Accepted Manuscript

Early endothelial nitrosylation and increased abdominal adiposity after long-termconsumption of frying used canola oil in Wistar rats

Rocío Bautista, Ph.D. Elizabeth Carreón-Torres, Ph.D. María Luna-Luna, B.Sc.Yukari Komera-Arenas, B.Sc. Martha Franco, M.D., Ph.D. José-Manuel Fragoso,Ph.D. Victoria López-Olmos, M.Sc. David Cruz Robles, Ph.D. Jesús Vargas-Barrón,M.D. Gilberto Vargas-Alarcón, Ph.D. Oscar Pérez-Méndez, Ph.D.

PII: S0899-9007(14)00076-8

DOI: 10.1016/j.nut.2014.01.010

Reference: NUT 9210

To appear in: Nutrition

Received Date: 10 June 2013

Revised Date: 7 January 2014

Accepted Date: 11 January 2014

Please cite this article as: Bautista R, Carreón-Torres E, Luna-Luna M, Komera-Arenas Y, Franco M,Fragoso J-M, López-Olmos V, Robles DC, Vargas-Barrón J, Vargas-Alarcón G, Pérez-Méndez O, Earlyendothelial nitrosylation and increased abdominal adiposity after long-term consumption of frying usedcanola oil in Wistar rats, Nutrition (2014), doi: 10.1016/j.nut.2014.01.010.

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Early endothelial nitrosylation and increased abdominal adiposity after long-term consumption

of frying used canola oil in Wistar rats

Rocío Bautista, Ph.D. a#, Elizabeth Carreón-Torres, Ph.D. b#, María Luna-Luna, B.Sc. b, Yukari

Komera-Arenas, B.Sc. b, Martha Franco, M.D., Ph.D. a, José-Manuel Fragoso Ph.D.b, Victoria López-

Olmos, M.Sc. b, David Cruz Robles Ph.D.b, Jesús Vargas-Barrón, M.D. c, Gilberto Vargas-Alarcón,

Ph.D. b, Oscar Pérez-Méndez, Ph.D. b *

a Nephrology, b Molecular Biology and c Echocardiography Departments, Instituto Nacional de

Cardiología “Ignacio Chávez”, Mexico, DF, Mexico.

# These authors contributed equally to this study

* Corresponding author: Oscar Pérez-Méndez. Department of Molecular Biology, Instituto Nacional de

Cardiología “Ignacio Chávez” Juan Badiano 1, Sección XVI, 14080 México D.F. México. Tel. (52-55)

55 73 29 11 ext. 1460; Fax (52-55) 55 73 09 26; E-mail: opmendez@yahoo.com

Running head title: Frying used canola oil induces endothelial dysfunction

Word count: 4,993 including abstract, text, references, tables and figures.

Number of tables: 2

Number of figures: 5

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Contributions

Conception and design of the study: OP-M, J.V-B, GV-A

Generation, collection, assembly, analysis and/or interpretation of data: RB, EC-T. ML-L, YK-A, MF,

J-MF, VL-O, DC-R, OP-M

Drafting or revision of the manuscript: OP-M, RB, EC-T

Approval of the final version of the manuscript: All authors.

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Abstract

Objective. To establish whether the long-term consumption of reused canola oil contributes to the

development of dyslipidemia, obesity, and endothelial function.

Research Methods & Procedures. Canola oil was used for one frying cycle (1FC) of corn flour dough

or reused 10 times (10FC). Rats received chow diet (control) or supplemented with 7% raw oil (RO),

1FC or 10FC oil (n=10 per group). Food consumption, blood pressure (BP), and body weight plasma

glucose, plasma lipids were monitored. Vascular reactivity was analyzed using aorta rings stimulated

with phenylephrine and acetylcholine. Nitrotyrosine presence in aorta rings was analyzed by

immunohistochemistry.

