http://informahealthcare.com/ijf ISSN: 0963-7486 (print), 1465-3478 (electronic) Int J Food Sci Nutr, Early Online: 1–9 ! 2015 Informa UK Ltd. DOI: 10.3109/09637486.2015.1025719

RESEARCH ARTICLE

Monounsaturated fatty acids-rich diets in hypercholesterolemic-growing rats Elisa V. Macri1, Fima Lifshitz2, Estefania Alsina1, Natalia Juiz1, Valeria Zago3, Christian Lezo´n4, Patricia N. Rodriguez1, Laura Schreier3, Patricia M. Boyer4, and Silvia M. Friedman5

Int J Food Sci Nutr Downloaded from informahealthcare.com by Kainan University on 04/02/15 For personal use only.

1

Department of Biochemistry, School of Dentistry, University of Buenos Aires, Buenos Aires, Argentina, 2Pediatric Sunshine Academics Inc, Santa Barbara, CA, USA, 3Laboratory of Lipids and Lipoproteins, Department of Clinical Biochemistry, Faculty of Pharmacy and Biochemistry, INFIBIOC, 4Department of Physiology, and 5Department of General and Oral Biochemistry, School of Dentistry, University of Buenos Aires, Buenos Aires, Argentina Abstract

Keywords

The effects of replacing dietary saturated fat by different monounsaturated fatty acid (!-9MUFA) sources on serum lipids, body fat and bone in growing hypercholesterolemic rats were studied. Rats received one of the six different diets: AIN-93G (control, C); extra virgin olive oil (OO) + C; high-oleic sunflower oil (HOSO) + C or atherogenic diet (AT) for 8 weeks; the remaining two groups received AT for 3 weeks and then, the saturated fat was replaced by an oil mixture of soybean oil added with OO or HOSO for 5 weeks. Rats consuming MUFA-rich diets showed the highest body fat, hepatic index and epididymal, intestinal and perirenal fat, and triglycerides. T-chol and non-HDL-chol were increased in HOSO rats but decreased in OO rats. Bone mineral content and density were higher in both OO and HOSO groups than in AT rats. This study casts caution to the generalization of the benefits of MUFA for the treatment of hypercholesterolemia.

Body fat distribution, bone mineral content, lipid profile, liver fat, MUFA

Introduction In the last three decades, epidemiological and experimental studies showed that there is a lower rate of death from CVD in countries consuming the so called Mediterranean diets, which are rich in olive oil (OO) (Frankel, 2011; Michas et al., 2014), unrefined cereals, fruit, vegetables, legumes and nuts; moderate to high fish content, moderate to low white meat and dairy products, low red meat and meat products, moderate wine associated with lifestyle habits, such as physical exercise and socializing (Aro´s & Estruch, 2013). Extra-virgin OO consumption provides health benefits due to the high content of oleic acid (80%) and a low proportion of saturated fat (10%). Recently, a multicenter trial performed in persons at high cardiovascular risk showed that a Mediterranean diet supplemented with extra-virgin OO or nuts reduced the incidence of major cardiovascular events (Estruch et al., 2013). Conversely, intake of dietary trans fatty acids (TFA), mainly from frying oils, margarine and industrial fats (shortenings) used in bakery and snack products, lead to alterations in serum lipids, vascular inflammation and development of vascular diseases (Carrillo Ferna´ndez et al., 2011; Srinath Reddy & Katan, 2004).

Correspondence: Silvia M. Friedman, Department of General and Oral Biochemistry, School of Dentistry, University of Buenos Aires, Marcelo T. de Alvear 2142 12 B, C1122 AAH Buenos Aires, Argentina. Tel: +5411 4964 1277. E-mail: [email protected], silviamfriedman@ hotmail.com

History Received 7 July 2014 Revised 4 February 2015 Accepted 15 February 2015 Published online 1 April 2015

Therefore, a variety of international recommendations have urged to replace or eliminate TFA from food products (Uauy et al., 2009). The introduction of HOSO was not a recommendation from FAO/WHO. However, FAO/WHO recommended avoiding TFA. Therefore, food industries in order to fulfill this requirement, have introduced monounsaturated fatty acids (!-9MUFA) oils based on the extra-OO benefits (Macri et al., 2007). High-oleic sunflower oil (HOSO) has been introduced for frying processes and baking (Valenzuela, 2008). HOSO has a similar composition of !-9MUFA as compared to OO, but differs in the amount and type of vegetable sterols and other nonnutritive components (Huang & Sumpio, 2008). It is well known that adipocytes and osteoblasts were derived from a common progenitor, the mesenchymal stem cell and there are agents that inhibiting adipogenesis, stimulated osteoblast differentiation and vice versa, influencing bone density (LeckaCzernik & Stechschulte, 2014). Diet composition, in particular fat content, has been shown to act negatively on bone status. Moreover, the quality of fat may also affect the skeleton characteristics. Diets based on virgin OO or fish oil but not on sunflower oil prevent age-related alveolar bone resorption (Bullon et al., 2013). In previous studies, we demonstrated that atherogenic diets (ATs) fed to healthy growing rats constituted a risk factor for bone modeling (Gamba et al., 2009; Macri et al., 2009). Saturated fat feedings decreased total skeleton bone mineral content and reduced the percentage of bone volume with an increased tibia cartilage growth plate thickness (Macri et al., 2009). Additionally, the mandible bone architecture was altered in young rats given saturated fat-enriched diets (Gamba et al., 2009).

2

E. V. Macri et al.

Int J Food Sci Nutr, Early Online: 1–9

Therefore, the objective of this study was to evaluate in an experimental model of growing hypercholesterolemic male rats, the effects of replacing dietary saturated fat by MUFA-rich fats on zoometrics, serum lipoprotein profiles, body fat content and distribution, and total skeleton bone mineral content and density.

Materials and methods Animals

Int J Food Sci Nutr Downloaded from informahealthcare.com by Kainan University on 04/02/15 For personal use only.

