Participants

Of 700 students living under boarding school-like conditions in a third-level technical college, 115 volunteers were screened for participation. Inclusion criteria were a body mass index (BMI) of less than 27 kg/m2, serum cholesterol concentrations below 7.76 mmol/l and triacylglycerol concentrations below 3.39 mmol/l. Of the 115 volunteers, one was excluded because of diabetes mellitus, three because of hyperlipidemia, five because of thyroid disease, two because of intake of vitamin supplements, four because of hyperuricemia, and 25 because of allergy, intolerance or aversion to foodstuffs contained in the study diets. Other exclusion criteria were smoking, drug or substance abuse and malabsorption syndromes. Of the 75 students who qualified for participation in the study, 69 (35 male, 34 female), aged between 18 and 43 y were chosen for inclusion by drawing lots. Six subjects withdrew during the study because of intercurrent illness and five withdrew because they were unwilling or unable to comply with the dietary regimen. The baseline characteristics of the 58 (31 male, 27 female) participants who finished the study are shown in Table 1. Twenty-one female participants who were taking oral contraceptives were instructed not to stop taking them and not to change to another pill. The participants were also asked not to change their regular lifestyles and their usual extent of physical activity throughout the study.

Table 1 Baseline characteristics of participants (n=58) Full size table

The protocol and the objectives of the study were explained to the subjects in detail. All gave written consent. The study protocol was approved by the Ethics Commitee of the University of Münster and was in accordance with the Helsinki Declaration of 1975, as revised in 1983.

Design and diets

The study was conducted in a parallel design and consisted of two consecutive dietary periods for each subject. All participants consumed a wash-in high-fat diet rich in SFA for 2 weeks and were then randomly divided into three groups. Each group received a high-fat diet containing refined olive oil (11 men, nine women), sunflower oil (10 men, 10 women) or rapeseed oil (10 men, eight women), respectively, as the principal source of fat for 4 weeks. These diets were identical in every respect apart from the fatty acid composition. Venous blood samples were obtained at the beginning of the study (visit 1), after the wash-in-period (visit 2), after 2 weeks of the study diets (visit 3) and at the end of the study (visit 4). All samples were drawn after an overnight fast of at least 12 h.

Before the study, the participants kept a careful dietary record for 3 days. This was used to estimate each subject's habitual energy and nutrient intake. The records were coded and calculated on the basis of German standard food tables (Bundeslebensmittelschlüssel). The study diets were calculated for 10 levels of energy intake ranging in steps of 0.84 MJ/day (200 kcal/day) from 7.52 to 15.05 MJ/day (1800–3600 kcal/day) by using a computer-based nutrient calculation program (EBIS, E&D Partner, Stuttgart, Germany). All participants were weighed twice a week while wearing light clothing and energy intake was adjusted when necessary to maintain a stable body weight. During the study the mean body weight decreased by 0.68±1.16 kg (mean±standard deviation; range −4.0 to +2.1).

The composition of the participants' habitual diet and the study diets are shown in Table 2. All study diets consisted of conventional mixed foods that were freshly prepared. Menus were changed daily. The kitchen and dining facilities were located in the school in which the students were trained and housed during the week. The participants were served breakfast, lunch and dinner from Monday morning to Friday noon. This food was immediately consumed in the school canteen under the direct supervision of one of the authors (MK). On Friday afternoons, participants were given hampers containing their entire food supply for the weekend. All foodstuffs were weighed. On the study diets, the basic menus were identical for all participants. All dietary items were low in fat, eg lean meat, skimmed milk and low-fat dairy products. This provided scope for the enrichment of these meals with the specific oils, which were provided in sauces, desserts and salad dressings. A margarine was specially manufactured based on these oils. This margarine contained 20% water, 20% hard stock (coconut fat, palm kernel fat and palm oil), and 60% refined olive oil, rapeseed oil, or sunflower oil, respectively. To ensure equal vitamin E intake in all groups, the margarines were supplemented with different amounts of vitamin E to compensate for the different amounts of vitamin E in the oils. We also used specially baked oil-enriched bread containing 10% oil (olive, rapeseed, or sunflower, respectively). To compensate for short-term differences in individual energy requirements, participants were provided on request with special bread rolls which were baked so as to contain exactly the same nutrient composition as that person's study diet. By means of these rolls, energy balance was ensured without changing the composition of the diets.

Table 2 Composition of the study dietsa Full size table

Participants were directly supplied with enough food to meet 90% of their mean daily energy requirements. The remaining energy was provided in the form of free-choice foodstuffs such as beverages or fruit which contained only trace amounts of fat, protein or cholesterol. These were chosen from a given list and were recorded in diaries as was any food that was not consumed and deviations from the diets. Based on these diaries, adherence was found to be very high. The intake of drugs and any signs of illness were also recorded in the diaries.

Laboratory measurements

Isolation of LDL.

For analyses of LDL susceptibility to oxidation, fatty acid composition and vitamin E content blood was drawn into tubes containing 1.6 mg EDTA/ml (Sarstedt, Germany). The blood samples were centrifuged at 1800 g for 10 min, EDTA-plasma was separated and frozen at −80°C after addition of 0.6% sucrose. This plasma was frozen for a maximum of 6.5 months. LDL was separated from this plasma in a single run of 2 h by density gradient centrifugation using the method of Chung et al (Chung et al, 1980) with the following minor modifications: the rotor used was a Beckman NVT 65 (Beckman, Fullerton, USA), EDTA plasma was adjusted to 1.26 g/ml, and the centrifugation conditions were 60 000 rpm for 2 h. After centrifugation, LDL was collected by using a syringe and a needle and filtered through a 0.22 µm sterile filter (Renner, Dannstadt, Germany) into sterile vacuum containers (Mallinckrodt Radio-Pharma, Hennef, Germany). This preparation was stored in the dark at 4°C. Susceptibility to oxidation was always measured on the following day. Before the oxidation was started, the level of potential preoxidation due to storage or centrifugation procedures was tested by adjusting the LDL to a concentration of 0.08 mg LDL-cholesterol/ml and measuring the absorption at 234 nm, which should be lower than 0.31 at this concentration for native LDL.

