A total of 35 young and healthy volunteers of both sexes, aged between 18–30 years old, were enrolled in this prospective, interventional, randomised study (study registered under ClinicalTrials.gov Identifier: NCT04564690; title: “Effect of Enriched QUARTET® Hen Eggs on Cardiovascular Function in Cardiovascular Patients and Healthy Individuals.”). Exclusion criteria for participants were prior history of hypertension, coronary artery disease, diabetes mellitus, renal impairment, cerebrovascular and peripheral artery disorder, hyperlipidaemia and use of oral contraceptives or drugs that could affect endothelium.

Participants were recruited by the researcher and were included using a survey questionnaire to meet the inclusion and exclusion criteria of the study. After recruitment, a simple randomization procedure was performed by an unbiased associate drawing A or B for each subject, i.e., determining the affiliation to a group—A (Control group) or B (4Nutri group). Volunteers who met mentioned criteria were given the codes by the researcher that didn’t contain any personal information: ZCI-Q-1-No. or ZCI-Q-2-No. Label ZCI-Q denotes the name of the project within which the study is conducted; number -1- or -2- denotes belonging to the group (control or 4Nutri, depending on the eggs to be consumed), and were assigned by an unbiased associate that performed simple randomization and divided the eggs among participants. No. is the ordinal number of study participants (1, 2, 3…). Labels indicating group affiliation were known only to the associate assigning the intervention, while neither study participants nor the researcher knew to which groups they belonged throughout the duration of the study.

The study was carried out at the Department of Physiology and Immunology, Faculty of Medicine in Osijek, Croatia and included two appointments. All study participants were instructed to eat three hard-boiled hen eggs (L commercial size) per day for three weeks (total of 63 eggs). Participants were divided into Control group (N = 14; W/M = 6/8) and 4Nutri group (N = 20; W/M = 9/11). Control group consumed regular hen eggs (n-3 PUFAs content ~438 mg/per day; selenium content ~0.05 mg/per day; lutein content ~0.33 mg/per day; vitamin E content ~1.79 mg/per day), while 4Nutri group consumed hen eggs enriched with four nutrients (n-3 PUFAs content ~1026 mg/per day; selenium content ~0.06 mg/per day; lutein content ~1.85 mg/per day; vitamin E content ~3.29 mg/per day).

Study participants were instructed to follow their usual meal schedule and to avoid any supplements (e.g., n-3 PUFAs or vitamin E, selenium and lutein). They were advised to consume only the eggs provided for them during the study, preferably in the mornings after boiling them for 10 minutes. Each respondent recorded what he/she consumed over the duration of the study protocol in a diet diary in form of 24 hours recalls, designed and previously published by our research group [20]. Blood samples were taken on the first and last day of the study protocol for the purpose of serum collection and peripheral blood mononuclear cell isolation. A CONSORT diagram is presented in Fig. 1; a CONSORT checklist is presented in Supplementary Table 1.

Fig. 1.

CONSORT 2010 Flow diagram.

Enrichment of hen eggs was executed by an associate research group from the Faculty of Agrobiotechnical Sciences Osijek, Josip Juraj Strossmayer University of Osijek. Eggs enriched with four nutrients were produced by replacing standard feedstock used in feeding mixtures fed to laying hens, with a mixture of fish oil (0.75%), flaxseed oil (1%), reptile oil (2%) and soy oil (1.25%), 0.5 mg/kg of organic selenium, 200 mg/kg of lutein and 200 mg/kg of vitamin E [19]. The edible part (~60 g) of hen egg enriched with four nutrients (n-3 PUFAs, selenium, lutein and vitamin E) contained approximately 342 mg of n-3 PUFAs (~189 mg of $\alpha$-linolenic acid, ALA; ~19 mg of eicosapentaenoic acid, EPA; ~135 mg of docosahexaenoic acid, DHA), 0.02 mg of selenium, 0.616 mg of lutein and 1.097 mg of vitamin E [19].

As stated above, this study is registered on Clinical Trials as a part of a larger research, therefore there are several primary and secondary outcomes. Primary outcomes for this particular participant group and results are the measurements of biomarkers for oxidative stress and antioxidant capacity, and serum protein concentrations of various pro- and anti-inflammatory cytokines and cell adhesion molecules. Secondary outcomes include measurement of blood pressure and serum lipid profile. A pilot study was performed on a total of 16 participants after the respective dietary protocols (N (Control group) = 8; N (4Nutri group) = 8), considering the outcomes. Effect and sample size were calculated before the recruitment of participants for the main study.

