The repeated deep-frying process has been documented to be deleterious to the stability of unsaturated fatty acids and other biochemical parameters such as peroxide content, polar material and acid value [32–34] of dietary cooking oil. Fats are usually oxidized by free radicals at the sites of unsaturated bonds in the fatty acid chains. Fats with higher number of unsaturated bonds are prone to oxidation. FSO had a higher ratio of unsaturated fatty acids, thus more susceptible to oxidation (shown in Table 1). Deep-frying oil contained relatively more saturated fatty acids with less unsaturated fatty acids. Peroxide value is usually used as an indicator of the extent of oxidative rancidity. From the results obtained, the extent of oxidation was affected by the number of frying. Repeatedly deep-fried oil also had a rancid odour and darkening of colour compared to the fresh oil.
Incorporation of thermally oxidized oil to the rat's diet was meant to simulate human daily dietary pattern and formed part of the balanced diet. A group fed with fresh oil was included in the study to normalize any effects of the lipid fortification that was not oxidative in origin. There was a significant increase in the body weight at the end of the study for all the groups. This finding suggests that prolonged feeding with fresh or heated soy oil did not affect the growth response. Body weight gained was comparable amongst the control and other test groups. In addition, rats fed with diet enriched with heated soy oil had lower body weight. This may be due to the fatty acid composition in the dietary frying oil. Soy oil is rich in polyunsaturated fatty acid. When the soy oil is repeatedly deep-fried, oxidation degrades oil quality , producing sensory changes such as taste, texture and odour that may responsible for the lower food intake in these rats. Additionally, digestibility and absorption of fatty acids might get affected .
In present study, rats fed FSO and 1HSO did not show any significant differences in the blood pressure compared to the control. In spite of this, FSO exhibited a tendency to lower blood pressure at the end of experimental period which was in agreement with a recent study performed by Ribeiro Junior et al. . Administration of repeatedly heated oil consisting of 2HSO, 5HSO and 10HSO for 24 weeks caused a significant elevation in blood pressure in the rats. This was in accordance with earlier investigations [28, 29] demonstrating reheated-palm oil fed group had a significantly greater elevation in blood pressure than the control and fresh-oil fed groups. An earlier study conducted on the cooking oils reported that repeatedly oxidized frying oil is an independent risk factor for hypertension .
Our data reported that heated soy oil significantly reduced plasma nitrite levels which are the by-products of NO metabolism. Heated soy oil has been demonstrated in our laboratory to have reduced vitamin E constituents such as α-tocopherol, γ-tocopherol and δ-tocopherol  that act as a natural source of antioxidant against generation of free radicals during the frying process. Repeated heating of the cooking oil could not prevent unsaturated fatty acids from oxidative damage through lipid peroxidation. The reduction in nitrite levels could be explained by the enhanced NO sequestration by ROS and inactivation of NO due to the imbalance of antioxidant status. Subsequently, this causes cellular injury and increases blood pressure.
On the contrary, FSO was found to increase nitrites level. FSO contains natural antioxidant which may provide some protective effect by reducing oxidative stress or improving in production of NO . The study by Mahn et al. showed that dietary soy protein enhanced expression of nitric oxide synthase (NOS) and antioxidant enzymes . NO production is catalyzed by NOS. It has been documented that a reduction in NO release maybe due to deficiency of NOS [42, 43]. Therefore, a decrease in NO production combined with antioxidant/oxidant imbalance may be responsible to the development of endothelial dysfunction .
The vascular endothelium may generate a variety of ROS, which under pathological conditions, plays an important contributory role in the pathogenesis of hypertension . ACh was used to study the effects of the heated oil diet on endothelial function. On the other hand, SNP was employed as an endothelium-independent vasodilator in vascular smooth muscle. In the present study, we observed that 5HSO and 10HSO attenuated the endothelium-dependent relaxation induced by ACh as well as the endothelium-independent relaxation induced by SNP in the aortic rings compared to other dietary groups. Alternatively, rats fed with FSO exhibited greater relaxant responses. Diet rich in soy has been reported to have beneficial effect on endothelial function with lower blood pressure . Furthermore, Tousoulis et al. were able to show that consumption of soy oil may improve endothelial function in human healthy subjects .
