Introduction
Interest in the Mediterranean diet (MD) has grown worldwide in the course of the past decade, even among nutritionists outside of the Mediterranean area. This is largely due to the fact that the consumption of the MD has been linked to greater longevity, improved quality of life and lower incidences of cardiovascular disease, cancer and age cognitive decline, in spite of being a dietary model with a high fat content, unlike the diets recommended for several decades by many experts in nutrition in other geographical areasReference Perez-Jimenez1. However, most of the fat content of the MD is derived from a single component of the diet, namely olive oil, which means that the diet is low in saturated fats and high in monounsaturated fatty acids (MUFA), particularly in oleic acid. Moreover, the gastronomic characteristics of this dietary component encourage a higher level of consumption of plant products such as fruit, vegetables, pulses and cereals, all of which are foods that contain a high proportion of low glycaemic index carbohydrates and that have important potential for promoting good healthReference Serra-Majem, Cruz, Ribas and Tur2. In the course of the past few years, thanks to modern technology, other types of oil with similar fat composition have become available to human nutrition. These include oils obtained from certain types of seed, some of whose varieties are high in oleic acid, such as high oleic sunflower, soya and rapeseed oils. This situation has generated a new concept of MD, according to which dietary oleic acid is not necessarily derived predominantly from olive oil but rather from seedsReference Singh, Dubnov, Niaz, Ghosh, Singh and Rastogi3, Reference de Lorgeril, Renaud, Mamelle, Salen, Martin, Monjaud, Guidollet, Touboul and Delaye4. However, unlike virgin olive oil, these must be refined to be intake, as a result they do not contain the non-fat microcomponents, present in virgin olive oil, much of them with a high biological potency, including the phenols (tyrosol and hydroxytyrosol), secoroids (oleuropein and its conjugate forms) and lignansReference Owen, Giacosa, Hull, Haubner, Wurtele, Spiegelhalder and Bartsch5. This is important, because in this study we will discuss the benefits of diets rich in virgin olive oil, the genuine MD, whose effects are due not only to its oleic acid content but also to its other potentially health-promoting components.
Traditionally, the benefits of MUFA-rich diets were linked to its effect on lipoprotein metabolism and, to a lesser extent, to other risk factors, but today we realise that there exists a whole sheaf of other benefits, including the components of haemostasisReference Keys6–Reference Visioli, Bogani, Grande, Galli and Sirtori12. The interest that this has aroused in the course of the past few years is perfectly understandable when we take into account that, in persons at high risk of cardiovascular disease, there may well be present a chronic activation of the mechanisms of thrombosis, resulting in what might be called a prothrombotic environmentReference Mutanen and Freese10. Thrombogenesis is a very complex process, which involves the platelets activation (primary haemostasis), the mechanisms of coagulation (secondary haemostasis) and fibrinolysis, a system that has been implicated in the reabsorption of recently formed fibrin thrombus (Fig. 1). However, all these mechanisms are capable of being modulated by olive oil, as it is summarised in Table 1.
Fig. 1 Coagulation and fibrinolysis cascade. Continuous lines represent stimulation. Discontinuous lines represent inhibition
Table 1 Haemostatic effects favoured by the intake of virgin olive oil-enriched diet
Platelet function
Some years ago, Sirtori et al. observed that in healthy persons, a diet rich in olive oil lowered the sensitivity of platelets to collagen-induced aggregation, while if the diet was enriched with corn oil, the threshold of aggregation to arachidonic acid was also raisedReference Sirtori, Tremoli, Gatti, Montanari, Sirtori, Colli, Gianfranceschi, Maderna, Dentone and Testolin13. It was subsequently confirmed that in comparison with another diet with an elevated content of polyunsaturates, a MUFA-rich diet diminished the urinary excretion of 11-dehydro-thromboxane B2, a metabolite of thromboxane B2(TXB2), which in turn is derived from TXA2, a product that encourages aggregation, generated by the action of the cyclooxygenase found in platelets and the endotheliumReference Lahoz, Alonso, Ordovas, Lopez-Farre, de Oya and Mata14. More recent studies of the functions of the platelets have produced varying results, even to the extent of suggesting that the ingestion of MUFA induces unfavourable actions that resemble those of saturated fatty acids, with an increase in the urinary excretion of TXB2 metabolitesReference Thijssen, Hornstra and Mensink15–Reference Turpeinen, Pajari, Freese, Sauer and Mutanen17. There are many potential explanations for the existence of such wide range of results, which is otherwise a fairly normal situation in intervention studies, given their great complexity and the special difficulties associated with studies carried out on human beings, who are so heterogeneous in their design and with features peculiar to different populations. We also need to take into account the methodological and experimental differences between individual studies, including the potential confusion that results from the wide range of foods and nutritional factors involved, whose ingestion may well be modified when we design a study in which the total content