effect of γ-tocotreenol on blood pressure, lipid peroxtoation and total antioxtoant status in...

CLIN. AND EXPER. HYPERTENSION, 2 1 (S), 1297-1 3 13 (1 999)

EFFECT OF y-TOCOTRIENOL ON BLOOD PRESSURE, LIPID PEROXIDATION AND TOTAL ANTIOXIDANT STATUS IN

SPONTANEOUSLY HYPERTENSIVE RATS (SHR)

Newaz MA and Nawal NNA Faculty of Medicine, International Islamic University Malaysia, 'Department of

Biochemistry, Universiti Kebangsaan Malaysia.

ABSTRACT The aim of this study was to determine the effects of y tocotrienol on lipid peroxidation and total antioxidant status of spontaneously hypertensive rats (SHR), comparing them withnormal Wistar Kyoto (WKY) rats. SHR were divided into three groups and treated with different doses of y tocotrienol (yl, 15mgkg diet; y2, 3Orngkg diet and y3, 15Omgkg diet). Normal WKY and untreated SHR were used as normal (N) and hypertensive control (HC). Blood pressure were recorded every fortnightly for three months. At the end of the trial, animals were killed and measurement of plasma total antioxidant status, plasma superoxide dismutase (SOD) activity and lipid peroxide levels in plasma and blood vessels were carried out following well established methods. Study shows that lipid peroxides were significantly higher in hypertensive plasma and blood vessels compared to that of normal rats (Plasma- N: 0.06+0.01, HC: 0.13+0.008;

Correspondence: Dr. Mohammad Ali Newaz Faculty of Medicine International Islamic University,Malaysia P.0 Box 141, Kuantan 25710. Pahang D.M Malaysia. Phone: 609-5 132797

E-mail: [email protected] Fax: 609-5133615

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1298 NEWAZ AND NAWAL

p<O.OOl, B1. Vessels - N: 0.47k0.17, HC: 0.96f0.37; p<O.OOl). SOD activity was significantly lower in hypertensive than normal rats (N=148.58h29.56 U/ml, HC=110.08*14.36 U/ml ; ~ 4 . 0 1 4 ) . After three months of antioxidant trial with y- tocotrienol, it was found that all the treated groups have reduced plasma lipid peroxides concentration but was only significant for group y l (yl: O.lOW0.026, HC: 0.13%0.008; p=0.034). On the other hand, lipid peroxides in blood vessels reduced significantly in all treated groups (yl; p<O.OS, y2; p<O.OOl, y3; pc0.005). All the three treated groups showed improve total antioxidant status (p<O.OOl) significantly. SOD activity also showed significant improvement in all groups (yl: p<O.OOl, y2: peO.05, y3: p<O.OOl). Correlation studies showed that, total antioxidant status (TAS) and SOD were significantly negatively correlated with blood pressure in normal rats @=0.007; p=0.008) but not in SHR control. This correlation regained in all three groups SHR’s after treatment with tocotrienol. Lipid peroxides in blood vessel and plasma showed a positive correlation with blood pressure in normal and SHR control. This correlation also remains in treated groups significantly except that in y3 where positive correlation with plasma lipid peroxide was not significant. In conclusion it was found that antioxidant supplement of y-tocotrienol may prevent development of increased blood pressure, reduce lipid peroxides in plasma and blood vessels and enhanced total antioxidant status including SOD activity. Key words: Free radical, Lipid peroxide, Antioxidant status, Superoxide dismutase,

Blood pressure.

INTRODUCTION

Lipid peroxidation is one of the oldest studied free radical chain reactions (1,2,3,4). It was

reported earlier that free radicals might have contribution in the pathogenesis of human

essential hypertension ($6). On the other hand vascular endothelium itself can produce

tiee radicals (7) by xanthine -xanthine oxidase system, which is 1000-1 0000 times higher

in endothelial cells then other tissues of the body (8). At the same time, hypertensive rats

were reported to have reduced antioxidant content compared to the normal WKY rats (9).

There is always a certain amount of free radical generation in the biological system due to

metabolic process (lo), but the body’s antioxidant defence is sufficient enough to

scavenge these in normal situation.

It is not clear whether it is increased free radical generation or a reduced defence

against these radicals, which is contributing to high blood pressure. Whatever the reason,

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LIPID PEROXIDE AND ANTIOXIDANTS IN HYPERTENSION 1299

an antioxidant intervention may provide more information regarding the involvement of

free radicals in hypertension. Tocopherol and tocotrienol, two different forms of vitamin

E differs in their hydrocarbon tail; Tocotrienol has an unsaturated isoprenoid tail while

tocopherol has a saturated one (1 1). It has been shown that different forms of vitamin E

exhibit different antioxidant protection. In vitro studies suggest that Tocotrienol possess a

40-60 times greater antioxidant activities than tocopherol against lipid peroxidation.

