Olive oil bioactive components

The major components of OO are glycerols (saponifiable fraction) which represent more than 98 % of the total oil weight and are mainly TAG esters of oleic acid (55–83 %), palmitic acid (7·5–20 %), linoleic acid (3·5–21 %) and other fatty acids such as stearic acid (0·5–5 %)⁽ Reference Harwood and Aparico ^{13 )}. Minor components (the unsaponifiable fraction) include aliphatic and triterpenic alcohols, sterols, hydrocarbons as squalene, volatile compounds, tocopherols, carotenes, chlorophyll and phenolic compounds⁽ Reference Harwood and Aparico ^{13 –} Reference Perez-Jimenez, Ruano and Perez-Martinez ^{15 )}.

Special attention has been given to the phenolic compounds only found in VOO and EVOO. The agronomic and technological aspects of OO production have an impact on the concentration of phenolic compounds, as do the pedoclimatic conditions and agronomic techniques (e.g. irrigation)⁽ Reference Tripoli, Giammanco and Tabacchi ^{4 ,} Reference Servili and Montedoro ^{14 )}. The main classes of phenolic compounds present in VOO are phenolic acids, phenolic alcohols (hydroxytyrosol and tyrosol), flavonoids, lignans and secoiridoids; Table 1.

Table 1. Main classes of phenolic compounds in virgin olive oil

Oleuropein and ligstroside, the most significant secoiridoids in O. europaea L., are esters of elenolic acid glucoside with hydroxytyrosol and tyrosol, respectively. During the mechanical extraction of the oil, fruit endogenous β-glucosidases⁽ Reference Servili and Montedoro ^{14 ,} Reference Di Maio, Esposto and Taticchi ^{16 )} are released leading to the secoiridoid aglycones formation, accounting for more than 50 % of phenolic content of the oil⁽ Reference Brenes, Garcia and Garcia ^{17 ,} Reference Hrncirik and Fritsche ^{18 )}. The most abundant secoiridoids of VOO are the oleuropein and ligstroside aglycons and dialdehydic forms of deacetoxy of oleuropein and ligstroside aglycons⁽ Reference Servili and Montedoro ^{14 )} also named oleacein and oleocanthal, respectively⁽ Reference Karkoula, Skantzari and Melliou ^{19 )}.

Phenolic compounds bioavailability and bioactivity

Once OO has been ingested, it produces a micellar solution composed of a lipid and an aqueous phase. Chemical hydrolysis of secoiridoids can take place in the acidic medium of the stomach⁽ Reference Corona, Tzounis and Assunta Dessi ^{20 )} or in alkaline conditions in the small intestine⁽ Reference Pinto, Paiva-Martins and Corona ^{21 ,} Reference Aranzazu Soler, Alba and Shikha ^{22 )} leading to an increase of free phenolic alcohols released into the aqueous phase. As a result OO phenolic compounds are further absorbed in the small intestine⁽ Reference Vissers, Zock and Roodenburg ^{23 )}. Measuring the bioavailability of these compounds in plasma and urine reveals that OO phenolics undergo a conjugation process of methylation, glucuronidation and sulfation indicating that there is phase 2 metabolism involved during the absorption of these compounds⁽ Reference Garcia-Villalba, Carrasco-Pancorbo and Nevedomskaya ^{24 –} Reference Miro Casas, Farre Albadalejo and Covas Planells ^{27 )}. The between-subjects variability in human absorption and metabolism of OO phenolics may explain differences in proportion of methyl, glucuronide and sulphate conjugates reported⁽ Reference Garcia-Villalba, Larrosa and Possemiers ^{28 –} Reference Preedy and Watson ^{30 )}.

