DIETARY (n-3) FATTY ACIDS INCREASE SUPEROXIDE DISMUTASE ACTIVITY AND DECREASE THROMBOXANE PRODUCTION IN THE RAT HEART

NufritionResearch,Vol. 17,No. 1, pp. 163-175, 1997 Copyright@ 1996 ElsevierScience Inc. Printedin the USA. All rightsreserved 0271-5317/97 $17.(XI+ .00

. .. 1. ELSEVIER

PII S0271-5317(%)002424

DIETARY (n-3) FATTY ACIDS INCREASE SUPEROXIDE DISMUTASE ACTIVITY AND DECREASE THROMBOXANE PRODUCTION IN THE RAT HEART Riitta Luostarinenl, MD, PhD, Rolf Wallin,PhD & Tom Saldeen, MD, PhD Department of Forensic Medicine, Universityof Uppsala,Dag Hammarskjoldsvag 17, S-75237 Uppsala, Sweden. Fax: 4618559053.

The aims of the present studies were to examine the effects of fish oil (containing (n3) polyunsaturated fatty acids) on myocardial thromboxane and prostacyclin production, superoxide dismutase (SOD) activity and malondialdehyde (MDA) production in the rat. Male rats were fed standard pellet diets and the same diets enriched with 770 (w/w) stabilized fish oil or 770 butter (saturated fat) for 2-6 wk. Myocardial production of thromboxanewas lower in rats given fish oil than in those fed standard pellets (P < 0.01) or saturated fat (P < 0.05) and the prostacyclin/ thromboxane ratio was higher than in rats fed standardpellets (P < 0.05). Myocardial SOD activity was higher in rats fed stabilizedfish oil than in those given saturated fat (P < 0.05). Supplementation of the stabilized fish oil with extra vitamin E did not have any major effect on thromboxaneand prostacyclin production or SOD activity. The percentage of arachidonic acid in the myocardial phospholipids was lower (P < 0.001) during fish oil than during saturated fat feeding, with no modifying effect of vitamin E supplementation. Feeding with the stabilized fish oiI did not alter the myocardial a–tocopherol concentration, but the myocardial MDA concentration in vitro was higher (P c 0.01) than after feeding with saturated fat. Supplementation of the stabilized fish oil with extra vitamin E resultedin a higher a-tocopherol (P < 0.05) and lower MDA concentration (P < 0.05) in the myocardium compared to the unsupplemented fish oil. Plasma MDA concentration was not changed by fish oil feeding. In conclusion,fish oil feedingresultedin highermyocardialSOD activity and lower thromboxane production. These changes may be contributory mechanisms underlyingthe antiarrhythmiceffect of fish oil. Copyright631996 Elsevier Science k.

Kev worm: (n-3) Fatty Acids, ThromboxaneB2, SuperoxideDismutase, Vitamin E, Malondialdehyde,Myocardium

IN R ODUCTIO~ Several animal studies have shown that dietary fat can affect the vulnerability of the myocardium to arrhythmia stimuli (l). Dietary supplementationwith fish oil, for instance, has been found to reduce the incidence and severity of cardiac arrhythmia (2-4) and decrease the mortality (4, 5) and myocdial damage (6) associated with coronaryartery occlusionin the rat. The highest frequency of arrhythmia and the greatest degree of myocardialdarnagewas found in animals fed saturated fat (2). Both (n-3) and (n-6) polyunsaturated fatty acids (PUFA) provided considerable protection against arrhythmia, (n-3) PUFA being the most effective, especially in reducing the degree of reperfusion arrhythmia (2). ITOwhom correspondenceshouldbe addressed.