All study participants were legal adults (18 years old) and signed informed consent before their inclusion in the study. The study protocol and procedures conformed to the standards set by the latest revision of the Declaration of Helsinki and were approved by the Ethical Committee of the Science Center of Excellence, Josip Juraj Strossmayer University of Osijek (Class: 602-04/14-08/06; Reg. No.: 2158-610714-114) and Ethics Committee of the Medical Faculty Osijek (Class: 602-04/20-08/07, Reg. No.: 2158-61-07-20147). Participants were all students who volunteered to participate, therefore no selection bias was present in our research. Compensation was provided for respondents in the form of travel expenses. Fresh eggs were delivered from a poultry farm (Marijančanka d.o.o.) to the laboratory and distributed to participants within a week.

The arterial blood pressure of participants was measured with an automatic oscillometer (HR P-3-1134, OMRON, Osaka, Japan); the average of three measurements in a sitting position after 10 minutes of rest, before the start and after the end of the protocol, was taken as the final value. During both visits, subjects had their Body Mass Index (BMI; kg/m${}^2$), calculated from the ratio of weight in kilograms to a square meter of height, and waist-to-hip ratio (WHR).

Venous blood samples were collected after half an hour of rest in a calm and quiet room, at both appointments. Samples were taken after an overnight fast and were analysed for the following: leukocytes, erythrocytes, haemoglobin, haematocrit, platelets, triglycerides, cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, C reactive protein (CRP) and fasting blood glucose, liver enzymes (AST, ALT, GGT), urea, creatinine, urate, sodium, potassium, total iron and transferrin.

The serum fatty acid profile was determined by gas chromatography–tandem mass spectrometry at the BIOCentre’s Bioanalytical Laboratory, BIOCentre — incubation centre for biosciences, Zagreb, Croatia (by Thermo Fisher GC Trace 1300 coupled with a TSQ 9000 Triple Quadrupole) with a solution of fatty acid methyl esters (FAME MIX) purchased as 30 mg/mL of the total concentration of fatty acids in methylene chloride (Supelco Inc., Bellefonte, PA, USA) [10, 21].

Selenium concentrations in serum samples were determined by inductively coupled plasma mass spectrometry (ICP-MS) (ICP-MS, Agilent 7500a, Agilent Technologies Inc., California, USA) after appropriate sample preparation. This protocol was optimized by partner institutions through project cooperation [22]. Each serum sample on the ICP was analyzed by internal pooled plasma control and reference material NIST 1567b (wheat flour, National Institute of Standards and Technology, Gaithersburg, MD, USA) was used for the control of the analytical method. All samples were analyzed in triplicate.

Lutein concentrations in serum samples were determined according to the protocol by Tzeng et al. (2004) [23, 24]. Deionized water and 0.01% ascorbic acid (Gram-mol, GRAM-MOL d.o.o., Zagreb, Croatia) dissolved in absolute ethanol (Gram-mol GRAM-MOL d.o.o., Zagreb, Croatia) was added to serum samples and mixed thoroughly. Hexane (Carlo Erba, CARLO ERBA Reagents GmbH, Milano, Italy) was added to the mixture, stirred and centrifuged at 2500 rpm/20 min. The supernatant was separated and evaporated, followed by lutein concentration determination using high-performance liquid chromatography (HPLC). A high-resolution end-capped HPLC column (Hypersil; particle size 5 $\mu$m; 4.6 $\times$ 250 mm) and a C18 analytical column (Shim-pack GIST; particle size 5 $\mu$m; 250 $\times$ 4.6 mm) were purchased. HPLC LC-30 NEXERA (Shimadzu, Japan, 2018) was used for the determination of concentrations.