Bioavailability of OO phenolic compounds differs according to the intake matrix. OO as the intake vehicle promotes absorption of hydroxytyrosol: the corresponding bioavailability of hydroxytyrosol in rats for aqueous and OO solutions were reported as 75 and 99 %⁽ Reference Tuck, Freeman and Hayball ^{31 )}, respectively. When a supplement containing hydroxytyrosol as a single oral dose (2·5 mg/kg) was fed to human subjets, the bioavailability was below 10 %⁽ Reference Gonzalez-Santiago, Fonolla and Lopez-Huertas ^{32 )}, whereas previous studies showed higher bioavailability for hydroxytyrosol supplementation in lipid vehicles⁽ Reference Visioli, Galli and Grande ^{33 )}. The addition of hydroxytyrosol to low fat yoghurt and administered to human subjects was also associated with a lower excretion of hydroxytyrosol when compared with OO⁽ Reference Visioli, Galli and Grande ^{33 )}. As OO phenolic compounds are mainly absorbed in the small intestine⁽ Reference Vissers, Zock and Roodenburg ^{23 )} the increase of hydroxytyrosol bioavailability, in OO, might be related to the rate of gastric emptying⁽ Reference Gonzalez-Santiago, Fonolla and Lopez-Huertas ^{32 )} and slow release of hydroxytyrosol from the oil matrix⁽ Reference Miro-Casas, Covas and Farre ^{26 ,} Reference Gonzalez-Santiago, Fonolla and Lopez-Huertas ^{32 )}. The presence of other antioxidants in OO might prevent breakdown of hydroxytyrosol before absorption in the gastrointestinal tract⁽ Reference Tuck, Freeman and Hayball ^{31 )}.

Secoiridoids that are not absorbed in the small intestine are degraded by the colonic microbiota with oleuropein producing hydroxytyrosol as the major product⁽ Reference Corona, Tzounis and Assunta Dessi ^{20 )}. In vitro colonic metabolism was evaluated on tyrosol, hydroxytyrosol, hydroxytyrosol acetate and oleuropein showing an increase in phenolic acids, stability of hydroxytyrosol and tyrosol and degradation of hydroxytyrosol acetate and oleuropein mainly to hydroxytyrosol⁽ Reference Mosele, Martin-Pelaez and Macia ^{34 )}. To evaluate OO phenolic metabolites produced from colonic fermentation, faecal samples were analysed before and after mid-term consumption of phenol-rich OO⁽ Reference Mosele, Martin-Pelaez and Macia ^{34 )}. A significant increase in hydroxytyrosol concentration (P < 0·05) was observed after phenol-rich OO intake. Although absorption of OO phenolic compounds mainly occurs in the small intestine a small proportion of hydroxytyrosol and its derivatives still pass into the large intestine⁽ Reference Vissers, Zock and Roodenburg ^{23 )}. This highlights the need to study the impact of OO phenolics in the colon, either with gut microbiota interaction or local activity due to its antioxidant and anti-inflammatory properties.

When assessing the chemical and in vitro biological antioxidant activities of these compounds, it is the glucuronide conjugates of hydroxytyrosol and tyrosol that must be assessed. These were tested in the range 0·01–10 μm against the radical 1,1-diphenyl-2-picrylhydrazyl. None of the glucuronides displayed significant antioxidant activities at the concentrations tested, whereas the parent aglycones did display antioxidant activity at these concentrations⁽ Reference Khymenets, Fito and Tourino ^{35 )}. This conflicts with the results of others⁽ Reference Tuck, Hayball and Stupans ^{36 )} with differences attributed to the fact that in one study reference standard material⁽ Reference Khymenets, Fito and Tourino ^{35 )} was used and in the other the glucuronide conjugates were extracted from urine samples⁽ Reference Tuck, Hayball and Stupans ^{36 )}, and likely contained impurities that had antioxidant activity. Hydroxytyrosol metabolites might act as ‘sinks’ of hydroxytyrosol that could be locally released in the cells after enzymatic hydrolysis⁽ Reference Kotronoulas, Pizarro and Serra ^{37 )}, thereby explaining the proposed hydroxytyrosol biological effects observed in vivo. Moreover, in situ deconjugation of hydroxytyrosol metabolites (into their free form) in erythrocytes was observed in rats after oral administration of an OO phenolic extract obtained from olive cake (1·5 g/kg body weight, equivalent to 34·4 mg hydroxytyrosol and derivatives), highlighting a potential protective mechanism against cell oxidative damage⁽ Reference Laura Rubió, Alba and Carme ^{38 )}.