and the proportion of dietary fats are alteredReference Rajaram18. At any rate, recent studies appear to confirm the initial results, which noted the beneficial effects of MUFA-rich diets on platelets. In one such study, 8 weeks of a diet high in rapeseed and sunflower oils, both of which have a high oleic acid content, reduced the aggregation to three agonists (arachidonic acid, collagen and ADP), although by the end of 16 weeks only the effect on the last-named of these was maintainedReference Kelly, Smith and Williams19. The same authors confirmed these data in a carefully designed study of two levels of ingestion of MUFA (18 and 15% of the total supply of calories) by comparing this dietary component with saturated fats (16%). Although the effect of the elevated consumption was greater in the short term (8 weeks), the results were identical after 16 weeks at both levels of ingestion, maintaining the lower level of aggregation induced by arachidonic acidReference Smith, Kelly, Fielding, Hauton, Silva, Nydahl, Miller and Williams20. Another interesting question concerns whether the richness in minor components of the oils utilised in these studies might be capable of having a specific influence on primary haemostasis. With this idea in mind, Visoli et al. observed that daily administration of 40 ml of virgin olive oil rich in phenolic compounds, compared with refined oil, was accompanied after 7 weeks by a lower TXB2 plasma levels contentReference Visioli, Caruso, Grande, Bosisio, Villa and Galli21. More recently, the same authors have confirmed these findings, in this case through experiments carried out during the postprandial phase of the ingestion of oils similar to those used in the previous studyReference Bogani, Galli, Villa and Visioli22. This recalls perfectly the in vitro data in experiments on endothelial cells from the umbilical cord, which secreted less TXB2 when they were exposed to postprandial particles obtained after the consumption of olive oil enriched with micronutrients, although this experiments did not identify with any degree of certainty just which components this effect could be attributed toReference Perona, Martinez-Gonzalez, Sanchez-Dominguez, Badimon and Ruiz-Gutierrez23. Finally, one component of the circulation in haemostasis, which favours the adherence of the platelets to the disrupted endothelium, i.e. the von Willebrand factor (vWF), may be decreased by diets rich in olive oil, although the biological significance of these facts is not well knownReference Rasmussen, Thomsen, Ingerslev and Hermansen24.
Coagulation pathway
One of the components of haemostasis that has aroused particular interest is factor VII (FVII), since it may play a central role in the development of coronary diseaseReference Ye, Liu, Higgins, Keavney, Lowe, Collins and Danesh25, Reference Kohler and Grant26. The first studies confirmed that the activity of fasting FVII plasma levels are increased with the regular ingestion of any type of fat, with the result that palmitic acid would raise the levels of the coagulant fraction (FVIIc)Reference Tholstrup, Marckmann, Jespersen and Sandstrom27, while MUFA would elevate activated FVII (FVIIa)Reference Sanders, Grassi, Millar and Humphries28. A study performed using oleic acid observed a possible sex-linked effect, in that only female subjects displayed decreased levels while they were consuming fatty acid-rich diets, compared with those containing saturated fatsReference Temme, Mensink and Hornstra29. This favourable effect has also been confirmed with the ingestion of MUFA derived from rapeseed oil by hypertriglyceridaemic patientsReference Junker, Pieke, Schulte, Nofer, Neufeld, Assmann and Wahrburg30, as well as in another population in which an olive oil-rich MD model was compared with two others, one of which was rich in carbohydrates while the other contained a high proportion of saturated fatsReference Gomez, Fernandez de la Puebla, Castro, Lopez-Miranda, Marin, Fuentes, Perez-Martinez, Velasco, Moreno, Torres and Perez-Jimenez31. It should be emphasised that in this case, FVII levels fell only with the MD. The possibility that MUFA has a special effect on FVIIc is reinforced by the fact that its level in the plasma of healthy person is reduced with a sunflower oil-rich diet that is high in oleicsReference Turpeinen and Mutanen32. Similarly, Larsen et al. did not observe any differences in either FVIIa or FVIIc in a comparison of various high-MUFA oils pressed from seeds (sunflower, olive and rapeseed), although in one postprandial study the increase in FVIIa was less following ingestion of virgin olive oil, suggesting that its micronutrients might modulate the fat-induced activation of FVII. An interesting fact that has recently emerged is that postprandial activation of FVII, following the acute ingestion of fat, may depend on the background diet prior to the acute ingestion phase, so that chronic consumption of olive oil would prevent the acute activation of FVIIReference Roche, Zampelas, Knapper, Webb, Brooks, Jackson, Wright, Gould, Kafatos, Gibney and Williams33. Similar results have been observed during a prolonged MUFA-rich diet (18% of the total energy provided by MUFA) compared with another diet with 15% from MUFA, derived from refined oils. This finding, confirmed by other authors, might be explained why the consumption of a background diet, enriched in MUFA, could lower the number of postprandial chylomicrons during the acute fat overload phase, reducing the activation of FVIIReference Silva, Kelly, Jones, Smith, Wootton, Miller and Williams34.
