In this study, we examined the effects of olive oils with low (ROO) or high (EVOO) phenolic content on plasma lipid profile and ex-vivo LPS-stimulated whole blood culture inflammatory cytokines. EVOO consumption exerted a mild LDL-C lowering effect, while it did not reduce sd-LDL-C and TC/HDL-C levels. EVOO also had an attenuating effect on plasma CRP and increased ex-vivo production of LPS-stimulated IL-10 in whole blood culture.
Both LDL-C concentration and size are directly associated with the risk of cardiovascular disease [25, 26]. Although there are several studies in which polyphenol consumption has not affected LDL-C concentration [13, 14], in some studies, olive polyphenols have reduced LDL-C concentration. Hernáez et al. showed that consumption of polyphenol-rich olive oil (366 mg/kg) in the amount of 25 mL/day for 3 weeks reduces serum LDL-C levels when compared to low-polyphenol olive oil (2.7 mg/kg) . In another similar study, Fernández-Castillejo and coworkers showed that consumption (25 mL/day) of polyphenol-enriched olive oil (containing 500 mg polyphenol/kg) have LDL-C lowering effect in hypercholesterolemic individuals, whereas, they did not find any decrease in serum sd-LDL-C particles . EVOO has also been shown to have a blunting effect on postprandial LDL-C elevation in an acute meal consumption study . The mechanisms underlying the LDL-C lowering effects of olive polyphenols are not well defined. However, animal data suggest that it may be mediated through increases in bile flow and biliary cholesterol and bile acid concentrations, leading to increased fecal excretion . In the present study, although EVOO consumption was associated with the reduction in LDL-C levels, the plasma sd-LDL-C, and TC/HDL-C did not improve. TC/HDL-C has been suggested to be a powerful and more accurate predictor of future cardiovascular disease risk and is associated with sd-LDL-C particles . Therefore, except for a slight effect on LDL-C, olive polyphenols do not appear to have a significant effect on the improvement of plasma lipid profile.
Immune‐modulatory and anti‐inflammatory effects of EVOO bioactive minor components, in particular, hydroxytyrosol [31, 32] and also the entire phenolic fraction  have been investigated through in vitro models. Phenolic compounds present in EVOO have been reported to possess anti-inflammatory actions in animal models of inflammation [34, 35]. In our patients, reduced plasma levels of CRP were observed after EVOO ingestion. The reduction of circulating CRP after EVOO compared to ROO intake has been reported in a previous study in which patients with coronary artery disease took a daily amount of 50 mL of EVOO and ROO sequentially over two 3-week periods . However, in another study on diabetic subjects, the consumption of EVOO (25 mL/day, 577 mg of phenolic compounds/kg) did not significantly alter the levels of circulating hs-CRP compared to refined olive oil .
Production of cytokines in humans may be studied in the circulation and ex vivo by stimulation of isolated cells. Circulating levels of cytokines may not necessarily reflect local cytokine production. Yet the isolation of cells is cumbersome and when isolated cells are cultured, they deprived of adjacent cells and serum components which may cause them to lose their in vivo characteristics. Cytokine production from whole-blood culture could be a valid and convenient assay for the measurement of cytokine production, particularly when stimulated with LPS . In the present study, despite the improved plasma CRP after EVOO ingestion, we found no changes in the ex-vivo production of LPS-stimulated IL-6. On the contrary, EVOO consumption produced a significant increase in LPS-stimulated IL-10. IL-10 is an important immunoregulatory cytokine with pleiotropic effects that appears to play a role in the function of regulatory T cells, which are recognized as potent suppressors of excessive immune responses . Administration of olive polyphenols containing mostly hydroxytyrosol has been associated with increased IL-10 levels in an animal model of paw inflammation induced by injecting carrageenan . Our finding is consistent with the results of Aparicio-Soto et al., which reported evidence of increased IL-10 production when olive phenolic compounds were added to the ex-vivo culture of peripheral blood mononuclear cells (PBMC) from healthy subjects . Furthermore, co-stimulation of human PBMCs with hydroxytyrosol and an allergen has been associated with an increase in the amount of IL-10 production . Moreover, acute ingestion of 50 ml EVOO has been reported to increase the IL-10 gene expression in PBMCs from healthy subjects .
The anti-inflammatory effects of olive bioactive minor compounds could be attributed to their capacity to reduce the reactive oxygen species generation [42-44]. However, in the current study, plasma MDA levels did not differ between the two treatments, although within-group analysis showed a significant decrease in EVOO treatment. In contrast to our findings, the antioxidant effects of olive oil polyphenols have been demonstrated in previous studies. In a crossover study in healthy participants, daily administration of 25 mL of olive oils (with low, medium, and high phenolic content) for 3 weeks, linearly decreased oxidative stress markers (including oxidized-LDL-C) with increasing phenolic content . One of the reasons for the lack of effect of EVOO on MDA in the present study may be related to the detection method of MDA (colorimetric TBA-based method) which has been claimed to be insufficiently sensitive .
There were some limitations to this study. The sample sizes in each group were rather small and the power to detect subtle changes is therefore restricted. Furthermore, the study would have benefited if continued for longer durations to assess maintenance. Moreover, cross-over design may have been a better choice for reducing the effects of confounding factors. However, since seasonal variations influence inflammatory biomarkers [47, 48], we decided to choose a parallel design. Moreover, we assessed a limited number of parameters related to inflammation.