Vitamin E concentrations in serum samples were determined according to the protocol by Jargar et al. [25]. Serum samples were slowly added to absolute ethanol with gentle shaking to denature the proteins. Sample tubes were shaken for 30 seconds while protected from light. Further, xylene was added to separate the supernatant from proteins. Once again, tubes were mixed thoroughly and centrifuged at 3000 rpm/10 minutes. The supernatant containing xylene was separated in a clean tube and followed by adding 2,2-bipyridyl (Acros Organics, Geel, Belgium, Lot: A0433287) and FeCl${}_3$ (Carlo Erba, CARLO ERBA Reagents GmbH, Milano, Italy, Batch no. V1L014201L), resulting in pink colouration. After incubation, the absorbance was measured using a spectrophotometer (PR 3100 TSC Microplate Reader, BioRad Laboratories, Hercules, CA, USA) at 492 nm, and the obtained absorbance was proportional to the serum vitamin E concentration. A five-point calibration curve was also made.

Approximately 16 mL of whole blood was collected in 10 mL vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) (BD Vacutainer, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and diluted with 1$\times$ Phosphate-buffered saline (PBS), 1:1 ratio. Diluted sample was layered on Ficoll-Paque® PLUS centrifugation media (GE Healthcare Bio-Sciences AB, Uppsala, Sweden, Lot:10296933). Samples were centrifuged at 800 G with brake off, for 25 minutes. Mononuclear cells visible as a cloudy ring separating two layers—yellow plasma and colourless media, were collected and rinsed twice in 1$\times$ PBS. Cell count and viability was estimated by staining with 0.4% Trypan blue solution (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany, Lot: RNBH9907) and using a Bürker-Türk counting chamber. Refrigerated reagents and buffers used in isolation were warmed up to room temperature (RT, ~20–25 °C) prior to isolation. Samples were processed within three hours of collection.

Mixture of dimethyl Sulfoxide (DMSO; Supelco, Merck KGaA, Darmstadt, Germany) and ethylenediaminetetraacetic acid (FBS) was used for cryopreservation purposes (1:9 ratio). Cryovials containing samples were stacked in Mr. Frosty freezing container (Nalgene Labware, Thermo Fisher Scientific, Waltham, MA, USA) containing isopropyl alcohol and placed in a –80 °C freezer for 24 hours before transferring them into labelled containers.

Oxidative stress was measured in the serum of all participants, before and after the respective diet, by spectrophotometric measurement of lipid peroxidation products (Thiobarbituric acid reactive substances, TBARS), while antioxidant capacity was measured in the same manner using the ferric-reducing ability of plasma (FRAP), according to the previously described protocols from our laboratory [26, 27, 28, 29]. Serum levels of myeloperoxidase (MPO) protein were measured in all participants, before and after dietary protocol, by commercially available in vitro enzyme-linked immunosorbent assay (ELISA) kit (Abcam, Cambridge, UK) on compact absorbance reader for 96-well microplates (BioRad PR 3100 TSC, Bio-Rad Laboratories, CA, USA). Spectrophotometric antioxidant enzyme activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) were measured before and after respective dietary protocols from serum samples of study participants practising previously described methods by our research group [26, 30, 31]. The activity of examined enzymes was expressed as U (mg protein) ${}^{-1}$. Measurements were performed using a Lambda 25 UV-Vis spectrophotometer with UV WinLab 6.0 software package (PerkinElmer for the Better, Waltham, MA, USA).

Total RNA was extracted with TRIzol reagent (Life Technologies, Carlsbad, CA, USA, Lot: 13901902) from homogenised stored PBMC samples of participants according to available protocols previously optimized in our laboratory [26, 32]. RNA concentrations and sample purity were determined using a Nanophotometer P300 UV/VIS, IMPLEN. Sample purification and cDNA preparation were performed according to the manufacturer’s instructions from Sigma-Aldrich and Applied Biosystems. Determination of gene expression of nitric oxide synthases (NOS); endothelial nitric oxide synthase (eNOS) - sequence: forward: 5′-CAACAGCATCTCCTGCTCAGA-3′ and reverse 5′-CCACTTCCACTCCTCGTAGC-3′; neuronal nitric oxide synthase (nNOS) - sequence: forward: 5′-ATTTATBCCTTTCCAGCC-3′, and reverse 5′-AGGATCCAGGCTTCAGGCTA-3′; inducible nitric oxide synthase (iNOS) - sequence: forward 5′-AGAGCCAGAAGCGCTATCAC-3′, and reverse 5′-TTGAAGTGGTGCACTCAGCA-3′), antioxidative enzymes SOD2 (sequence: forward: 5′-GGCCTACGTGAACCTGA-3′, and revers 5′-CAGGTTTGATGGCTTCCAGC-3′), GPx1 (sequence: forward: 5′-TATCGAGAATGTGGCGTCCC-3′, and reverse 5′-TCTTGGCGTTCTCCTGATGC-3′) and GPx4 (sequence: forward: 5′-TTCCGGTGTAACCAGTTCGG-3′, and revers 5′-GTGGAGAGACGGTGTCCAAA-3′) was performed by real-time quantitative PCR (Bio-Rad CFX96) from stored PBMC samples of participants. Gene expression was normalized to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH - sequence: forward 5′-ACAGTCAGCCGCATCTTCTT-3′, and revers 5′- AATTTGCCATGGGTGGAAT-3′) gene.