Tocotrienol also protects cytochrome p450 against oxidative damage 6.5 times better than

tocopherol (12). a-Tocopherol has been reported to scavenge free radicals, which can

directly or indirectly initiate or propagate lipid chain oxidation (13,14). Besides this, there

are reports of reducing blood pressure in SHR by tocopherols and tocotrienols (1 5). This

study was designed to compare lipid peroxides and antioxidant status of the normal and

SHR and to evaluate the effect of y-tocotrienol supplementation on their blood pressure.

MATERIALS AND METHODS

Chemicals. A basal diet of rat chow was purchased from Gold Coin Co., Klang, Malaysia.

y-Tocotrienol (Natural, 90% pure) used in t h s study was extracted from palm oil and

supplied by Palm Oil Research Institute of Malaysia (PORIM). Kit for total antioxidant

status (TAS) and superoxide dismutase (SOD) was purchased fi-om Randox laboratories

Ltd. UK (Cat no: NX2332 & SD125). All other reagents used were of the highest grade

commercially available and obtained from Sigma Chemical Co. (St Louis, MO) unless

otherwise specified.

Animal treatm.ent. Thirty three male SHR, 150-200 gm, 8-10 weeks old, were caged (1-2

rat /cage) and maintained on normal or treated rat chow and water ad libitum for the

duration of the 12 weeks study. The rats were divided into 4 groups consisting of the

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control (C), y l (y-tocotrienol 15 mgkg diet), 72 (30 mgkg diet) and y3 (150 mgkg diet).

Seventeen age and weight matched normal Wister Kyoto (WKY) rats were used as the

normal control.

After 12 weeks of treatment, all the animals were sacrificed by cervical dislocation.

Blood sample were collected by cardiac puncture under anaesthesia induced by Zoletil

(Tileramine + Zolazepum) 0.5 mYrat intra-muscular prior to cervical dislocation.

Approximately 4 cm of thoracic aorta were excised and cleaned off very gently from the

surrounding fats. Tissues were homogenised in distilled water and centrihged at 1000 x g

for 20 min. The Supernatant was used for the analysis. Plasma and cells from blood

sample were separated by centrifuging at 1000 x g for 10 minutes.

Blood pressure monitoring. Blood pressure was measured every fortnightly in all animals

using standard tail cuff method (16,17). Tails of the animal were occluded with an

appropriate size metal tubular tail cuff (7116 inch) and pulse was detected as the cuff

pressure was lowered. The pressure at which the first pulse appears was the measurement

of systolic blood pressure. The occluding tubular cuff together with the pneumatic pulse

transducer (Narc0 Bio Systems, USA) was connected to electrophysiograph (Narc0 Bio

Systems, USA) cuff outlet. The rat-tails were pre-warmed by lamp before every

measurement of blood pressure and the average of three readings was taken as the final

reading. CV of Blood pressure measurement was 4.4.

Measurement of Total Antioxidant Status. TAS was measured in plasma sample using the

Randox kit (18). Twenty pl of plasma samples or standard (6-hydroxy-2,5,7,8-

tetramethylchroman-2-carboxylic acid 2.5 mmoV1) or reagent blank (Double deionized

&O) were mixed with 1 ml of chromogen (Metmyoglobin 6.1 pmoM & 2,2’-Azino-di[3-

ethylbenzthiazoline sulphonate, 610 pmoV1) and after mixing the initial absorbance (Al)

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was read at 600 nm wavelength at 37OC. Substrate (hydrogen peroxide, 250pmoY1) of

volume 200 p1 was added to the sample/standard/blank and the absorbance was read

exactly after 3 minutes (A2). Subtraction of A2 from A1 gave the absorbance of

sample/standard/blank. The following formula were used to get the TAS: Total

antioxidant status: = Factor x (absorbance of blank-absorbance of sample) mmoYl.

Factor= Concentration of standard/(absorbance of blank-absorbance of standard).

