Effect of the Consumption on Buriti Oil on the Metabolism of Rats Induced by Iron Overload

Objectives: To compare the effect of the consumption of buriti oil and soybean oil on the metabolism of rats under stress induced by iron overload. Materials and methods: A total of 28 rats were ran-domized into control groups who consumed diet added of soybean (CS) or buriti oil (CB) and gavage with saline and two experimental groups who consumed diet added of soybean (ES) or buriti oil (EB) and daily gavage with iron II sulfate as stress inducer. The fatty acid profile of diets was analyzed. Body weight and diet consumption were evaluated every two days. The lipid profile and liver weight of animals were evaluated at the end of the experiment. Results: Diet added of soybean oil showed higher percentage of polyunsaturated fatty acids (45.6%) and diet with buriti oil was rich in mono-unsaturated fatty acids (66.9%). There were no differences in food intake, total cholesterol, HDL-cholesterol and LDL-cholesterol among groups (p > 0.05). However, animals fed with diet supplemented with buriti oil showed intermediate triglyceride levels (CB: 65 mg/dL; EB: 68.7 mg/dL) compared to ES group (102.5 mg/dL). The liver of rats from the CS group had higher weight (2.06 ± 0.2 g) compared to the CB group (1.56 ± 0.1 g). Conclusion: Buriti oil consumption was able to minimize some changes related to iron overload.


INTRODUCTION
F ree radicals are naturally generated in aerobic me tabolism, behave as unstable reactive species and have in their last orbital a mismatch of electrons, being therefore capable of reacting with other reactive mo lecules (1).As protection against this effect, the organ ism has a defense mechanism comprising the action of endogenous enzymes or the action of antioxidants usually obtained from the consumption of foods, such as phenolic compounds, vitamin C, betacarotene and vitamin E (2).However, despite the proven antioxidant effect of these compounds, the antioxidant therapy in different pathologies may have some negative effects such as prooxidante action on lipids, proteins and DNA from excess of these compounds or indicate the pres ence of high oxygen concentrations (24).
The imbalance between the formation of reactive species and the body's defense mechanism generates a process called oxidative stress (5).Oxidative stress participates in the pathophysiology of dyslipidemia through the oxidation of LDLC molecules.When these molecules are absorbed by the scavenger recep tor, the appearance of foam cells and autoimmune reac tions will take place, which will lead to the formation of thrombi that can trigger serious and sometimes fatal cardiovascular events (6).There are several ways to in duce oxidative stress in animals in order to assess the impact of food consumption on this event.The use of dyslipidemic diet (7) over training (8) and iron over load (9) are among the most important.Excess iron in the body is associated with increased generation of free radicals by the Fenton reaction, which culminates in the formation of extremely reactive hydroxyl radical (10).
Due to the important effect of oxidative stress in sev eral diseases, there has been a great interest on research of foods with antioxidant properties, especially vegetable oils.Nagajaru and Belur (11) used blends of coconut oil with peanut oil or olive oil in the diet of rats and obtained

Metabolic action of buriti oil
improvement in the action of antioxidant enzymes, re duced lipid peroxidation and susceptibility to the oxida tion of LDLcholesterol.Pieszka and cols.( 12) observed a reduction in the activity of superoxide dismutase and glutathione peroxidase enzymes in animals fed with di ets supplemented with strawberry and raspberry seed oils and concluded that these oils can improve the antioxi dant status and thus reduce oxidative stress.These results have increased the population's interest in the consump tion of vegetable oils with functional properties.
In this context, buriti oil has antiinflammatory and healing applications (13), and is considered a functional food due to its high concentration of monounsaturated fatty acids and antioxidants, such as tocopherols and ca rotenoids (14,15), being consumed by the population as an ingredient in various preparations (16).Given the above, this study aimed to compare the effect of buriti oil and soybean oil consumption on the metabolism of rats under oxidative stress induced by iron overload.

