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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2009 Jun;90(3):321–327. doi: 10.1111/j.1365-2613.2009.00658.x

A detailed microscopic study of the changes in the aorta of experimental model of postmenopausal rats fed with repeatedly heated palm oil

Siti Khadijah Adam *, Srijit Das †, Kamsiah Jaarin *
PMCID: PMC2697554  PMID: 19563614

Abstract

Hypercholesterolaemia, increase in lipid peroxidation and hyperhomocysteinaemia may contribute to the pathogenesis of atherosclerosis. This study was performed to examine the effects of repeatedly heated palm oil mixed with 2% cholesterol diet on atherosclerosis in oestrogen-deficient postmenopausal rats. Ovariectomy causes disruption of tunica intima layer of the rat aorta simulating a postmenopausal condition in females. Twenty-four ovariectomized female Sprague–Dawley rats were divided into four groups. The control group received 2% cholesterol diet without palm oil. A diet with 2% cholesterol content fortified with fresh, once-heated and five-times-heated palm oil was given to the other treatment groups. The rats were sacrificed at the end of 4 months of study and the aortic arch tissue was processed for histomorphometry and electron microscopy. On observation, there was disruption of the intimal layer of the ovariectomized rat aorta. There was no obvious ultrastructural change in the aorta of the rats fed with fresh palm oil. The ultrastructural changes were minimal with once-heated palm oil, in which there was a focal disruption of the endothelial layer. The focal disruption was more pronounced with five-times-heated palm oil. The results of this study show that the ingestion of fresh palm oil may have a protective effect on the aorta but such a protective action may be lost when the palm oil is repeatedly heated. The study may be clinically important for all postmenopausal women who are susceptible to atherosclerosis.

Keywords: aorta, atherosclerosis, electron microscopy, heating, ovariectomy, palm oil

Atherosclerosis is one of the leading causes of illness and death in all the developed countries (Chan-Park et al. 2008). Considered to be a slowly progressive disease, it begins in childhood with the development of fatty streaks. The incidence of atherosclerosis and cardiovascular disease is lower in women as compared with men of similar age (Zago et al. 2004). Oestrogen level decreases after menopause thereby predisposing to atherosclerosis. Oestrogen has been reported to exhibit cardioprotective effects during pre-menopausal stage (Deroo & Korach 2006). Administration of oestrogen has proved to decrease low-density lipoprotein (LDL) levels and increase high-density lipoprotein (HDL) levels in postmenopausal women (Herrington et al. 2002). Interestingly, past studies have shown that oestrogen exhibits antioxidant properties that are responsible for the protective action against lipid peroxidation (Badeau et al. 2005).

Atherosclerotic lesions in human and animals appear to be related to elevated plasma total cholesterol (TC), LDL and decreased HDL. The mechanism behind the hypothesis as how hypercholesterolaemia can cause atherosclerosis, is not well understood. LDL in the blood moves inside the vessel wall thereby resulting in lipid deposition. This LDL is then oxidized and will contribute to atherogenic process (Moreno & Mitjavila 2003). The oxidized LDL (oxLDL) is the sole responsible factor altering the structure and function of the endothelial cell which attract monocytes and macrophages by chemotactive action which then develop into the lipid laden foam cells of an atheromatous plaque (Turk et al. 2005).

The fat and protein consumed in our diet is always exposed to heat during processing. It has been observed that in deep frying, cooking oil may attain a high temperature of 150–190 °C (Choe & Min 2007). Surprisingly, the heating process generates lipid peroxidation products that may affect human health (Lapointe et al. 2006). The common practice of repeatedly using the oil for frying may generate free radicals which are considered to be harmful to our health. Such practices are followed to cut down the cost of cooking with the consumers being unaware of its side effects.

Several studies had demonstrated the adverse effects of oxidized dietary fats on human and experimental animals. There are research reports which depict that consumption of oxidized oil caused liver dysfunction (Hayam et al. 1995; Owu et al. 1998). Elevation of plasma TC levels, with decrease in level of HDL and increase in level of LDL has been documented (Narasimhamurthy & Raina 1999). In fact, one of our earlier research studies had demonstrated the biochemical and histological changes (light microscopy) as a result of heated palm oil consumption (Leong et al. 2008).