163

164

R. LUOSTARINENet al.

Furthermore, intake of fatty fish or fish oil in humanshas been reported to reduce the mortrdityfrom ischemic heart disease (7, 8), probably by reducing lethal arrhythmia. The mechanisms underlying the antiarrhythmic effect of fish oil are not fully understood. The findings that thromboxane is arrhythmogenic and that prostacyclin may act as an endogenousantiarrhythmic substance (9) imply that the myocardial prostanoid balance maybe one important factor involved in cardiac arrhythmia. Further, increasing evidence suggests that oxygen free radicals and endogenous antioxidant defense systems such as superoxide dismutase (SOD) play a major role in the pathogenesis of reperfusion injury (10, 11). In the present studies, the effects of fish oil on myocardialthromboxaneand prostacyclin production, SOD activity, and in vitro malondialdehyde(MDA)productionin the rat were investigated.

Animals Male pathogen-free Sprague-Dawley rats, weighing 175-200 g (B&K Universal AB, Sollentuna, Sweden) were housed two in each cage in a room of controlled temperature (20-21”C), lighting (lights on between 0600 and 1800 h) and humidity (50-70%). All procedures and care of animals were approved by the local Animal Care and Use Committee.The animals were allowed free access to tap water and standard pellet (Rat& Mouse Standard Diet, B&K Universal AB) or fish oil diets throughoutthe experiments. After 2 (experimentI) or 6 wk (experimentII) the rats were anesthetized with pentobarbital sodium (40 m@kgbody weight) given intraperitoneallyand exsanguinate via the abdominal aorta. Plasma was preparedimmediatelyby centrifugationof the blood for 10 min at 500 g and 4°C in tubes containing EDTA and then sto~d at -70”C.The heart was removed, weighed, and stored at -70°C. Experiments

Exuerimew 1. In an initial serie of experiments the effects of dietary fish oil on myocardial thromboxane and prostacyclin production, and the effect of dietary fish oil containing different amounts of vitamin E on myocardial in vitro MDA productionin the rat was studied. In these initial experiments rats fed standard pellet diets were used as controls. Ex~erimtzLL In the next study, the effects of a stabilized fish oil with and without extra vitamin E on myocardial thromboxane, prostacyclin and in vitro MDA production, and SOD activity was studied using rats fed butter as controls. Diets Rats were fed standard pellet diets containing57% carbohydrates, 17%protein, 1.4% fat and 74 mg vitamin E /kg. In experiment I the standard pellet diet was enriched with 51Z0(w/w) fish oil and in fish oil and 7% butter (saturatedfat, SAT). The fish oil contained 0.2 mg, 1.2 experiment II with 7?40 mg (FO, ESKIMO-3, Cardinova, Uppsala, Sweden) or 3.3 mg (FO+E) D-a–tocopheryl acetate/g fish oil in experiment I and 1.2 mg (FO) or 3.3 mg (FO+E) D-a–tocopheryl acetate/g fish oil in experiment II. The butter contained 0.02 mg vitamin E/g butter. The fish oil preparation contained about 37Y0 (n-3) PUFA, of which 1870 was eicosapentaenoic acid (20:5, EPA) and 12% docosahexaenoic acid (22:6, DHA). Supplementationof the diet with 7% (w/w) fat corresponded to 16 energy percent (en%) fat and to 6 energy % (n-3) PUFA in the fish oil diet. The fish oil was well stabilized against auto-oxidation with tocopherolsand other antioxidants and had low peroxide (c 1 mEq/kg) and anisidine (< 7) values which did not increase after exposure of the oil to air at room temperature for 24 h. The cholesterol concentrationwas less than 3 mg/g both in the butter and fish oil supplements. The fatty acid compositions of the supplements are shown in Table 1. The supplements contained approximatelyequal amounts of monounsaturatedand (n-6) fatty acids. The saturated fat in the SAT diet was replacedby (n-3)fatty acids in the fish oil diet. All diets were stored in sealed containers under nitrogenat 4°C in the dark and suppliedto the animals once daily.