Tissue factor (TF) is expressed in the arterial wall cells and its presence increases coagulatory activity in atheroma plaques, encouraging the emergence of acute coronary syndrome. The quantity and quality of fatty acids in the diet regulate its expression. We already know that the ingestion of polyunsaturated fatty acids inhibits the stimulating effect of lipopolysaccharides in mononuclear cells, while saturated fats raise postprandial levels and diets that are low in fat and the MD reduces its level of activity in mononuclear cellsReference Tremoli, Eligini and Colli35–Reference Motton, Mackman, Tilley and Rutledge37. Tissue factor pathway inhibitor (TFPI) is an activated factor X-dependent inhibitor of TF-induced coagulation. The principal role of TFPI appears to be to inhibit the activity of small amounts of TF, which are probably essential for the maintenance of normal haemostatic balance. It has been shown that TFPI in the plasma of crab-eating macaques rises markedly in response to a high-cholesterol dietReference Abumiya, Nakamura and Takenaka38. Furthermore, we have shown that the isocaloric replacement of a palm oil-enriched diet or a low-fat diet by a MD had the effect of reducing plasma TFPI. Although the decrease in TFPI levels is difficult to interpret, it has been suggested that it may reflect an increased presence of the protease in the endothelium, which would have a regulatory effect on thrombogenesis. In this way, the decrease in plasma TFPI levels would therefore be interpreted as protective effect against thrombogenesisReference Perez-Jimenez, Castro, Lopez-Miranda, Paz-Rojas, Blanco, Lopez-Segura, Velasco, Marin, Fuentes and Ordovas39.
Data concerning fibrinogen are less definite, possibly because the fact that it is a marker of inflammation means that changes may depend principally on the underlying inflammation rather than on a specific effect of nutrients. In a study in which subjects consumed a diet rich in MUFA derived from refined oils, plasma levels of fibrinogen rose, while a MD reduced them in parallel with other parameters of inflammationReference Chrysohoou, Panagiotakos, Pitsavos, Das and Stefanadis40. More studies are needed in order to clarify these effectsReference Poppit41.
Fibrinolysis pathway
This key mechanism is fundamental to the maintenance of adequate haemostatic equilibrium, since it avoids thrombogenesis on blood vessel walls. Its fibrinolytic action depends on the action of tissue plasminogen activator (tPA), an enzyme that favours the production of plasmin, which hydrolyses recently created fibrin, thus hindering the growth of the thrombus. Its most important mechanism of regulation depends on the equilibrium in the plasma between tPA and PAI-1 (tPA inhibitor type 1), and it is known that the level of the latter substance rises with the ingestion of diets that are high in palmitic acidReference Temme, Mensink and Hornstra42. A study performed by our group has already demonstrated that a MD, rich in olive oil, reduced levels of PAI-1 in comparison with another carbohydrate-rich, low-fat diet, independently of the dietary cholesterol content in the dietReference Lopez-Segura, Velasco, Lopez-Miranda, Castro, Lopez-Pedrera, Blanco, Jimenez-Pereperez, Torres, Trujillo, Ordovas and Perez-Jimenez43. However, there exist data that disagree with these findings, as have been provided by a study carried out using refined high oleic seed oils, which found no changes in PAI-1 levels. However, more recently, the use of a diet rich in virgin olive oil in hypertensive patients confirmed our previous data, reducing PAI-1 plasma concentration in comparison with soya oilReference Trifiletti, Scamardi, Gaudio, Lasco and Frisina44. Other subsequent studies using diets rich in virgin olive oil have reconfirmed its beneficial effects, leading us to speculate that its micronutrients may have a specific action, so that the possible reduction in PAI-1 may be dependent on such components rather than on its content of oleic acidReference Perez-Jimenez, Castro, Lopez-Miranda, Paz-Rojas, Blanco, Lopez-Segura, Velasco, Marin, Fuentes and Ordovas39, Reference Avellone, Di Garbo, Cordova, Scaffidi and Bompiani45.