Eighty male weanling Wistar rats (aged 21 days), initial body weight ¼ 39.90 ± 4.78 g (mean ± SD), were obtained from the animal laboratory of the Department of Biochemistry, Faculty of Dentistry, University of Buenos Aires, Argentina. Animals were housed in galvanized cages with meshed floors in order to maintain hygienic conditions and to avoid coprophagy. Rats were kept in individual cages and exposed to a 12-h light/dark cycle throughout the study. Room temperature was maintained at 21 ± 1  C with a humidity of 50–60%. Ethics The rats were maintained in accordance with the USA National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocols for these experiments were approved by the University of Buenos Aires, Ethics Committee (UBACyT 20020100100613). Diets There were four control diets: C, OO-C, HOSO-C and AT. They differed in fat content and composition as shown in Table 1. The control diet, C, was an AIN-93G (Reeves et al., 1993) that contained 7% of soybean oil (SO). Meanwhile, OO-C and HOSOC diets were rich in MUFA oils and contained 20 g of fat per 100 g of diet; 7% from SO and 13% from OO or HOSO, respectively. C and OO-C or HOSO-C, provided 16% and 40% of total calories from lipids, respectively. AT diet contained 20% of fat per 100 g of diet, but it was rich in saturated fatty acids (12.3 g%) and cholesterol (4 g%) (Macri et al., 2007). The experimental diets were OO and HOSO and contained 20% of fat per 100 g of diet and 11.82% and 12.9 g%, respectively, of MUFA, and 4% of cholesterol. All the diets contained the same type and concentration of calcium (Calcium carbonate anhydrous at 40.04% Ca, equal to 143 mg/kg mix), phosphorus (Potassium phosphate monobasic at

22.76% P, equal to 44.60 mg/kg mix) and Vitamin D (Vitamin D-3, cholecalciferol, 400 000 IU/g equal to 250 mg/kg mix) (AIN93G) (Reeves et al., 1993); 0.6 mg alpha-tocopherol equivalents/g polyunsaturated fatty acids were added to the high fat diets as recommended by Valk & Hornstra (2000). Diets were prepared every 2 days and stored at 4  C until fed. Fresh diets were offered daily and food containers were cleaned before being refilled. Food cups were refilled once a day, and food consumption was measured with a Mettler scale PC 4000 (accuracy ± 1 mg). Daily food intake was recorded as kcal per 100 g of body weight and per day (kcal/100 g W/day). The oils compositions were as follows: (1) Extra virgin OO, OO composition was: Saturated Fat: 13.3%; Monounsaturated Fat: 76.7%; Polyunsaturated Fat: 10%; TFA: 0%; phenolic compounds: 1328 mg/kg. The sterol composition (expressed as% of desmethylsterols among total sterols) was as follows: Cholesterol: 0.5; Brassicasterol: 0.1; Campesterol: 4.0; Stigmasterol: Less than campesterol; Delta-7-stigmastenol: 0.5 and Beta-sitosterol+ Delta-5-avenasterol +Delta-5-23-stigmastadienol clerosterol+++ Delta-sitostanol 5-24-stigmastadienol: 93.0%. (2) High-oleic sunflower oil, HOSO composition: Saturated Fat: 7.2%; Monounsaturated Fat: 85%; Polyunsaturated Fat: 7.8%; Cholesterol: 0%; TFA: 0% and Tocopherol: 54 mg%. Experimental design Forty-eight rats were randomly assigned to one of six different groups according to dietary intake and fed ‘‘ad libitum’’, for 8 weeks. Three groups were controls: C (AIN93-G), OO-C and HOSO-C. OO-C and HOSO-C were included based on fat content and MUFA source for OO and HOSO groups (Table 1). The remaining groups received AT diet for 3 weeks and subsequently in two of them (OO and HOSO groups), the source of saturated fat was replaced by an oil mixture of SO (7%) added with OO or HOSO (13% each) for 5 weeks, meanwhile the other group (AT group) continued on AT diet. At the end of 8-week experimental period, zoometrics and dual-energy X-ray absorptiometry (DXA) analyses were performed under light anesthesia. Then, animals were euthanized under anesthesia (ketamin hydrochloride: 0.1 ml/100 g bodyweight, Holliday Lab. and Xilasyn: 0.02 ml, Konig Lab. Buenos Aires, Argentina, by intramuscular injection. Immediately, blood was drawn by cardiac puncture, allowed to clot and the serum was stored at 20  C until biochemical assays were performed.

Table 1. Composition of the diets given to the different groups. Diets Component % (w/w) Energy (kcal) Protein (g) Carbohydrate (g) Total fat (g) Saturated Monounsaturated Polyunsaturated Cholesterin (g) Vitamins, minerals and fiber

C

OO-C

384.6 17.4 63 7 1.30 1.85 3.85 –

429.6 17.4 45 20 3.03 11.82 5.15 –

HOSO-C 429.6 17.4 45 20 2.24 12.90 4.86 – According to AIN-93G

AT 465.6 17.4 45 20 12.30 6.78 0.92 4 per 100 g

OO

HOSO

465.6 17.4 45 20 3.03 11.82 5.15 4

465.6 17.4 45 20 2.24 12.90 4.86 4

The energy and fat content of C diet was lower than that of the other diets. Oils were provided by Molinos Rio de la Plata, Argentina. Protein: as Potassium Caseinate, containing 87% of protein mesh 90. Provided by Inmobal Nutrer SA, Argentina. Carbohydrate: corn dextrin from corn refinery. Provided by Food SA, Argentina. Vitamins, minerals and fiber: to meet rat requirements during growth, according to AIN 93 G. Manufactured by the Department of Food Science School of Biochemistry, University of Buenos Aires. Choline chloride salt (C-6556), Anedra SA, Buenos Aires, Argentina. Cholesterine provided by Parafarm Saporiti SA, Argentina. C, control diet; OO-C, olive oil control diet; HOSO-C, high-oleic sunflower oil control diet; AT, atherogenic diet; OO, olive oil diet; HOSO, high-oleic sunflower oil diet.

DOI: 10.3109/09637486.2015.1025719

Biochemical determinations, hepatic index, body fat content were performed in all the rats. Additionally, 32 weaning rats were randomly assigned into four groups (C, OO-C, HOSO-C and AT3) in order to get basal values parameters (3 weeks), before to switch to MUFA-rich diets. AT3 is another control group in order to analyze at 8 weeks the metabolic effects of introducing MUFA oils. Zoometrics

Int J Food Sci Nutr Downloaded from informahealthcare.com by Kainan University on 04/02/15 For personal use only.

Body weight (W) and body length (L) were measured, after a fasting period of 2–4 h (Kale et al., 2009). A Mettler PC 4000 scale was used to measure W; L was determined in slightly anesthetized rats with a scaled ruler in mm from the nose tip to the hairline of the tail. W and L gain velocities (WV ¼ [(Final W  Initial W)/(Final W + Initial W)/2]  100 and LV ¼ [(Final L  Initial L)/(Final L + Initial L)/2]  100) were expressed as g W/100 g W rat and L/10 cm rat, respectively. Biochemical determinations At week 8, blood samples were drawn from the heart after 4–6 h fasting; serum was isolated and immediately stored at 20  C for biochemical analysis. Total serum cholesterol (T-chol in mg per dl of serum), high-density lipoprotein-cholesterol (HDL-chol in mg per dl of serum) and triglycerides (TG in mg of TG per dl of serum) were determined by standardized methods (Roche Diagnostics GmbH, Mannheim, Germany) in a Hitachi 917 autoanalyser (Hitachi, Tokyo, Japan). As circulating serum HDLchol levels in rats is usually higher than the low-density lipoprotein-cholesterol (LDL-chol in mg per 100 g of serum), the measurement of this parameter loses sensitivity; therefore non-HDL cholesterol (mg per dl of serum) was calculated as the difference between the T-chol and HDL-chol. Non-HDL-chol represents the set of atherogenic lipoproteins rich in apoB that constitute the criteria for human intervention and treatment (Zago et al., 2010). The fasting serum insulin (ng/ml) and glucose levels (mg/ml) were also measured. Hepatic index The liver was removed immediately after death to avoid dehydration, and weighed with an electronic analytical scale. Data were used to calculate the hepatic index, expressed as liver weight/total body weight  100 (% LiW/TBW); the liver of each rat was normalized to the percentage of TBW to minimize the individual differences in body size. This index was used to determine enlargement of the liver (hepatomegaly) and estimate the liver steatosis (Qin & Tian, 2010; Wang et al., 2002). Body fat tissue Fat content from epididymal, intestinal and perirenal areas were identified, removed and weighed in order to evaluate visceral adiposity. Chemical carcass analysis was performed as described elsewhere (ATP III, NCEP, 2001). Total body fat content was determined by Soxhlet extraction method (AOAC, 1990). Data were expressed as percent of body weight.

