Original Communication | Published:

Effects of dietary fatty acids on the composition and oxidizability of low-density lipoprotein

European Journal of Clinical Nutrition volume 56, pages 7281 (2002) | Download Citation

Subjects

Abstract

Objective: The objective of this study was to compare the effects of dietary monounsaturated fatty acids (MUFA), n-6 and n-3 polyunsaturated fatty acids (PUFA) on LDL composition and oxidizability.

Design, setting and subjects: Sixty-nine healthy young volunteers, students at a nearby college, were included. Six subjects withdrew because of intercurrent illness and five withdrew because they were unable to comply with the dietary regimen.

Interventions: The participants received a 2-week wash-in diet rich in saturated fatty acids (SFA) followed by diets rich in refined olive oil, rapeseed oil or sunflower oil for 4 weeks. Intakes of vitamin E and other antioxidants did not differ significantly between the diets.

Results: At the end of the study, LDL oxidizability was lowest in the olive oil group (lag time: 72.6 min), intermediate in the rapeseed oil group (68.2 min) and highest in the sunflower oil group (60.4 min, P<0.05 for comparison of all three groups). Despite wide variations in SFA intake, the SFA content of LDL was not statistically different between the four diets (25.8–28.5% of LDL fatty acids). By contrast, the PUFA (43.5%–60.5% of LDL fatty acids) and MUFA content of LDL (13.7–29.1% of LDL fatty acids) showed a wider variability dependent on diet.

Conclusions: Enrichment of LDL with MUFA reduces LDL susceptibility to oxidation. As seen on the rapeseed oil diet this effect is independent of a displacement of higher unsaturated fatty acids from LDL. Evidence from this diet also suggests that highly unsaturated n-3 fatty acids in moderate amounts do not increase LDL oxidizability when provided in the context of a diet rich in MUFA.

Sponsorship: This work was supported by the Central Marketing Agency of the German Agricultural Industry (CMA), the German Union for the Promotion of Oil- and Protein Plants (UFOP), the Austrian Science Foundation, project F00709 (to P.M.A.) and the Brökelmann Ölmühle Company, Hamm, Germany.

Introduction

Worldwide, cardiovascular diseases are now the commonest cause of death, having outstripped infectious diseases for the first time in 1997 (World Health Organization, 1997). A major underlying cause of cardiovascular mortality and morbidity is atherosclerosis of the coronary arteries. Evidence exists from observational (Keys, 1980) and interventional (de Lorgeril et al, 1994) studies of a link between the amount and type of dietary fat and the risk of coronary heart disease.

Dietary fat may influence the risk of coronary heart disease by several mechanisms. One such mechanism is the effect of dietary fat on the susceptibility of LDL to oxidation. According to the oxidation hypothesis, one of the initial steps in atherogenesis is the oxidative modification of lipoproteins, mainly LDL, in the arterial wall. The oxidized LDL is taken up by macrophages, which in turn become lipid-laden foam cells (Westhuyzen, 1997). Although the relevance of this hypothesis in vivo has not been fully established, much supportive data has been generated in the last decade. LDL has been shown to be oxidized in vivo (Palinski et al, 1989), autoantibodies to oxidized LDL have been found in humans (Mironova et al, 1996), and LDL extracted from human atherosclerotic lesions demonstrates many of the physical, chemical and biological properties of in vitro oxidized LDL (Ylä-Herttuala et al, 1989). Furthermore, the measurement of the susceptibility of LDL to oxidation has been shown to correlate independently with the extent of atherosclerosis (Regnstrom et al, 1992).

It might be assumed that diets rich in saturated fatty acids (SFA) would lead to the production of LDL that are relatively resistant to oxidation, while the susceptibility of LDL to oxidation can be expected to increase with an increasing amount of unsaturated fatty acids in the diet. Several studies have indeed shown that diets rich in monounsaturated fatty acids (MUFA) lead to the production of LDL that is more resistant to oxidation than that found in persons consuming a diet rich in n-6 polyunsaturated fatty acids (PUFA) (Berry et al, 1991; Reaven et al, 1991, 1993; Mata et al, 1996; Turpeinen et al, 1995; Abbey et al, 1993; Bonanome et al, 1992). However, in some regards the interaction of dietary fat and LDL composition and oxidizability is poorly understood. It is still unclear if the protective effect of dietary MUFA is a specific feature of these fatty acids, or if they act by simple displacement of PUFA (Reaven, 1996). Furthermore, investigations on the impact of n-3 PUFA on LDL oxidizability are sparse and show contradictory results (Mata et al, 1996; Turpeinen et al, 1995; Brude et al, 1997; Nestel et al, 1997; Sorensen et al, 1998; McGrath et al, 1996).