SUPEROXIDE DISMUTASEACTIVITY IN RATS

165

Myocardial phospholipid fatty acid composition The heart muscle was homogenizedin methanoland extractedwith chloroform/methanolas described earlier (12). Butylated hydroxytoluene (BHT, 0.23 mmol/L) was added to the solvents to avoid decomposition of PUFA during sample processing. The phospholipidswere separated by thin layer chromatographyand, after transmethylation,the fatty acid methylesters were identified by gas liquid chromatography.The results are expressedas percentageof all fatty acids detected. Myocardial concentration of a-tocopherol The concentration of a-tocopherol in the heart muscle was assayed as described elsewhere (13). Briefly, 100 mg heart muscle was first homogenized in 1 mL distilled water containing 10 pL of e~anolic BHT (227 ynmol/L).Thereafter, 1 mL SDS (0.1 mol/L) was added and homogenization was continued. The sample was transferred to a test tube and the homogenizer was rinsed with ethanol, which was then combinedwith the sample.The mixture was vortexed for 30s, 2 ML hexane was added, and the mixture was again vortexed for 2 min. After centrifugation for 5 min at 1000 g, the hexane layer was removed and evaporated to dryness under nitrogen and redissolved in 2 mL methanol. The tocopherol isomers were separated by RP-HPLC on a 10 cm column packed with Nucleosil C18 (5 ~m). The mobile phase was methanolpumped at a flow rate of 1.0 mL/min and the a–tocopherol level was detected and quantified at 292 nm. The results are expressed as nmol/g wet tissue. The coefficient of variation in samples taken from a single batch of myocarchd homogenate was about 2-4 ~0 (n = 30 in 3 separate experiments). The recovery of a–tocopherol added to myocardialhomogenatewas >9090 as comparedto a direct injectioninto the RP-HPLC system. TABLE 1 Fatty Acid Compositions of the Supplements Fatty acid

Butter

Fish oil

4:0-12:0 14:0 16:0 16:1 18:0 18:1 18:2 (n-6) 18:3(n-3) 20:5 (n-3) 22:5 (n-3) 22:6 (n-3)

14.6 11.1 27.4 3.1 10.5 23.6

5.5 15.8 9.7 2.9 12.9

minor monounsaturated minor (n-6) minor (n-3) others Z saturated X monounsaturated Z polyunsaturated ~ (n-6) X (n-3)

::: — — — — i.7

63.6 26.7 3.0 2.6 0.4

::: 17.8 2.4 11.7 2.6 ::: 7.2 26.2 25.2 41.4 3.9 37.5

The standard pellet diet (1.4~0 fat, WIW,contammg about 3670 lmolelc acid) was ennched with 770 (w/w) butter or fish oil. The fatty acid composition of the butter was reported by the manufacturer and that of the fish oil was a mean of 8 differentbatchanalysesfrom this laboratory.