Results. After 10 weeks of follow-up, visceral adipose tissue was significantly more abundant in 1FC

(7.4 ± 0.6 g) and 10FC (8.8 ± 0.7 g) than the RO (5.0 ± 0.2 g, P = 0.05 vs. 10FC group) or control

group (2.6 ± 0.3 g, P = 0.05 vs. all groups). In spite of similar plasma cholesterol, triglycerides, and BP

among groups, a significantly reduced acetylcholine-induced vascular relaxation was observed in the

three groups receiving oil-supplemented diet (47.2 ± 3.6, 27.2 ± 7.7, and 25.9 ± 7.6 % of relaxation, for

the RO, 1FC and 10FC, respectively, P < 0.05 for all vs. 62.4 ± 9.7 % of the control group). Endothelial

dysfunction was concomitant with the presence of nitrotyrosine residues at a higher extent in the groups

that received heated oils as compared to the RO group.

Conclusion. High canola oil intake during 10 weeks was associated with increased adipose tissue and

early endothelial dysfunction probably induced by peroxinitrite formation. Such deleterious effects

were significantly potentiated when the consumed oil had been used repeatedly for frying.

Key words: nitrotyrosine, canola oil, endothelial dysfunction, lipoproteins, high-fat diet

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Introduction

Reuse of vegetable oils for frying food is a common practice in the culinary tradition in several

countries. However, numerous potential toxic compounds are produced when vegetable oils are heated

or reheated during long periods and/or high temperatures: including unsaturated aldehydes [1], cyclic

fatty acids [2], and trans-fatty acids [3]. These compounds have been suggested to produce liver

damage [2,4], altered body weight gain [5], endothelial dysfunction [6,7], increased oxidative stress [8],

vascular inflammation [9], lipoprotein abnormalities [10,11], and coronary heart disease risk [12].

Previous studies have demonstrated that chronic consumption during six months of reused vegetable

oils was associated with hypertension in rats [6,7]. In contrast, these results have not been consistently

confirmed during shorter periods (10 weeks), not even on spontaneously hypertensive rats [8],

suggesting that clinical manifestations appear only after long periods of chronic consumption of reused

vegetable oils. Nevertheless, it cannot be discarded that reused oils for frying induce early

cardiometabolic effects before the detectable clinical symptoms. Thus, in this study we explored the

possible early damage to endothelial cells, and occurrence of obesity or dyslipidaemia derived from the

ingestion of reused canola oil in Wistar rats.

Materials and Methods

Animals and study design

Forty-eight male Wistar rats weighing 300 to 330 g were randomly assigned to four dietary groups

comprised of 12 animals each. Animal manipulations followed with the recommended guidelines; the

Scientific and Ethics Committees of the Instituto Nacional de Cardiología “Ignacio Chávez” approved

all procedures.

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The rats were maintained at a temperature of 25 °C with a 12 h light dark cycle. All rats had free access

to food and water during the study period. Each group of rats received during 10 weeks one of the

following diets: group 1 was given only commercial rat chow diet (control group); the other groups

were fed with the chow diet supplemented with 7% weight/weight of raw canola oil (RO group), canola

oil used once (1 cycle) for frying (1FC group), or used 10 times (10 cycles) for frying (10FC group).

Systolic blood pressure was measured using a non-invasive method previously described [13] at

baseline and at 1-week intervals along the study. Body weight and caloric consumption were monitored

periodically along the study. Blood samples were drawn from the tail vein for periodic biochemical

analyses. After 10 weeks of treatment, under pentobarbital anesthesia, blood samples were drawn from

the hepatic artery; then, animals were euthanized and abdominal aortas were collected for histological

analysis and vascular reactivity studies as described below. The blood samples were immediately

processed for total cholesterol, HDL-cholesterol, triglycerides, and glucose plasma concentrations, and

aliquots were stored at -70°C for further studies. Abdominal adipose tissue was carefully dissected,

washed in isotonic saline solution, gently wiped and weighed on an analytical balance.

