*3.5. HDL Monolayer Fluidity*

HDL monolayer fluidity was analyzed by a determination of steady-state anisotropy of 1,6-diphenyl-1,3,4-hexatriene (DHP) (Table 5). High-polyphenol content olive oil (HPCOO) [15] and extra VOO [18] consumption were found to increase HDL monolayer fluidity. However, different VOO interventions (VOO, FVOO and FVOOT) did not show effect over HDL monolayer fluidity [22–25].


**Table 5.** High-density lipoprotein (HDL) monolayer fluidity.

LPCOO, low-polyphenol content olive oil; HPCOO, high-polyphenol content olive oil; VOO, virgin olive oil; FVOO, functional virgin olive oil; FVOOT, functional virgin olive oil with thyme; NS, no significant change.

> Correlation models between HDL monolayer fluidity and HDL functionality-related parameters found that free HDL-C and triglyceride contents and HDL size were the main determinants of HDL monolayer fluidity [23].

#### *3.6. HDL Composition*

HDL lipidome is modulated by diet, even by a short-time dietary intervention. After two 4-day dietary interventions, HDL lipidome was widely modulated and fatty acids contained in HDL phospholipids were the most variable lipids by diet [17]. The 4-day MD intervention increased the quantity of phosphatidylcholine with very long chain and double bonds fatty acids, compared to baseline and to the 4-day fast food diet intervention. Fatty acid length, which was found to increase after the 4-day MD intervention, was directly related to MUFA and PUFA consumption [17]. In addition, an oleic acid rich diet, as MD, increased oleic acid and its derivatives in HDL phospholipids [19,23]. A linoleic acid rich diet increased linoleic HDL quantity, but linoleic acid binding capacity was lower than oleic acid [19].

Phenolic compounds contained in OO were found to present ability to bind to HDL particles. HDL showed higher phenolic compounds after HPCOO intervention [15] and after VOO, FVOO and FVOOT diets [23], specifically α-tocopherol, β-cryptoxanthin and coenzyme Q [23]. Lycopene rich and supplemented diets modulated HDL composition, higher HDL lycopene quantity was found after lycopene interventions [16].

HDL proteome showed to be influenced by nutritional interventions. After three interventions with VOO (VOO, FVOO and FVOOT), the HDL proteome was remodeled: 15 HDL metabolism-related proteins were identified as the most importantly modulated after the interventions, including PON3, apolipoprotein (apo)-AI, apoA-II and apoD [25]. However, no change in apo-AI quantity was found after interventions with HPCOO, LPCOO [15] and lycopene [16]. While, higher apo-AI quantity was found after a traditional MD-enriched diet enriched with nuts and VOO [14] and after three diets with VOO (VOO, FVOO and FVOOT) compared to baseline [25].

#### *3.7. HDL Size*

HDL size range between 8–10 nm of diameter and two groups are defined in terms of HDL size: large and small, also named as HDL3 and HDL2, respectively. The effect of different OO interventions on HDL size is shown in Table 6. Large HDL number was found to be increased after a MD enriched with VOO and with nuts [14] and after HPCOO [15] consumption. In addition, HPCOO consumption showed to decrease small HDL number. When HPCOO consumption is compared with LPCOO, large HDL number was increased and small HDL number decreased [15]. However, different VOO enriched with polyphenols (FVOO and FVOOT) showed to decrease large HDL number and to increase small HDL number [22–25]. On the other hand, extra VOO did not show capacity to modulate HDL size [18].

**Table 6.** High-density lipoprotein (HDL) size variation against baseline.


MD, Mediterranean diet; VOO, virgin olive oil; LPCOO, low-polyphenol content olive oil; HPCOO, high-polyphenol content olive oil; FVOO, functional virgin olive oil; FVOOT, functional olive oil with thyme; HDL, high-density lipoprotein; NS, no significant change. <sup>a</sup> Significant change compared to LPCOO. <sup>b</sup> Significant change compared to VOO.

#### **4. Discussion**

HDL-C is clinically considered a CVD protective factor. However, pharmacological strategies that led to an increase in HDL-C did not reduce CVD risk [26]. The lack of effect of HDL-C raising strategies puts into question HDL-C concentrations as a protective factor against CVD. HDL-C should be in the spotlight as a CVD lowering risk factor and HDL modulations should be considered in order to predict CVD risk [3].