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In vitro malondialdehyde production in the myocardium Malondialdehyde (MDA) was determined as the thiobarbitunc acid (TBA)-MDA complex (14). Briefly, a piece of heart muscle (1:10) was homogenizedin Tris/KCl buffer (0.2 (mol/L)/O.16mol/L, pH 7.4) and incubated in a water bath (37°C) for 1 h. The incubated heart homogenate (0.5 g) was boiled with 0.75 mL phosphoric acid (O.15 mol/L) and 0.25 mL TBA (42 mrnol/L) in a water bath (1OO”C)for 1 h and then placed on ice. After addition of 0.5 mL of a methanol-NaOHsolution to 0.5 mL of sample, the samples were centrifuged at 9500 g for 5 min at 4°C, and the supernatants were analyzed in a spectrophotometer at 532 nm using tetraethoxypropane as standard. The results are expressed as nmol/g wet tissue. The MDA concentrationin the rat myocardium measured by HPLC or spectrophotomerncallydid not differ significantly(12rats fed standardpellet or fish oil diets). Myocardial SOD activity SOD activity in the myocardium was determined from its ability to inhibit the auto-oxidation of pyrogallol (0.2 mmol/L) by a method described elsewhere (15). A piece of heart muscle (100 @) was homogenized in 50 mmol/L Tris-HCl containing 1 rnmol/Ldiethylenetriaminepentaacetic acid, pH 8.20. The homogenate was centrifuged at 2300 g at 4°C for 20 min and the supernatant was freshly analyzed for SOD activity spectrophotometricallyat 25°C at 420 nm in the presence of 0.2 Lmol/L catalase. Mn SOD activity was determinedin the presence of 1 mmoliL KCN, which inhibits CuZn SOD activity (1 mmol/L KCN decreasedpyrogallolauto-oxidationby about 25%). Myocardial production of thromboxane Bz and 6-keto-prostaglandin FIa After washing a piece of heart muscle (about 100mg) in.cold Hanks’balanced salt solution (HBSS, pH 7.4) without calcium and magnesium, the muscle was incubated in HBSS with calcium and magnesium in the presence or absence of indomethacin (0.3 mmol/L) in a water bath (37”C). The samples were gently shaken manually every 5 rein, and after 30 min the incubation medium was removed and stored at -70”C until the concentrations of thromboxane (Tx) B2 and 6-ketoProstaglandin (PG) FIa, the stable hydrolysisproducts of TxA2 and prostacyclin, respectively, were determined by radioimmunoassay using 3H-labeled antigen and anti-rabbit antibody as described previously (16). The sensitivity of the assays was about 108 pmol/g. The samples incubated with indomethacin contained about 9090less prostacyclinand thromboxane,indicating that the measured levels are primarily referable to in vitro productionand not to release of preformed prostanoids. The cross-reactivity of 6-keto-PGFla with PGF2a was 2.6Y0,with PGE1 1.9’ZO, with TxB2 1.490,with PGE2 1.1%, and with PGFla 0.870, and that with other compounds was < 0.5%. The crossreactivity of thromboxane B2 with PGD2was 3.970and with other compounds < 0.5%. The results are expressed as nmol/g wet tissue. Triglycerides, cholesterol, glucose, fibrinogen and malondialdehyde in plasma Triglyceridesand total cholesterolin plasmawere measuredby enzymaticcolonmetric methodsat 500 nm (Peridochrom@Triglycerides GPO-PAP and Monotest@Cholesterol CHOD-PAP, Boehringer Mannheim, Sweden). Glucose in plasma was assayed immediately after blood sampling, with a glucose oxidase method (Reflotron@-Glucose,Boehnnger Mannheim). Fibrinogen in plasma was determined as clottable fibrinogen as described elsewhere (17). Lipid peroxidation in plasma was measured by determining the concentration of MDA by HPLC as described elsewhere (14). Plasma (1 mL) was immediatelymixed with 10 pL ethanolicBHT (227mmol/L) and then stored at -70”Cfor analysis. Statistical methods Student’stwo-tailed ttest for unpaired observationswas used in experiment I and Fisher’sPLSD in experiment II to compare values in the different dietary groups. Means * standard error of means (SEM) are shown unless otherwise stated, and P values <0.05 were adopted as significant.

SUPEROXIDE DISMUTASEACTIVITY IN RATS

4Zmri~nt I Myocardial production of thromboxane B2 and 6-keto-prostaglandin FIa Myocardial production of TxB2 was 63Y0lower in rats fed fish oil for 2 wk than in those given standard pellet diets (P <0.01, Fig. 1). Although 4WZ0 lower, the production of 6-keto-PGFla was not significantlydifferent. The ratio betweenprostacyclinand thromboxanewas higher in fish oil-fed rats (P < 0.05). In vitro malondialdehyde production in the myocardium After a dietary period of 2 wk the myocardialMDA productionin vitro (Fig. 2) was higher in rats fed fish oil with a low (FO+E 0.2, P c 0.001) or moderate (FO+E 1.2 = FO diet, P c 0.01) vitamin E concentration, but not in those fed fish oil containing extra vitamin E (FO+E 3.3 = FO+E diet), compared to rats given standardpellet diets.

