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Effect of ingestion of virgin olive oil on human low-density lipoprotein composition


Objective: To measure the incorporation of oleic acid and antioxidants (phenols and vitamin E) to low density lipoprotein (LDL) after acute and short-term ingestion of virgin olive oil. To study whether this incorporation contributes to an increase in LDL resistance to oxidation.

Setting: Department of Food and Nutrition, University of Barcelona, Spain and Department of Lipids and Cardiovascular Epidemiology, IMIM, Barcelona, Spain.

Subjects: Sixteen healthy volunteers aged 25–65 y.

Design and interventions: To observe the change in the fatty acid profile, vitamin E, phenolic compounds and LDL oxidation-related variables after the postprandial phase and after daily ingestion of olive oil for one week.

Results: Few changes were observed in the postprandial phase. However, after a week of olive oil consumption there was an increase in oleic acid (P=0.015), vitamin E (P=0.047), phenolics (P=0.021) and lag time (P=0.000), and a decrease in the maximum amount of dienes (P=0.045) and oxidation rate (P=0.05).

Conclusion: After ingestion of virgin olive oil, an increase in antioxidants and oleic acid in LDL was observed as well as an improvement of LDL resistance to oxidation. Our results support the idea that daily ingestion of virgin olive oil could protect LDL from oxidation.

Sponsorship: This study was supported by a research grant from Spain (ALI 97-1607-C02-02).


There is increasing evidence that oxidative modification of low density lipoprotein (LDL) plays a key role in the development of atherosclerosis (Esterbauer et al, 1991; Princen et al, 1995; Nicolaïew et al, 1998; O'Byrne et al, 1998) and the effect of dietary fatty acids and antioxidants on the resistance of lipoprotein to oxidation is well known. A lower incidence of coronary heart disease (CHD) in Mediterranean countries has been correlated with a diet rich in fruit, vegetables, legumes and grains. In these countries at least 35% of daily energy is derived from fats because of the high consumption of monounsaturated fatty acids (MUFA) in olive oil (Serra-Majem et al, 1995). Virgin olive oil, obtained exclusively by physical procedures, is much more than a monounsaturated fat because it contains relatively high amounts of antioxidants, mainly phenolic compounds and vitamin E. However, these antioxidants are lost when the oil is refined (Litridou et al, 1997).

Tissue membranes that are rich in MUFA are less susceptible to oxidation by free radicals than membranes rich in polyunsaturated fatty acids (PUFA; Parthasarathy et al, 1990; Esterbauer et al, 1992). Linoleic acid (C18:2) accounts for 90% of the PUFAs present in LDL and is the major substrate for oxidation. Therefore diets rich in this fatty acid may increase the risk of LDL oxidation. On the other hand diets rich in oleic acid generate LDL particles that appear to be more resistant to this process.

However, apart from their fatty acid profile, the formation of oxidized LDL depends on the amount of cholesterol and also on antioxidants such as vitamin E and phenols present in LDL (Esterbauer et al, 1992; Fuller & Jialal, 1994; Caruso, 1999). Vitamin E (α-tocopherol) is the main lipid-soluble antioxidant present in LDL (Dieber-Rotheneder et al, 1991; Jialal & Grundy, 1992). On the other hand, phenolic compounds which can bind to LDL are good candidates in preventing lipid peroxidation and atherosclerotic processes. They have antioxidant properties both in vitro (Visioli & Galli, 1994; Visioli et al, 1995; Fitó et al, 2000) and in vivo (Nigdikas et al, 1998; Bonamone et al, 2000). The importance of phenols is evident in the case of olive oil. Although all kinds of olive oils protect LDL from oxidation, virgin olive oil shows increased antioxidant activity because of its high phenolic content (Fitó et al, 2000). Recent studies have shown the bioavailability of some phenols from wine or onions (Lapidot et al, 1998; Manach et al, 1998; Bell et al, 2000) but data on olive oil are scarce. Moreover, most of these studies have been done with rats or rabbits (Wiseman et al, 1996; Coni et al, 2000), but few with humans (Paganga & Rice-Evans, 1997; Miró-Casas et al, 2001).