This study was designed to answer the following questions: (1) what are the effects of diets containing different amounts of MUFA, n-6-PUFA and n-3-PUFA on LDL oxidation? (2) Do dietary fatty acids differ in their propensity for incorporation into LDL and hence in their ability to influence the susceptibility of LDL to oxidation? (3) Do dietary MUFA result in reduced susceptibility of LDL to oxidation simply because they displace PUFA, or are there other protective mechanisms?

Subjects and methods

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)

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

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 CuSO4 (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 CuSO4 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 Na2WO4 and 1 M MgCl2 in H2O 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 C18-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%.

Results

Effect of the diets on concentrations of circulating lipoproteins

During the diet phase (visit 2 to visit 4), the concentration of LDL cholesterol fell significantly with all three diets (Table 3), with a more distinct fall on the sunflower oil-based diet compared to the olive oil-based diet (P<0.05). A comparable pattern was seen with respect to HDL cholesterol. The fall in HDL cholesterol was most pronounced on the sunflower oil diet and the rapeseed oil diet and less pronounced on the olive oil diet (P<0.05 compared to sunflower oil).

Table 3: Influence of the study diets on serum concentrations of total, LDL- and HDL-cholesterol and triacylglycerols

Effect of the diets on susceptibility of LDL to oxidation

Lag time was longer on both MUFA-rich diets (olive oil and rapeseed oil) compared to the sunflower oil diet (Figure 1a). The rate of propagation was lowest on the olive oil diet, intermediate on the rapeseed oil diet, and greatest on the sunflower oil diet (Figure 1b). This also applied to the measurements of the maximum amount of conjugated dienes, which was greatest on sunflower oil, intermediate on rapeseed oil, and lowest on olive oil (Figure 1c). On the wash-in diet, with SFA as the main source of fat, the susceptibility of LDL to oxidation increased significantly with respect to the rate of propagation and the maximum amount of conjugated dienes (Figure 1b and c). We observed no effect of gender or weight changes on this parameter.

Figure 1
Figure 1

Effect of diets rich in SFA (wash-in, n=56), olive oil (n=18), rapeseed oil (n=18), or sunflower oil (n=20), respectively, on lag time until start of LDL oxidation (a), rate of propagation (b) and maximum amount of conjugated dienes (c) (mean±standard error of the mean). 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). *P<0.001 visit 4 vs visit 2; †P<0.01 visit 4 vs visit 2; ‡ P<0.001 visit 2 vs visit 1; § P<0.05 visit 4 vs visit 2. The significance levels for comparison between the diets are shown on the right. For reasons of legibility, siginificances of visit 3 vs visit 2 are not shown.

Effect of the diets on composition of LDL

The main changes during the wash-in diet were a decreased amount of stearic acid (C18:0) and oleic acid (C18:1) in LDL, while the amount of linoleic acid (C18:2) increased (Figure 2). On the olive oil diet, the amount of oleic acid increased markedly (+68.3 µg/mg LDL-cholesterol (+52%)) while the content of nearly all other fatty acids decreased with the greatest drop in linoleic acid (−54.4 µg/mg LDL-cholesterol (−18%)). On the sunflower oil diet, the amount of linoleic acid in LDL increased (+79.9 µg/mg LDL-cholesterol (+26%)), while the content of nearly all other fatty acids, mainly oleic acid (−32.5 µg/mg LDL-cholesterol (−25%)), decreased. On the rapeseed oil diet, there was a significant increase in oleic acid (+32.2 µg/mg LDL-cholesterol (+26%)), α-linolenic acid (+4.7 µg/mg LDL-cholesterol (+188%)) and eicosapentaenoic acid (+3.5 µg/mg LDL-cholesterol (+69%)) with a decrease in palmitoleic acid only. The increase of oleic acid content was more pronounced on the olive oil diet than on the rapeseed oil diet (P<0.001). We observed no effect of gender or weight changes on the LDL fatty acid composition.

Figure 2
Figure 2

Fatty acid composition of LDL in subjects receiving diets rich in SFA (a–c, visit 1 to visit 2), olive oil (a, visit 2 to visit 4, n=18), sunflower oil (b, visit 2 to visit 4, n=20) or rapeseed oil (c, visit 2 to visit 4, n=18). All values mean±standard deviation except C18:3 and C20:5 for which the median value and the 25th and 75th percentile are shown. 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). *P<0.05 visit 2 vs visit 1, †P<0.05 visit 4 vs visit 2. For reasons of legibility, significances of visit 3 vs visit 2 are not shown. Note that, because of the wide range of fatty acid concentrations, the y-axis shows two scales.

The LDL vitamin E content increased slightly and to a similar extent from visit 2 to visit 4 in all three groups (between+8% and+12%, Table 4), probably reflecting the increased dietary intake in this phase. There were no significant differences in the increase in LDL vitamin E content between the three groups.

