Effects of phenol-enriched virgin olive oils on hdl functionality in hypercholesterolemic subjects
- Autores: Sara Fernández Castillejo
- Directores de la Tesis: Rosa Solà Alberich (dir. tes.), Rosa María Valls Zamora (dir. tes.)
- Lectura: En la Universitat Rovira i Virgili ( España ) en 2017
- Idioma: inglés
- Tribunal Calificador de la Tesis: Josep Ribalta Vives (presid.), Judit Marsillach López (secret.), Joan Carles Escolà i Gil (voc.)
- Programa de doctorado: Programa Oficial de Doctorado en Biomedicina
- Calificación: Sobresaliente Cum Laude
- Materias:
- Enlaces
- Tesis en acceso abierto en: hdl.handle.net TDX
- Resumen
INTRODUCTION: Mounting epidemiologic evidence has confirmed that high-density lipoprotein (HDL) cholesterol levels is a strong negative risk marker for cardiovascular disease (CVD). However, conflicting data has cast doubt on such association since therapeutic strategies that increase HDL cholesterol (HDL-C) levels in circulation have not yielded clinical benefits on CVD.
This paradoxical association between low HDL-C levels and high CVD risk can be well explained by the fact that HDL pool encompasses a heterogeneous population of HDL particles that differ not only in the physicochemical properties but also in function quality irrespective of its cholesterol content. Therefore, alternative therapeutic strategies should be developed to properly modify HDL functionality rather than quantity to tackle CVD.
In November 2011 the EFSA released a health claim concerning beneficial properties of phenolic compounds (PC) from virgin olive oil (VOO) on maintenance of normal HDL-C levels. However, phenolic concentration in most VOOs available on the market is too low to allow enough consumption of VOO phenols to support the health claim. A good approach to ensure the optimal intake of PC in the context of a balanced diet is to tailor a phenol-enriched functional VOO. Combining PC from different classes and sources might allow increasing phenol consumption to optimize likely effects observed with the consumption of a single type of PC.
HYPOTHESIS:The sustained intake of functional VOO enriched with its own PC, predominantly secoiridoids such as hydroxytyrosol and its derivatives, or with them plus additional complementary ones from thyme, such as flavonoids, may modify the physicochemical properties of HDL particles towards a cardioprotective mode, and may promote changes in HDL subclasses distribution leading to the consequent enhancement of HDL functionality in hypercholesterolemic subjects. The secondary hypothesis is that several HDL physicochemical properties, such as composition, fluidity, oxidative status, and size, might be the main determinants of the capacity of HDL for promoting ChE from cells.
OBJECTIVES: Primary objective To assess whether the sustained intake of phenol-enriched VOOs could enhance HDL functionality in hypercholesterolemic subjects by modifying: 1. HDL metabolism and maturation and therefore subpopulations distribution, 2. antioxidant content (i.e. fat-soluble antioxidants and phenolic metabolites), 3. cholesterol efflux (ChE) and HDL characteristics influencing ChE (i.e. fluidity), and 4. paraoxonase (PON) enzymes family.
Secondary objectives 1. To assess the effect of the sustained intake of phenol-enriched VOOs on several CVD risk parameters such as very-low density lipoprotein (VLDL) and low-density lipoprotein (LDL) subpopulations distribution, and LDL/HDL particle number (LDL-P/HDL-P), HDL-C/HDL-P and small-HDL/large-HDL (s-HDL/l-HDL) atherogenic ratios.
2. To devise a high-throughput in vitro ChE assay to screen efficiently large numbers of specimens such in the case of clinical trials, avoiding the use of radiolabeled cholesterol.
3. To assess whether the acute intake of phenol-enriched VOOs could instigate short-term modifications in PON-related variables.
4. To investigate the mechanisms of action involved in the PON system in an in vivo animal model.
5. To define which parameters can be the best predictors for HDL ChE capacity (CEC), and its interrelations with fluidity of HDL monolayer after VOO intake.
METHODS:Two randomized, controlled, double-blind, and crossover interventions were conducted within the frame of the “Virgin Olive Oil and HDL Functionality” (VOHF) study: Acute intake study: 12 healthy participants ingested a single dose of 30 mL of raw VOOs enriched with their own PC but differing in the phenolic content: a. Low-functional VOO (L-FVOO) enriched with 250 ppm of PC b. Medium-functional VOO (M-FVOO) enriched with 500 ppm of PC c. High-functional VOO (H-FVOO) enriched with 750 ppm of PC A natural VOO with 80 ppm of PC was used as the matrix to prepare these phenol-enriched VOOs by adding a freeze-dried olive cake extract rich in PC from olive oil. Interventions were separated by 1-week washout period.
Sustained intake study: 33 hypercholesterolemic participants ingested 25 mL/day of three VOOs differing in PC source and content: a. A natural control VOO with 80 ppm of PC b. A functional VOO (FVOO) enriched with 500 ppm of PC from olive oil c. A functional VOO (FVOOT) enriched with 500 ppm of total PC from olive oil and complementary ones from thyme in a 1:1 ratio.