Vasorelaxation induced by ACh involves increased bioavailability and release of NO from the endothelium. In contrast, SNP molecules undergo chemical transformation to generate NO which activates cyclic GMP-dependent relaxation in the aortic rings. Heated soy oil attenuated the endothelium-dependent relaxation induced by ACh. However, SNP-induced relaxation was similar in all the groups, indicating the ability of vascular smooth muscle to relax in response to exogenous NO was not impaired in heated oil-fed rats. Heated oil diet selectively impaired endothelium-dependent vasodilatation induced by ACh.
According to the present study findings, it is apparent that heated soy oil diet enhanced PE-induced contraction compared to the control and FSO groups. This indicates an increased in vascular reactivity which would contribute to increasing vascular tone. Generation of free radicals such as superoxide anion has been associated with increased vascular contractile reactivity . This effect may be mediated by reducing NO bioavailability in the aorta of heated oil groups as observed in the present work. In addition, antioxidant protective effect may be diminished when the oil is repeatedly heated.
Measurement of plasma nitrite indirectly indicated there was reduced NO bioavailability either due to reduced NO released from the endothelium or increased inactivation of NO by ROS. Published reports showed that chronic ingestion of repeatedly heated palm oil similarly impaired endothelial ex-vivo[29, 47]. A limitation in the present work was the absence of measurement of ROS levels to correlate with the endothelial dysfunction.
HO plays a major role in the modulation of blood pressure and vascular tone. It has been postulated that HO-dependent by-products, biliverdin and carbon monoxide have cytoprotective effects against oxidative stress [48, 49]. In present experiment, HO level was found to be decreased in all the experimental groups. Nevertheless, repeatedly heated soy oil showed a higher percentage of reduction in plasma HO concentration. Previous research studies reported that over-expression of HO-1 inhibits lipid peroxidation and affects NO metabolism [50, 51]. Furthermore, high expression of HO-1 has been linked to an increased in HO enzyme activity and a reduction in blood pressure . In present work, we postulate that chronic consumption of heated soy oil suppressed HO enzyme activity and consequently resulted in unheeded ROS generation.
The present results showed that plasma ACE level was significantly elevated in all heated oil treated groups with 10HSO group showing the highest values. ACE converts inactive Ang I to potent vasoconstrictor, Ang II and raising blood pressure. Ang II increases generation of superoxide free radicals via NADPH/NADH oxidase system. Ang II has a dual role in elevating blood pressure, direct vasoconstrictor effect and increasing production of free radicals which reduces bioavailability of NO and indirectly attenuating endothelium-dependent relaxation responses.
Our results indicated that heated soy oil increased blood pressure and ACE levels with a reduction in NO content. These findings were contradictory to a past study that had reported no influence on blood pressure, ACE activity and an increased in NO concentration . The results could be due to differences in duration of study, the method of oil preparation, the age and strain of the rats. For present study, heated soy oil was fed to adult Sprague-Dawley rats instead of 7-week old spontaneously hypertensive rats and Wistar Kyoto rats . Secondly, the animals in earlier study  were fed for 10 weeks compared to our rats which were fed for 24 weeks in present study. In addition, the heating procedure was differed in terms of fried food, duration of frying and cooling of the oil.
Our previous study  had used palm oil, with its saturated fatty acid to unsaturated fatty acid ratio close to one compared to soy oil with higher level of polyunsaturated fatty acid. We had found that repeatedly heated palm oil showed a higher percentage of elevation in blood pressure and reduction in nitrite level compare to the control and fresh-oil fed groups. Nevertheless, reheated soy oil showed greater adverse effects as observed in present work. In addition, vasorelaxation in response to ACh was further attenuated with repeatedly heated soy oil compared to palm oil. Previous results from our laboratory showed that consumption of repeatedly heated soy oil and palm oil had caused deterioration in bone histomorphometric properties  and lipid peroxidation [55, 56] of ovariectomized rats. From these studies, it was concluded that repeatedly heated soy oil worsens the bone histomorphometric changes and increases lipid peroxidation more than the recycled palm oil.