Effect of frying on vitamin E content in virgin olive oil. Values are means ± SE for six oil samples. Each value is significantly different from all other values not showing the same letter on the top of the bar ( P < 0 . 05), i.e., the same letter on the top of two or more bars means no statistically significant differences between the corresponding values. 

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... diet): 26.7 casein, 13.53 starch, 45.29 sucrose, 8.0 edible oil, 3.68 mineral supplement, 1.0 vitamin supplement, 1.84 cellulose, 0.09 choline, and 0.30 methionine. Min- eral and vitamin supplements were designed following the AIN criteria (AIN76 supplements) (American Institute of Nutrition, 1977). Groups only differed in the type of edible oil used to feed the rats on. A group was fed on nonfried oil and fried oil group was fed on the four cycles fried oil. At the end of the experiment, the rats were killed by decapitation at the same time of the day in all cases (between 12:00 and 13:00 h) to avoid circadian fluctuations. The protocols were approved by the Ethical Committee of the Interministerial Commission of Science and Technol- ogy. Animals were handled according to the guidelines for care and use of laboratory animals of the Spanish Society for Laboratory Animal Sciences. Blood was collected in EDTA-coated tubes, and the plasma was centrifuged at 1750 g for 10 min. All samples were stored at − 80 ◦ C until analyzed. Liver mitochondria were isolated following Fleischer et al. (1979) procedure. Triglycerides and total cholesterol in plasma were determined by enzymatic methods, using Boehringer- Mannheim kits (Munich, Germany). Mitochondrial membrane cholesterol was determined according to Röschlau et al. (1974). The protein concentrations of mitochondrial samples were determined according to the method of Lowry et al. (1951), using bovine serum albumin as a standard. Fatty acid profile of plasma and liver mitochondrial membranes was measured by gas–liquid chromatography according to Lepage and Roy (1986) as described above for the oil samples. The ferrous-oxide xylenol orange (FOX2) method was used for determining hydroperoxides (HP) in mitochondrial membrane. HP levels were assayed according to the principle of the rapid peroxide-mediated oxidation of Fe 2 + to Fe 3 + under acidic conditions (Nourouz-Zadeh et al. , 1994) using tryphenylphosphine (TPP), an agent that avoids artifactual color generation in samples that might contain substantial quantities of loosely available iron. Briefly, mitochondria (0.1 mg) were incubated at 37 ◦ C for 30 min with and without 1 mM TPP. Then FOX2 reagent was added to each sample and incubated again at 37 ◦ C for 30 min in a water-shaking bath. After centrifugation (2000 g for 5 min) the supernatants were monitored at 560 nm. TBARS in mitochondrial membrane were determined according to Orrenius et al. (1977). Analyzes of coenzyme Q 9 and α -tocopherol in mitochondrial membranes were carried out according to Lang and Packer (1987), by high-performance liquid chromatography with in-line Diode Array Detector, model 168 (Beckman Instruments, Inc. Fullerton, CA). Plasma coenzyme Q 9 and α -tocopherol were also assayed by HPLC according to MacCrehan (1990). The concentration of mitochondrial cytochrome c + c 1 , b , and a + a 3 was evaluated by differential spectra in a λ 16-Perkin Elmer double-beam spectrophotometer according to Vanneste (1966) and Nicholls (1976). Ex- actly 200 μ L of sodium deoxycholate 10% (w/v) plus the sample (the equivalent volume to 2 mg of mitochondrial protein), together with 7 mM KH 2 PO 4 until a fi- nal volume of 1.7 mL, were gently mixed in a spectrophotometer cuvette. After 10 μ L of 20 mM potas- sium ferricyanide were added to the mix, to allow the total oxidation of the cytochromes, the oxidized spectra between 650 and 500 nm was recorded. Afterward, 20 μ g of sodium dithionite were added to reduce the cytochromes completely, acquiring again the spectra between 650 and 500 nm. The differential spectra (reduced minus oxidized) were recorded to calculate the concentration of cytochromes, using the following extinction coefficients: ε (A –A ) = 20 mM − 1 · cm − 1 ; b (A 561 –A 575 ) 25 mM cm ; a + a 3 (A 605 –A 630 ) 24 mM − 1 · cm − 1 . Cytochrome c oxidase activity was assayed at 25 ◦ C using cytochrome c (90 μ M; in 10 mM Tris) reduced by sodium dithionite. After reduction, cytochrome c was pu- rified in a Sephadex G-25 column (Battino et al. , 1986; Degli Esposti and Lenaz, 1982), so that the ratio between the extinction at 500 and 565 nm was between 8 and 10. Cytochrome c was then mixed with 10 mM Tris, 50 mM KCl, 1 mM EDTA, and added with 0.3 mg/mL of antimicine A. Samples were poured into the cuvette and mixed, monitoring the absorbance decrease of cytochrome c upon oxidation at 417–409 nm every 10 s for 2 min; the extinction coefficient used for cytochrome c was 40.7 mM − 1 · cm − 1 (Battino et al ., 1986). The results represent the mean ± SEM of eight animals. Significant ( P < 0 . 