!-9MUFA-rich diets in hypercholesterolemic-growing rats

3

The coefficients of variation (CV) evaluated by repeated measurements of five subjects of similar age and gender on five consecutive days were 3.0% and 0.9% for total skeleton BMC and BMD, respectively. The same operator to avoid inter-assay error performed all the DXA measurements. Statistical analysis The results were expressed as mean values with their standard deviations (SDs). One-way analysis of variance (ANOVA) was used to compare data among groups. When a statistically significant difference was encountered a Student’s–Newman– Keul’s test or Dunn (not parametric test) was performed. In all the analyses, Bartlett’s test for homogeneous variances was done. The Kolmogorov–Smirnov test was used to verify whether data had normal distribution. Significance was set at the p50.05 level (Glantz, 1992). The Statistical Product and Service Solutions for Windows 11.5 (SPSS, Inc., Chicago, IL) and Graphpad Prism (version 5.0) statistical package (Graphpad) were used for statistical analyses.

Results Comparison of basal values parameters among groups (3 weeks) Energy intake, body weight, body length, total body fat and body fat distribution, serum lipid profile and DXA measurements are shown in Table 2. AT (AT3) rats had higher body weight and length, fat body mass and hepatic index, epididymal fat, intestinal fat and perirenal fat than C, OO-C or HOSO-C groups (p50.0001). Serum T-chol, Non-HDL-chol and triglycerides were significantly increased in AT3 as compared to the other groups. There were no significant differences for serum HDL-chol (p ¼ 0.070). Bone parameters BMC, BMC/W and BMD in AT3 rats were lower than the other groups (p50.0001). Comparison of values parameters at the end of the experimental period, among groups (8 weeks) Effects on zoometrics Energy intake, final body weight, body weight and length velocities are shown in Table 3. Rats fed MUFA diets containing either extra virgin OO or HOSO did not show significant differences in energy intake as compared with C group, despite the differences in energy content among the diets (40% versus 16%). AT group showed the highest intake as compared to C and HOSOC groups (p ¼ 0.006). In the beginning of the experiment, all the animals in the six groups had similar body weights. At the end of the experimental period, the weights were significantly lower in the HOSO-C, AT, OO and HOSO rats as compared with the C and OO-C rats. Final body weights of AT, OO and HOSO groups were consistent with decrease in body weight gain velocity (p ¼ 0.001). However, the type and amount of fat in the diets did not alter linear body growth over the duration of the experiment (p ¼ 0.19), among groups. Effects on serum lipid profile

Dual-energy X-ray absorptiometry measurements At the end of the study, total skeleton bone mineral content (BMC) and bone mineral density (BMD) were assessed in vivo under light anesthesia, using a total body scanner with software designed specifically for small animals (DPX Alpha 8034, Small Animal Software, Lunar Radiation Corp, Madison, WI), as previously described (Zeni et al., 2002). All the rats were scanned using an identical procedure as reported by Zeni et al. (2001).

Lipid profiles of the six groups are shown in Figure 1. All the rats fed the AT diet showed an altered serum lipoprotein profile as compared to C, OO-C and HOSO-C groups. However, the HOSO rats showed the highest serum T-chol (Figure 2a) and non-HDLchol (Figure 2d) levels, and the lowest HDL-chol (Figure 2c) concentrations (p50.001). The HOSO serum TG levels (Figure 2b) were lower than those of the AT rats (p50.001). At the end of the experimental period (T8), there was a 40% increase

4

E. V. Macri et al.

Int J Food Sci Nutr, Early Online: 1–9

Table 2. Basal values at 3 weeks. Groups Parameters

C

Energy intake (kcal per 100 g body weight/rat/day) Body weight (g) Body length (cm) Total body fat (%) Hepatic index (g per 100 g) Epididymal fat (g per 100 g) Intestinal fat (g per 100 g) Perirenal fat (g per 100 g) T-chol (mg/dl) HDL-chol (mg/dl) Non-HDL-chol (mg/dl) Triglycerides (mg/dl) BMC (g) BMC/W (g per 100 g) BMD (g/cm2) Int J Food Sci Nutr Downloaded from informahealthcare.com by Kainan University on 04/02/15 For personal use only.

OO-C a

HOSO-C b,c

49.1 ± 5.8 122.5 ± 14.9b 17.43 ± 0.63a 7.9 ± 0.92 1.95 ± 0.60 2.23 ± 0.75 1.44 ± 0.70 2.00 ± 1.12 83.4 ± 6.7a 44.0 ± 4.8 22.8 ± 4.6a 58.7 ± 8.4a 0.97 ± 0.07b 0.78 ± 0.04b 0.217 ± 0.015b

45.7 ± 3.1 124.5 ± 7.2b 17.84 ± 0.54a 8.2 ± 1.3 2.15 ± 0.50 2.13 ± 0.33 1.87 ± 0.49 1.80 ± 0.69 78.6 ± 9.5a 41.8 ± 10.8 25.9 ± 6.6a 61.3 ± 14.0a 1.29 ± 0.24c 1.04 ± 0.08c 0.245 ± 0.016c

AT

a

43.9 ± 4.2 113.98 ± 13.9a 16.99 ± 0.76a 8.1 ± 0.81 2.22 ± 0.25 1.79 ± 0.84 1.51 ± 0.39 1.23 ± 0.6 75.3 ± 6.7a 41.7 ± 6.9 24.7 ± 4.5a 64.1 ± 6.5a 0.87 ± 0.11b 0.72 ± 0.07c 0.224 ± 0.013b

ANOVA c

p ¼ 0.0051 p50.0001 p50.0001 p ¼ 0.1459 p ¼ 0.0927 p ¼ 0.500 p ¼ 0.1116 p ¼ 0.1538 p50.0001 p ¼ 0.070 p ¼ 0.0097 p50.0001 p50.0001 p50.0001 p50.0001

54.1 ± 7.8 152.3 ± 17.2c 19.93 ± 1.22b 9.02 ± 0.90 2.50 ± 0.17 2.20 ± 0.0.5 1.95 ± 0.26 2.11 ± 0.74 128.0 ± 11.0b 52.0 ± 10.1 32.0 ± 5.0b 106.0 ± 17.9b 0.69 ± 0.22a 0.46 ± 0.02a 0.197 ± 0.016a

Data are means ± SD. Values with different superscript letters denote statistical significance between diets (p50.05), ‘‘a’’ being the lowest value. C, control diet; OO-C, olive oil control diet; HOSO-C, high-oleic sunflower oil control diet; AT, atherogenic diet; HOSO, high-oleic sunflower oil diet; OO, olive oil diet.