Measurement of superoxide dismutase: SOD was measured using a kit supplied by

Randox Laboratories Ltd. Aliquots of whole blood (0.5 ml) were centrifuged at 1000 x g,

washed 4 times with 0.9% NaCl and lysed in a total volume of 1 ml ice cold double de-

ionised H,O. The lysate was then diluted accordingly so that the % inhibition falls

between 30%-60% with Ransod diluting buffer. This preparation was used for

measurement of SOD. The reaction was measured at 505 run, using 50 p1 of sample in a

total reaction volume of 2 ml. SOD was expressed as SOD unit/ml of whole blood (19)

Lipid peroxides. Lipid peroxides were measured in thoracic aorta and plasma as the

thiobarbituric acid reaction product by spectrofluorometry following well established

procedure (20) and was expressed as nmol MDA (Malondialdehyde) equivalent / ml of

plasma . l,l,3,3-Tetra ethoxy propane (TEP) was used as the standard. Samples (100 pL

of plasma / tissue homogenate) were diluted with 400 pL distilled water. The diluted

sample or standard (500 pl) was mixed with 2.5 ml of TCA/HCl (1.22M Trichloroacetic

acid in 0.6 M HC1) and left at room temperature for 15 minutes. A volume (1.5 ml) of

TBA/NaOH (Thiobarbituric acid 0.67% on .05 M NaOH) was mixed with the

sample/standard and incubated at 100°C for 30 minutes. Samples were cooled with tap

water and mixed with 4 ml n-Butanol. After extensive mixing samples were centrifuged

at 1000 x g for 10 minutes. The n-Butanol layer was separated out and fluorescence was

read at 515nm and 553nm excitation and emission respectively. MDA equivalent was

calculated using the TEP standard curve.

Amount of tissue protein was measured by procedure described earlier (21).

Overall CV of the biochemical analysis was maintained within the range of 4-7.

Statistical analysis. The results obtained were analysed via ANOVA and Student's t-test

for significance difference. A value of pCO.05 was considered as significant. Computer

program STA'TISTICA were used for analysis.

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* * *

* T

I I 1 m + A

j O !

2 w k 4 w k 6 w k 8 w k 1Owk 12wk Owk

Study period (w k)

Fig. 1 Comparison of blood pressure between SHR control and SHR treated with different doses of y-Tocotrienol. SHR-C = SHR control, Gama-Trl= y- Tocotrienol 15 mgkg diet, Gama-Td= y-Tocotrienol 30 mgkg diet, Gama-Tr3= y-Tocotrienol 150 mgkg diet. *=p<0.05 when compare with hypertensive control.

RESULTS

From this study we found that treating with different doses of y-tocohienol has

significantly prevented the age related increase of blood pressure in SHR (Fig. 1). This

reduction was more marked in group treated with y-tocotrienol 15 mg/kg diet (N:

110.29k16.3; HC: 209.56f8.47; y l : 122.67-+18.87; y2: 143.56k10.62; y3: 136.5-12.78;

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Table 1 Comparison of Parameters between Normal Wister Rats and SHR. Values are Mean f SD.

Parameters Normal (n=17) SHR-Control (n=8) P Value LP(BV) 0.47 f 0.17 0.96 f 0.37 p<O.O001* W P ) 0.06 f 0.01 0.13 f 0.01 p<O.O001* TAS 0.88 f 0.05 0.83 f 0.02 p<0.05* SOD 172.93 f 46.91 110.08 f 14.38 p<O .005 * Note: LP= Lipid peroxide (nmol MDA equiv./mg protein), TAS= Total antioxidant

status (mmol/L), SOD= superoxide dismutase (unitlml whole blood), BV= Blood vessels, P=Plasma. n= Number of animals, *=p<0.05 statistically significant.

p<O.OOl when compared with HC). Biochemical analysis showed that SHR has

significantly higher lipid peroxides in blood vessels and plasma and a reduced TAS and

SOD activity (Table 1). After 12 weeks of antioxidant therapy, results showed that all

three doses of y-tocotrienol significantly reduced the lipid peroxidation in blood vessels

and for plasma it was only significant in groups treated with yl (Table 2 ) . Results also

showed that ytocotrienol increased TAS and SOD in all treated groups significantly

compared to their control SHR (Table 2). Correlation study showed that in normal rat,

mean blood pressure had a significant positive correlation with lipid peroxide in both

plasma and blood vessels together with a significant negative correlation with TAS and

SOD (Table 3). In S H R control group, this correlation was significant only with lipid

peroxide in blood vessels and plasma. In treated groups, TAS and SOD maintained a

negative correlation with blood pressure in all three groups. Lipid peroxides in blood

vessels gave significant positive correlation with blood pressure in all three treated

groups, whereas for plasma lipid peroxide it was significant only with groups y l and y2

(Table 3). However lipid peroxide in blood vessels gave significant positive correlation

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effect of γ-tocotreenol on blood pressure, lipid peroxtoation and total antioxtoant status in...

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