05) interaction terms were evaluated by a Student t test. Data were analyzed using the SPSS/PC statistical software package (SPSS for Windows, 9.0.1, 1999, SPSS Inc. Chicago, IL, U.S.A.). Nonfried oil showed a typical fatty acid composition (Mataix et al. , 1998), and we did not find changes in the fatty acids profile of the oils after frying (Table I). Figure 1 shows the vitamin E content present in the oil submitted to different frying times. Nonfried oil showed an amount of 400 mg/kg of vitamin E, which is normal for this type of edible oil. The frying procedure led to a decrease in the levels of vitamin E after 60 min frying period but not for 15, 30, or 45 min. The total content in phenolic compounds is shown in Fig. 2. Initial level of nonfried oil is around 1500 mg/kg and this amount is progressively decreasing with the frying. The lowest amount is found for the 60-min fried oil, which has around 740 mg/kg. Figure 3 represents the antioxidant capacity of the oils measured by ESR. This parameter shows that frying decreases the antioxidant capacity of the oil by about 50% at the end of the study. The decrease in the antioxidant capacity is progressive. Finally, Fig. 4 shows the percentage of total polar material, which is a specific marker of damage in the oil produced by frying. The content in polar materials start to increase in our model after 45 min of frying and remain constant for the 60-min fried oil. Dietary intake did not vary significantly among groups. Body weight was similar for all groups through- out the whole study and the weight of the liver was not affected by experimental treatments (data not shown). Table II shows the determinations done on plasma of rats fed for 8 weeks on fried or nonfried olive oil. Concerning total cholesterol and triglycerides, normal values were found in both groups with no differences between them. The level of the antioxidants vitamin E and coenzyme Q 9 were significantly lower in fried-oil fed group. Concerning the fatty acid profile of plasma, no differences were found for total monounsaturated fatty acids (MUFA), diunsaturated fatty acids (DUFA), polyunsaturated fatty acids n − 6 (PUFA n − 6), PUFA n − 3 or the ratio between saturated fatty acids and monounsaturated fatty acids (SFA:MUFA). However, the level of total SFA was higher in fried-oil fed animals and the level of total unsaturated fatty acids (UFA) and unsaturation index (UI) were lower in fried-oil fed animals. The results on the mitochondrial membrane of rats are showed in Table III. No differences were found in mitochondrial membrane content of cholesterol. The levels of coenzyme Q 9 were higher in fried-oil fed animals and no differences were found between two groups for vitamin E. Concerning lipid peroxidation markers, both lipid hydroperoxides and TBARS were higher in the group of animals on the diet formulated with fried-oil fed for 8 weeks. Concerning the lipid profile in the mitochondrial membranes, similar changes to those described for plasma fatty acids were found, i.e., lower UFA, lower UI, and higher SFA in fried-oil fed rats than in nonfried-oil fed rats. The rest of the fatty acid parameters (MUFA, DUFA, SFA:MUFA, PUFA n − 6, and PUFA n − 3) did not show differences between both groups. Figure 5 shows the levels of cytochromes b , c + c 1 , and a + a 3 as well as the cytochrome c oxidase activity in liver mitochondrial membranes. No differences were found for cytochrome b after frying. However, the levels of cytochrome c , a + a 3 , and the cytochrome c oxidase activity was higher in rats fed on fried oil compared with those fed on nonfried oil. Oxidative injury is assumed to play a crucial role in the development of several chronic diseases, e.g., coronary heart disease (CHD) and cancer, and the possibility that dietary antioxidants may protect against LDL oxidation and oxidative injury has received growing attention in the past few years (Halliwell and Gutteridge, 1999). Dietary fatty acids can influence the susceptibility of cells to oxidative stress, probably also by changing cell membrane fatty acid composition (Battino et al. , 1999). Cells enriched with MUFA have been shown to be less susceptible to oxidative damage, whereas n − 6 PUFA increased the susceptibility to oxidative damage. Moreover, since some aldehydic products are damaging to hu- man health (Grootveld et al. , 1998), the results of several investigations indicate that the dietary ingestion of thermally stressed PUFA-rich culinary oils promotes the induction, development, and progression of CHD. Despite the availability of much epidemiological and experimental evidence related to the dietary consumption of virgin olive oil in limiting the development and progression of several pathologies, the toxicological hazards associated with the ingestion of unheated and/or thermally stressed virgin olive oil is partially lacking. This is particularly interesting if we considered that, as we recently demonstrated (Quiles et al. , 2001), the dietary fat type may modulate the composition and function of mitochondrial respiratory chain components. The data we are discussing indicate that the virgin olive oil used in the rat diet had not suffered any changes in its fatty acid pattern after frying (Table I). This has to be ascribed to the short-time deep fat frying ...