Table 3. Zoometrics, body composition, dietary intake and hepatic index at 8 weeks. Groups Parameters Energy intakey (kcal/100 g rat/day) Final bodyy weight (g) Body weight velocityy (g/100 g) Body length velocityy (cm/10 cm) Final body fat (% body weight) Hepatic index (g/100 g)

C

OO-C a

39.8 ± 3.9 325.2 ± 11.1b 78.6 ± 5.7c 2.46 ± 0.26 10.3 ± 0.7a 3.00 ± 0.30a

HOSO-C a,b

46.4 ± 3.6 330.8 ± 27.9b 80.8 ± 10.9c 2.50 ± 0.39 10.8 ± 2.0a 3.20 ± 0.22a

a

38.9 ± 2.3 266.2 ± 30.6a 74.2 ± 9.2b,c 2.52 ± 0.34 10.4 ± 1.1a 3.26 ± 0.25a

AT

OO b

50.3 ± 9.9 264.7 ± 19.7a 67.6 ± 7.3b 2.73 ± 0.18 11.5 ± 1.3a 5.30 ± 0.20b

HOSO a,b

44.9 ± 7.3 262.4 ± 7.7a 68.1 ± 11.8b 2.84 ± 0.28 14.4 ± 1.7b 5.93 ± 0.55c

ANOVA a,b

47.1 ± 7.4 265.5 ± 18.2a 61.9 ± 7.5a 2.66 ± 0.49 14.2 ± 0.9b 5.84 ± 0.73c

p ¼ 0.006 p ¼ 0.001 p ¼ 0.001 p ¼ 0.19 p ¼ 0.001 p ¼ 0.001

C was fed AIN-93G, OO-C was fed AIN-93G plus OO, HOSO-C was fed AIN-93G plus HOSO and AT group was fed an atherogenic diet plus cholesterol for 8 weeks. HOSO and OO were fed AT diet for 3 weeks followed by HOSO or OO diet, for 5 weeks. Data are means ± SD. Values with different superscript letters denote statistical significance between diets (p50.05), ‘‘a’’ being the lowest value. y8 weeks versus baseline. C, control diet; OO-C, olive oil control diet; HOSO-C, high-oleic sunflower oil control diet; AT, atherogenic diet; HOSO, high-oleic sunflower oil diet; OO, olive oil diet.

(202.8 ± 29.9 mg/dl versus 144.0 ± 20.1 mg/dl; p50.001) in the serum T-chol levels of the HOSO rats, compared with the AT group; the non-HDL-chol showed an increase of 65% (172.1 ± 26.0 mg/dl versus 103.9 ± 17.8 mg/dl; p50.001) in the HOSO rats versus the AT group. In contrast, the OO group had a decrease T-chol of 12% (p ¼ 0.029) compared to control rats; and the non-HDL-chol showed a decrease of 16% (87.9 ± 20.2 mg/dl versus 103.9 ± 17.8 mg/dl; p ¼ 0.018) compared to AT rats. The control rats (C, HOSO-C and OO-C) showed no significant differences in their serum lipoprotein levels within the three groups. However, the serum triglyceride levels were higher among the MUFA-fed rats that the levels of the C group. There were no significant differences of serum insulin levels between groups (C ¼ 4.8 ± 1.7; OO-C ¼ 5.3 ± 2.3; HOSOC ¼ 4.5 ± 1.9; AT ¼ 4.9 ± 2.1; OO ¼ 5.6 ± 2.7; HOSO ¼ 4.7 ± 2.0 ng/ml; p ¼ 0.935). Similarly, the fasting glucose levels did not denote any significant differences between groups (C ¼ 99.0 ± 4.7; OO-C ¼ 103.0 ± 3.9; HOSO-C ¼ 101.2 ± 5.9; AT ¼ 98.4 ± 2.3; OO ¼ 102.2 ± 3.8; HOSO ¼ 99.8 ± 4.1 mg/ml; p ¼ 0.217).

various diets. Rats consuming MUFA-rich diets, OO or HOSO showed the highest body fat content (p ¼ 0.001) as compared with the AT rats and the three control groups. Moreover, the hepatic index was elevated in AT, OO and HOSO as compared with the three control groups (p ¼ 0.001). The control rats fed diets C, OO-C and HOSO-C did not show any differences among them (p40.05) (Table 2). Body fat distribution is shown in Figure 2. The percentage of intestinal fat exhibited the highest values for the MUFA-rich experimental groups (OO: 1.40 ± 0.24 g% and HOSO: 1.37 ± 0.28 g%), the differences were significant as compared with the other rats (p50.001). The percentage of epididymal fat in HOSO rats was higher as compared with the other groups (p50.029), followed by the percentage found among the OO and AT rats. The other groups did not show any epididymal fat differences among them (p40.05). The percentage of perirenal fat in HOSO, OO and AT rats was higher as compared with the control groups (p50.05), being similar within controls and the experimental groups (p ¼ 0.05). Effects on bone mineral content

Effects on total body fat and body fat distribution The final body fat and hepatic index are shown in Table 2. The percentage of total body fat differed among the rats fed the

The results of total skeleton BMC, BMC related to body weight (BMC/W) and total skeleton BMD, are shown in Figure 3. Rats fed AT and MUFA diets had decreased BMC and BMD as

!-9MUFA-rich diets in hypercholesterolemic-growing rats

DOI: 10.3109/09637486.2015.1025719

T-chol

5

HDL-chol

c

200

200 b

100

a

150

b mg/dL

mg/dL

150

a

100

a

50

50 0 C

OO-C HOSO-C

AT

OO

b

b

b

b

AT

OO

a

0

HOSO

C

Triglycerides

200

200

150

150

OO-C HOSO-C

Non-HDL-chol

HOSO

d

100

d b

50

a

c

c

a,b

mg/dL

c mg/dL

Int J Food Sci Nutr Downloaded from informahealthcare.com by Kainan University on 04/02/15 For personal use only.