Acceleration of fatty streak formation has been reported in rabbits fed oxidized lipid (Hur et al. 2005). It has also been reported that oxidized fats in the diets can cause excessive hair loss, heart, kidney and liver damage (Gabriel et al. 1977). Keeping those facts in mind, this study was performed to determine the effects of thermally oxidized palm oil taken together with 2% cholesterol diet in ovariectomized animal model of oestrogen-deficient female rats. The study assumes an important role considering the fact that heated palm oil which is widely used as cooking oil may have an influence on the development of atherosclerosis in postmenopausal women. To the best of our knowledge, this is the first study to demonstrate the electron microscopic changes in the aorta as a result of consumption of heated palm oil.

Materials and methods

Experimental animals and diet

Thirty healthy and mature female rats of Sprague–Dawley strain (200–250 g) were obtained from the Animal Unit, Universiti Kebangsaan Malaysia. The rats were housed in stainless steel cages at room temperature 27 ± 2 °C and a 12-h light cycle. The experiments were performed after having proper ethical clearance from the UKM Animal Ethic Committee. The animals had free access to water and were administered 150 g of test diet/week. Body weight was checked every week on the same scale.

Palm oil (Lam Soon Edible Oil, Malaysia) used was fresh, once-heated or five-times-heated as described by Owu et al. (1998) and Isong et al. (2000). The heating process involved using 2.5 L of the palm oil to fry 1 kg (approximately 25 pieces) of ‘keropok lekor’ (fish-flavoured chips) in a metal wok. The temperature of the heated oil reached about 180 °C, and the cooking process lasted for 10 min (Adam et al. 2008b). To heat the oil five times, the oil was cooled for 5 h in between heating, then the whole frying process was repeated with a fresh batch of ‘keropok lekor’. No fresh oil was added between batches to make up for any loss due to uptake of the oil by the frying material. This protocol was in accordance with our earlier experimental procedures (Adam et al. 2008b).

We used 2% cholesterol diet obtained from MP Biomedicals, (MP Biomedicals, Inc., Seven Hills, Australia). A mixture of test diet with 15% (w/w) of palm oil and ground 2% cholesterol diet was made. The mixture was made into pellets which were dried overnight at 80 °C in an oven (Adam et al. 2008b).

Study design

The rats (n = 24) were allowed to acclimatize for 1 week prior to treatment and were ovariectomized ahead of the study. They were randomly divided equally into four groups; group I was fed with 2% cholesterol diet (OVXC), group II, III and IV were administered 2% cholesterol diet fortified with fresh palm oil (FPO), once-heated palm oil (1HPO) and five-times-heated palm oil (5HPO) respectively, for 4 months (Adam et al. 2008b). Another group (n = 6) i.e. the normal control group was administered normal rat chow throughout the study period (NC). Their food intake and body weight were measured carefully, every week. After 4 months of feeding with respective diet, all the rats were killed. The proximal portion of ascending aorta and the arch of aorta were taken for histomorphometric (light microscopic study with Verhoeff van Gieson stain) and electron microscopic studies respectively.

Histomorphometric study (light microscopic study with Verhoeff van Gieson stain)

The aorta tissues were removed and preserved in 10% formalin and underwent standard tissue processing techniques. The cross sections of the aorta were then subsequently stained with Verhoeff van Gieson stain. The intimal and medial thickness was measured at 0, 90, 180 and 270° of each of the cross section using computerized image analyser with software (Image-Pro Plus version 5.0.2.9, Media Cybernetics, Inc., Bethesda, USA), in conjunction with microscope (Eclipse 80i, Nikon Corporation, Tokyo, Japan) and camera (Qimaging MicroPublisher 5.0, Surrey, Canada). Their average reading was taken for statistical analysis.

Electron microscopic study

Pieces of less than 0.5 mm thickness were immersed for 12–16 h at 4 °C in glutaraldehyde fixative. The samples were washed three times in 0.1 M phosphate buffer; treated with 1% buffered osmium tetroxide for 1–2 h and washed in distilled water for three times. Then, they were bulk-stained with uranyl acetate for 30 min; dehydrated in an ascending series of ethanol solutions; infiltrated in propylene oxide and embedded in resin for 24 h at 60 °C. Ultrathin sections were collected and stained with 3% uranyl acetate and Reynold’s lead citrate. These specimens were examined with a transmission electron microscope (Philips HMG 400, Philips, Eindhoven, The Netherlands) and micrographs were taken.