To date, dietary studies that have analyzed the effects of fatty acids and antioxidants on LDL composition have used capsules or liquid formulas and not ‘natural’ foods. Furthermore most of the experimental diets were relatively high in fat or used doses of antioxidants that are incompatible with the recommendations and with a usual diet. There are few clinical studies on the effects of olive oil as a natural source of monounsaturated fat on lipoprotein metabolism (Bonamone et al, 2000). The present study aimed to follow the incorporation of some components of virgin olive oil into LDL in a population of healthy men and women on a regular solid-food diet. It also aimed to test whether LDL enrichment in ‘anti-atherogenic’ compounds decreases the risk of LDL oxidation.



Sixteen (nine men and seven women) healthy volunteers (aged 25–65) were recruited. The ethical committee CEIC-IMAS (register no. 98/798/I) approved the protocol and participants signed an informed consent form. On the basis of physical examination and standard biochemical and hematological tests, all volunteers were considered healthy. Subjects had an average weight of 75±13.47 kg and a body mass index (BMI) of 25±3.1 kg/ m2.

Olive oil composition

The only oil used in the study was of extra-virgin quality and belonged to the appellation of Catalonia Les Garrigues. Acidity value, peroxide index and UV spectrophotometric index (k270) were determined following the analytical methods described in Regulations EEC/2568/91 of the European Union Commission. Fatty acids were transformed into methylesters and analyzed by gas chromatography (EEC/2568/91). α-Tocopherol and β-carotene were determined by HPLC as previously described (Gimeno et al, 2000). Phenolic compounds were measured by the Folin–Ciocalteau method (Swain & Hillis, 1969).

Study design

Volunteers followed a phenolic-free diet for 4 days (washout period) before the administration of an acute dose of virgin olive oil. A nutritionist instructed them on excluding several foods from their diet (coffee, tea, fruit, vegetables, wine and olive oil). At 8 am on day 5, subjects were given 50 ml (44 g) of extra-virgin olive oil in a single dose either alone or with bread. Subjects did not ingest any food or drink other than water in the first 6 h after the meal and olive oil was the sole source of phenols over the following 24 h. Blood samples were drawn on day 1 before the washout period (baseline 1), on day 5 after washout (baseline 2) and at 2, 4, 6, 8 and 24 h after administering virgin olive oil. Thereafter, the participants followed their habitual diet supplemented by 25 ml/day (22 g; individual dose provided to each subject) of the same olive oil for a week. Dietary fats from other sources were minimized. Subjects were instructed to exclude butter, margarine, cooking oil, nuts, visible fat on meat, chocolate, baked goods, eggs and poultry skin from their diets. Another blood sample was taken on day 12 at 8 am. All blood samples were stored at −80°C until lipoprotein separation.

Dietary survey

Nutrient intakes were calculated from 12 day dietary records using the software Diet Analysis Nutritionist IV (N Squared Computing, San Bruno, SA).

Laboratory measurements

Blood from healthy volunteers was collected in tubes containing 1 g/l of EDTA and plasma was separated by centrifugation at 1000 g at 4°C for 15 min. Very-low-density lipoproteins (VLDL) and LDL were isolated by sequential flotation ultracentrifugation (Havel et al, 1995). All samples were stored under −80°C until analysis.

Total cholesterol, HDL-cholesterol and TG levels were measured by standard enzymatic methods. LDL-cholesterol was calculated using the Friedewald equation. The fatty acid composition of LDL was determined following the method described by Bondía et al (1994) in which fatty acids are transformed into methyl esters and analyzed by gas chromatography. For the determination of α-tocopherol, an aliquot of sample was deproteinized with ethanol, which contained α-tocopherol acetate as internal standard, and the analyte was then extracted with hexane. The solvent was evaporated to dryness under a stream of nitrogen and the residue was diluted in methanol and injected directly into an HPLC system (Gimeno et al, 2001). Phenolic compounds in LDL were also determined by HPLC-DAD as described previously (Lamuela-Raventós et al, 1999). Briefly, acidulated LDL was applied to a Waters Oasis™ HLB extraction cartridge and washed with water and 5% aqueous methanol. Phenols were eluted with methanol, which was evaporated under a stream of nitrogen. The residue was dissolved in the mobile phase and injected into the HPLC system. The chromatogram was monitored at 280 nm and the phenolic areas were expressed as caffeic acid equivalents (CAE).