Preparation of oil for dietary supplement

Canola oil used in this study was a local commercial trademark available in most local stores. It was

used either fresh, used for frying once, or reused 10 times following the method described by Leong et

al. [7] with some slight modifications. Briefly, 1 L of oil was heated at 190 ± 5 °C in a stainless steel

wok and used to deep-fry 100 g of cornmeal dough. The heating process lasted for 10 min. The hot oil

was then left to cool at room temperature for 30 min. This corresponds to the canola oil of one frying

cycle (1FC). The cooled oil was used to deep-fry another new batch of 100 g of corn flour dough and

the process was repeated up to 10 times. This corresponds to the canola oil with 10 frying cycles

(10FC). The frying process was carried out without replenishing with fresh oil. The experimental diets

were prepared twice a week by spreading the oil on the pellets of the laboratory rodent diet 5001

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(LabDiet, San Louis, MO, USA) at a final proportion of 7% w/w. Used oils and supplemented pellets

were kept under nitrogen atmosphere at 4 °C until use.

Biochemical analyses

Glucose, triglycerides, and cholesterol plasma concentrations were determined by enzymatic

colorimetric assays commercially available (Randox, England). LDL- and HDL-cholesterol were

determined in plasma after ultracentrifugation, since rat’s apo B containing lipoproteins, do not

precipitate quantitatively with the polyanion solutions usually used for human samples [14-16].

Vascular reactivity of aorta rings

Rats were anesthetized with pentobarbital (45 mg/kg of body weight, i.p.), the thoracic aorta was

dissected free from surrounding tissues and cut into rings of 3 mm in length. The preparation was then

transferred into organ baths with 10 mL of Krebs solution (composition, mM: NaCl 118.1, KCl 4.7,

MgSO4 1.2, KH2PO4 1.20, CaCl2 2.5, NaHCO3 25.0, glucose 11.1 at pH 7.4), continuously bubbled

with 95% O2-5% CO2, at 37 °C. Each aortic ring was mounted between two L-shaped stainless steel

hooks. One of the hooks was mounted at the bottom of the bath while the other was connected to a

force displacement transducer and 1 g basal tension was applied to all experiments. After an

equilibrium period of 2 h, endothelium integrity was verified by the presence of the relaxant response

(over 80% relaxation) to 3 µM acetylcholine at the start of each experiment. The contractile capacity of

vascular smooth muscle cells was evaluated through the dose-response curve to phenylephrine (PE,

6.25 × 10–9 to 1 × 10–4 M). The dose-response curve to acetylcholine (6.25 × 10–9 to 1 × 10–4 M) was

determined in rings pre-contracted by 3 x 10–6 M of PE. Vasorelaxation is expressed as the percentage

of pre-contraction with PE. Contractions were measured isometrically with an FT-03 Grass Force

Displacement Transducer and recorded on a Grass Polygraph (Model 7D, Grass Medical Instruments,

Quincy, MA, USA) [17].

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Immunohistochemistry

Immunohistochemistry was performed to determine the relative extent of protein nitration (a stable

biomarker of endogenous peroxynitrite formation), using an antibody raised against 3-nitrotyrosine (3-

NT) [18]. Aorta samples were cut and prepared as paraffin-embedded sections. The sections were then

dewaxed in xylene for 5 min and dehydrated in graded alcohol. The slides were immersed in H2O2

(3%) for 15 min at room temperature to block endogenous peroxidase activity. Nonspecific protein

binding was blocked by incubation with 1.5 % goat serum in phosphate buffer saline 10mM, pH 7.4 for

one hour. The slides were incubated overnight at 4 °C with anti-3-NT (1:200 dilution, Upstate

Biotechnology, Lake Placid, NY, USA). Biotinylated secondary goat anti-mouse was then applied

(1:200) followed by horseradish peroxidase complex reagent (ABC staining system, Santa Cruz, CA,

USA). Positive immunoreactivity was visualized through the development of diaminobenzidine (DAB)

chromogen. Hematoxylin was used for nuclear counterstaining. Unmodified images were analyzed

using research-based image analysis software (Image-Pro Plus, Media Cybernetics Inc., Bethesda, MD,

USA.). Ten endothelial regions of each aortic ring were systematically captured at 200x, representing

90% of the total vascular endothelium circumference.