Ewe riment11 The body weight gain after a dietary period of 6 wk did not differ between the FO, FO+E and SAT groups (235 * 20, 240&25 and 250 f 30 g, respectively, NS). The heart weights were also similar in these groups (1.19* 0.13, 1.24* 0.07 and 1.13 * 0.13 g, respectively, NS). Myocardial phospholipid fatty acid composition After 6 wk of dietary supplementationwith fish oil, the percentages of EPA (20:5) and DHA (22:6) were higher (P < 0.001) and those of arachidonic (20:4) and linoleic acid (18:2) in myocardial phospholipids were lower than those after a similar period on a SAT diet (P < 0.001), with no differences between rats fed FO and FO+E (Table2). Myocardial SOD activity Myocardial Mn SOD activity was higherin rats fed FO for 6 wk (P<0.05) and tended to be higher in rats fed FO+E (P = 0.06) compared with SAT (Fig. 3). The total SOD and CuZn SOD activities did not differ significantlybetween the dietarygroups(data not shown). Myocardial production of thromboxane B2 and 6-keto-prostaglandin Fla Feeding FO for 6 wk resulted in lower myocardislproductionof TxB2 (P <0.05,Table 3) compared to SAT. ‘he production of 6-ke~-PGFla, althoughlower, did not differ significantly between these two dietary groups (P = 0.06, Table 3). There was a tendency towards a smaller decrease in myocardialprostanoidproductionin rats fed FG+E comparedto those that receivedFO (Table 3). Malondialdehyde and a-tocopherol in the myocardium After 6 wk of diet the myocardial MDA productionin vitro was higher in rats fed FO than in those given FO+E (P < 0.05) or SAT (P <0.01,Table 3, Fig. 4). The concentration of a–tocopherol in the myocardium was higher after 6 wk in the FO+E groupthan in the FO group (P <0.05,Table 3). Triglycerides, cholesterol, glucose, fibrinogen and malondialdehyde in plasma The plasma triglyceride and cholesterol concentrations were significantly lower in rats fed fish oil than in those given the SAT die~ with no significantdifferencebetweenFO and FO+E (Table 3). The plasma glucose concentration was significantlyhigher in the FO group compamxlto that in the SAT and FO+E groups (Table 3). Fibrinogenand MDA (Fig. 4) in plasma did not differ among the dietary groups (Table 3).

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TABLE 2 Fatty Acid Composition in Myocardial Phospholipids of Rats fed the different Diets for 6 weeks

Diets Fatty acid

SAT

16:0 18:0 18:1

14.2 k 0.5 23.2 h 0.4 7.4 * ().7

14.6 A 0.7 24.1 t 1.0 6.6 * 0.3*

18:2 (n-6) 20:4 (n-6) 20:5 (n-3) 22:5 (n-3) 22:6 (n-3)

12.6 + 0.6 16.8 * 0.6 0.4 * ().()1 2.0 & 0.2 16.1 t 1.0

10.3 * I. O** 12.9 f 1.3*** 2.7 + 0.3*** 2.4 f 0.3 20.1 * 1.1***

others

7.3

6.4

20.2 * 1.2*** 6.8

X saturated

37.4 * 0.7 7.4 * ().7 47.9 k 1.3 29.4 & 0.2 18.5 * 1.1

38.6 & 1.3 b.fj * ().3* 48.4 f 1.3 23.2 k 1.2***

38.9 * 0.7* 6.5 & o.5 47.9 * 1.0 22.7 A 0.8***

25.2 t 1.1***

25.2 * 1.3***

FO

FO+E

Percent

Z monounsaturated Z polyum~urated (n-3)

14.6 A 0.7 24.3 k 0.6*

6.5 f o.5* 9.7 & o.8*** 12.9 t 0.6*** 2.5 k o.2*** 2.4 k 0.2*

Values are means ~ SD for 4 (SAT), 6 (F(3)and 5 (FO+E)rats. *P< 0.05, **P< 0.01 and ***P< 0.001 compared to SAT. SAT refers to diet enrichedwith butter;FO and FO+E refer to fish oil diets containing 1.2 and 3.3 mg D-a-tocopheryl acetate/gfish oil, respectively.