Table 4: Influence of the study diets on α-tocopherol content of low density lipoproteina

Discussion

In the present study, we compared the effects of diets rich in MUFA, n-6 and n-3 PUFA on serum lipid concentrations, LDL composition and susceptibility of LDL to oxidation.

With regard to the effects of dietary fatty acids on serum lipid concentrations, our study essentially confirms the results of other studies. In short, n-6-PUFA appear to have a greater potential to lower LDL-cholesterol than MUFA as well as a greater HDL-cholesterol lowering effect. Thus, their overall effect on lipoprotein metabolism is considered to be similar (for a more detailed review see Mensink and Katan, 1992; Gardner and Kraemer, 1995).

The effect of dietary MUFA and PUFA on LDL oxidizability has been investigated in several studies (Berry et al, 1991; Reaven et al, 1991, 1993; Mata et al, 1996; Turpeinen et al, 1995; Abbey et al, 1993; Bonanome et al, 1992). These studies show that compared to PUFA-rich diets, diets rich in MUFA lead to LDL that is more resistant to oxidation. Several groups have investigated the effect of n-3 PUFA on LDL oxidizability (Mata et al, 1996; Turpeinen et al, 1995; Brude et al, 1997; Nestel et al, 1997; Sorensen et al, 1998; McGrath et al, 1996). Although the results of these studies have been conflicting, one point to emerge is that n-3 PUFA in large amounts (eg 20 g per day in the study by Nestel et al, 1997) or in the context of a diet rich in PUFA (Mata et al, 1996) increase the susceptibility of LDL to oxidation. However, recently published studies on the effect of dietary n-3 PUFA on the urinary isoprostane excretion, which is a good measure of in vivo lipid peroxidation, show that intake of n-3 PUFA is not associated with an increase in this parameter (Higdon et al, 2000; Mori et al, 1999, 2000). In contrast, these investigators unanimously observed a reduced urinary concentration of isoprostanes after administration of n-3 PUFA, indicating that n-3 PUFA are not necessarily detrimental with regard to oxidative processes.

Our study confirms previous reports that MUFA-rich diets lead to LDL that is less oxidizable than that found on a diet rich in PUFA. It is notable that this finding was consistent for all three parameters of LDL oxidizability, ie lag time, rate of propagation and maximum amount of conjugated dienes. This indicates that LDL do not only exert a lower tendency to become oxidized, but also that progression of the oxidation process once started is slower and that a lower amount of conjugated dienes are formed during oxidation after MUFA-rich diets compared to PUFA-rich diets.

A new and surprising finding was that on the MUFA-rich diets the oxidizability of LDL was lower even than that on the initial SFA-rich diet. It must be noted, however, that our study was not designed to compare the SFA-rich diet with the oil diets, so that a cause-and-effect conclusion cannot necessarily be drawn. The reduction in the susceptibility of LDL to oxidation might, for example, have been due to changes in unmeasured lifestyle factors during the 4 weeks of the oil diets. However, based on the diaries filled in by the participants, no differences in lifestyle variables known to influence oxidative processes were found. It might also be argued that the 2 week wash-in period was too short to allow saturation of LDL with SFA. However, during the oil phase of the study nearly all the changes in LDL composition occurred between weeks 0 and 2, with very little change between weeks 2 and 4, indicating that a potential of the wash-in diet to increase the incorporation of SFA into LDL would have become apparent within these 2 weeks. Furthermore, the changes in LDL fatty acid composition provide a reasonable explanation for the finding that MUFA are more beneficial than SFA with regard to LDL oxidizability. In contrast to MUFA and PUFA, SFA were not incorporated into LDL to a degree commensurate with their presence in the diet. Despite a more than two-fold variation in SFA intake (between 24.5 and 53.1% of dietary fatty acids), the SFA content of LDL was similar on all four diets (25.8–28.5% of LDL fatty acids). Thus, our SFA-rich wash-in diet did not result in the least oxidizable LDL. In fact, this diet was actually associated with an increase in the susceptibility of LDL to oxidation. This may have been due to the slightly reduced relative amount of MUFA in our wash-in diet (29.9% of fatty acids) compared to the habitual diet of the participants (33.0% of fatty acids), which resulted in a reduced amount of oleic acid in LDL. Although the relative amount of dietary linoleic acid did not change during the SFA-rich wash-in phase compared to the habitual diet, the reduced relative amount of oleic acid in the diet may have provided scope for further enrichment of LDL in linoleic acid. Our study was not designed to investigate the effect of the SFA-rich diet on LDL oxidizability. Therefore conclusions regarding causal relationships between diet and LDL oxidizability during this phase remain tentative. Nevertheless, several lines of evidence support the concept that MUFA reduce the oxidizability of LDL even compared to SFA.