HDL composition is demonstrated to change in different physiological and pathological conditions [27,28]. Diseases associated to higher CVD risk, such as obesity and diabetes, remodel HDL composition and functionality. Diabetic people showed glycosylated and oxidized HDLs that presented lower HDL CEC, antioxidant and anti-inflammatory activities [29]. Moreover, lifestyle remodels HDLs and influences on their biological activities. In particular, unhealthy diets contribute to the development of dysfunctional HDLs, even in

people without diseases [27]. In addition, healthy diets such as MD, have shown capacity to remodel HDL functionality even in pathological conditions, like obesity [28].

In a whole view of the data analyzed in this review, MD showed capacity to produce changes in HDL by modulating HDL functionality, oxidation, composition, and size. MD-induced changes in HDL are variable between records analyzed and not all data were comparable because the same measurements were not always performed. This variability could be influenced by population differences, healthy and high cardiovascular risk populations. Moreover, despite all records performed interventions with MD, different foods or dietary patterns were used in each record, which may contribute to different changes in HDLs, highlighting the need for greater consistency between studies in the amount of foods and nutrients administered as part of a MD. Interestingly, intervention time in the studies included in this review was found not to be a limiting factor, because even after short interventions HDL were remodeled. Several studies were conducted over different samples of the PREDIMED population, which demonstrated that HDL are widely altered by diet and that MD improves HDL quality [13,14,21].

A set of 5 studies made an intervention with VOO [13,14,18], OO or components isolated from OO, specifically polyphenols [15,22–25] and oleic acid [19]. HDL CEC was the most OO-enhanced HDL function [13–15,18,22–25], due, at least in part, to OO polyphenols [15,22–25]. CETP was not significantly decreased after OO interventions, with the exception of a traditional MD-enriched with VOO [14]. However, when the study population was subdivided, the new VOO group did not show changes in CETP activity [13]. LCAT activity showed significant change only after FVOOT when compared to VOO [22–25], which suggests that LCAT activity is specially modulated by polyphenols. However, a HP-COO diet did not increase LCAT activity, against baseline neither against LPCOO [15]. In addition, lycopene rich and supplemented diets were the only interventions that showed an improvement of LCAT activity compared to baseline [16]. FVOOT differs from FVOO and HPCOO in that it is enriched in thyme polyphenols. Thus, LCAT activity may be susceptible to thyme polyphenols when compared to other OO and OO polyphenols.

In regards to antioxidant functions of HDL, a traditional MD enriched with VOO and an oleic acid enriched diet lowered HDL oxidation [14,19], but no significant change in oxidation rate was found after phenol enriched VOO diets (FVOO and FVOOT). However, PON1 activity was higher after a FVOOT intervention [22–25], but no significant change was found after a traditional MD enriched with VOO [14]. FVOOT was demonstrated to increase HDL antioxidant function but not to alter HDL oxidation rate. On one hand, a traditional MD enriched with VOO decreased HDL oxidation but did not change PON1 activity. PON1 is not the only HDL antioxidant component, so the reduction on HDL oxidation rate described after a traditional MD-enriched with VOO could be due to other HDL antioxidant components, or to a MD-induced lower pro-oxidant environment, which in addition to VOO leads to lower HDL oxidation rate. On the other hand, PON1 activity could be increased by the higher presence of polyphenols on FVOOT, not altering other HDL oxidation parameters or the environment. The study population is not widely differential between interventions, since high cardiovascular risk people were included in both. However, the number of participants in the traditional MD enriched with VOO intervention was higher compared to the intervention with FVOOT, and the HDL oxidation rate was differently measured. It would be necessary to determine HDL oxidation rate after FVOO and FVOOT interventions as malondialdehyde production to be able to compare.

A HPCOO diet and an extra VOO intervention increased HDL fluidity [15,18]. There was no change in HDL monolayer fluidity in phenol enriched VOO (FVOOT and FVOO) and VOO diets [22–25]. Opposed evidences are exposed in terms of VOO and polyphenols influence on HDL monolayer fluidity. However, it is important to highlight that there were differences between the study populations. When the study population was healthy, HDL monolayer fluidity was increased by OO, but no change was found when the study population was hypercholesterolemic. OO does not benefit HDL monolayer fluidity when hypercholesterolemia is present.