b

b

100 50

0

a

a

a

0 C

OO-C HOSO-C AT

OO

HOSO

C

OO-C HOSO-C AT

OO

HOSO

Figure 1. Serum lipid-lipoprotein profile, at 8 weeks. Data are means ± standard deviation of the mean (SD). Values with different letters denote statistical significance between groups (p50.05), ‘‘a’’ being the lowest value. C, control diet; OO-C, olive oil control diet; HOSO-C, high-oleic sunflower oil control diet; AT, atherogenic diet; OO, olive oil diet; HOSO, high-oleic sunflower oil diet; T-chol, total cholesterol; triglycerides; HDL-chol, high-density lipoprotein cholesterol; Non-HDL-chol.

compared with the rats of three control groups (p50.001). However, OO and HOSO groups showed a significant increased in BMC/W and BMD as compared with AT (p50.05). Total skeleton BMC and BMD of rats fed control diets were similar among the three control groups (Figure 3a–c) and were higher than those of the three groups with hypercholesterolemia. Comparison of OO and HOSO values parameters at 8 weeks versus AT3 basal values OO and HOSO rats at 8 weeks were compared with AT group at 3 weeks (AT3) in order to evaluate the effects of replacing saturated fat by MUFA oils. Although no significant differences were observed for % of total body fat (AT3 ¼ 14.1 ± 0.90 versus OO8 ¼ 14.4 ± 1.7 and HOSO8 ¼ 14.2 ± 0.9; p ¼ 0.884), an increased in liver fat (HI) (OO8 ¼ 5.93 ± 0.55 and HOSO8 ¼ 5.84 ± 0.73 versus AT3 ¼ 4.50 ± 0.34; p50.001) and an significant decreased in epididymal and HOSO8 ¼ 1.33 ± 0.42 versus (OO8 ¼ 1.09 ± 0.33 AT3 ¼ 2.20 ± 0.05; p50.001), intestinal (OO8 ¼ 1.40 ± 0.24 and HOSO8 ¼ 1.36 ± 0.28 versus AT3 ¼ 1.95 ± 0.28; p ¼ 0.002 and perirenal (OO8 ¼ 1.23 ± 0.36 and HOSO8 ¼ 1.11 ± 0.56 versus AT3 ¼ 2.11 ± 0.74; p ¼ 0.0042) fats were detected. The replacement of saturated fat by OO diet resulted in important diminutions of 42% in serum T-chol, (127.1 ± 24.7 versus AT3 ¼ 243.0 ± 18.5 mg/dl), 53% in Non-HDL-chol (87.9 ± 20.2 versus 188.3 ± 17.9 mg/dl) and 41% in triglycerides (62.4 ± 7.3 versus 106.0 ± 17.9 mg/dl). The HOSO diet also decreased in

some serum lipids but in a smaller proportion (T-chol ¼ 16.5%; 202.8 ± 29.9 mg/dl and triglycerides ¼ 40%; 63.8 ± 7.7 mg/dl) but non-HDL-chol (172.1 ± 26.0 mg/dl) remained unchanged. Bone parameters BMC (OO8 ¼ 4.490 ± 0.325 and HOSO8 ¼ 4.360 ± 0.390 versus AT3 ¼ 3.550 ± 0.520 g; p ¼ 0.0004) and BMD (OO8 ¼ 0.257 ± 0.002 and HOSO8 ¼ 0.259 ± 0.001 versus AT3 ¼ 0.197 ± 0.016 g/cm2; p50.0001) were higher under OO and HOSO diets.

Discussion These experiments demonstrated that rats fed an AT diet fail to gain adequate weight and also develop alterations in the serum lipoprotein profiles with increased body fat content and decreased BMD and BMC. These data were similar to previous studies (Macri et al., 2007). Substituting MUFA for saturated fat in the diet ameliorated these alterations; however these rats continued to demonstrate hypercholesterolemia, increased body fat and elevated hepatic index, as well as decreased BMD and BMC. These results may serve as a note of caution to the recommendations of the use of MUFA substitutes for the dietary intervention in the treatment of hypercholesterolemia. It is known that intake off at-rich diets promote weight gain regardless of the caloric density of the diet (Mani et al., 2007). However, in these experiments, the body weight attained by OO and HOSO rats was lower than the C group. Previously, we reported that consumption of diets rich in saturated fat had lower digestibility and resulted in significantly lower body weight gain

6

E. V. Macri et al.

Int J Food Sci Nutr Downloaded from informahealthcare.com by Kainan University on 04/02/15 For personal use only.

Figure 2. Percentage of total body fat and distribution, at 8 weeks. Values are means ± SD. Values with different letters denote statistical significance between percent of total body fat among diets (p50.05), ‘‘a’’ being the lowest value. Percentage of Epididymal fat: HOSO (1.32 ± 0.43)4OO (1.09 ± 0.33), p50.05 and AT (1.12 ± 0.15), p50.05 and C (1.01 ± 0.12), p50.01, groups. Percentage of intestinal fat: HOSO (1.36 ± 0.28) and OO (1.40 ± 0.24)4C (0.85 ± 0.13), p50.01. Percentage of perirenal fat: AT (1.22 ± 0.31) and HOSO (1.11 ± 0.56) and OO (1.23 ± 0.36)4C (0.92 ± 0.18), p50.05 and C-HOSO (0.94 ± 0.36), p50.05 and C-OO (0.67 ± 0.33) (p50.01). C, control diet; OO-C, olive oil control diet; HOSO-C, higholeic sunflower oil control diet; AT, atherogenic diet; OO, olive oil diet; HOSO, higholeic sunflower oil diet.