Relationship between thrombosis and the endothelium
The initial alteration that precedes the development of arteriosclerosis is the activation of the endothelium, through which the cells that line the vessel walls express, on the surface of their lumen, a combination of molecules that encourage the adhesion and migration of the circulating mononuclear cells to the sub-endothelial space. This forms the initiation of the inflammatory process, one of the consequences of which is the loss of endothelial functions. Among these functions are the well-known vasodilatory response that is dependent on the production of nitric oxide, and its capacity to reduce thrombogenesis, which is the subject of this review. The cellular mechanism, which mediates the expression of the genes involved in the inflammatory response, both in the endothelium and in other cells that contribute to the inflammation of the vascular walls, depends on the cytoplasmic expression of the transcription factors. Among them, NF-κB is particularly interesting as the mediator sensitive to oxidative changes and as that which induces the activation of the genes that are involved in the synthesis of the adhesion molecules. Of special interest in this respect is the demonstration that oleic acid buffers the inflammatory process that leads to endothelial dysfunctionReference Toborek, Lee, Garrido, Kaiser and Hennig46–Reference Bellido, Lopez-Miranda, Blanco-Colio, Perez-Martinez, Muriana, Martin-Ventura, Marin, Gomez, Fuentes, Egido and Perez-Jimenez48. Although we now find ourselves in fairly speculative field, where we do not even know what initiates the initial phenomenon of wall lesions, the anti-inflammatory effect of olive oil needs to be considered in the context of the interaction produced during the ingestion of high-energy diets that are capable of promoting the overproduction of reactive species of oxygen and inducing changes in fraction 3 of the complementReference Charo and Ransohoff49, Reference van Oostrom, Alipour, Plokker, Sniderman and Cabezas50. Data that exist suggest that fats with antioxidative or membrane-stabilising capacity might be capable of protecting endothelial cellsReference Henning, Toborek and McClain51. In this context, olive oil has such an ability, both through its high content of MUFAReference Rodriguez-Villar, Perez-Heras, Mercade, Casals and Ros52–Reference Mata, Varela, Alonso, Lahoz, de Oya and Badimon54 and via the antioxidative effect of its microcomponents, particularly of phenolic compounds, among which the typical is the oleuropein, an aglycone the hydrolysis of which generates tyrosol and hydroxytyrosol, which in their free, secoroid and conjugated forms make up some 80% of the phenolic compounds of virgin olive oil. These products are absorbed by the intestine in human beings, as has been demonstrated by experimental studies, possess antioxidative and anti-inflammatory properties, and are also capable of modifying haemostasis, inhibiting platelet aggregation and displaying antithrombotic propertiesReference Owen, Mier, Giacosa, Hull, Spiegelhalder and Bartsch55.
In summary, the MD is an alimentary model with a high content of monounsaturated fats that is capable of inducing a wide range of biological effects on the cardiovascular system. Of these effects, haemostasis is of particular importance with regard to both thrombogenesis and fibrinolysis. These beneficial effects are united to their capacity to protect the endothelium, an ability that is linked with MUFA, their predominant type of fat species and to their elevated content of other components that display a wide range of biological activities. In the future, the application of modern focuses of study based on nutrigenetics will enable us to understand in greater depth this interesting biological effect, which will probably turn out to play a key role in the prevention of cardiovascular disease.
Acknowledgements
This work was supported by research grants from the Plan Andaluz de Investigación (Proyecto de Excelencia AGR 922; Consejería de Innovación Ciencia y Empresa de la Junta de Andalucía) and from the Plan Nacional de Investigación (Ministerio de Educación y Ciencia; SAF2003-05 770) to FPJ.