MATERIALS AND METHODS
Crude buriti oil was acquired from the popular trade of the city of Picos -PI, Brazil, which was extracted from mature fruits by cooking them in water for 20 minutes at temperature of ± 60ºC, subsequently separating the oily from the aqueous fraction.For all experiments, 3 L of crude oil were used, which were refined following degumming, neutralization, washing and drying steps according to methodology adapted from Aquino and cols (14).Soybean oil of the same trade mark was used to be added to the control diet, being purchased from a local supermarket.
Twentyeight male Wistar rats aged ± 21 days and initial weight of ± 60 g were used and kept in individual cages with water and diet ad libitum, temperature of 22 ± 1ºC, relative humidity between 50% and 55% and light/dark cycle of 12 h.
The contents of macro and micronutrients in the di ets offered to animals were calculated according to rec ommendations of the American Institute of Nutrition (AIN) (17).The animals were randomized and allocated into four groups: control group with daily gavage with saline and AIN 93M diet added of soybean oil (CS, n = 7), control group with daily gavage with saline and AIN 93M diet added of Buriti oil (CB, n = 7), experimental group with daily gavage with iron II sulfate and AIN 93M diet supplemented with soybean oil (ES; n = 7) and experimental group with daily gavage with iron II sulfate and AIN 93M diet supplemented with buriti oil (EB; n = 7).The entire experimental protocol was ap proved by the Ethics Committee of Animal Research -CEPA -UFPE under number 23076.001000/201029 and followed recommendations of the Brazilian College of Animal Experimentation -COBEA.
Diets for CS, ES, CB and EB groups (Table 1) were prepared on a weekly basis and offered daily in suffi cient amounts to ensure ad libitum intake during the 17day experimental period.Oxidative stress was induced by daily gavage for 17 days with iron II sulfate (60 mg) for experimental groups, with a volume of 2 mL/100 g body weight (18,19).Animals' body weight and diet consumption were evaluated every two days during the 17day ex perimental period.
The fatty acid profile of diets offered to animals was performed by esterification to obtain methyl esters (20).Gas chromatograph model GCMaster (Ciola & Gre gori Ltda., São Paulo, Brazil) with flame ionization de tector was used to identify and quantify methyl esters.To perform the analysis, the following chromatographic conditions were met: polyethylene glycol fused silica col umn (Carbowax 20M) with 30 m in length, 0.53 mm in diameter and 0.25 µm of stationary phase film thickness.Temperatures were adjusted to 150ºC for the vaporizer, 200 ºC for the detector and the oven was programmed at 80ºC for 3 minutes, 10ºC/minute up to 120ºC, remaining at 200ºC for 6 minutes subsequently decreas ing 3ºC/minute up to 180ºC.The mobile phase was

Metabolic action of buriti oil
Arch Endocrinol Metab.
hydrogen, with flow rate of 5 mL/minute.The volume injected was 1 µm with a split ratio of 1:25.The charac terization of fatty acids was performed by comparing the mass spectrum obtained with that of standards that were also injected into the chromatograph (14).
After the 17day experimental period and a 12h fast ing period, the animals were anesthetized with ketamine (75 mg/kg) associated with xylazine (10 mg/kg) in tramuscularly and blood was collected by direct cardiac puncture for the collection of 4 mL from each animal.Serum was obtained after centrifugation (807 × g/10 min/20ºC) and maintained at temperature of 25ºC for carrying out cholesterol (enzyme), HDLc (polyethyl ene glycolPEG), and triglyceride measurements (enzy matic) (Dolles Kit).After blood collection, the animals were euthanized by cervical dislocation and liver was removed, washed in NaCl solution (0.9 g/100 mL), dried on absorbent paper and weighed.
All results are expressed as mean and standard devia tion and submitted to analysis of variance (ANOVA) and TukeyKramer posttest using the Graph Pad Prism Software version 5.0, considering 5% significance level.

RESULTS
The fatty acid profile found in diets added of soybean oil was quite distinct from that found in diet added of buriti oil because diet added of soybean oil had higher proportion of polyunsaturated fatty acids, especially li noleic acid, while diet added of buriti oil showed higher amounts of monounsaturated fatty acids, mainly oleic acid (Table 2).
Animals showed irregular food consumption (Figure 1A), considering that in the two weeks of experiment ES group (AIN 93M diet added of soybean oil and gavage with iron II sulfate) consumed 88.48 ± 9.70 g in the first week and 108.14 ± 10.1 g in the second week and CB group (AIN 93M diet added of buriti oil and gavage with saline) consumed 106.26 ± 10.3 g diet in the first week and 121.72 ± 10.6 g in the second week (p > 0.05).
Body weight increased, on average, 72 g over the 17 days of experimental period (Figure 1B).Body weight varied among groups from 50.3 ± 1.86 to 54.5 ± 5.85 g on the first day of the experimental period (day 0), 65.4 ± 8.66 to 73.7 ± 3.5 in eighth day and from 107.9 ± 12.0 to 118.6 ± 8.9 g on the last day of the experi mental period (day 17); however, with no difference among groups (p > 0.05).