Statistical analysis

Data are expressed as the mean ± SEM if not otherwise indicated. Comparisons among groups were

made using ANOVA test and LSD post-hoc analysis. Significance of differences was set at P < 0.05.

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Results

Body weight evolution and caloric intake

The nutritional facts of the chow diet and the supplemented diet with 7% of canola oil are detailed in

Table 1. The supplemented diet provided 10% more calories by gram with a different balance of

calories from proteins, carbohydrates and lipids, and contained more vitamin E than the chow diet.

Figure 1A depicts the body weight evolution of experimental groups along the 10 weeks of the study.

Despite the lack of significant weight gain, the mean daily caloric intake during the study was

significantly different among all groups (ANOVA P < 0.001, Figure 1B); the group that received raw

oil had the lowest mean caloric intake per rat (74.9±4.4 cal/d), followed by the control group (83.8±1.9

cal/d), and the highest caloric intake was observed in the groups that received the used frying oils

(86.7±1.8 and 89.9±2.1 cal/d per rat for the 1FC and 10FC, respectively). The difference of the caloric

intake was particularly important during the last week of the study; the caloric intake during the 10th

week in RO group (55.6 ± 1.41 cal/d) was 28, 33, and 31% lower than the control, 1FC and 10FC,

respectively (P < 0.001 for all). Moreover, the abdominal adipose tissue was significantly higher in

groups that received the used oils as compared with the rats that received the raw oil or the control

group (Figure 2).

Biochemical profile

Glucose plasma concentration was significantly higher in 10FC group as compared to the RO group

(Table 2). The 10FC group had a mean triglycerides plasma concentration 44% higher than the group

that received the raw oil (Table 2). Plasma cholesterol concentration was significantly higher in the

10FC when compared to the RO group. LDL- and HDL-cholesterol were similar among groups.

Blood pressure

Evolution of blood pressure is depicted in Figure 3; 10FC group showed maximum mean values on the

3rd and 4th weeks, and the difference reached statistical significance when compared to controls. The

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raw oil group also had a significant maximum mean blood pressure at week 3 than that of the control

group. Nevertheless, mean systolic blood pressure of all groups was similar at the end of the 10 weeks

(Figure 3).

Vascular reactivity

Our initial hypothesis was that endothelial damage might occur early after the consumption of used

vegetable oil for frying foodstuff. We therefore performed studies of endothelial-mediated vascular

reactivity in aorta rings obtained from rats after 10 weeks of treatment as described in the Methods

section. Figure 4A depicts the phenylephrine-induced vasoconstriction of aorta rings; contraction of

aorta rings was comparable among the four groups. In contrast, aorta rings obtained from rats that

received either 1 or 10FC oil showed a significant lower vasodilation at almost all tested acetylcholine

concentrations (from 1.125 × 10-8 M to 1 × 10-6 M, Figure 4B). Vasodilation of aorta rings from the RO

group was also lower than in controls, but the differences were significant only at higher doses of

acetylcholine (from 5 × 10-8 M). The post hoc analysis demonstrated that the maximum response to

acetylcholine of aorta rings from the RO group (47.2 ± 3.6 %) was higher than that observed in the 1FC

or 10FC groups (27.2 ± 7.7 and 25.9 ± 7.6 %, respectively, P < 0.05 for both).