Serum concentrations of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), endothelial-leukocyte adhesion molecule 1, E-selectin (CD62E), type 1 membrane glycoprotein endoglin (CD105), tumour necrosis factor-alpha (TNF-$\alpha$), pro- and anti-inflammatory cytokines including interleukin 17A (IL-17A), interleukin 6 (IL-6) and interleukin 10 (IL-10), were measured before and after respective diet, using the Invitrogen ProcartaPlex antibody-based, magnetic bead reagent kits (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) and panels for multiplex and simplex protein quantitation on the Luminex 200 instrument platform (Luminex Corp., Austin, TX, USA). Measurements were performed in the Laboratory of Molecular and HLA Diagnostics of the University Hospital Osijek (Osijek, Croatia). Data were analysed by using ProcartaPlex Analyst free software (eBioscience, Affymetrix by Thermo Fisher Scientific, Waltham, MA, USA) and are expressed as a concentration in picograms per millilitre.

Serum concentrations of leukotriene B4 (LTB4) (Cusabio, Houston, TX, USA, LTB4 Cat. No. CSB-E08033h) and B5 (LTB5) (MyBioSource, MyBioSource Inc., CA, USA, LTB5 Cat.No. MBS3800320), prostaglandins E2 (PGE2) and E3 (PGE3) (MyBioSource, MyBioSource Inc., San Diego, CA, USA, PGE2 Cat. No. MBS007171, PGE3 Cat.No. MBS9310830) were measured by commercially available enzyme-linked immunosorbent assay (ELISA) kits on compact absorbance reader for 96-well microplates (BioRad PR 3100 TSC, Bio-Rad Laboratories, Hercules, CA, USA).

Statistical analyses were performed using Graph Pad Prism v6.01 (GraphPad Software, San Diego, CA, USA) and Microsoft Excel 2016 (Microsoft Office 365, Microsoft Corporation, Redmond, WA, USA). GPower v3.1.9.7 software (Heinrich Heine University Düsseldorf, Düsseldorf, Germany) was used for sample size calculation. Cohen’s d ($\mathrm{\Delta }$ / SD) effect size was determined according to the primary outcome of the study. A pilot study was conducted during the preparation of the study. The sample size required to show a potentially significant effect was calculated based on preliminary data collected from a total of 16 subjects. To detect differences in primary outcomes recorded in this study with a significance level of 0.05 and a statistical power of 80% for a paired t-test, the required sample size was 14 subjects per group.

The normality of distributions was tested by Shapiro–Wilk test. Data are presented as average $\pm$ standard deviation (SD). Student’s t-test and Mann–Whitney tests were used for group comparisons, while Paired t-test and Wilcoxon rank-sum tests were used to test the differences between the measurements within a group. Correlations between paired datasets were determined by the Spearman rank test. Two-tailed p 0.05 was considered significant.

The content of n-3 PUFAs, selenium, lutein and vitamin E in the edible part of regular and enriched hen eggs consummated by study participants is shown in Table 1.

Initial anthropometric and hemodynamic values of all participants enrolled in the study are presented in Table 2. There were no significant differences regarding the age of participants between the control (21.9 $\pm$ 2.6 years old) and 4Nutri group (21.4 $\pm$ 2 years old). According to the average baseline BMI of both control (24.33 $\pm$ 3.6 kg/m${}^2$) and the 4Nutri group (23.49 $\pm$ 3.1 kg/m${}^2$), study participants fit in the normal BMI range according to the WHO criteria for European population weight classification (18.5–24.9 kg/m${}^2$).