Int J Food Sci Nutr, Early Online: 1–9

diets HOSO

b b

OO

AT

a a

HOSO-C

a

OO-C

Perirenal Epididym al Intestinal

a

C 0.0

and decreased percentage of body fat (Iossa et al., 2000). Others have shown that consumption of high-energy dense diets could increase the thermogenic capacity and decline energy efficiency altering energy balance, liver oxidative capacity (Majima et al., 2008) and fat oxidation (Krishnan & Cooper, 2014). HOSO and OO groups exhibited increases in the percentage of body fat mass related to the type of dietary fat (MUFA versus SF) or the MUFA source (Zaman et al., 2011). The increased energy content in the diet with the vegetable oils (HOSO and OO) increased the body fat and deposits of abdominal fat, with more pronounced liver weights, despite the absence of hyperphagia and of excess body weight gain (da Costa et al., 2011). In the present study, the highest increase in the amount of epididymal fat was detected in rats fed the enriched MUFA diets. This increase may denote a higher risk of CVD (Altintas et al., 2011; Jensen, 2008; Mathieu et al., 2010; Yamaguchi et al., 2012) and decreased bone health (Macri et al., 2012). In previous studies, we demonstrated that feeding an AT diet increased liver weights almost entirely at the expense of lipid deposits with micro- and macrovesicular fat deposits (Macri et al., 2007). Fat accumulation in the liver is of concern (Fassini et al., 2011; Qin & Tian, 2010). The oleic acid predominant in these diets provides cholesteryl esters (Chang & Huang, 1999; Landau et al., 1997). The pool of cholesteryl oleate in the liver reflects the type of fat intake (MUFA-enriched diets). However, the individual cholesterolemic status may be determined, at least in part, by the activity of the hepatic enzyme acyl-CoA: cholesterol-O-acyl transferase 2 (ACAT2), defined as an isoform of plasma ACAT. This is primarily responsible for the increased hepatic synthesis of cholesteryl oleate and secretion into the apoB-containing lipoproteins, as reflected in serum Non-HDL-C levels in the OO and HOSO groups. The accumulation of cholesteryl oleate in the liver promotes an accumulation of plasma LDL particles enriched in cholesteryloleate and the accumulation of cholesteryl esters in coronary arteries (Bell et al., 2007; Ochoa-Herrera et al., 2001; Vazquez et al., 2000). Saturated and monounsaturated fatty acids also share a common atherogenic link through the hepatic formation of the oleil CoA, with strong effect on apoB-containing particles (Dixon & Ginsberg, 1993; Pe´rez-Jime´nez et al., 2002). In the present study, the HOSO group showed higher TG/ HDL-chol ratios compared with OO (p50.001), suggesting that OO may have beneficial effects not related to the fatty acid composition (Degirolamo et al., 2009; Di Benedetto et al., 2010). It is well known that the Mediterranean diet, rich in MUFA, primarily extra virgin OO, is associated with a low incidence of

2.5

5.0

15

20

% body fat

CVD. Epidemiological studies have shown that Mediterranean countries share a geographical and climatic condition and a similar food culture (Degirolamo & Rudel, 2010; Sales et al., 2009). This has led to generalize the beneficial effects of MUFA and to overestimate their utility as replacement of saturated fatty acids. The cardio protective effects that MUFA may confer may be related to other bioactive compounds present in the oil rather than MUFA per se. Similarly, the beneficial effects of OO on coronary heart disease risk factors attributed to its high MUFA content may be related to other components, i.e. phenolics that could reduce the oxidative lipid damage (Fito´ et al., 2000). HOSO, which has an average MUFA of 85%, is similar to extra virgin OO with the lowest concentrations of SF (max. 8%). These oils retain most of their lipophilic components with strong antioxidant properties, among them the a-tocopherol, betacarotene, flavonoids and phenolics with (Frankel, 2011; Huang & Sumpio, 2008). Previous studies have also shown that diets with high fat content have an adverse effect on bone mineralization due to lipid oxidation and lipid composition alterations (Covas, 2008). This results in low bone mass and poor bone quality in growing animals (Lac et al., 2008). It is also known that AT diets fed to healthy growing rats constitutes a risk factor for bone modeling (Gamba et al., 2009; Macri et al., 2009). The replacement of saturated fat by MUFA-rich diet in the present experiments improved bone mass, BMC, BMC/W and BMD. However, after 5 weeks on HOSO- or OO-rich diets, the detrimental effects of the high-saturated animal fat intake were not sufficient to ameliorate the consequences of hypercholesterolemia on bone modeling. Rats fed PUFA-rich diets showed altered tibiae static-histomorphometry analysis and low bone alkaline phosphatase activity. They had low BMC and an inadequate subendochondral ossification and mineralization (Macri et al., 2009, 2012); though virgin OO diets may inhibit osteoblastic differentiation (Tintut et al., 2002). The total skeleton mineral content in hypercholesterolemic rats could infer the existence of possible structural bone alterations. Bone with a faster turnover rate (modeling), could reveal potential adverse effects of inappropriate types of fat intake on the skeletal and biomechanical bone properties of a growing animal (Byers et al., 2000). Studies in vitro demonstrated that a minimum oxidation of LDL-chol, as influenced by the type of dietary oil and its oxidation, might affect bone mineralization and bone marrow differentiation (Mani et al., 2007; Parhami et al., 1999, 2001). In the present study, the reduction in bone mass was more

!-9MUFA-rich diets in hypercholesterolemic-growing rats

DOI: 10.3109/09637486.2015.1025719

BMC

b

BMC/W

b

(g)

b a

a

4.5

a

b

3.0 1.5 0.0

C

OO-C

b

2.5

b

g per 100g rat

6.0

7

HOSO-C

AT

OO

2.0

b

a,b

a,b

OO

HOSO

a 1.5 1.0 0.5 0.0

HOSO

C

OO-C

b

b

OO

HOSO

HOSO-C

AT

BMD

c

c

c a

g/cm2

Int J Food Sci Nutr Downloaded from informahealthcare.com by Kainan University on 04/02/15 For personal use only.

0.30

0.25

0.20 0.2 0.0

C

OO-C

HOSO-C

AT

Figure 3. Effects on bone mass at 8 weeks. Total skeleton body mineral content (BMC), total skeleton body mineral content per body weight (BMC/W) and total skeleton body mineral density (BMD) at 8 weeks. Data are means ± SD. Values with different letters denote statistical significance between diets (p50.05), ‘‘a’’ being the lowest value. C, control diet; OO-C, olive oil control diet; HOSO-C, high-oleic sunflower oil control diet; AT, atherogenic diet; OO, olive oil diet; HOSO, high-oleic sunflower oil diet.

related to the presence of a MUFA-rich diet, rather than to the source of MUFA. However, there may be other variables rather than MUFA that may play a prominent role in determining the risk for cardiovascular and bone effects of these oils intake. A comprehensive metabolic profile status of such diets was not determined in this study; though fasting serum glucose and insulin levels were not altered by the various diets. Potential alterations of such variables would be pertinent to be considered as metabolic syndrome risk factors (Casavalle et al., 2014). Also, the potential atherogenic role of LDL and non-LDL particles including cell cholesterol accumulation would need to be ascertained along with HDL functionality, cholesterol efflux capacity, more than the HDL quantity to fully assess the CVD and bone risks and protective effects of PUFA diets (Favari et al., 2013; Khera et al., 2011).

Conclusion It is relatively well accepted to recommend MUFA-rich diets in place of SFA-rich diets and trans products in CVD prevention; this experimental study casts caution to the generalization of the benefits of MUFA for the dietary intervention for the treatment of hypercholesterolemia. Substituting MUFA for saturated fat in the diet ameliorated some of the alterations induced by an AT diet, although these rats continued to demonstrate elevated: cholesterol, body fat and hepatic index, along with decreased BMD and BMC. Hypercholesterolemic-growing rats showed that the potential beneficial effects of extra virgin OO intake on serum T-chol, HDL-chol and non-HDL-chol could not be demonstrated when they replaced an AT.

Acknowledgements The authors would like to thank Ricardo Orzuza of the Department of Biochemistry and Oral Biology, School of Dentistry, University of Buenos Aires, for his technical support and animal care.