Statistical analysis

The data were presented as the mean ± standard error of mean (SEM). Normally distributed data were analysed using parametric tests, i.e. Student’s paired t-test and analysis of variance (anova) followed by Tukey HSD post hoc test. Data which were not normally distributed were analysed using non-parametric tests, i.e. Kruskal–Wallis, Mann–Whitney U-test and Wilcoxon-Signed Rank tests. A value of P < 0.05 was considered significant. All analysis was conducted using Statistical Product and Service Solutions (spss) software.

Results

Relation of food consumed and body weight

We observed that the oil-fed groups had lesser food intake as compared with NC and OVXC. The body weight for all the groups increased significantly but the highest increase in body weight was observed in 5HPO group (Figure 1).

Figure 1.

Figure 1

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Average food intake per week and body weight gain of rats fed respective diets after 4 months. Results are mean ± SEM (n = 6). a, significant compared to NC (P < 0.05); b, significant compared to OVXC (P < 0.05); c, significant compared to FPO (P < 0.05); d, significant compared to 1HPO (P < 0.05). NC, normal control; OVXC, ovariectomized control; FPO, fresh palm oil treated group; 1HPO, once-heated palm oil treated group; 5HPO, five-times-heated palm oil treated group.

Histomorphometric study

Table 1 shows the average intimal thickness, medial thickness as well as intima/media ratio. There was no significant difference of intimal thickness among the treatment groups. However, all ovariectomized groups showed significant reduction of medial thickness compared with the normal control. FPO showed an increase in medial thickness compared with OVXC while 5HPO showed a reduction compared with NC and FPO. When the ratio of intima/media was calculated, only 5HPO showed a significant difference compared with the other groups.

Table 1.

Intimal thickness, medial thickness and intima/media ratio of rats according to group

Group Intimal thickness (μm) Medial thickness (μm) Intima/media ratio
NC 4.05 ± 0.39 141.03 ± 5.07 0.029 ± 0.003
OVXC 3.40 ± 0.22 92.35 ± 1.99* 0.037 ± 0.002
FPO 2.82 ± 0.14 106.29 ± 3.79* 0.027 ± 0.001
1HPO 3.50 ± 0.31 92.73 ± 3.44* 0.037 ± 0.002
5HPO 3.83 ± 0.19 83.07 ± 2.84*‡ 0.046 ± 0.002*†‡§
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Results are mean ± SEM (n = 6).

NC, normal control; OVXC, ovariectomized control; FPO, fresh palm oil treated group; 1HPO, once-heated palm oil treated group; 5HPO, five-times-heated palm oil treated group.

*

Significant compared with NC (P<0.05).

†

Significant compared with OVXC (P<0.05).

‡

Significant compared with FPO (P<0.05).

§

Significant compared with 1HPO (P<0.05).

Electron microscopic study

Ovariectomized control showed disruption in the intimal layer compared with the normal control (Figure 2a,b respectively). Condensation of cytoplasm and karyopyknosis was observed in the ovariectomized control. Corrugation was present in the internal elastic lamina (→). However, FPO did not show any obvious ultrastructural changes (Figure 2c). Endothelial cell was seen similar to normal control. Both 1HPO and 5HPO showed disruption of the intimal layer (Figure 2d,e respectively), but the 5HPO had severe damages as compared with the 1HPO group. The subendothelial width increased with more collagen fibrils (c) and some vacuolization (v) were observed near the endothelial cells. Mononuclear cells were also observed in the intimal layer.

Figure 2.

Figure 2

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(a) shows normal control rat aorta, (b) shows ovariectomized control rat aorta, (c) shows rat aorta fed with fresh palm oil, (d) shows rat aorta fed with once-heated palm oil and (e) shows rat aorta fed with five-times-heated palm oil. Note the corrugation in the IEL (→), collagen fibrils (c) and vacuolization (v) in the subendothelial layer. L, lumen; TI, tunica intima; IEL, internal elastic lamina; TM, tunica media; EC, endothelial cell; MNC, mononuclear cell. (7700×).