Participants were lean and normotensive, and there were no differences in these values after consumption of regular nor enriched eggs compared to respective baseline values as there were no significant differences between the two experimental groups. Based on the medical history and biochemical characteristics presented in Table 3, all participants had normal fasting blood glucose, hsCRP, and fasting lipid profile levels. There were no significant differences in measured parameters after both diet protocols when compared to respective baseline values.

Following the three-week dietary protocol, serum levels of EPA, DHA, vitamin E and lutein significantly increased only in the 4Nutri group compared to the baseline levels following the dietary protocol as presented in Table 4.

Three-week consumption of enriched eggs did not significantly alter serum TBARS or FRAP in the 4Nutri group compared to respective baseline values, neither did regular eggs in Control group (Fig. 2). MPO serum values were significantly increased in the 4Nutri group after consumption of enriched eggs compared to baseline values (Fig. 3). MPO serum values in the Control group remained unaltered after consumption of regular eggs. Antioxidative enzyme activity was measured for superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT). There were no significant differences detected in the Control or 4Nutri group after respective dietary protocols compared to the respective baseline values, although there was a slight trend towards an increase of CAT activity (p = 0.08) in the 4Nutri group after dietary protocol (Table 5).

Fig. 2.

Effects of regular (Control group) and enriched hen eggs (4Nutri group) consumption on the serum levels of oxidative stress (TBARS) (A) and antioxidant capacity (FRAP) (B) after three-week diet protocol. Effects of regular (Control group) and enriched hen eggs (4Nutri group) consumption on the serum levels of oxidative stress (TBARS) (A) and antioxidant capacity (FRAP) (B) after three-week diet protocol. Both diets didn’t significantly alter neither marker after respective diet protocols. Paired t-test; significance level p 0.05; before vs. after within the group (Control or 4Nutri).

Fig. 3.

Effects of regular (Control group) and enriched hen eggs (4Nutri group) consumption on the serum levels of MPO after three-week diet protocol. Paired t-test; significance level p 0.05; before vs. after within the group (Control or 4Nutri).

mRNA expression of only nNOS was significantly increased in PBMCs of participants in 4Nutri group after dietary protocol compared to the baseline levels (Fig. 4E). mRNA expression of antioxidative enzymes remain unchanged (Fig. 4A–D, 4F).

Fig. 4.

Effects of regular (Control group) and enriched hen eggs (4Nutri group) consumption on relative gene expression of GPx1 (A), GPx4 (B), SOD2 (C), iNOS (D), nNOS (E) and eNOS (F) in PBMCs after three-week diet protocol. GPx, glutathione peroxidase; SOD, superoxide dismutase; iNOS, inducible nitric oxide synthase; nNOS, neural nitric oxide synthase; eNOS, endothelial nitric oxide synthase. Paired t-test; significance level p 0.05; before vs. after within the group (Control or 4Nutri).

Serum concentrations of pro- and anti-inflammatory cytokines (IL-17A, IL-10) and tumour necrosis factor-alpha in the control and 4Nutri group, before and after dietary protocols, are presented in Fig. 5. There were no significant changes of IL-10 or TNF-$\alpha$ (Fig. 5A,B) observed in any of the groups after respective diet protocols. There was a significant decrease in IL-17A serum concentration after the consumption of eggs enriched with four nutrients compared to the baseline values (p = 0.031) (Fig. 5C).

Fig. 5.

Effects of regular (Control group) and enriched hen eggs (4Nutri group) consumption on the serum concentrations of tumor necrosis factor alpha (TNF-$\alpha$) (A), anti- inflammatory cytokine interleukin 10 (IL-10, B) and pro-inflammatory cytokine IL-17A (C) after three-week diet protocol. TNF, tumor necrosis factor; IL, interleukin. Paired t-test; significance level p 0.05; before vs. after within the group (Control or 4Nutri).

Serum concentrations of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), endothelial-leukocyte adhesion molecule 1 E-selectin (CD62E), type 1 membrane glycoprotein endoglin (CD105) before and after consumption of regular eggs and eggs enriched with four nutrients, are presented in Table 6. There were no significant changes observed in either control or 4Nutri group or between groups before or after respective diet protocols or compared to baseline values.