Declaration of interest The authors declare that they have no conflict of interest. The University of Buenos Aires, grant 20020100100613, supported this study. F.L. was supported by Pediatric Sunshine Academics Inc. E.A. also received support from Pediatric Sunshine Academics Inc.

References Altintas MM, Rossetti MA, Nayer B, Puig A, Zagallo P, Ortega LM, Johnson KB, et al. 2011. Apoptosis, mastocytosis, and diminished adipocytokine gene expression accompany reduced epididymal fat mass in long-standing diet-induced obese mice. Lipids Health Dis 10: 198–210. AOAC. 1990. Official methods of analysis of the Association of Official Analytical Chemists. 15th ed. Washington, DC: Association of Official Analytical Chemists. Aro´s F, Estruch R. 2013. Mediterranean diet and cardiovascular prevention. Rev Esp Cardiol (Engl Ed) 66:771–774. ATP III, NCEP. 2001. Expert panel on detection, evaluation and treatment of high blood cholesterol in adults (Adult Treatment Panel III). Executive summary of the third report of the National Cholesterol Education Program (NCEP). JAMA 285:2486–2497. Bell TA, Kelley K, Wilson MD, Sawyer JK, Rudel LL. 2007. Dietary fatinduced alterations in atherosclerosis are abolished by ACAT2deficiency in ApoB100 only, LDLr/ mice. Arterioscler Thromb Vasc Biol 27:1396–1402. Bullon P, Battino M, Varela-Lopez A, Perez-Lopez P, Granados-Principal S, Ramirez-Tortosa MC, Ochoa JJ, et al. 2013. Diets based on virgin

Int J Food Sci Nutr Downloaded from informahealthcare.com by Kainan University on 04/02/15 For personal use only.

8

E. V. Macri et al.

olive oil or fish oil but not on sunflower oil prevent age-related alveolar bone resorption by mitochondrial-related mechanisms. LoS One 8:e74234. Byers S, Moore AJ, Byard RW, Fazzalari NL. 2000. Quantitative histomorphometric analysis of the human growth plate from birth to adolescence. Bone 27:495–501. ´ lvarez JR, Sola` Carrillo Ferna´ndez L, Dalmau Serra J, Martı´nez A Alberich R, Pe´rez-Jime´nez F. 2011. Grasas de la dieta y salud cardiovascular. An Pediatr (Barc) 74:192. e1–e16. Casavalle P, Lifshitz F, Romano L, Pandolfo M, Caaman˜o A, Boyer PM, Rodrı´guez PN, Friedman SM. 2014. Prevalence of dyslipidemia and metabolic syndrome risk factor in overweight and obese children. Ped Endocrinol Rev 12:213–223. Chang NW, Huang PC. 1999. Comparative effects of polyunsaturated- to saturated fatty acid ratio versus polyunsaturated- and monounsaturated fatty acids to saturated fatty acid ratio on lipid metabolism in rats. Atherosclerosis 142:185–191. Covas MI. 2008. Bioactive effects of olive oil phenolic compounds in humans: reduction of heart disease factors and oxidative damage. Inflammopharmacology 16:216–218 [review]. da Costa CA, Carlos AS, dos Santos Ade S, Monteiro AM, Moura EG, Nascimento-Saba CC. 2011. Abdominal adiposity, insulin and bone quality in young male rats fed a high-fat diet containing soybean or canola oil. Clinics (Sao Paulo) 66:1811–1816. Degirolamo C, Rudel LL. 2010. Dietary monounsaturated fatty acids appear not to provide cardioprotection. Curr Atheroscler Rep 12: 391–396. Degirolamo C, Shelness GS, Rudel LL. 2009. LDL cholesteryl oleate as a predictor for atherosclerosis: evidence from human and animal studies on dietary fat. J Lipid Res 50:S434–S439. Di Benedetto R, Attorri L, Chiarotti F, Eusepi A, Di Biase A, Salvati S. 2010. Effect of micronutrient-enriched sunflower oils on plasma lipid profile and antioxidant status in high-fat-fed rats. J Agric Food Chem 58:5328–5333. Dixon JL, Ginsberg HN. 1993. Regulation of hepatic secretion of apolipoprotein B-containing lipoproteins: information obtained from cultured liver cells. J Lipid Res 34:167–179. Estruch R, Ros E, Salas-Salvado´ J, Covas MI, Corella D, Aro´s F, Go´mezGracia E, et al. 2013. Primary prevention of cardiovascular disease with a Mediterranean diet. N Engl J Med 368:1279–1290. Fassini PG, Noda RW, Ferreira ES, Silva MA, Neves VA, Demonte A. 2011. Soybean glycinin improves HDL-C and suppresses the effects of rosuvastatin on hypercholesterolemic rats. Lipids Health Dis 10:165. Favari E, Ronda N, Adorni MP, Zimetti F, Salvi P, Manfredini M, Bernini F, et al. 2013. ABCA1-dependent serum cholesterol efflux capacity inversely correlates with pulse wave velocity in healthy subjects. J Lipid Res 54:238–243. Fito´ M, Covas MI, Lamuela-Ravento´s RM, Vila J, Torrents L, de la Torre C, Marrugat J. 2000. Protective effect of olive oil and its phenolic compounds against low density lipoprotein oxidation. Lipids 35: 633–638. Frankel EN. 2011. Nutritional and biological properties of extra virgin olive oil. J Agric Food Chem 59:785–792. Gamba CA, Macri V, Gonzalez Chaves M, Orzuza R, Zago V, Rodriguez P, Mandalunis P, et al. 2009. Rat mandible bone response to atherogenic diet [abstract]. Bone 45:S156. Glantz SA. 1992. Primer of biostatistics. 3rd ed. Nueva York: Mc Graw Hill. p. 358. Huang CL, Sumpio BE. 2008. Olive oil, the mediterranean diet and cardiovascular health. J Am Coll Surg 207:407–416. Iossa S, Lionetti L, Mollica MP, Crescenzo R, Barletta A, Liverini G. 2000. Effect of long-term high-fat feeding on energy balance and liver oxidative activity in rats. Br J Nutr 84:377–385. Jensen MD. 2008. Role of body fat distribution and the metabolic complications of obesity. J Clin Endocrinol Metab 93:S57–S63. Kale VP, Joshi GS, Gohil PB, Jain MR. 2009. Effect of fasting duration on clinical pathology results in Wistar rats. Vet Clin Pathol 38:361–366. Khera AV, Cuchel M, de la Llera-Moya M, Rodrigues A, Burke MF, Jafri K, French B, et al. 2011. Cholesterol efflux capacity, high-density lipoprotein function, and atherosclerosis. N Engl J Med 364:127–135. Krishnan S, Cooper JA. 2014. Effect of dietary fatty acid composition on substrate utilization and body weight maintenance in humans. Eur J Nutr 53:691–710.