Although there are a number of biological effects for OO phenolic compounds, most cannot be achieved via normal dietary exposure to OO. This has led to development of enriched products with natural OO phenolic compounds. OO by-products such as olive mill wastewater⁽ Reference Hamden, Allouche and Damak ^{39 )} and olive pomace⁽ Reference Sanchez de Medina, Priego-Capote and Luque de Castro ^{40 ,} Reference Suarez, Romero and Motilva ^{41 )} are potential sources of natural bioactives which could be used to supplement OO. The development of new OO products such as pomace OO or refined olive oil enriched in natural bioactives opens new perspectives in the field.

Chronic inflammation

Rheumatoid arthritis is a major inflammatory, autoimmune, disease characterised by chronic joint inflammation⁽ Reference O'Connor ^{47 ,} Reference Waterman and Lockwood ^{48 )}. Hydroxytyrosol has been studied for its anti-inflammatory effects in a rheumatoid arthritis animal model. We reported that it provided beneficial effects in the evolution of the disease⁽ Reference Silva, Sepodes and Rocha ^{49 )}, with 0·5 and 5 mg/kg doses in rats, after gavage administration, using refined olive oil as vehicle (human-equivalent of 4·9 and 49 mg/d, respectively, for a 60 kg adult), Fig. 1. Significant effects, on paw oedema reduction, were observed for a human-equivalent dose of 49 mg/d, a dose ten times higher than the approved European Food Safety Authority (EFSA) dose for phenolic compounds in relation to protection of lipid oxidation^{( 50 )}. The same hydroxytyrosol dose was effective on colitis, another chronic inflammatory disease⁽ Reference Sanchez-Fidalgo, Sanchez de Ibarguen and Cardeno ^{51 )}. This dose would only be achievable through nutraceutical supplementation of OO with hydroxytyrosol, and the use of this functional food on a daily basis.

Fig. 1. Chronic inflammation model and impact on rats paw oedema. ANOVA, *P < 0·001 v. positive control – Rheumatoid Arthritis, ⁺ P < 0·01 v. Refined Olive Oil; OHTYR, hydroxytyrosol. Adapted from Silva et al. ⁽ Reference Silva, Sepodes and Rocha ^{49 )}.

To further evaluate the anti-inflammatory mechanisms involved with hydroxytyrosol, we studied COX-2 and inducible NO synthase expression⁽ Reference Silva, Sepodes and Rocha ^{49 )}. The treatment at 5 mg/kg dose significantly decreased histological damage, COX-2 and inducible NO synthase expression (P < 0·001 v. positive control), markedly reduced the degree of bone resorption, soft tissue swelling and osteophyte formation, improving articular function in the treated animals. Moreover, at the same dose there was a significant decrease (P < 0·005 v. positive control and refined olive oil) in TNF-α serum levels. These results are in line with others that reported benefits on rheumatoid arthritis, in animal models, after oral administration of an EVOO extract⁽ Reference Rosillo, Alcaraz and Sanchez-Hidalgo ^{52 )}, intraperitoneal administration of oleuropein aglycone⁽ Reference Impellizzeri, Esposito and Mazzon ^{53 )} or polyphenol supplemented VOO diets⁽ Reference Martinez-Dominguez, de la Puerta and Ruiz-Gutierrez ^{54 )}. The reports highlight the effects on rheumatoid arthritis of OO phenolic compounds either administered as isolated compounds or as an extract. However, dose comparison between animal studies have to take in consideration not only differences in species (rats v. mice), but also routes of administration. Compared with intraperitoneal administration, an oral dose has an extra pass through the liver with consequent metabolism through the first-pass effect.