Discussion

Research reports depict that oestrogen therapy is essential in postmenopausal women to protect themselves against atherosclerosis and cardiovascular diseases (Naessen & Rodriguez-Macias 2006). This study was conducted on the postmenopausal rat model to observe the ultrastructural changes in the aorta due to the ingestion of heated palm oil. Palm oil was chosen in this study because of its wider usage as cooking oil in Malaysia. The oil is often used repeatedly for deep-frying at many food outlets to save cost incurred in cooking. Unfortunately, the general public are unaware of such harmful effects caused due to repeated heating.

We used 2% cholesterol diet because one of our previous studies had shown that 2% cholesterol diet was atherogenic to rabbits (Kamsiah et al. 1994). The addition of palm oil in the cholesterol diet had caused lower food intake as compared with normal diet or 2% cholesterol diet alone. However, heating process did not have any significant effects on the food intake. Despite the difference in the degree of heating process, the food intake of the treatment groups were similar and it was associated with similar increase in their body weight. It is notable to highlight that the body weight gain in the five-times-heated group showed significant changes as compared with the fresh and once-heated group. The reason for this is not well understood, but we assume that increase in fat deposition in adipose tissue might have contributed to the increase in weight.

Ageing is responsible for the differential changes in the tunica intima (TI) and tunica media (TM) thickness and the development of atherosclerosis (Naessen & Rodriguez-Macias 2006). In this study, we had performed the electron microscopic study on the arch of aorta as this is considered to be the most susceptible site for any haemodynamic changes in the vessel leading towards atherosclerosis (Schwenke 1995). We found that the TI layer of the treatment groups were unaffected. However, the TM layer became thinner in the ovariectomized groups as compared with the normal control (Table 1). Interestingly, increase in the tunica intima/tunica media (TI/TM) ratio is an early indicator of atherosclerosis (Zureik et al. 2000; Rodriguez-Macias et al. 2006). Considering the significant increase in TI/TM ratio, this study may also denote an early stage of atherosclerosis. Even though the TI thickness did not exhibit any changes, the TM thickness decreased substantially and this might contributed to the significant increase of the TI/TM ratio in 5HPO.

Ovariectomy represents an oestrogen-deficient state where the muscle growth may be deficient. After the rats were ovariectomized, the administration of fresh palm oil together with cholesterol diet exhibited its protective effects. The protective effects of the palm oil were lost while it was heated once and five times which was evident from the reduction of TM thickness. There is a possibility that presence of oestrogen and the antioxidant properties of the fresh palm oil could have promoted muscular growth via the release of growth factors. The muscle mass in the TM may be dependent on the oestrogen activity. Earlier research reports suggested that oestrogen produces muscle growth (Kamanga-Sollo et al. 2008). It has also been found that oestrogen has a vasodilator effect on the cardiovascular system (Sharpe 1998). Thus, the protective effect of the oestrogen might have been lost in the ovariectomized state. The tocotrienol-rich fraction (TRF) in the palm oil is known to exhibit cardioprotective effects (Das et al. 2008). This may explain why the fresh palm oil acted as a protective agent. Repeated heating of palm oil may predispose the oil to oxidation thus making it darker in colour. The uncontrolled oxidation leads to peroxidation and the resultant action is cytotoxic thereby causing hypertension, increase basal metabolic rate and tissue damage (Ebong et al. 1999). In this study, the repeated heating of the palm oil may have resulted in oxidative damage to the muscular wall of the vessel and this is accordance with earlier research findings.

To the best of our knowledge, no past studies had reported the electron microscopic findings of the effect of heated palm oil on the vascular structure in postmenopausal rat model. Majority of the studies looked into the biochemical parameters of heated oil such as lipid peroxidation, lipid profile and liver profile with no mention of electron microscopic study. In our earlier studies, it was observed that oxidative stress caused by heated oils may modulate lipid peroxidation and lipoprotein level changes thereby predisposing to atherosclerosis (Adam et al. 2008a,b).