I

150 +

* T

r

I

a I

Prostacyclin

-

■

CONTROL

W

FISH OIL

T

50H Thromboxane -

Ratio PC/Tx

“

Effectsof 2 weeks of dietarysupplementation withfish oil (n= 13) on FIG. 1 in vitro production of thromboxaneand prostacyclin (nmol/g wet weight) and prostacyclinhhromboxane (PC/Tx)ratio(x1O)in theratheti. Meres * SEM. * P < 0.05 and **P c 0.01 compared to control (standardpellets, n = 25),

SUPEROXIDE DISMUTASEACTIVITY IN RATS

169

70 ***

6050

■

40

W FO+EO.2

30

■ ❑

CONTROL

FO+E 1.2 FO+E 3.3

20 10 0 ,

Malondialdehyde FIG. 2 Effects of 2 weeks of dietary supplementationwith fish oil containing different amounts of vitamin E on in vitro malondialdehydeconcentration in the rat heart (nmol/g wet weight). Means ~ SEM, FO refers to fish oil and E 0.2 (n = 5), E 1.2 (n =9) and E 3.3 (n =4) refer to 0.2, 1,2 and 3.3 mg D-a–tocopheryl acetate/g fish oil, respectively. **P <0.01 and ●**P c 0.001 compared to control (standard pellets, n = 10).

TABLE 3 Plasma and Heart Metabolize Concentrations in Rats Fed the different Diets for 6 weeks

Diets SAT

2.85f 0.51 1.73* 0.11 9.1 * 0.1 3.1 * ().1

Triglycerides, mmollL Cholesterol, mmollL Glucose, mmo\fL Fibrinogen, mmollL MDA, /lmollL

0.72 k 0.04

a–tocopherol MDA Thromboxane Prostacyclin

80.8 * 1.97 44.3 k 2.25 57.8 * 19.4 391.8 k 54.0

FO

Pl&na 1.15t 0.1 I*** 1.20 f 0.09** 9.8 &0.2* 2.9 A 0.1 0.61 t 0.03

FO+E 1.08f 0.09*** 1.37 * O.1O* 9.2 k 0.2” 3.1 * 0.1 0.67 A 0.04

Myocardial(nmollgwetweight)

~

77.8 k 2.74 58.2 k 2.57** 21.1 f 4.0* 201.7 f 60.7

87.5 k 3.34* 50.2 k 1.80” 25.1 & 5.7A 247.3 k 79.4

***pc o.001comp~edtoSAT, ●P c 0.05 comparedto FO. SAT refers to diet enriched with butter; FO and FO+E refer to fish oil diets containing 1.2 and 3.3 mg D-a-tocopheryl acetate/g fish oil, respectively. A P =0.06compared to SAT.

R. LUOSTARINENet al.

170

200 190

!

T

170

I ■ ❑

E

160 150 140

,

130

1---

120

BUTTER FO

FO+E

1

100 Mn SOD FIG. 3 Effects of 6 weeks of dietarysupplementationwith fish oil or butter (control)on ratmyocardialMn superoxidedismutase(MnSOD)activity(units/gwet weight). MeansY SEM.FO (n = 6) andFO+E(n =6) referto fish oil containing1.2 and 3.3 mg D-a–tocopherylacetate/gfish oil, respectively.* P c 0.05 compared to control (n = 4).

ION After dietary supplementationwith fish oil, the relative proportionsof individurd(n-3) and (n6) PUFA in myocardial phospholipidswere substantiallyaltered. The percentages of EPA and DHA were higher and those of arachidonic and linoleic acid lower, with no difference between FO and FO+E. The latter finding is in conformity with studies in humans, in which EPA was increased and arachidonic acid decreased in neutrophil phospholipids to the same extent after dietary supplementation with FO and FO+E (unpublished observations). These results indicate that supplementation of fish oil with extra vitamin E, at least in a moderate dose (as in FO+E), does not influence the fatty acid incorporation into membrane phospholipids. Interestingly, although SAT contained much more saturatedfatty acids and less PUFAthan the fish oil die~ an equal proportionof saturated fatty acids and PUFA was found in myocardialphospholipidsof rats fed the two respective diets in our study. Similar observations have been made in myocardial and various other animal tissues (18) and it seems that the myocardial membranes of rats maintain constant proportions of saturatedand unsaturatedfatty acids regardlessof the type of dietaryfat. In the present study, myocardial Mn SOD activity was higher in rats fed fish oil than in those given saturated fat. Supplementationof the stabilized fish oil (FO) with extra vitamin E (FO+E) did not have any major influence on the results. To the best of our knowledge,increased SOD activity in the myocardium after intake of fish oil has only been reportedin one other study (19). The activity of another antioxidant enzyme, glutathione peroxidase, has been reported to be higher in the rat heart after intake of fish oil (20). The mechanismby which intake of (n-3) PUFA may increase myocardial SOD activity is unknown. In a recent study in mouse kidney, feeding the animals fish oil resulted in higher mRNA levels of several antioxidantenzymes, such as SOD (21).The highly unsaturated fatty acids in fish oil could, by increasing oxidative stress in the myocardium, influence gene expression