Measurement of LDL susceptibility to oxidation.

In two subjects, LDL concentrations at visits 3 and 4 were too low to allow separation of sufficient amounts of LDL from blood. Susceptibility to oxidation was measured from LDL of the remaining 56 participants by the method of Esterbauer et al (Esterbauer et al, 1989). Briefly, LDL was desalted by gel-filtration on an Econo-Pac 10DG column (Bio-Rad, Munich, Germany) and stored on ice until the oxidation was started. The concentration of the desalted LDL solution was assessed by measurement of cholesterol content using a commercially available assay (CHOD-PAP, Boehringer-Mannheim, Mannheim, Germany). LDL was diluted to 0.08 mg LDL-cholesterol/ml by adding the appropriate amount of desalted LDL to phosphate buffered saline. The oxidation was started at 37°C by the addition of CuSO 4 (final concentration 1.6 µM) exactly 1 h after desalting. The formation of conjugated dienes was monitored by measurement of the change in absorbance at 234 nm in a Uvikon 922 photometer (Kontron, Neufahrn, Germany) for 3 h, resulting in a curve. A tangent to this curve was drawn at the point of inflexion. The lag time was defined as the time from the addition of CuSO 4 until the intersection of this tangent with the baseline. The rate of propagation was calculated from the slope of the tangent, and the maximum amount of conjugated diene formation was determined as the height of maximum absorbance above baseline. All four samples of each participant were measured in a single run. The pooled plasma of six healthy volunteers was used as an internal standard.

Measurement of LDL fatty acid composition.

The total fatty acid composition of LDL was measured by gas chromatography using the method of Lepage and Roy (1999) with the following minor modifications: C21:0 was used as an internal standard. Methanol-benzene was replaced by methanol–toluol. Analysis was conducted on a Dani 8250a gas chromatograph (Dani, Mainz, Germany) equipped with a CP Sil 88 column (50 m, 0.32 mm, 0.2 µm, 100% cyanopropyl-phase; Chrompack, Middelburg, The Netherlands), a temperature-programable vaporizer and a flame ionization detector (FID). Gas chromatograph parameters were: injector temperature, 60–240°C; oven temperature, 80°C for 2 min, increased in steps of 6°C/min to 140°C and maintained for 4 min, then increased in steps of 3°C/min to 225°C; FID temperature, 240°C; flow rate, 1.7 ml He/min, split 1:10.

Measurement of LDL tocopherol content.

LDL was stored at −80°C in brown tubes until analysis. Before analysis 50 µl of tocopheryl acetate (internal standard, 0.1 mg/ml), 1 ml of ethanol containing 0.1 mg BHT and 0.1 mg EDTA and 20 µl of a solution containing 0.6 M Na 2 WO 4 and 1 M MgCl 2 in H 2 O were added to the LDL. Water (500 µl) was added to the supernatant and the solution was extracted twice with 500 µl n-hexane-dichlormethane-isopropanol (80:19:1, v/v/v). The pooled organic phases were evaporated to dryness under nitrogen and the residue was dissolved in 50 µl ethanol and injected into the high performance liquid chromatograph (HPLC) by means of a Rheodyne 7125 (Cotati, CA, USA) 50 µl loop injector. All extraction steps were performed on ice in a laboratory with fluorescent lamps.

HPLC analysis was performed on a Kontron (Neufahm, Germany) liquid chromatograph (pump, model 422; column oven model 480; diode array detector, model 440). Separation was carried out on a 5 µm C 18 -resolve column (30 cm×3.9 mm i.d; Waters, Milford, USA; column temperature, 30°C) and a flow rate of 1.2 ml/min (solvent, acetonitrile–dichlorethane–methanol (85:10:5, v/v/v)+0.05% ammoniumacetate, isocratic). Column eluates were monitored by UV absorption at 292 nm.

Measurement of serum lipid parameters.

Total serum cholesterol, triacylglycerols and HDL cholesterol were measured using enzymatic assays (Röschlau et al, 1974) and (for HDL cholesterol) a precipitation method from Boehringer Mannheim (based on Burstein & Samaille, 1960), Germany, on a Hitachi 737 autoanalyzer. These methods are validated by regular analyses of reference sera supplied by the national German INSTAND proficiency testing program and the international quality assurance program of the US Centers for Disease Control and Prevention. LDL cholesterol was calculated by the Friedewald formula.

Statistics

All statistical calculations were performed using the Statistical Package for the Social Sciences (SPSS, version 10.0) computer program. Comparisons of the visits were done using a Wilcoxon matched-pairs signed-ranks test and the between-group-comparisons were done using a Mann–Whitney U-test. All tests were two-tailed and the level of significance was P<0.05. Due to the multiple test situation in the between-group comparisons, a Bonferroni correction was done in these tests.

The coefficients of variation for all automated lipid measurements were below 5%, that of the measurement of LDL tocopherol was 5%, those for the measurement of lag time, rate of propagation and maximum amount of conjugated dienes were 7, 10 and 6%, respectively, and those for the measurement of the LDL fatty acids were between 2 and 7%.