Serum concentrations of pro-inflammatory eicosanoids (LT B4 and PG E2) from AA, and inflammation-resolving oxylipins (LT B5 and PG E3), before and after the dietary protocols, are shown in Fig. 6. Pro-inflammatory LT B4 serum concentrations significantly increased in the Control group after dietary protocol (Fig. 6A), while anti-inflammatory LT B5 serum concentrations remained unchanged in both groups compared with respective baseline values (Fig. 6B). Serum concentrations of PGE2 (Fig. 6C) significantly increased while PGE3 concentrations remained unchanged (Fig. 6D) following consumption of regular hen eggs. Leukotriene B4/B5 (Fig. 7A) and prostaglandin E2/E3 (Fig. 7B) ratios were calculated and compared across the measurements. Both ratios remained unchanged in the 4Nutri group, while the prostaglandin E2/E3 ratio significantly increased in Control group (p = 0.008).

Fig. 6.

Effects of regular (Control group) and enriched hen eggs (4Nutri group) consumption on the serum concentrations of lipid mediators LTB4 (A), LTB5 (B), PGE2 (C) and PGE3 (D), after respective dietary protocols. LTB4, leukotriene B4; LTB5, leukotriene B5; PGE2, prostaglandin E2; PG E3, prostaglandin E3. Paired t-test; significance level p 0.05; before vs. after within the group (Control or 4Nutri).

Fig. 7.

Effects of regular (Control group) and enriched hen eggs (4Nutri group) consumption on leukotriene B4/B5 (A) and prostaglandin E2/E3 (B) serum ratio after respective dietary protocols. LTB4, leukotriene B4; LTB5, leukotriene B5; PGE2, prostaglandin E2; PGE3, prostaglandin E3. Paired t-test; significance level p 0.05; before vs. after within the group (Control or 4Nutri).

Correlation analysis was performed to assess the relationship between all measured parameters: biochemical and anthropometric measurements, serum concentrations of PUFAs derived lipid mediators, cytokines, adhesion molecules, oxidative stress parameters and antioxidative enzymes activity/expression. Results are as follows: BMI of study participants correlated positively with triglyceride serum levels in the Control group (r = 0.643; p = 0.0001) and with WHR in the 4Nutri group (r = 0.550; p = 0.0002).

In the Control group, serum MPO concentrations positively correlated with PGE2 serum concentrations (r = 0.646; p = 0.002) and negatively with SOD activity (r = –0.590, p = 0.009). Furthermore, lipid mediators PGE2 and LTB4 concentrations correlated positively after regular eggs consummation (r = 0.546, p = 0.002). There was also a significant positive correlation between serum concentrations of TNF-$\alpha$ and ICAM-1 (r = 0.587; p= 0.02), as well as between E-selectin and LDL-cholesterol (r = 0.643; p = 0.004). There was a strong positive correlation noticed between nNOS and iNOS in the Control group (r = 0.796; p 0.0001).

In the 4Nutri group, serum TNF-$\alpha$ concentrations significantly negatively correlated with ICAM-1 concentration (r = –0.665; p = 0.003) while positively correlated with iNOS expression (r = 0.585; p = 0.007). A significantly negative correlation was observed between serum IL-17A concentrations and endoglin (r = –0.627; p = 0.005), and serum IL-17A and HDL-cholesterol (r = –0.432; p = 0.045), while serum IL-17A positively correlated with antioxidant potential in samples (FRAP) (r = 0.445; p = 0.038). nNOS expression in PBMCs of 4Nutri group negatively correlated with cholesterol (r = –0.458; p = 0.01) and LDL-cholesterol serum levels (r = –0.464; p = 0.009). Further, ICAM-1 in serum negatively correlated with PGE2 concentration (r = –0.548; p = 0.02).

The study population in the present study consisted of mainly students. There were no major differences between study groups regarding their daily dietary habits. Participants mainly failed to adhere to recommended guidelines for consuming at least 5 meals/per day ($>$70% of participants). Approximately 54% of participants consumed three or fewer fruit/vegetable units per day which are below the recommended daily intake of five units. Also, their intake of n-3 PUFAs-rich food is low. More than 90% of participants consume dairy fermented products. Only a small part of study participants (~30%) include foods rich in n-3 PUFAs in their weekly diet plan. The results of this dietary assessment are consistent with previously published results by our research group regarding the dietary habits of students [15].