Acute inflammation

Acute inflammation has been commonly induced using carrageenan in animals to evaluate the effects of non-steroid anti-inflammatory drugs⁽ Reference Bignotto, Rocha and Sepodes ^{55 )}. We studied the effect of hydroxytyrosol-supplemented OO on acute inflammation, induced by carrageenan in rats, at 0·5 and 5 mg/kg⁽ Reference Silva, Sepodes and Rocha ^{49 )} dose, after gavage administration which occurred 30 min before the challenge with carrageenan. Both doses significantly reduced paw oedema (P < 0·001 v. positive control) with the lowest effective dose being achievable through OO daily intake. Previous studies in rats⁽ Reference Gong, Geng and Jiang ^{56 )} also showed inhibition of carrageenan; acute inflammation of an aqueous hydroxytyrosol formulation (HT-20, 22 % hydroxytyrosol), and significant effects were obtained at a 22 mg/kg hydroxytyrosol dose. Differences in dose effect might be related to the administration vehicle with refined olive oil or OO being better vehicles than water.

Impact of olive oil constituents on lipoproteins and atherosclerosis

Cardioprotective mechanisms of oleic acid

OO intake has been related with a decrease on blood pressure with oleic acid regarded as being a major contributor to this effect, as evidenced in animal models⁽ Reference Teres, Barcelo-Coblijn and Benet ^{83 )}. Chronic oral administration of VOO (rich in oleic acid), triolein (a TAG with three oleic acid moieties) or oleic acid over 14 d significantly reduced systolic blood pressure in rats (−26 (sem 4) for VOO and −21 (sem 3) mm Hg for triolein, P < 0·001 and −17 (sem 1·9) mm Hg for oleic acid P < 0·05) when compared with the control group that received water. Similarly acute (2 h) treatments with either VOO or triolein also significantly reduced systolic blood pressure when compared with the control group (−20 (sem 0) mm Hg, P < 0·001 and −14 (sem 2) mm Hg, respectively; P < 0·05) with oleic acid again significantly reducing systolic blood pressure (−13·0 (sem 0·3) mm Hg; P < 0·001). In contrast, chronic treatment with the trans-MUFA elaidic (18 : 1n-9) or the SFA stearic acid (18 : 0) did not significantly affect blood pressure. Results show that saturation and cis/trans double-bond arrangement are implicated with the cardioprotective effect of the long-chain fatty acid in this animal model at high-dose levels⁽ Reference Teres, Barcelo-Coblijn and Benet ^{83 )}. Similar significant results were obtained after VOO and oleic acid intake in an animal model of hypertension using spontaneously hypertensive rats⁽ Reference Teres, Barcelo-Coblijn and Benet ^{83 )}.

The molecular mechanisms were evaluated by measuring signalling proteins involved in the control of blood pressure in the aorta. OO intake increases oleic acid levels in membranes, which regulate membrane lipid structure and impact on G-protein-mediated signalling, causing a reduction in blood pressure⁽ Reference Yang, Alemany and Casas ^{84 )}. Unlike its analogues elaidic and stearic acid, oleic acid, due to its cis-18 : 1n-9 structure, regulates cellular membrane lipid structure and the α₂ receptor system involved in the control of blood pressure (α_2A/D – adrenoreceptor/G protein/adenylyl cyclase-cAMP/PKA) as demonstrated in vitro ⁽ Reference Yang, Alemany and Casas ^{84 )} and in vivo ⁽ Reference Teres, Barcelo-Coblijn and Benet ^{83 )}. Oleic acid can also contribute to heart health via intramyocardial TAG turnover⁽ Reference Lahey, Wang and Carley ^{85 )}, which is reduced in pressure-overloaded failing hearts. In this situation, oleate (derivative of oleic acid) upregulated TAG dynamics when compared with palmitate (derivative of palmitic acid and major SFA of palm oil). This result underscores the importance of the intracellular lipid storage type on nuclear receptor signalling and contractility⁽ Reference Lahey, Wang and Carley ^{85 )} in diseased hearts.