To determine the resistance of LDL to oxidation, native LDL was dialyzed by molecular size exclusion chromatography in a G25 Sephadex column (Pharmacia, Upsala, Sweden), with 2.7 ml phosphate buffered saline (PBS) 0.01 M, pH 7.4 under gravity feed at 4°C. To initiate oxidation, dialyzed LDL (0.05 g protein/l) was incubated with copper sulfate (5 µM) in PBS at a final volume of 1 ml. Absorbance at 234 nm was continuously monitored at 2 min intervals for 5 h at 30°C (Puhl et al, 1994) using a spectrophotometer (Hewlett Packard, Palo Alto, USA) fitted with a heater and equipped with a seven-position automatic sample change. Three variables were used to study the resistance of LDL to oxidation: lag time, maximum amount of dienes and maximal oxidation rate. The length of the lag phase (min) was determined as the intercept of the propagation phase tangent with the extrapolated line for the slow reaction. Maximum diene content was calculated by the maximum increase of absorbance (µmol dienes g−1 of LDL protein), and maximal rate of oxidation was derived from the slope of the propagation phase tangent (µmol dienes min−1 g−1 of LDL protein). LDL protein content was determined by the red pyrogallol method (Sigma, St Louis, MO, USA).

Statistical analysis

The assays were carried out in duplicate. The values in the tables are given as means±s.d. The discussion is based on the one-way analysis of variance (ANOVA). Homogenicity of variances was tested by Levene's test. Logarithmic transformation was performed to normalize non-parametric variables. The differences between two groups were assessed by the Student's t-test for paired samples. A P-value<0.05 was considered statistically significant. The SPSS statistical package was used (SPSS Incorporated Co., Chicago, IL, USA).


The daily caloric intake during the week of sustained olive oil ingestion ranged from 1465 to 2284 kcal (mean 1748±331) and consisted of 44.2% of carbohydrates (±5.1%), 18.3% of proteins (±2.7%) and 37.5% of lipids (±3%), of which 47.8% were MUFA, 39.2% were SFA (saturated fatty acids) and 12.9% were PUFA.

The composition of the oil used in the experiment is shown in Table 1. Quality parameters were within the limits accepted by its appellation (EEC/2081/92).

Table 1 Composition of the olive oil used in the study

Table 2 shows the mean plasma lipid concentrations at the beginning and at the end of the study. Total cholesterol content was unchanged, but there was a significant increase in triglycerides after a week of virgin olive oil consumption. While LDL-cholesterol decreased significantly, the increase in HDL-cholesterol was not significant.

Table 2 Levels of cholesterols and tryglicerides in plasma after 1 week of virgin olive oil comsumption

The fatty acid composition of LDL is shown in Table 3. Significant enrichment in oleic acid (C18:1 n-9) was observed after a week of virgin olive oil consumption (P=0.015) and the same trend was seen in the relation MUFA/PUFA (P=0.028) and MUFA/SFA (P=0.011). However no significant differences were detected for palmitic (C16:0), stearic (C18:0), linoleic (C18:2 n-6) or arachidonic (C20:4 n-6) acids.

Table 3 Changes In the fatty acid composition of the LDL (%)

There was a decrease of antioxidants in LDL between baseline 1 and 2 (post-washout), and an increase after olive oil consumption (Table 4). For vitamin E, there were significant differences between baseline 1 and 2 (P=0.045), and between baseline 2 and after a week (P=0.047). The decrease in phenolic compounds between baseline 1 and 2 was not significant but the increase after a week was considerable (P=0.021).