The olive oil diet led to an increase in LDL oleic acid and a concomitant decrease in LDL linoleic acid, thereby reducing the susceptibility of the LDL particles to oxidation compared to the SFA-rich diet. However, the rapeseed oil diet led to enrichment of LDL with the n-3 PUFAs α-linolenic acid and eicosapentaenoic acid (while linoleic acid remained unchanged), but nevertheless reduced susceptibility of LDL to oxidation. The most likely reason for this effect is that the increase in oleic acid more than compensated for the increase in highly oxidizable n-3 PUFA found within LDL on this diet. This finding has never been reported before and may be important, because it implies that an increase in LDL oleic acid content reduces LDL in vitro oxidizability independently of a displacement of PUFA from LDL. Furthermore, n-3 PUFA in moderate amounts appear not to exert adverse effects with regard to LDL oxidation when provided in the context of a diet rich in MUFA. However, caution must be warranted in the interpretation of these findings, as the spectrophotometric measurement of lipid oxidation at 234 nm may not be optimal when trienoic acids are present.

A factor that must also be taken into account when discussing LDL oxidizability is the dietary and LDL content of α-tocopherol. In our study the LDL α-tocopherol content increased slightly and to a similar extent in all three groups. This may have had some influence on LDL oxidation. However, previous studies indicate that the influence of vitamin E in such low amounts is small and affects mainly lag time (Jialal et al, 1995; Princen et al, 1995). Additionally, even very high doses of vitamin E in the range of 1200 mg/day do not mask the effects of dietary fatty acids on LDL susceptibility to oxidation (Reaven et al, 1994). The amounts of tocopherol isomers other than α-tocopherol in diets or in LDL has not been measured as their contribution to the LDL total tocopherol content and thus its antioxidative capacity is very low (Esterbauer et al, 1992).

Our study investigated the effects of diet on the intermediate risk factors, and not its effect on arteriosclerosis or coronary heart disease. Nevertheless, our results and those of the literature (for review see Grundy, 1997; Kris-Etherton, 1999) generally indicate that MUFA are more salubrious than SFA or n-6 PUFA, although some epidemiological studies found a greater CHD risk reduction with a high PUFA intake (Ma et al, 1997; Hu et al, 1997). However, final proof of a protective effect of MUFA on atherogenesis will depend on well-controlled long-term primary prevention studies in humans.

In the healthy persons in our study, MUFA were clearly more beneficial than SFA and n-6 PUFA in terms of their effects on LDL oxidizability. They also prevented highly unsaturated n-3 fatty acids from exerting their potential negative effects in this regard. Within the limitations of our study, our results indicate that consideration should be given to increasing the ratio of MUFA to n-6 PUFA in dietary recommendations.

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Acknowledgements

We are indebted to B Pieke, B Berning, J Harmsen and particularly W Hanekamp and E Gramenz for excellent technical assistance; to R Schmidt, Dr R Junker and Dr G Bannenberg for performing the venipunctures; to Dr Arnold von Eckardstein for valuable discussion; to M Nestola and J Ackermann at the Bildungszentrum der Bundesfinanzverwaltung for their generous cooperation; to E Ostermann and the Camphill Werkstätten, Steinfurt, for supplying the oil-enriched bread and cake; to M Stennecken and Dr H Schulte for statistical analyses; to the Homann Company, Dissen, Germany, and particularly W Heimhalt for supplying the specially manufactured margarine; and last but not least to the study subjects for participation.

Author information

Author notes

    • M Kratz

    Guarantor: M Kratz.

    • M Kratz
    • , P Cullen
    • , F Kannenberg
    • , A Kassner
    • , M Fobker
    • , PM Abuja
    • , G Assmann
    •  & U Wahrburg

    Contributors: MK participated in designing and planning the study, calculated the diets, headed the investigation during the dietary period, measured the parameters of LDL oxidizability and wrote the paper together with PC, who also contributed to designing and planning of the study. FK and AK measured LDL fatty acid composition, MF measured LDL tocopherol levels, PMA contributed to the measurement of the parameters of LDL oxidizability and was involved in designing and planning the study, GA contributed to designing the study and provided the infrastructure, and UW initiated the project.

Affiliations

  1. Institute of Arteriosclerosis Research at the University of Münster, Münster, Germany

    • M Kratz
    • , P Cullen
    • , F Kannenberg
    • , A Kassner
    • , M Fobker
    • , G Assmann
    •  & U Wahrburg
  2. Institute of Clinical Chemistry and Laboratory Medicine, University of Münster, Münster, Germany

    • P Cullen
    • , M Fobker
    •  & G Assmann
  3. Institute of Biochemistry, University of Graz, Graz, Austria

    • PM Abuja
  4. University of Applied Sciences, Münster, Germany

    • U Wahrburg

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Correspondence to M Kratz.

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https://doi.org/10.1038/sj.ejcn.1601288