Body weight (g)
There was difference in the weight of liver of rats from CS and CB groups (2.06 ± 0.2 g vs. 1.56 ± 0.1g; p = 0.006) as shown in figure 2.

Metabolic action of buriti oil
Arch Endocrinol Metab.
There was no difference among groups for param eters total cholesterol, HDLC and LDLC.Total cho lesterol values ranged from 81 ± 6.6 to 87.8 ± 4.7 mg/ dL; HDL cholesterol levels from 45.9 ± 8.7 to 52.2 ± 2.7 mg/dL and LDL cholesterol from 15.02 ± 2.9 to 27.7 ± 6.3 mg/dL.A difference (p = 0.004) in the tri glyceride levels between CS (41.2 ± 22.0 mg/dL) and ES groups (95.2 ± 27.2 mg/dL) was found.However, groups that consumed diet supplemented with buriti oil showed similar triglyceride levels, of 65.0 ± 17.8 mg/dL and 68.6 ± 25.5 mg/dL, respectively.saturated fatty acids and 24 g/day of polyunsaturated fatty acids (23).Thus, it was observed that diet added of buriti oil showed adequate composition in relation to polyunsaturated fatty acids, according to the lipid consumption recommendation.
The increase in diet consumption justifies the weight gain of animals throughout the experimental period.With increasing age, animals started to consume grea ter amounts of diet, which led to weight gain.Almeida and cols.(22) observed no changes in food consump tion of rats that ingested different lipid sources in their diets (soybean, fish and pork oil, butter or margarine).As observed by Yatmark and cols.(24), iron intake did not influence the weight gain of animals.
No changes were observed in the weight of liver of animals receiving iron overload by gavage (ES and EB), although this organ is important in iron metabo lism.These results corroborate those by Almeida and cols.(22), who also found no changes in the weight of liver of rats treated with iron and those who were not.However, the present results differ from those obtained by Yatmark and cols.(24), who found higher weight of liver of rats (2.6 ± 0.8) submitted to iron overload compared with the control group (1.5 ± 0.3).Impor tantly a reduction in the weight of liver of rats from CB group compared to those from CS was observed (p < 0.05), which can be explained by the difference in the composition of dietary lipids consumed by each group, which directly influences the weight of liver, fatty acid composition of liver and the deposition of antioxidant vitamins A and E, which source is buriti oil (15,21,25).
The presence of high percentage of linoleic fatty acid in soybean oil could indicate a reduction in total cholesterol of groups that consumed diet added of this oil (26).However, the present study did not detect any change in this parameter in the lipid profile of animals.Similarly, HDLc and LDLc levels were not affected by the consumption of buriti oil or iron overload.The results of the quantification of cholesterol and lipopro teins in this study differed from the results obtained by Aquino and cols.(15), in which a reduction in total cholesterol, LDLC, VLDLC and HDLC levels was observed in rats that consumed refined buriti oil added to the diet in the percentage of 7% for 28 consecutive days.
Although no change in the LDLc fraction among groups was observed, literature has shown that cells submitted to oxidative stress may have accumulation of oxidized LDLc particles, a process called lipid per Control group fed with AIN 93M diet supplemented with soybean oil and gavage with saline (CS); control group fed with AIN 93M diet supplemented with buriti oil and gavage with saline (CB); experimental group fed with AIN 93M diet supplemented with soybean oil and gavage with iron II sulfate (ES); experimental group fed with AIN 93M diet supplemented with buriti oil and gavage with iron II sulfate (EB).* CS vs. CB = there was significant difference (p < 0.05).