Taken all data together, OO showed ability to modulate HDL functionality, and polyphenols may be very influencing components. Despite controversial results were found in HDL oxidation and monolayer fluidity, and the missing data of some enzyme activities, HDL functions could be modulated by OO. The remodeling pattern needs to be clarified, suggesting the need for further research.

EPA is an essential fatty acid, which has showed anti-inflammatory functions (complete). Fish is an EPA-rich food highly consumed in MD. An EPA enriched diet showed ability to increase HDL CEC and PON1 activity [20]. In addition, a traditional MD enriched with fish increased HDL CEC and PON1 activity and decreased CETP activity [13]. Dyslipidemia did not alter EPA-related HDL functionality improvement. Whereas, the data is not contradictory compared to interventions with VOO enriched with polyphenols (FVOO and FVOOT) in hypercholesterolemia, because PON1 activity and CEC were improved after FVOOT intervention and VOO, FVOO and FVOOT, respectively. It would be interesting to study the effect of EPA in hypercholesterolemic population over HDL oxidation rate as malondialdehyde production, to clarify if high CVD risk population is not susceptible to improve HDL oxidation rate by diet.

All records in which HDL compositional change was analyzed, HDL composition was modulated by a MD. HDL seem to be susceptible to the kind of lipid consumed. HDL lipid composition was enriched on dietary lipids. However, there is not a HDL composition change pattern by MD and no correlations could be made between HDL composition and HDL functionality.

HDL do not only vary in composition and functionality, they are subdivided by size in different types of HDLs. HDL size is variable due to HDL metabolism, since HDLs bind different components and modulate their size, as a result of HDL synthesis and catabolism, and it is also reflected in the HDL functions. HDL type in terms of size seems to influence CVD risk. Owing to the relation between HDL size and HDL functionality development, MD could change HDL size distribution. Results found by different researches are controversial, since VOO and nuts enriched diets increased large HDL quantity [14], and a HPCOO intervention increased large HDL quantity and decreased small HDL quantity, compared to baseline and to a LPCOO diet [15]. Nevertheless, two diets based on VOO enriched with polyphenols (FVOO and FVOOT) showed to have the contrary effect [22–25]. HDL size could be differently modulated when the study population is hypercholesterolemic, since hypercholesterolemia could alter OO benefits over HDL functionality.

#### **5. Conclusions**

In conclusion, this systematic review shows that MD influences HDL functionality, composition, and size. HDL functionality is improved by MD, in terms of enzymatic activity and CEC, also HDL antioxidant properties are improved, as HDL oxidation is reduced. There is a need to clarify MD-derived modulation of HDL size to determine HDL components which fundamentally are influenced by MD. In addition, further research is needed to determine MD specific food HDL-modulating abilities and the effect of the global MD. While, it would be interesting to clarify the MD abilities over healthy and hypercholesterolemic or dyslipidemic population separately. Taken together, MD has demonstrated to be an influencing factor over HDL quality, which indicate that MD could be a target to improve cardiovascular health via HDL modulation.

**Author Contributions:** Conceptualization, E.G.-C., L.M.V., M.E.M., B.B., S.M.-d.l.P.; data collection and analysis: E.G.-C.; data interpretation: E.G.-C., L.M.V., M.E.M., B.B., S.M.-d.l.P.; writing-original draft preparation: E.G.-C. with the assistance from S.M.-d.l.P. and L.M.V.; writing-review and editing, E.G.-C., L.M.V., M.E.M., B.B., S.M.-d.l.P.; funding acquisition, B.B. and S.M.-d.l.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the research Grant US-1263458 (Andalusian Ministry of Economy, Knowledge, Business, and University, Government of Andalusia, Spain) into the European Regional Development Fund Operational Programme—2014 to 2020.

**Institutional Review Board Statement:** This research was exempt from ethics approval because the work was carried out on published documents.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** L.M.V. acknowledges financial support from VI PPIT-US (University of Seville).

**Conflicts of Interest:** The authors state no conflict of interest.

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