Int J Food Sci Nutr, Early Online: 1–9

Lac G, Cavalie H, Ebal E, Michaux O. 2008. Effects of a high fat diet on bone of growing rats. Correlations between visceral fat, adiponectin and bone mass density. Lipids Health Dis 7:16. Landau JM, Sekowski A, Hamm MW. 1997. Dietary cholesterol and the activity of stearoyl CoA desaturase in rats: evidence for an indirect regulatory effect. Biochim Biophys Acta 1345:349–357. Lecka-Czernik B, Stechschulte LA. 2014. Bone and fat: a relationship of different shades. Arch Biochem Biophys 561:124–129. Macri EV, Gonzales Chaves MM, Rodriguez PN, Mandalunis P, Zeni S, Lifshitz F, Friedman SM. 2012. High-fat diets affect energy and bone metabolism in growing rats. Eur J Nutr 51:399–406. Macri EV, Juiz N, Gonzales Chaves M, Ponce G, Lanata E, Rodriguez P, Mandalunis P, et al. 2009. Effect of high saturated-fat diets on bone mass and endochondral ossification in healthy growing rats. FASEB J 23:Abs 2550. Macri EV, Zago V, Ramos C, Gamba C, Suarez C, Rodrı´guez P, Schreier L, Friedman SM. 2007. Aceite de Girasol alto oleico: ¿Es un coadyuvante adecuado en la Hipercolesterolemia de origen nutricional? Actualizacio´n Nutricio´n 8:269–281. Majima T, Shimatsu A, Komatsu Y, Satoh N, Fukao A, Ninomiya K, Matsumura T, Nakao K. 2008. Increased bone turnover in patients with hypercholesterolemia. Endocr J 55:143–151. Mani A, Radhakrishnan J, Wang H, Mani A, Mani MA, Nelson-Williams C, Carew KS, et al. 2007. LRP6 mutation in a family with early coronary disease and metabolic risk factors. Science 315:1278–1282. Mathieu P, Lemieux I, Despre´s JP. 2010. Obesity, inflammation, and cardiovascular risk. Clin Pharmacol Ther 87:407–416 [review]. Michas G, Micha R, Zampelas A. 2014. Dietary fats and cardiovascular disease: putting together the pieces of a complicated puzzle. Atherosclerosis 234:320–328. Ochoa-Herrera JJ, Huertas JR, Quiles JL, Mataix J. 2001. Dietary oils high in oleic acid, but with different non-glyceride contents, have different effects on lipid profiles and peroxidation in rabbit hepatic mitochondria. J Nutr Biochem 12:357–364. Parhami F, Jackson SM, Tintut Y, Le V, Balucan JP, Territo M, Demer LL. 1999. Atherogenic diet and minimally oxidized low density lipoprotein inhibit osteogenic and promote adipogenic differentiation of marrow stromal cells. J Bone Miner Res 14:2067–2068. Parhami F, Tintut Y, Beamer WG, Gharavi N, Goodman W, Demer LL. 2001. Atherogenic high-fat diet reduces bone mineralization in mice. J Bone Miner Res 16:182–188. Pe´rez-Jime´nez F, Lo´pez-Miranda J, Mata P. 2002. Protective effect of dietary monounsaturated fat on arteriosclerosis: beyond cholesterol. Atherosclerosis 163:385–398. Qin Y, Tian YP. 2010. Preventive effects of chronic exogenous growth hormone levels on diet-induced hepatic steatosis in rats. Lipids Health Dis 9:78–89. Reeves P, Nielsen F, Fahey G Jr. 1993. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr 123:1939–1951. Sales C, Oliviero F, Spinella P. 2009. The mediterranean diet model in inflammatory rheumatic diseases. Reumatismo 61:10–14. Srinath Reddy K, Katan MB. 2004. Diet, nutrition and the prevention of hypertension and cardiovascular diseases. Public Health Nutr 7:167–186. Tintut Y, Parhami F, Tsingotjidou A, Tetradis S, Territo M, Demer LL. 2002. 8-Isoprostaglandin E2 enhances receptor-activated NFkappa B ligand (RANKL)-dependent osteoclastic potential of marrow hematopoietic precursors via the cAMP pathway. J Biol Chem 277: 14221–14226. Uauy R, Aro A, Clarke R, Ghafoorunissa, L’Abbe´ MR, Mozaffarian D, Skeaff CM, et al. 2009. WHO Scientific Update on trans fatty acids: summary and conclusions. Eur J Clin Nutr 63:S68–S75. ´ cidos grasos con isomerı´a trans. Situacio´n de Valenzuela BA. 2008. A consumo en Latinoame´rica y alternativas para su sustitucio´n. Rev Chil Nutr 35:172–180. Valk EE, Hornstra G. 2000. Relationship between Vitamin E requirement and polyunsaturated fatty acid intake in man: a review. Int J Vitam Nutr Res 70:31–42. Vazquez CM, Zanetti R, Santa-Maria C, Ruiz-Gutierrez V. 2000. Effects of two highly monounsaturated oils on lipid composition and enzyme activities in rat jejunum. Biosci Rep 20:355–368. Wang H, Storlien LH, Huang XF. 2002. Effects of dietary fat types on body fatness, leptin, and ARC leptin receptor, NPY, and

DOI: 10.3109/09637486.2015.1025719

Int J Food Sci Nutr Downloaded from informahealthcare.com by Kainan University on 04/02/15 For personal use only.

AgRP mRNA expression. Am J Physiol Endocrinol Metab 282: E1352–1359. Yamaguchi T, Miyashita Y, Saiki A, Watanabe F, Watanabe H, Shirai K. 2012. Formula diet is effective for the reduction and differentiation of visceral adipose tissue in zucker fatty rats. J Atheroscler Thromb 19: 127–136. Zago V, Lucero D, Macri EV, Cacciagiu´ L, Gamba C, Miksztowicz V, Berg G, et al. 2010. Circulating very-low-density lipoprotein characteristics resulting from fatty liver in an insulin resistance rat model. Ann Nutr Metab 56:198–206.

!-9MUFA-rich diets in hypercholesterolemic-growing rats

9

Zaman MQ, Leray V, Le Bloc’h J, Thorin C, Ouguerram K, Nguyen P. 2011. Lipid profile and insulin sensitivity in rats fed with high-fat or high-fructose diets. Br J Nutr 106: S206–S210. Zeni SN, Gregorio S, Gomez AC, Somoza J, Mautalen C. 2002. Olpadronate prevents the bone loss induced by cyclosporine in the rat. Calcif Tissue Int 70:48–53. Zeni SN, Wittich C, Di Gregorio S, Casco C, Oviedo A, Somoza J. 2001. ´ sea. Utilidad Clı´nica de los Marcadores de Formacio´n y Resorcio´n O Acta Bioquı´m Clı´n Latinoam XXXV:3–36.