An important driver of vasorelaxation is NO, a free radical that readily reacts with fats and proteins. Nitro-fatty acids are mediators of cardiovascular signalling actions⁽ Reference Rudolph, Rudolph and Schopfer ^{86 )} as these compounds relax blood vessels, attenuate platelet activation and reduce inflammation⁽ Reference Coles, Bloodsworth and Clark ^{87 ,} Reference Coles, Bloodsworth and Eiserich ^{88 )}.

Both oleic acid and linoleic acid are unsaturated fatty acids that after reaction with nitrite may form nitro-fatty acids. Nitro-oleic acid-mediated antihypertensive signalling actions were shown in a mouse model⁽ Reference Charles, Rudyk and Prysyazhna ^{89 )}. The mechanism was attributed to the inhibition of soluble epoxide hydrolase by nitro-fatty acids, thus lowering blood pressure in an angiotensin II-induced hypertension⁽ Reference Charles, Rudyk and Prysyazhna ^{89 )}. It is however unclear how the extent of nitrite in the human diet may contribute to nitration of dietary fat, and the physiological relevance of this finding.

Role of phenolic compounds on endothelium protection

Oxidative stress and reactive oxygen species have been implicated in endothelial damage, progression to atherosclerosis, injury in sustained myocardial infarction and ischaemia reperfusion⁽ Reference Khurana, Venkataraman and Hollingsworth ^{76 ,} Reference Dhalla, Temsah and Netticadan ^{90 –} Reference Raedschelders, Ansley and Chen ^{92 )}. Monocytes and macrophages are critical cells that are involved in atherosclerosis. These cells produce proinflammatory cytokines, such as IL-1β, TNF-α and C-reactive protein, which induce the expression of adhesion molecules such as intercellular adhesion molecule-1, vascular-cell adhesion molecule-1 and E-selectin⁽ Reference Ross ^{93 )}.

Meanwhile, oxidative stress through reactive oxygen species production promotes the expression of the adhesion molecules on the endothelium⁽ Reference Dell'Agli, Fagnani and Galli ^{94 )}.

Expression of adhesion molecules attracts circulating monocytes, inducing their adherence to the endothelium. OO phenolic compounds have been shown to act on endothelium protection as evidenced in in vitro assays with typical OO phenolic compounds and less on in vivo circulating metabolites. OO phenolic extract, oleuropein aglycone or homovanillic alcohol (metabolite of hydroxytyrosol) had inhibitory effects on vascular-cell adhesion molecule-1, intercellular adhesion molecule-1 and E-selectin surface expression in human umbilical vascular endothelial cells, using TNF-α as pro-inflammatory stimulus⁽ Reference Dell'Agli, Fagnani and Mitro ^{95 )}.

Endothelium dysfunction refers to an impairment of endothelium-dependent vasorelaxation caused by a loss of NO bioactivity in the vessel wall. In animal models with rats oral hydroxytyrosol administration was tested on NO production and platetet function⁽ Reference Gonzalez-Correa, Navas and Munoz-Marin ^{96 )}. Results showed that hydroxytyrosol administration (100 mg/kg daily) increased vascular NO production by up to 34·2 % (P < 0·01) and inhibited platelet aggregation for 50 % inhibitory dose of 48·25 mg/d for hydroxytyrosol (P < 0·01) when compared with control group (treated with isotonic saline solution). Animal dose translation to human subjects allowed us to conclude that the effective hydroxytyrosol doses tested would be above the expected intake through OO daily. The reported benefits would only be achievable through nutraceutical supplementation.

Endothelium repair: matrix metalloproteinases and olive oil

Matrix metalloproteinases (MMP) play a role in endothelium repair. Macrophages resident in human and experimental atherosclerosis co-localise with and release active MMP, including the gelatinase MMP-9, which is specialised in the digestion of basement membrane collagens and elastin, and is implicated in atherogenesis, unstable coronary syndromes and in aortic aneurysms⁽ Reference Dollery and Libby ^{97 )}. Accumulating evidence points to the MMP as major molecular mediators of arterial diseases⁽ Reference Dollery and Libby ^{97 )}. Collagens, types 1 and 3, are the main proteins in arterial walls being also present in the thickened intima of atherosclerotic lesions⁽ Reference Barderas, Vivanco and Alvarez-Llamas ^{98 ,} Reference von Zur Muhlen, Schiffer and Zuerbig ^{99 )}. Fragments of collagens found in urine are present as a result of proteolytic activity in arterial walls and other vascular structures. Collagen type 1 or 3 fragments were upregulated in urine in coronary artery disease (CAD) patients⁽ Reference Zimmerli, Schiffer and Zurbig ^{100 )}. Increase in collagen degradation is related with an increase on collagenases circulation, such as MMP-9, as shown in patients with CAD⁽ Reference Kalela, Koivu and Sisto ^{101 )}.