Immunohistochemistry

In order to gain more insight about the etiology of endothelial dysfunction, we determined through

immunohistochemistry the presence of nitrotyrosine in aorta rings; results are shown in Figure 5. Aorta

rings from both, 1FC and 10FC groups, had a significantly higher reactivity with the nitrotyrosine

antibody than the RO group. In contrast, aortas from control rats were negative for nitrotyrosine

immunostain (data not shown).

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Discussion

In this study we demonstrate early endothelial damage induced by used or re-used canola oils

associated to an augmented visceral adipose tissue, in the absence of dyslipidemia or increased body

mass.

Taking into account the higher caloric content of the supplemented diet than that of the chow diet, it

was expected that any of the three groups that received canola oil would present increased weight gain

as compared to control animals. Unexpectedly, the lowest daily mean caloric intake was observed in

the RO group, whereas the 1FC and 10FC had the highest caloric consumption, as expected. Such

differences may be related to organoleptic characteristics of the used oils; it is likely that the raw oil

was less palatable than heated oils. However, a negative feedback for caloric consumption induced by

raw oil cannot be excluded [19, 20], an effect that could be attenuated by the heating process. This

potential beneficial effect of raw canola oil should be specifically addressed in future studies.

Independently of the mechanism that induced to eat less, it is remarkable that RO group gained weight

at the same rate than the other groups with a lower caloric intake; recent studies have demonstrated that

oleic acid and other polyunsaturated fatty acids (PUFAs) inhibit heat production in the mitochondria by

a mechanism poorly understood [21,22]. Considering that the canola oil contains 61% of oleic acid and

31% of PUFAs, it is likely that RO rats dissipated less energy as heat, (then used for ATP synthesis),

than 1FC and 10 FC groups, because the former consumed less fatty acids than the latter. Therefore, in

spite of the caloric intake, the body mass was comparable among groups along the 10 weeks of the

study. However, the visceral adipose tissue was significantly higher in the groups that received canola

oil when compared to the control group. The accumulation of adipose tissue is not only a matter of

increased caloric intake since, as above mentioned, the lowest daily caloric intake was observed in the

RO group. Therefore, it seems that adipose tissue accumulation is related mainly to a high-fat diet

intake [23,24].

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The increased abdominal adipose tissue suggests the presence of dyslipidemia, i.e.,

hypertriglyceridemia and hypoalphalipoproteinemia [25]; in fact, triglycerides plasma concentrations in

the 10FC group were higher than those observed in the control group and tended to be higher than in

the RO group. Elevated triglycerides plasma levels are usually associated with low HDL-cholesterol,

due to an enhanced exchange of cholesterol from HDL by triglycerides from apo B-containing

lipoproteins; such exchange is facilitated by cholesteryl ester plasma protein (CETP). Nevertheless,

HDL-cholesterol was not decreased in the 10FC group despite the high triglycerides plasma levels in

this group. The rat lacks of CETP [26], and as a consequence, high triglycerides are not linked to low

HDL-cholesterol plasma levels in this species as observed in humans [25]. Furthermore, at equivalent

fat content per gram of food, 10FC group had higher plasma levels of plasma cholesterol than the RO

group, emphasizing the deleterious effect of reused canola oil. Whether the cholesterol increase is

related to the toxic compounds formed during the heating process of the vegetable oil remains to be

addressed in specific studies.

Our results showed that blood pressure transitory increased within the groups that received RO and

10FC oil, but these pressures returned to levels similar to the basal values at the end of the 10 weeks of

the study. Previous studies reported increased blood pressures in rats after 10 weeks of commercial

high-fat diets consumption [27,28]. In contrast, our study as well as in other related works with

reheated vegetable oils [6,8], the fat-enriched diet is not associated with hypertension in young rats.

This apparent controversy is likely related to the quantity of consumed fat; the commercial of high-fat

diets provide 45 to 60% of the daily intake of calories by fat [22,23], whereas in our study fat provided

only 28% of the daily caloric intake. Moreover, the commercial high-fat diets contain 35% more

cholesterol (270 µg/g) than the diet supplemented with vegetable oil of our study (200 µg/g of

cholesterol, Table 2) [24]. Other fat features, such as saturated fatty acids content may also be involved

in the increased blood pressure observed in other studies [24].