SUPEROXIDE DISMUTASEACTIVITY IN RATS

J

171

** o

I J

I 1“ Plasma MDA

‘

E ■

I

BUTTER

(/#j FO

■

FO+E

Myocardial MDA

FIG. 4 Effects of 6 weeks of dietarysupplementationwith fish oil or butter (control) on rat plasma (nmol/L, x1OO)and myocardial (nmol/g wet weight) malondialdehyde(MDA)concentrations.Meansf SEM. FO (n = 6) and FO+E (n = 6) refer to fish oil containing 1.2 and 3.3 mg D-ct-tocopheryl acetate/g fish oil, respectively. 0 P <0.05 compared to FO+E, ** P <0.01 compared to control (n = 4). for SOD, a free radical scavenger. There is growing evidence that (n-3) PUFA are able to regulate gene expression (22). It might be possible that a transcription factor (nuclear factor kappa B) is involved. This multiprotein complex can be activated by oxidative stress and induces a variety of genes involved in early defense reactions (23). Interestingly, tumor necrosis factor (TNF) has been shown to induce mRNA for Mn SOD, possibly throughinductionof free radicals (24). The question as to whetherthe incremedmyoeardialMn SOD activityis a specificeffect of (n3) fatty acids or a general effect of PUFA, cannot be answered on the basis of the present results. However, we have found that rats fed saffloweroil (containing(n-6) PUFA) have higher myocardial Mn SOD activity than those fed saturatedfat but lower than rats fed fish oil (unpublishedresults). In contrast to our findings, intake of a high level of dietary (n-3) PUFA (20%, w/w) for 16 wk has been reported to decrease Mn SOD activityin the weanlingrat heart comparedto a control diet containing lard/corn oil (25). It may be possible that high levels of dietary (n-3) PUFA in that study led to a consumption of endogenousantioxidantsin the heart, resulting in decreased SOD activity. In contrast, a moderate level of dietary (n-3) PUFA in the present study probably did not result in consumption of endogenous antioxidants, as indicated by an unaltered myocardial ce-tocopherol concentration.A moderate increasein oxidativestressmay thereforelead to inductionof SOD without any substantial consumption. The discrepancies between the results may also be explained by differences in the stability of the oils; the fish oil used in our studybeing very stable against oxidation (26). Formation of excessive amountsof oxygenfree radicalsplays a major role in the pathogenesis of reperfusion injury (10, 11), and therefore the activity of myocardial endogenous antioxidative defense systems such as that of SOD maybe an important factor in determining the vulnerability of the myocardiumto arrhythmia.

