortexed for 30 seconds. Thus, 20 µL of this solution was injected
in the HPLC. After the end of the analysis, α-tocopherol concentration was obtained by the
quantification of chromatograms where the area of this vitamin is divided by the area of the
internal standard (DL-α-tocopherol).
Analysis of α-tocopherol in the diet. The diet was analyzed according to a previous study (24). Five mL of H2O was added to 10 g of diet and then swirled for 1 minute. Extraction
solution [30 mL; hexane/acetone/ethanol/toluene (50:35:30:35)] was added and swirled for
1 minute, followed by 16 hours in the dark at room temperature; 4 mL of 40% methanolic
KOH and hexane (30 mL) were added and the mixture was left in the dark at room
temperature. After 1 hour, 10% Na2SO4 (30 mL) was added, shaken and left in the dark at
room temperature. After 1 hour the hexane layer was collected and volume was recorded.
Aliquots of 200 µL were evaporated under N2 and the residue redissolved in 100 µL of
ethanol, vortexed and sonicated for 30 seconds. Aliquots of 50 µL of the final extract were
injected into the HPLC system.
Tissue Preparation and Extraction. Heart (left ventricle) was harvested, weighed and
analyzed after saponification as previously described (24). Briefly, samples (150g) were cut