In an in vitro study hydroxytyrosol (1–10 μm) reduced MMP-9 (IC₅₀ = 10 μm/l, P < 0·05) and COX-2 induction in activated human monocytes, with phorbol myristate acetate⁽ Reference Scoditti, Nestola and Massaro ^{102 )}. These effects were mediated by inhibition of transcription factor NF-κB and protein kinase Cα and protein kinase C β1 activation⁽ Reference Scoditti, Nestola and Massaro ^{102 )}. Results are in line with previous in vitro reports that showed inhibition of MMP-9 on endothelial cells by OO phenolics namely hydroxytyrosol in phorbol myristate acetate-induced cells⁽ Reference Scoditti, Calabriso and Massaro ^{103 )} and oleuropein aglycone in TNF-α-induced cells by acting on NF-κB⁽ Reference Dell'Agli, Fagnani and Galli ^{94 )}. No hydroxytyrosol activity on MMP-9 was found in TNF-α-induced cells⁽ Reference Dell'Agli, Fagnani and Galli ^{94 )}.

The discriminatory polypetides that increase in CAD includes collagen type 1 and 3 fragments with a C-terminal GxPGP motif⁽ Reference Delles, Schiffer and von Zur Muhlen ^{104 )}. Increase on these polypeptides would come from a protease decrease activity possibly related with chemical change of the substrate (e.g. oxidative damage) thus inhibiting it acting at a specific site, or a decrease in circulating levels by lack of enzyme activation. MMP-2 is secreted in an inactive form (pro-MMP-2) and several factors can promote its activation such as plasmin⁽ Reference Monea, Lehti and Keski-Oja ^{105 )} and thrombin⁽ Reference Lafleur, Hollenberg and Atkinson ^{106 )}. Other mechanisms that involve proteinases or oxidative stress can also activate MMP-2⁽ Reference Rajagopalan, Meng and Ramasamy ^{107 )}. Therefore antioxidants, as phenolic compounds, might have a role on MMP-2 activation and published data indicate phenolic compounds from red wine⁽ Reference Oak, El Bedoui and Anglard ^{108 )} and green tea⁽ Reference El Bedoui, Oak and Anglard ^{109 )} as acting on prevention of thrombin-induced activation of MMP-2 in vascular smooth cells.

We evaluated the impact of a 6-week OO supplementation in healthy adults on urinary proteomic biomarkers of CAD in a randomised, parallel, controlled, double-blind study⁽ Reference Silva, Bronze and Figueira ^{110 )}. The present study was the first to describe the significant impact of daily OO supplementation on highly specific disease biomarkers for CAD. Analysis of urinary proteomic profiles at baseline and endpoint enabled the identification of twelve sequenced peptides that were significantly regulated towards healthy scoring. Eight of them included four collagen α−1(I) chain, one α−2 (1) chain, one α−2(V) chain and one α−2(VI) chain fragments. Changes in circulating concentrations of collagenases may mediate these changes in the urinary fingerprint. Therefore with more data or in future intervention studies with OO, it would be interesting to link urinary fragments to the proteases involved in their generation. This predictive analysis would enable looking at the peptide cleavage sites studying the MMP up or downregulated with OO intervention.