For the sustained-intake study, the same parental VOO used for the acute-intake study was used as a control condition and as a matrix to prepare both functional VOOs. Intervention periods were of 3 weeks separated by 2-week washout periods.
Study 1: “Polyphenol rich olive oils improve lipoprotein particle atherogenic ratios and subclasses profile: A randomized, crossover, controlled trial” Lipoprotein particle counts and subclasses distribution measurements were performed by nuclear magnetic resonance (NMR) in a Vantera clinical spectrometer, produced by LipoScience. LipoProfile-3 algorithm was performed to quantify the average particle size and particle concentrations of VLDL, LDL, and HDL. Subparticle concentrations were determined for three VLDL subclasses (large or chylomicrons: >60 nm; medium: 35–60 nm; and small: 27–35 nm); three LDL subclasses (intermediate-density lipoprotein (IDL): 23–27 nm; large: 21.2–23 nm; and small: 18–21.2 nm); and three HDL subclasses (large: 8.8–13 nm; medium: 8.2– 8.8 nm; and small: 7.3–8.2 nm). Lipoprotein Resistance Index (LP-IR), a lipoprotein particle-derived measure of insulin resistance was also assessed. These analyses were performed in the samples of the sustained-intake study.
Study 2: “Phenol-enriched olive oils improve HDL antioxidant content in hypercholesterolemic subjects. A randomised, double-blind, cross-over, controlled trial” HDL fatty acids, fat-soluble antioxidants and phenolic metabolites were analyzed by gas chromatography (GC), liquid chromatography (HPLC), and ultra-performance liquid chromatography-tandem mass spectrometer (UPLC/MS/MS), respectively.
HDL particle fluidity was measured by determination of the steady-state anisotropy of 1,6-diphenyl-1,3,5-hexatriene fluorescent probe stimulated with a polarized light.
ChE was assessed in an in vitro cellular model that implies macrophages labelling with fluorescent cholesterol and incubation with HDL from the volunteers. All these analyses were performed in the samples of the sustained-intake study.
Study 3: “Phenol-enriched olive oils modify paraoxonase–related variables: a Randomized, Crossover, Controlled Trial” PON3 and PON1 protein concentrations were measured in human sera by in-house Enzyme-Linked ImmunoSorbent Assay (ELISA) with rabbit polyclonal antibodies. PON1-associated paraoxonase and lactonase activities were assessed in human sera by measuring the rate of hydrolysis of paraoxon and 5-thiobityl butyrolactone, respectively. These analyses were performed in the samples of both the acute- and the sustained-intake study.
Mechanistic studies were carried out in rats, which followed a diet enriched with the appropriate phenolic extract for 21 days: phenolic extracts from olive oil, from thyme, or both from olive oil and thyme. A control group was also included in the study. Activation of mitogen-activated protein kinases and additional related kinases was assessed in rat liver homogenates. Furthermore, gene expression of Pon1, Pon3, and chemokine (C-C motif) ligand 2 (CCL-2), along with the transcription factors aryl hydrocarbon receptor, peroxisome proliferator-activated (PPAR)α, δ, and γ were also analyzed in rat liver homogenates.
Study 4: “Determinants of HDL cholesterol efflux capacity after virgin olive oil ingestion: Interrelationships with fluidity of HDL monolayer” Post-hoc analysis were performed with samples from participants in the sustained-intake VOHF study to assess the relationship of the 3-week changes in HDL-related variables after the three intervention periods. Mixed linear models, adjusted by age and sex, were fitted with variables related to CEC and fluidity in univariate analyses.
RESULTS:Study 1: “Polyphenol rich olive oils improve lipoprotein particle atherogenic ratios and subclasses profile: A randomized, crossover, controlled trial” FVOO decreased LDL-C and the LDL-P/HDL-P ratio, and improved the lipoprotein subclasses by decreasing total ApoB100-containing lipoproteins, total LDL, small LDL, IDL and s-HDL particle concentrations, as well as LDL and VLDL particle size. Both, FVOO and FVOOT decreased medium VLDL particles, increased l-HDL particles and HDL size, decreased s-HDL/l-HDL and HDL-C/HDL-P ratios, and the LP-IR.
Study 2: “Phenol-enriched olive oils improve HDL antioxidant content in hypercholesterolemic subjects. A randomised, double-blind, cross-over, controlled trial” The sustained intake of FVOO and FVOOT increased both lipophilic and hydrophilic compounds with antioxidant properties in HDL. Regarding fat-soluble antioxidants, FVOO and FVOOT intake increased ubiquinol and the carotenoids lutein and β-cryptoxanthin. FVOO increased retinol while FVOOT increased α-tocopherol. Concerning hydrophilic compounds, FVOOT intake increased the phenolic metabolites caffeic acid sulfate, hydroxyphenylpropionic acid sulfate, and thymol sulfate, while hydroxytyrosol acetate sulfate increased after FVOO intake.