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Despite the lack of augmented blood pressure, it cannot be discarded that the peaks of blood pressure

observed in the 10FC group at the 3rd and 4th weeks are the result of early endothelial damage.

Therefore, we performed the study of endothelial function in vitro using aorta rings. These studies

demonstrated an impaired endothelium response to acetylcholine in the three groups that received a

supplement of canola oil; such effect was more important in the two groups that were supplemented

with reused oils than in the RO group. Thus, the increased intake of lipids in the diet led to early

endothelial impairment in our animal model, and such impairment became more important when lipids

were reused for frying before ingestion. Dyslipidemia has been frequently related to endothelial

dysfunction [25,26]; however, as above stated, only the 10FC group was slightly hypertriglyceridemic

and hypercholesterolemic, whereas both, the 1FC and 10 CF groups, showed the same level of

endothelial dysfunction. Therefore, these results suggest that the impaired endothelial response to

acetylcholine may be related to toxic compounds derived from fatty acids during the cooking process

that have been reported in previous studies [1-5]. Interestingly, those toxic compounds appear even

after brief heating periods (10 min), thus suggesting that they were produced through the heating

process of canola oil performed in our study.

Among the toxic compounds derived from polyunsaturated fatty acids (PUFAs), the α,β-unsaturated

aldehydes have been described as alkylating agents that react with amino groups in proteins and

sulfhydryls as in glutathione, thus promoting protein inactivation and oxidative stress [1,27]. Taking

into account that canola oil contains 31% of PUFAs, it could be assumed that the amount of alkylating

aldehydes was considerable in the heated canola oils of this study. Therefore,, the impaired response of

aorta rings to acetylcholine could result from modifications of membrane proteins in endothelial cells.

In order to explore this possibility, we determined the relative extent of protein nitration in aorta

samples by immunohistochemistry. The increased presence of nitrotyrosine in 1FC and 10FC groups as

compared to the RO group is consistent with the observed endothelial-dependent vasodilation of aorta

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rings; the extent of endothelial dysfunction matched the amount of nitrotyrosine residues in aorta rings.

Quantification of tissue 3-nitrotyrosine has been considered to be a footprint of peroxynitrite-mediated

protein damage [15]. Peroxynitrite oxidizes tetrahydrobiopterin, a cofactor of nitric oxide synthases

(NOS) [28], which in its oxidized form causes uncoupling of constitutive NOS enzymes [29] resulting

in O�2- production and endothelial dysfunction. These evidences, together with our results, suggest that

chronic consumption of canola oil used or re-used for frying induces early endothelial impairment by

NOS uncoupling. However, other mechanisms of tissue damage induced by heated canola oil, such as

decreased antioxidant enzyme activities, inflammation, and anti-inflammatory mechanisms impairment

should be specifically explored in further studies.

In summary, Wistar rats that consumed canola oil used once, or re-used for frying, during a period of

10 weeks, developed endothelial nitrosylation and dysfunction, as well as increased abdominal adipose

tissue; at equivalent doses, the deleterious effects are lesser with raw oil. The endothelial dysfunction

observed in vitro seems to be related with NOS uncoupling but other mechanisms cannot be ruled out.

Conclusion

A high-canola oil diet during only 10 weeks induced increased adipose tissue and endothelial

dysfunction probably induced by peroxinitrite formation, without blood pressure increases. Such

deleterious effects were significantly potentiated when the consumed canola oil had been used

repeatedly for frying.

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Figure legends

Figure 1. Body weight evolution ± SEM (A) and mean daily caloric intake per rat (B) along the 10

weeks of the study. RO, raw oil group; 1FC, one-cycle frying group; 10FC, ten-cycle frying group.