The majority of studies of dietary intake of proposed bioactive foods asses the activities of these foods based on the major risk factors of CVD. However, markers such as lipoprotein profile, blood pressure, endothelial function, inflammation and oxidative stress have no direct link to the disease itself but are merely associated with it. There is a great need for more biomarkers that appear as a direct result of the disease itself⁽ Reference Ruiz-Canela and Martinez-Gonzalez ^{63 ,} Reference Covas, Konstantinidou and Fito ^{67 )}.

Proteomic in vitro studies on olive oil phenolic compounds

Proteomics has been applied in a number of studies of OO phenolic compounds on cardiovascular health using animal and in vitro studies. The in vitro effects of alperujo extract, an OO production waste product containing phenolic compounds present in olive fruits, were studied on platelet aggregation and changes in the platelet proteome⁽ Reference de Roos, Zhang and Rodriguez Gutierrez ^{131 )}. Nine proteins were differentially regulated by the alperujo extract upon platelet aggregation underlying the anti-platelet effects of the extract. However, like a number of previously mentioned in vitro studies, the effective concentrations (40–500 mg/l) were far above the physiologically concentrations achievable by dietary intake.

The effects of EVOO, with low and high phenolic content, were evaluated in the hepatic proteome in ApoE^−/− mice that spontaneously develop atherosclerosis⁽ Reference Arbones-Mainar, Ross and Rucklidge ^{132 )}. For 10 weeks the mice were fed with a high-fat, high-cholesterol diet supplemented with 0·15 % (w/w) cholesterol and either 20 % (w/w) low phenolic EVOO or 20 % (w/w) high phenolic EVOO v. a control group fed with 0·15 % (w/w) cholesterol and 20 % (w/w) palm oil. Within this work a range of hepatic antioxidant enzymes differentially regulated by OO⁽ Reference Arbones-Mainar, Ross and Rucklidge ^{132 )} were identified. The authors concluded that the upregulation of a large array of antioxidant enzymes might explain anti-atherogenic mechanisms of EVOO⁽ Reference Arbones-Mainar, Ross and Rucklidge ^{132 )}. Again the dose level was above what could be achieved through dietary intake and translation from an animal model to human use has also to be considered.

Urinary proteomics biomarkers, olive oil and CVD

Atherosclerosis is a process of chronic inflammation, characterised by the accumulation of lipids, cells, and fibrous elements in medium and large arteries⁽ Reference Barderas, Vivanco and Alvarez-Llamas ^{98 )}. The extent of inflammation, proteolysis, calcification and neovascularisation influences the development of advanced lesions (atheroma plaques) on the arteries⁽ Reference Barderas, Vivanco and Alvarez-Llamas ^{98 )}.

Classical risk factors in atherosclerosis (hypertension, LDL-cholesterol, C-reactive protein, ageing, smoking, male gender, among others) do not actually measure disease initiation or progression. As such, they cannot be used directly to identify individuals who have developed atherosclerosis and prevent a fatal event⁽ Reference Barderas, Vivanco and Alvarez-Llamas ^{98 ,} Reference Ge and Wang ^{133 )}. Other, more recent markers that indicate changes in vascular structure can still only be detected once CVD has progressed to an advanced stage where drug or surgical intervention is required⁽ Reference Sharma, Cosme and Gramolini ^{134 )}.

The analysis of urine samples from diseased and healthy individuals has been used to establish a database of naturally occurring urinary peptides, making a basis for the definition and validation of biomarkers for diagnosis/prognosis/monitoring of a wide range of diseases using proteomic biomarker patterns⁽ Reference Coon, Zurbig and Dakna ^{128 )}, such as CAD⁽ Reference Zimmerli, Schiffer and Zurbig ^{100 )}, emphasising that non-invasive proteomics analysis could become a valuable addition to assess CVD alongside other biomarkers that are indicators of cardiovascular risk.

The first time that urinary proteomics was applied to assess cardiovascular health improvements of OO consumption in human subjects, was in a randomised, parallel, controlled, double-blind study designed to evaluate the impact of a 6-week OO supplementation in healthy adults on urinary proteomic biomarkers of CAD⁽ Reference Silva, Bronze and Figueira ^{110 )}. The impact of the supplementation with OO was also studied on urinary proteomic biomarkers of CKD and diabetes.