Table 4 Levels of antioxidants in LDL and oxidation parameters

The lag time of conjugated dienes decreased after the post-washout period (P=0.026) and slowly increased, becoming significant after 24 h (P=0.025) and after a week of the ingestion of virgin olive oil (P=0.000). The oxidation rate (OR) showed the opposite trend, decreasing and reaching the lowest levels at 8 h. Significant differences were observed between baseline 1 and 2 h (P=0.05), 4 h (P=0.042), 6 h (P=0.028), 8 h (P=0.03), 24 h (P=0.026) and after a week (P=0.05). Although diene content tended to increase after ingestion, there were no significant differences in the dienes formed during LDL oxidation. However after a week the maximum amount of dienes was significantly lower compared with the baseline 1 value (P=0.045).


In our study, the consumption of virgin olive oil for 1 week led to a significant decrease in LDL cholesterol. Furthermore, this LDL has been found to be rich in oleic acid, phenolic compounds and vitamin E. This enrichment could account for the decrease in LDL oxidation observed during this period.

The effects observed on plasma LDL cholesterol and on susceptibility of LDL to oxidation are consistent with those reported in previous studies with MUFA-enriched diets (Mata et al, 1992; Reaven et al, 1994), but these studies were longer (3–4 weeks). Other studies have also showed that extra-virgin olive oil increases resistance to oxidation in animals (Wiseman et al, 1996), in healthy subjects (Carmena et al, 1996), and in subjects with cardiovascular disease (Ramirez-Tortosa et al, 1999). It has been reported (Van der Vijver et al, 1998) that oxidation rate of conjugated diene formation in LDL is inversely related to the percentage of MUFA in LDL. In a review, Parthasarathy et al, (1999) explain that the susceptibility of LDL to oxidation depends on the MUFA/PUFA ratio, their oleic acid and antioxidant contents and also on the size of LDL particle. It has been reported (Reaven et al, 1994) that small dense lipoprotein particles are more susceptible to oxidation and that oleic acid promotes the formation of larger particles.

The intake of virgin olive oil in a single dose did not affect the postprandial LDL composition; however, after only one week of sustained realistic doses, the levels of MUFA and antioxidants in LDL increased significantly. This could explain the benefits of the Mediterranean diet, in which olive oil is ingested every day. Therefore this study presents new insights into the biochemical basis of the beneficial effects associated with long-term dietary MUFA intake, which may explain the lower rates of coronary mortality in Mediterranean regions.

 However, little is known about the effects of MUFA intake on postprandial lipoprotein metabolism. It is now recognized that the postprandial triacylglycerol concentration is an important factor in the development of cardiovascular disease (CAD). Roche et al, (1998) showed that the alteration in postprandial triacylglycerol response depended on the type of oil used. Southern Europeans, whose habitual dietary MUFA intake was significantly greater, had greater initial postprandial triacylglycerolemia but concentrations quickly returned to preprandial values. In contrast, Northern Europeans, with a SFA-enriched diet, showed a more prolonged increase in postprandial concentrations. Therefore they suggested that triacylglycerol clearance was faster in a MUFA-enriched diet.

Another interesting point is the rate of incorporation of the different fatty acids into the plasma lipids. Sadou et al, (1995) provide quantitative evidence that the incorporation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into plasma lipids is quite different. Whether the effect of regioselective distribution of these fatty acids can be extended to other fatty acids deserves particular attention.

Choudhury et al, (1995) show that synthetic fats have fatty acids randomly attached to the glycerol molecule. This contrasts with natural fats, which have a specific fatty acid distribution and the positions of these fatty acids along the glycerol molecule can modulate the effect of different fats on cholesterol metabolism. Therefore liquid formula diets fail to evoke the characteristic cholesterolemic response in humans.

Lipid peroxidation is involved in the pathogenesis of atherosclerosis through the formation of oxidized LDL. Thus, apart from the fatty acid composition, exogenous antioxidants could be used to prevent this disease (Esterbauer et al, 1992). In quantitative terms, α-tocopherol is the major antioxidant among those present in LDL, thus it is considered the first line of defense against oxidation (Reaven et al, 1994). LDL resistance varies greatly among subjects and this variation is caused, in part, by differences in the α-tocopherol content of LDL, but it also depends on other variables that have not been identified as yet.