Endothelial cells in the normal rat aorta are usually elongated or elliptical in shape (Flaherty et al. 1972). However, it was found that there was disruption of the intimal layer of the ovariectomized rat aorta (Figure 2b). This may be attributed to the deficiency of oestrogen due to ovariectomized state. There was no obvious ultrastructural change observed in aorta of the rats fed with fresh palm oil. A uniform endothelial lining and the endothelial cell shape resembled that of a normal control. No corrugation was observed in the internal elastic lamina (IEL). These findings suggest that fresh palm oil which is rich in antioxidant contents protects the rat aorta from any damage caused by ovariectomy and high cholesterol diet, which are stress oxidative conditions. Research studies have shown that oxidants play an important role in the pathogenesis of atherosclerosis (Siekmeier et al. 2007). Vitamin E has important constituents like α-tocopherol, which has protective effect on the tunica media of the artery. It has been found that α-tocopherol helps to protect the cell membranes from lipid peroxidation by trapping peroxyl radicals (Dutta & Dutta 2003). Thus, postmenopausal women may be protected from developing atherosclerosis by palm vitamin E, but it is important that the oil should not lose its potential because of repeated heating. The reason for the harmful effects of the heated oil may be attributed to the reduction of vitamin E content which was observed in one of our earlier study (Adam et al. 2007). We had observed that heating reduced α-tocopherol, α-tocotrienol, γ-tocotrienol and δ-tocotrienol (Table 2). These may be deleterious to rat aorta and may predispose to the development of atherosclerosis.

Table 2.

Percentage of difference of PO1H and PO5H on α-tf, α-tt, γ-tt and δ-tt compared with POF (n = 6)

Oil Vitamin E fractions Percentage difference (compared with POF) (%)
PO1H α-tf −55.70
α-tt −57.47
γ-tt −25.94
δ-tt −14.32
PO5H α-tf −98.13
α-tt −97.97
γ-tt −87.80
δ-tt −48.69
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Reproduced from Malaysian Journal of Biochemistry and Molecular Biology with due permission.

POF, fresh palm oil; PO1, once-heated palm oil; PO5, five-times-heated palm oil.

The ultrastructural changes were minimal in the once-heated group but it was more severe in five-times-heated group. There was increase in the width of subendothelial layer containing intercellular materials such as collagen fibrils. Vacuolization was observed near the endothelial cells. There was presence of mononuclear cells which may be migrated from the TM layer into the TI layer. These cells could be differentiated from endothelial cells due to their location being nearer to the IEL while endothelial cells were located near to the lumen. The aorta of ovariectomized control also showed severe damage similar to 5HPO group. In addition, the nucleus of endothelial cell had denudated and corrugation was also observed in the IEL. The cytoplasm of the nucleus was condensed and karyopyknosis was observed in the ovariectomized control indicating an apoptosis (Shi et al. 2007). According to Moreno and Mitjavila (2003), an excess of oxLDL in macrophages during progression of atherosclerosis can induce necrosis or apoptosis that may contribute to the development of advanced atherosclerotic lesions. There was also mononuclear cell, which we assume has penetrated into the intimal layer from circulation. These findings showed that ovariectomy caused intimal disruption of rat aorta and feeding with palm oil offered some degree of protections which was lost gradually when the oil was repeatedly heated.

Often, the procedure of repeated heating is adopted at house or in the restaurants to curb cooking expenses. Unfortunately, the harmful effects of repeated heating are never considered. Vitamin E is considered to be an important constituent of palm oil and repeated heating may predispose the oestrogen-deficient postmenopausal individuals to atherosclerosis. Based on the observations, we opine that thermally oxidized palm oil generates free radicals that enhance the oxidative stress secondary to oestrogen deficiency and ingested high cholesterol diet. Fresh palm oil, may offer some protection in postmenopausal rat model but its protective effect is lost when the oil is being repeatedly heated.

Acknowledgments

This study was funded by a grant received from Ministry of Science, Technology and Innovation. The authors wish to thank Dr Faizah Othman from Department of Anatomy and Puan Sinar Suriya Muhamad from Department of Pharmacology, UKM for their valuable assistances. We would also like to thank Mr Megat Radzi Abdul Rani from Department of Pathology, UKM and Mr Rafiuz Zaman Haroun from Institute of Bioscience, Universiti Putra Malaysia for their technical assistances. The authors are grateful to the Editor of Malaysian Journal of Biochemistry and Molecular Biology for having allowed to reproduce the Table 2 from an earlier published article in that journal.

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