Figure 2. Visceral adipose tissue mass in the four groups of study after 10 weeks of feeding with chow

diet (control), supplemented with raw canola oil (RO), canola oil used for 1 frying cycle (1FC), and

canola oil used for 10 cycles of frying (10FC). Tissues were dissected, washed in isotonic saline

solution, gently wiped, and weighed. Bars represent the mean weight for each group ± SEM (n = 10 per

group). * P < 0.05 vs. control, # P < 0.05 vs. RO group.

Figure 3. Systolic blood pressure along the 10 weeks of feeding with chow diet (Control),

supplemented with raw canola oil (RO), canola oil used for 1 frying cycle (1FC), and canola oil used

for 10 frying cycles (10FC). Data represent mean ± SEM (n = 10 per group). * P < 0.05 vs. control

Figure 4. Effect of raw or reused canola oils on vascular reactivity of isolated rat aortic rings.

Phenylephrine-induced contraction (A) after equilibrium time, and acetylcholine-induced endothelium-

dependent vasodilation (B) of pre-contacted rings by 3 × 10–6 M phenylephrine. Data represent

mean ± SEM (n = 6 per group, 2 segments analyzed per each experimental animal).

Figure 5. Representative immunohistochemistry of 3-nitrotyrosine staining, a marker of peroxynitrite

mediated oxidative stress in aortic rings from RO (A), 1FC (B), and 10 FC animals. The images were

analyzed at 400-fold magnification. Panel D: Densitometry analysis of positive staining area for

nitrotyrosine in RO (white bar), 1FC (black bar), and 10FC (gray bar) aorta ring samples. Data

represent mean ± SEM (n = 4 per group, 6 optical fields analyzed per each experimental animal).

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Table 1 Chemical composition and nutrional facts of the rat chow diet and the supplemented diet with 7% of canola oil.

Nutriment Chow diet* Supplemented

with 7%canola oil **

Protein, % 23.9 22.2

Fat, % 5.7 12.3

Cholesterol, ppm 200 186

Linoleic Acid, % 1.22 2.54

Linolenic Acid, % 0.10 0.83

Total Saturated

Fatty Acids, % 1.56 1.92

Total Monounsaturated

Fatty Acids, % 1.60 2.89

Fiber (Crude), % 5.1 4.7

Nitrogen-free extract, % 48.7 45.3

Starch, % 31.9 29.6

Glucose, % 0.22 0.20

Fructose, % 0.30 0.27

Sucrose, % 3.70 3.44

Lactose, % 2.01 1.87

Energy, kcal/gm 4.07 4.40

Calories provided by:

Protein, %

Fat (ether extract), %

Carbohydrates, %

28.507

13.496

57.996

23.694

28.100

48.205

Vitamin E, IU/kg 42 55

* As reported by the manufacturer (LabDiet, San Louis MO, USA)

** Calculated on the basis of the 93% chow diet and 7% of canola oil

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Table 2. Biochemical profile of the four groups included in the study after 10 weeks of treatment Control

n =12

RO

n =12

1FC

n =12

10FC

n =12

mg/dL

Glucose 81.9 ± 6.1 73.1 ± 4.5 82.5 ± 6.0 95.4 ± 7.5 &

Triglycerides 50.3 ± 4.9 56.9 ± 5.7 63.1 ± 7.1 82.1 ± 7.4 *

Cholesterol 46.6 ± 5.8 37.3 ± 6.1 49.2 ± 5.4 63.7 ± 4.3 #

LDL-cholesterol 13.0 ± 1.3 14.2 ± 1.4 14.1 ± 1.1 13.0 ± 2.0

HDL-cholesterol 28.0± 1.2 29.5± 1.1 28.5± 1.4 31.2± 1.5

ANOVA post hoc analysis *P = 0.017 vs. control, #P = 0.006 vs. RO group, and &P = 0.032 vs. RO group.

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