172

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The present studies also showed that myocardial thromboxaneproduction was lower in rats fed (n-3) PUFA than in control rats. Furthermore, the ratio between prostacyclin and thromboxane was higher in rats fed (n-3) PUFA than in those given standard pellet diets. Our results are in line with those of another study in which an increasein the above ratio in the rat heart was observed after several months of fish oil feeding (27). Supplementationof FO with extra vitamin E did not have any major impact on the results, despite a minor tendencyto a smallerdecrease in myocardial prostanoid production. In light of the fact that thromboxaneis arrhythmogenicand prostacyclin may act as an endogenous antiarrhythmic substance (9), the lower myocardialproduction of thromboxane and the higher ratio between prostacyclin and thromboxane during feeding with fish oil may also be of importancefor the antkwrhythmiceffect of fish oil. Intake of (n-3) PUFA (especially EPA) may reduce eicosanoid synthesis from arachidonic acid by several mechanisms: inhibition of the synthesis of arachidonic acid from linoleic acid, competition with arachidonic acid for a position in membrane phospholipids, and inhibition of the cyclooxygenaseenzyme (28). In the present study, the most probable cause for the lower production of myocardial prostanoids during fish oil feeding was a substrate(arachidonicacid) deficiency, since the percentage of arachidonic acid in myocardial phospholipidswas lower (both after FO and after FO+E) than in the control group. However, recent studiesin rats indicate that changes in prostanoid production cannot simply be explained on the basis of the phospholipid fatty acid composition (27, 29). For example, the regulatory role of lipid hydroperoxides in prostaglandin formation is well known (30, 31). In the present study, supplementation of FO with vitamin E resulted in lower formation of lipid peroxides measured as in vitro MDA formationin the myocardium. However, the prostaglandin formation did not differ between rats fed FO+E compared to those fed FO. Therefore, tissue peroxide tone seemed not to have any major influenceon the results in our sudy. It is well established that in myocardialcell membraneshighly unsaturated lipids, such as (n3) PUFA, are more prone to lipid peroxidationthan more saturated lipids (20, 32-36). In the present study, myocardial lipid peroxidationmeasuredas in vitro MDA productionwas signitlcrmtlyhigher in rats fed fish oil with a low or moderate vitamin E concentration than in those given control diets. Addition of extra vitamin E to fish oil (FO+E) resulted in a lower in vitro MDA and higher a– tocopherol concentration in the myocardium compared to the FO diet. Thus, a relatively modest increase in the concentration of dietary vitamin E in fish oil could significantlydiminish in vitro lipid peroxidation in the heart membranes. This may be of pathophysiological importance, since the present in vitro myocmiial MDA assay (withoutadditionof antioxidantsto the assay) may mimic an in vivo situation with strong free radical formationand consumptionof endogenousantioxidaots such as occurs during ischemia-reperfusion. Our results are in agreement with those of another study in which increasing amounts of vitamin E in fish oil reduced the in vitro lipid peroxidation in rat heart tissue slices (33). Interestingly, the fish oil diet in the present study did not alter the plasma MDA concentration, indicating that the MDA production in tissues may increase even though the plasma MDA concentrationis unchanged. The blood glucose concentration was higher in rats fed FO compared to that in the SAT and FO+E groups. This is in accordance with findings in human studies, where addition of extra vitamin E to fish oil counteracted the FO-inducedincrease in blood glucose (37), and maybe explained by a more adequate pancreatic insulin responseto glucoseduringintake of FO+E. Our data indicate that a stabilized fish oil and the same fish oil supplemented with extra vitamin E are approximatelyequally effectivein decreasingmyocardialthromboxaneproduction and increasing SOD activity. However, extra vitamin E in fish oil may result in lower oxidative stress in the myocardium, as indicated by a lower myocardial MDA concentration. Concerning this latter finding, it is possible that in situations with increased oxidative stress, such as during ischemiareperfusion, a higher dose of vitamin E in fish oil (as in FO+E) would be preferable. Also, in contrast to treatment with FO, the blood glucoseconcentrationwas not altered after intake of the FO+E diet. In conclusion, the present investigation showedhigher SOD activity and lower thromboxane production in the rat heart after intake of fish oil. These changesmay constitutepossible mechanisms underlyingthe reported antiarrhythmiceffect of fish oil.

SUPEROXIDE DISMUTASEACTIVITY IN RATS

173

ow~

This work was supported by grants from the Swedish Medical Research Council. We wish to thank Birgitta Alving and Ritva Jokela for expert technicalassistance.

1.

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Accepted for publicationJuly 5, 1996.