The increase or decrease in the concentration of the peptides in the biomarker determines the scoring value of each disease biomarker. The CAD proteomic biomarker developed for clinical diagnosis produces a CAD scoring system from 1 (CAD case) to −1 (healthy artery). A scoring of disease absence, presence and severity is provided, based on the concentration of a group (panel) of urinary peptides measured by capillary electrophoresis coupled with MS, allowing monitoring of progression and/or effect of treatment⁽ Reference Fliser, Novak and Thongboonkerd ^{135 ,} Reference Julian, Suzuki and Suzuki ^{136 )}. In the present study, self-reported healthy participants were randomly allocated to supplementation with a daily dose of OO either low or high in phenolic compounds. For 6 weeks, they consumed a daily dose of 20 ml OO (not heated or cooked) as a supplement (no specific time during the day, single intake, equivalent to 6 mg hydroxytyrosol and derivatives for the high phenolic OO), in line with the EFSA and Food and Drug Administration recommendations. The impact of supplementation with OO was evaluated on urinary proteomic biomarkers of CAD with biomarkers being measured at baseline and 3 and 6 weeks. Consumption of both OO significantly improved the proteomic CAD score at endpoint compared with baseline, moving the CAD biomarker pattern in a healthy profile direction (Table 2). No differences were observed for CKD or diabetes proteomic biomarkers, Table 2.

Table 2. Scores of CAD, CKD and diabetes proteomic biomarkers at baseline, middle (3 weeks) and end of intervention (6 weeks)

CAD, coronary artery disease; CKD, chronic kidney disease (adapted from Silva et al. ⁽ Reference Silva, Bronze and Figueira ^{110 )}).

Compared with corresponding baseline value: *P < 0·005, **P < 0·001. There were no significant differences in changes between groups.

A repeated-measures ANOVA test was used with statistical significance at P < 0·05.

In a placebo-controlled intervention, irbesartan (angiotensin II receptor antagonist used for the treatment of hypertension) taken at 300 mg/d over 2 years in hypertensive type 2 diabetes patients, using the CAD 238 biomarker panel, led to a 0·35 point reduction in the CAD score for the drug-controlled group⁽ Reference Delles, Schiffer and von Zur Muhlen ^{104 )}, which saw a significant reduction in incidents of CAD in this group. In the nutritional intervention⁽ Reference Silva, Bronze and Figueira ^{110 )}, the CAD score change in the intervention was significant for both OO tested, using the same CAD 238 biomarker, leading to a similar degree of change as observed for irbersartan over a 6-week period. This evidence highlights the importance of the CAD biomarker as a tool for nutrition and health intervention studies. This type of urinary biomarker enabled the measurement of health effects induced by a change in diet that could not be detected by monitoring the conventional risk markers of CAD such as plasma TAG, oxidised LDL, and LDL-cholesterol. The overall change in CAD score in a short period of time is more likely due to OO major components, such as fatty acids. However, the role of other OO minor components other than phenolic compounds should also be taken into account. Squalene, a polyunsaturated triterpene which makes up 60–75 % of the unsaponifiable fraction of OO⁽ Reference Perona, Cabello-Moruno and Ruiz-Gutierrez ^{137 )}, reduced atherosclerotic lesion size in male mice⁽ Reference Guillen, Acin and Navarro ^{138 )} and further investigation is needed to clarify its role on CVD.

Our results emphasise further the potential role of nutrition in the prevention or delay of CVD and offer new perspectives on OO applications. These results are highly translatable to guidelines for nutritional recommendations. The biomarkers were originally developed to detect early signs of diseases in clinical setting and to inform clinicians as to the effectiveness of treatment. However, the technology also provides a sensitive tool for the assessment of potential bioactive foods in cardiovascular health, CKD and diabetes, with a range of additional tests under development. Further testing of reportedly bioactive foods can now be carried out which will allow better nutritional health advice to be advanced and could also lead to better food labelling, so that the public can make informed choices on their food purchases.