DISCUSSION
The control diet showed higher percentage of polyun saturated fatty acids, especially linoleic acid and diet added of buriti oil was rich in monounsaturated fatty acids, mainly oleic acid.These results may be explained by the composition of vegetable oils used, as buriti oil has 4.6% saturated fatty acids, 91.6% monounsaturated fatty acids and 3.8% polyunsaturated fatty acids and soybean oil shows 31.7% saturated fatty acids, 23.6% monounsaturated fatty acids and 44.7 % polyunsatura ted fatty acids (14,15,21).Diets added of soybean oil and buriti oil of high concentration of polyunsaturated and monounsatu rated fatty acids, respectively, should be consumed because they have antiatherogenic, vasodilator and an tiplatelet action (22).The guidelines for proper fatty acids consumption suggest consumption according to the total caloric value of the diet from lipids with 7% of calories from saturated fatty acids, 15% from monoun Metabolic action of buriti oil Arch Endocrinol Metab.oxidation (27).Then, an inflammatory response oc curs, which ultimately results in endothelial dysfunc tion, platelet aggregation, formation of foam cells and thrombogenesis (28).This is the first step in athero sclerotic plaque formation, culminating in the emer gence of atherosclerosis.It is noteworthy that among the various factors that influence the development of atherosclerosis, oxidative and inflammatory pathways are the most frequently observed and can effectively be involved in atherogenesis (29).However, Yatmark and cols.(24) found no difference in the weight of heart of animals induced to iron overload.
The triglyceride levels were increased in the ES group compared to the CS group.Iron overload in duction might have caused an increase in triglyceride levels in the ES group, since there are data in literature indicating the presence of correlation between iron overload and elevated triglyceride levels (30,31), as previously reported by Chaparro and cols.(32), who observed increase in triglyceride levels of animals sub mitted to oxidative stress induction.Due to that the fact that buriti oil is a source of monounsaturated fatty acids and vitamins A and E. (14,15,21) may have pre vented this increase in the triglyceride levels even in the group that received iron overload (EB), as it showed intermediate and similar TAG levels when compared to CB group, indicating a possible action of this oil on this biochemical parameter.The maintenance of the levels of triglycerides in groups that consumed buriti oil add ed to the diet (CB, EB) can be justified by the mech anism of vitamin A and vitamin E absorption, which after absorption is transported by chylomicrons and lipoproteins containing other dietary lipids (such as tri glycerides, phospholipids, cholesterol, and cholesteryl esters) and apolipoprotein B (apoB), which can quan titatively reduce the transport of these lipids (33,34).
Reduced levels of triglycerides was observed in ani mals fed with refined buriti oil added to the diet, com pared to healthy animals who consumed soybean oil or crude buriti oil, indicating that depending on the consumption duration and conditions, this type of oil can improve the levels of this parameter in healthy rats (15).In addition to intake of foods containing anti oxidants such as phenolics, carotenoids, vitamin C and E, the animal organism presents an important enzyme system with enzymes such as catalase, superoxide dis mutase and glutathione peroxidase, which are involved in the neutralization of reactive oxygen species (ROS) formed during normal metabolism or pathogen, or from physical and chemical exogenous sources (35).However, the antioxidant therapy in different patholo gies must be used with caution, since the pharmaco logical and physicochemical properties compounds are obtained from natural sources and may not have the expected effect in clinical trials (3,4).
The consumption of buriti oil showed higher ca pacity to minimize some metabolic changes, even in animals submitted to iron overload, since it maintained liver weight and triglyceride levels in these animals, indicating that this oil may be a promising source of bioactive compounds to be inserted in the human diet.

Figure 2 .
Figure 2. Liver weight of rats submitted to iron overload and fed with diets added of soybean or buriti oil.

Figure 3 .
Figure 3. Triglycerides, total cholesterol and fractions of rats submitted to iron overload and fed with diets added of soybean or buriti oil.Control group fed with AIN 93M diet supplemented with soybean oil and gavage with saline (CS); control group fed with AIN 93M diet supplemented with buriti oil and gavage with saline (CB); experimental group fed with AIN 93M diet supplemented with soybean oil and gavage with iron II sulfate (ES); experimental group fed with AIN 93M diet supplemented with buriti oil and gavage with iron II sulfate (EB).Different letters in the same line indicates statistical difference (p < 0.05) among groups.CT: total cholesterol; HDL-C: high density lipoprotein cholesterol; LDL: low density lipoprotein cholesterol; TG: triglycerides.* CS vs. ES = there was significant difference (p < 0.05).

Table 1 .
Composition of diets offered to control and experimental groups Control group fed with AIN 93M diet supplemented with soy oil and gavage with saline (CS); control group fed with AIN 93M diet supplemented with buriti oil and gavage with saline (CB); experimental group fed with AIN 93M diet supplemented with soybean oil and gavage with iron II sulfate (ES); experimental group fed with AIN 93M diet supplemented with buriti oil and gavage with iron II sulfate (EB).

Table 2 .
Fatty acid profile of AIN 93M diet added of soybean oil or modified by the addition of buriti oil Food intake and body weight of rats submitted to iron overload and fed with diets added of soybean oil or buriti oil.
b Different letters in the same line indicates statistical difference (p < 0.05) among samples.Figure 1. oil and gavage with iron II sulfate (EB).* There was no significant difference among groups (p > 0.05).