Here we used HPLC to analyze phenols in LDL instead of the Folin–Ciocalteau method because any substance containing a phenolic group could interfere. Preliminary results indicate that tyrosol, hydroxytyrosol and oleuropein are bioavailable (Visioli et al, 2000). We have recently described the bioavailability of tyrosol from 50 ml of virgin olive oil in humans (Miró-Casas et al, 2001). The average daily intake of phenols from olive oil and their plasmatic concentration in populations consuming olive oil is unknown. Although studies should be carried out into this area, the observation that intakes of a few mg/day of flavonoids is correlated with a lower incidence of CHD (Paganga & Rice-Evans, 1997) indicates that the daily intake of olive oil phenols in the Mediterranean area is probably within that range. Whether the antioxidant effect of vitamin E is greater than that of phenolic compounds is controversial. Nigdikas et al, (1998) showed that the increase in lag time with vitamin E was significant (covariance analysis) being 4–5-fold greater than with red wine polyphenols.

Results of a recent study (Bonamone et al, 2000) suggest that phenolic compounds in olive oil are absorbed by the intestine and may exert a significant effect in vivo in the postprandial phase. In our study, the washout period may have produced an imbalance in the antioxidant pool and a delay in recovering the initial levels. Consequently, we did not observe a significant increase in the postprandial phase. In addition, we must consider the very high variability in the absorption of antioxidants observed among subjects. After the post-washout period there is a significant decrease in oleic acid and vitamin E but not for phenolic compounds. During the postprandial phase vitamin E, phenols and oleic acid tended to increase. Vitamin E was the most depleted antioxidant in the washout period; consequently it took one week to recover its initial levels. However, the phenolic content showed a clear tendency to increase quickly after the washout period, which strongly suggests that these compounds are absorbed more quickly than lipophilic antioxidants, such as vitamin E. Nevertheless, although the differences for each individual parameter were not significant, they may act synergically, because the oxidation rate was already significantly lower at 4 h and decreased considerably after 8 h.

In a previous report (Covas et al, 2000) tyrosol was the only olive oil phenol able to bind to LDL in vitro and it was significantly associated with the delay in LDL oxidation. In this report it was suggested that olive oil phenols protect other phenols bound to LDL from oxidation. However we cannot discard the role of other olive oil phenols, or other antioxidants such as vitamin E on the delay in LDL oxidation (Visioli et al, 1995). Moreover, the mechanism by which phenolic compounds protect LDL from oxidation is unknown. Lamuela-Raventós et al (1999) identified two phenolics in LDL but did not detect any phenol of virgin olive oil in the LDL particle. Thus, further studies with higher doses are needed to elucidate whether phenols from olive oil can directly bind to LDL.

On the other hand, because of an almost complete absence of changes in LDL during the postprandial phase, we carried out preliminary studies in VLDL. These lipoproteins showed a clear increase in oleic acid and vitamin E content after 6 h of acute ingestion of olive oil. In view of these results, we can assume that the major changes in LDL could happen at 10–12 h. Therefore in further studies we must choose the periods of blood extraction more appropriately.


In addition to the LDL-lowering effect of virgin olive oil, our results suggest that an intake of 25 ml/day could increase the resistance of LDL to oxidation, because it becomes richer in oleic acid and antioxidants. These benefits could be achieved by including virgin olive oil daily in our diet. Further research is needed before we could safely incorporate conclusions about oxidation of lipoproteins into nutritional recommendations for the general population.


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We thank all the volunteers for their co-operation and the staff of the Hospital del Mar de Barcelona for their assistance. We also thank Robin Rycroft for revising the English manuscript.

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Gimeno, E., Fitó, M., Lamuela-Raventós, R. et al. Effect of ingestion of virgin olive oil on human low-density lipoprotein composition. Eur J Clin Nutr 56, 114–120 (2002).

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  • virgin olive oil
  • LDL
  • postprandial
  • α-tocopherol
  • phenols
  • oleic acid
  • oxidation

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