ChE tended to increase after FVOOT versus its baseline and it increased versus FVOO.
No significant changes were observed in HDL monolayer fluidity nor in fatty acids after any intervention.
Study 3: “Phenol-enriched olive oils modify paraoxonase–related variables: a Randomized, Crossover, Controlled Trial” Acute and sustained intake of PC from olive oil (through VOO and FVOOs intake) decreased PON1 protein and increased PON1-associated activities, while FVOOT sustained intake yielded opposite results rather than producing synergic effects. PON3 protein levels increased only after sustained consumption of VOO.
Mechanistic studies performed in rat livers showed that intake of isolated PC from olive oil or from thyme modulate mitogen-activated protein kinases and peroxisome proliferator-activated receptors regulating PON synthesis, while a combination of these PCs cancels such regulation.
Study 4: “Determinants of HDL cholesterol efflux capacity after virgin olive oil ingestion: Interrelationships with fluidity of HDL monolayer” An increase in the HDL fluidity and in the concentration of apolipoprotein (Apo) A-I in HDL, and a decrease in HDL oxidative status were found to be the main determinants for HDL CEC. Changes in HDL lipid composition (in particular a reduction in free cholesterol (FC) and an increase in triglycerides (TG) in HDL), as well as a decrease in s-HDL particle number or an increase in HDL mean size, were the main determinants for the fluidity of the HDL monolayer. CONCLUSIONS: 1. Sustained consumption of phenol-enriched VOOs, that is FVOO and FVOOT, shifted lipoprotein subclasses distribution and their associated ratios to a less atherogenic pattern: 1.1. Both phenol-enriched VOOs enhanced HDL maturation, since they modified HDL size and subclasses distribution towards larger and more mature HDL particles.
1.2. FVOO decreased LDL and VLDL parameters commonly associated with the risk of CHD and with dyslipemia in individuals with T2DM.
1.3. Both phenol-enriched VOOs decreased several atherogenic ratios.
1.4. FVOO could be better at improving lipoprotein subclasses distribution, since FVOOT had milder impact on HDL and VLDL subclasses distribution and on atherogenic ratios than FVOO, and no impact on LDL particles distribution.
2. Sustained consumption of both phenol-enriched VOOs improved the HDL antioxidant content, since they increased lipophilic and hydrophilic antioxidants in HDL. The co-existence of these antioxidants linked to HDL may confer additional benefits by protecting lipids and proteins from oxidative damage via different antioxidant mechanisms. However, FVOOT intervention could be better at improving HDL antioxidant activity, because it increased α-tocopherol, the major antioxidant in human plasma. Moreover, FVOOT also increased phenolic acids in HDL, which are known to regenerate α-tocopherol.
3. Decreases in HDL oxidative status and increases in HDL monolayer fluidity and in ApoA-I content in HDL are major determinants of CEC. In turn, monolayer fluidity is determined by HDL lipid composition (not least by an increase in TG and a reduction in FC) together with a decrease in the concentration of s-HDL particles or an increase in HDL mean size.
4. Sustained intake of FVOOT increased ChE versus FVOO intake despite no changes were observed in HDL monolayer fluidity.
5. Other factors than monolayer fluidity may be responsible for the ChE enhancement observed after FVOOT. Those include a) higher presence of antioxidants in HDL that may confer better antioxidant protection to ApoA-I enhancing therefore ChE, b) decrease of FC in the HDL monolayer, and c) modulation of HDL subclasses distribution.
6. Acute and sustained intake of phenol-enriched VOOs modify PON enzymes family towards a cardioprotective mode according to the phenolic content and source. In particular, FVOO intake may be better at modulating PON enzymes family, since it promoted changes that are indicative of a proper oxidative balance, supporting HDL function enhancement. On the other hand, FVOOT induced opposite results due to the combination of PC from OO and from thyme, rather than the sole intake of PC from thyme. Several mechanisms are involved in the PON system modulation such as PON synthesis together higher content of antioxidants in HDL and enhanced HDL maturation.
GLOBAL CONCLUSION The present thesis confirms the hypothesis that the sustained intake of functional VOOs enriched with its own PC or with them plus additional complementary ones from thyme modifies the physicochemical properties of HDL particles towards a cardioprotective mode, and promotes changes in HDL subclasses distribution leading to the consequent enhancement of HDL functionality in hypercholesterolemic subjects. These changes are performed according to phenol content and source in the VOOs tested.
The enrichment of VOO with PCs is a way of increasing VOO healthy properties without increasing the individual’s caloric intake. Therefore, the tailoring of functional VOO is an interesting and useful strategy to enhance the functional quality of HDL and thus a complementary tool for the management of hypercholesterolemic individuals.
