Lipid Peroxidation in Different Tissues: Effect of High Cholesterol and Fish Oil in the Diet
SHARMILA UPADHYA*, KAVITHA, PRASHANTI, RAJYALAKSHMI, ROHINI P., SEETHA, SUCHARITHA M. AND SUBRAMANYA UPADHYA**
*Department of Biochemistry,
Kasturba Medical College,
Manipal – 576 119
**Department of physiology
Kasturba Medical College,
Manipal – 576 119
(Received on August 28, 2001)

Abstract: Malonyldialdehyde was measured in erythrocytes, aorta and spleen on feeding mice with high cholesterol diet in presence and absence of fish oil. Mice were grouped as:

Group I            :           Control laboratory diet

Group II           :           0.16% cholesterol (sunflower oil)

Group III          :           1.16% cholesterol (sunflower oil)

Group IV         :           1.16% cholesterol (fish oil)

After 7 weeks on their respective diets, erythrocytic, and splenic MDA levels were significantly higher in group III compared to controls. Also, MDA levels in aorta and spleen showed a significant increase in group IV males compared to group III males. However in group IV the erythrocyte MDA levels were almost equal to that in controls. This suggests that high cholesterol diet increases lipid peroxidation in erythrocytes, spleen and aorta. Addition of fish oil in the diet further increases lipid peroxidation in aorta and spleen, but not in the erythrocytes.

Key words:      lipid peroxidation                                  malonyldialdehyde       

fish oil                                                   high cholesterol diet

Introduction
Methods
Results
Discussion
References

INTRODUCTION

Different dietary habits have been reported to be associated with the risk of atherosclerotic plaque formation. It has been observed that diets rich in fish are associated with a decrease in the incidence of atherosclerosis and that is related to the n-3 fatty acid content of fish (1). Lipid peroxidation has been implicated in a variety of processes including atherogenesis (2). It has been suggested that an increased intake of polyunsaturated fatty acids increases the lipid peroxidation in plasma and in lipoproteins. The n-3 fatty acids present in fish oil have been shown to increase lipid peroxidation by various authors (3, 4, 5). Others have reported no increase in lipid peroxidation in women on dietary fish oil, but have reported an increase in thiobarbituric acid reactive substances in plasma on dietary fish oil compared to other oils (6). Introduction of dietary fish oil has reported to induce an oxidative stress in hepatocytes in rats (7). In the present study, we have made an attempt to evaluate the extent of lipid peroxidation by estimating the malonyldialdehyde (MDA) in various tissues.
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The study was designed to evaluate the influences of varying the dietary cholesterol content and the addition of fish oil on the levels of MDA in various tissues in mice. Swiss albino mice of male and female sex were included in the study separately as the serum cholesterol levels of females were influenced by hormones (8). Male animals were divided into four groups:

Group I (n = 10): Normal laboratory diet containing 8% of the calories provided by lipid and containing negligible amounts of cholesterol.

Group II (n = 10) : 0.16 gm% cholesterol diet and 1.6 gm% as sunflower oil per 100 gm of diet with 25% of calories provided by dietary lipids (25 Cals/1 100 Cals).

Group III (n = 10): 1.6 gm% cholesterol diet and 1.6 gm% as sunflower oil per 100 gm diet with 25% of calories provided by dietary lipids.

Group IV (n = 9): 1.6 gm% cholesterol diet and 1.6 gm% as fish oil per 100 gm diet with 25% of total calories provided by dietary lipids.

Female mice were similarly divided into the four groups (n =4, n = 8, n = 6, n = 6) respectively. All the diets were prepared in the laboratory considering the normal daily requirements of various nutrients. The diets were started once the mice were four weeks old. Serial body weights were measured once every week to monitor the weight gain in the animals. The animals were sacrificed after seven weeks on their respective diets. Blood was obtained by decapitation using citrate as the anticoagulant. Blood samples were preserved in ice until analysis. Aorta was obtained from the root of the aorta until the bifurcation of the aorta. Aorta and spleen obtained were preserved in chilled normal saline until homogenization. Analysis of MDA as thiobarbituric acid reacting substances in erythrocytes, aorta and spleen were performed on the same day.

MDA in erythrocytes was estimated by the method of Jain et al (9). Hemoglobin concentration was estimated by using the Drabkin’s reagent (10). MDA was estimated by using the extinction coefficient of MDA-TBA complex at 532 nm = 1.56 x 10⁵/cm/M. MDA values were expressed in nanomoles/gm of hemoglobin. MDA was estimated in the aortic and splenic homogenates (10% w/v) as TBA reacting substances (11). Tissue protein content was estimated by Folin Lowry’s method. MDA was expressed as nanomoles/mg of protein. Serum cholesterol was estimated by the cholesterol oxidase peroxidase method (Ranbaxy kit method).

Statistical analyses were performed by using Statview version 3 software package. Analyses were done between the groups by the unpaired ‘t’ test. Correlation coefficient was used for correlation analysis. Correlation was also confirmed by Fisher’s r to z test.
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The serum cholesterol and MDA levels in spleen, aorta and erythrocytes (mean ± SD) in males and females in all groups of animals are given in Tables II and I. Increasing the dietary cholesterol content has increased the serum cholesterol level in both males and females (P = 0.001, P = 0.01, group II vs. group III). In mice fed with high cholesterol diet (Group III), there was an increase in serum cholesterol compared to controls. However, there was a significant decrease in serum cholesterol in mice on fish oil diet (Group IV) compared to group III (P = 0.0001, P = 0.0001) and Group I (P = 0.0001, P = 0.01) in both males and females.

The MDA levels in erythrocytes have significantly increased in mice of Group III compared in group I in males and females indicating that increasing cholesterol content of the diet has increased the lipid peroxidation in the erythrocytes. However, in the fish oil group (group IV), the MDA levels are significantly lower compared to group III, despite the high dietary cholesterol content in group IV (P = 0.0004, P = 0.003), in males and females. Also, the mean MDA levels in erythrocytes of group IV are not significantly different when compared to control in males and females (P = 0.58, P = 0.09). Significant correlation between serum cholesterol and MDA in erythrocytes was observed in both males and females as shown in Fig. 1. The high dietary cholesterol has increased the MDA in erythrocytes, indicating that erythrocytes are exposed to an oxidative stress in the presence of cholesterol. And also, it has been observed that dietary fish oil has a hypocholesterolemic effect as well as a protective effect against lipid peroxidation in erythrocytes.

Fig.1 click for full view

TABLE I: The serum cholesterol, MDA levels in erythrocytes, spleen and aorta (Mean ± SD) in males of all groups is given.

All groups were compared to controls (group I)

*     P=0.01-0.02

**   P=0.001-0.008

*** P= <0.0001

Group I: Controls

Group II: 0.16% cholesterol, 25% fat

Group III: 1.6% cholesterol, 25% fat

Group IV: 1.6% cholesterol, 25% fat (fish liver oil)

TABLE II: The serum cholesterol, MDA levels in erythrocytes, spleen and aorts (Mean ± SD) in females of all groups in given.

All groups were compared to controls (group I)

*     P=0.01-0.05

**   P=0.001-0.005

*** P= <0.0003

Group I: Controls

Group II: 0.16% cholesterol, 25% fat

Group III: 1.6% cholesterol, 25% fat

Group IV: 1.6% cholesterol, 25% fat (fish liver oil)

The MDA levels in aorta have increased in group III compared to group I and group II (P = 0.0002, P = 0.0001) in both males and females. In contrast to the MDA in erythoryctes, the MDA in aorta has increased in mice of the fish oil group (group IV) compared to group I in males. However in females the increase in MDA levels in group IV is not statistically significant compared to group I. The aortic MDA levels in group IV mice are significantly higher than in group III in males (P = 0.02). However, in females the increase in aortic MDA levels in group IV is not statistically significant compared to group III.

Splenic MDA levels are also increased in mice of group III and they have further increased in group IV compared to controls in both males and in females. The MDA levels in spleen are significantly higher in mice of group IV compared to group III (P = 0.01) in males, however such a difference was not observed in females.

The above results indicate that a high dietary cholesterol content increases the MDA levels in erythrocytes, spleen and aorta, implying an increase in lipid peroxidation in the presence of cholesterol. There is an increased lipid peroxidation observed in the tissues such as spleen and aorta in the presence of fish oil. But erythrocytes are protected from peroxidation in the presence of fish oil in the diet.
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In our study we have observed a significant decrease in serum cholesterol in mice on a diet containing fish oil. However, the effect of fish oil on serum cholesterol level is varied. Many authors (3, 12, 13) have observed a decrease in serum cholesterol in the presence of dietary fish oil. An increase in serum cholesterol has been observed on a diet rich in n-3 fatty acids (14). Higgins et al have reported that, in low doses fish oil has no effect on serum cholesterol (15). Falx seed, which is a rich source of n-3 fatty acids, has been reported to decrease the severity of atherosclerosis without decreasing serum cholesterol level (16).

We have found an increase in MDA levels in aorta and spleen in animals on dietary fish oil, indicating that incorporation of fish oil in the diet increases lipid peroxidation in these tissues. This may be because of the incorporation of polyunsaturated fatty acids present in fish oil, into these tissues. These polyunsaturated fatty acids are more prone to oxidative damage, thus increasing MDA levels in these tissues. Similar increase in MDA levels in aorta has been reported to be increased on feeding animals with high cholesterol diet (17, 18) and in the presence of fatty streak lesions in aorta (19). There are also reports that endothelial cells (20) and membranes (21) were more susceptible to oxidant damage in the presence of dietary fish oil. There is also an increased susceptibility to the oxidative modification of LDL in the presence of fish oil in the diet (4, 22). An increase in MDA level in spleen homogenates in mice fed fish oil diets has also been reported by Avula et al (5). Such lipid peroxidative damage may be decreased by the incorporation of vitamin E which is an antioxidant suggested by some authors (21, 22). Bruckner has suggested that incorporation of fish oil in the diet without adequate vitamin E supplementation may be deleterious to the vascular endothelium because of the oxidative stress imposed by the polyunsaturated fatty acid content of fish oil (22). Brown and Wahle reported an increase in plasma TBA reacting substances in group with fish oil supplementation and with the addition of vitamin E (23). There are also reports of an increase in initial foam cells in the aorta of animals on fish oil (24).

However, in our study, we have found that the erythrocytes are not influenced by the presence of fish oil in the diet. In fact the MDA levels in the erythrocytes are significantly decreased despite the high dietary cholesterol content in the fish oil group, implying that dietary fish oil is protective against lipid peroxidative damage caused by the high cholesterol diet. Contrary to our findings, Konukoglu et al have reported no significant difference in MDA levels in RBC of hypercholesterolemic and normocholesterolemic subjects (25).

In our study there has been a significant decrease in serum cholesterol level in mice fed with fish oil when compared to controls. This could be due to the fact that polyunsaturated fatty acids in fish oil increase the utilization and uptake of cholesterol. Intake of fish oil increased the tendency for lipid peroxidation in spleen and aorta but spares the erythrocytes.
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