Cardio-metabolic and Immunological Impacts of Extra Virgin Olive Oil Consumption in Overweight and Obese Older Adults

A Randomized Controlled Trial

Mitra Rozati; Junaidah Barnett; Dayong Wu; Garry Handelman; Edward Saltzman; Thomas Wilson; Lijun Li; Junpeng Wang; Ascensión Marcos; José M. Ordovás; Yu-Chi Lee; Mohsen Meydani; Simin Nikbin Meydani


Nutr Metab. 2015;12(28) 

In This Article

Subjects and Methods


Participants for this study were recruited by the Recruitment and Volunteer Services Department at the Jean Mayer USDA Human Nutrition Research Center on Aging (HNRCA) at Tufts University by inviting individuals within the specified age and body mass index (BMI) ranges in the HNRCA recruitment database, advertising in various local newspapers, in media sources, at the Tufts University Boston campus, Tufts Medical Center clinics, and on public bulletin boards in the downtown Boston area and neighboring towns. A total of 960 responses were received. After telephone pre-screening, 799 individuals were considered ineligible because they either were no longer interested or did not meet study criteria. Following laboratory screenings, an assessment of medical history and a physical examination performed by a study nurse practitioner in the Metabolic Research Unit (MRU) at the HNRCA, an additional 117 of the remaining 161 individuals were found ineligible to participate.

Subjects were excluded if they reported that they did not speak English, were blind or deaf, were on a vegetarian diet, ate more than 3 meals per week at a restaurant, had chronic eating disorders, were HIV+, or had autoimmune diseases or cancers (except for non-melanoma skin cancer), used chemotherapy or immunosuppressive drugs, or had any major illnesses including uncontrolled CVD, liver disease, renal disease, diabetes, hypertension, asthma, a history of splenectomy, were on dialysis or had any conditions associated with maldigestion or malabsorption including pancreatitis, celiac disease, gastric bypass or surgery for weight loss. Additionally, they were excluded if they were diagnosed with or were in treatment for psychosis and had obtained a BDI-II (Beck Depression Inventory-II) score ≥ 20 or MMSE (Mini Mental State Examination) score ≤ 25. Finally, they were excluded if they consumed more than 2 glasses of alcoholic drinks/day or had a history of smoking; further, they were excluded if any of the following occurred prior to study blood draws and skin tests: they had used nicotine within 6 months, were on antibiotics or had infections within 2 weeks, received flu vaccination within 3 weeks and tetanus immunization within 6 weeks.

Subjects of both genders were included if they were 65 y or older, had a BMI between 25–35 kg/m 2, were willing to stop using dietary supplements (except vitamin D and calcium) including multivitamin and minerals, fish oil, olive oil, and canola oil, 30 days before and during the study. Additionally, they were consuming and were required to continue consuming a typical American diet. The diet composition of the typical American diet has been determined by National Health and Nutrition Examination Survey (NHANES) as follows: carbohydrates make up 50 % of daily energy requirements and include refined and processed grain products, fruits and vegetables and dairy products. Protein constitutes 15 % of daily energy requirements provided from sources such as meats and dairy products. Fat makes up 35 % of daily energy requirements, which represents 86 g of fat/day in a 2200 calorie diet. The fatty acid composition includes polyunsaturated fatty acids (PUFA), saturated fatty acid (SFA), and monounsaturated fatty acids (MUFA) at the ratio of 1:2:2. Dietary fiber and cholesterol intake are 12–14 g/day and ≥400 mg/day, respectively.[46,47]

The forty-four subjects who were eligible to participate in the study signed the informed consent form, and 41 participants completed the study. Three (3) participants dropped out of the study: one was prescribed an iron supplement, one refused to have DTH skin test implant, and one had an accident unrelated to the study (Fig. 1).

Figure 1.

Study profile for recruitment and enrollment

Study Design

We conducted a randomized, single-blinded, and placebo-controlled trial in overweight and obese older adults between 2011 and 2013 to determine the impact of replacing substitutable oils in a typical American diet with extra virgin olive oil. The study protocol and consent form were approved by the Tufts University/Tufts Medical Center Institutional Review Board. Eligible participants were randomized into either the CON or OO group in a 1:1 ratio.

The coding for the CON and treatment groups was assigned by the study statistician, who had no contact with subjects and had no role in data collection. The study oils (CON or OO) were labeled with the subjects' names by the study dietitian, who also held the randomization code; all other investigators and the study nurses were blinded to this information. Participants, however, could not be blinded since they might recognize the smell and taste of EVOO.

The oils were provided in a bottle or as a spread. To minimize bias, both study oils were provided to participants in the same type of bottle (oil) and the spread in the same type of container (spread). The CON oil was a blend of 10 % corn oil and 90 % soybean oil, and the CON spread was butter; these were defined as the oils consumed in a typical American diet.[48] The EVOO used for the study was provided by the Deoleo Company in Cordoba, Spain. Prior to screening, participants were asked to taste the oil/spread and to confirm they were willing to consume these oils while on the study ( Table 1 shows the composition of fatty acids in both the CON and OO groups).

Participants consumed the assigned oils for 3 months. During the study, both groups continued their typical American diet with only one change: they replaced substitutable oils in their diet (cooking oil, spread, and oils in dressings) with the study oil/spread provided to them. The study oils were distributed to the participants by the dietitians at the MRU of the HNRCA.

Baseline and Month 3 Study Visits

Subjects were asked to come to the center for 4 consecutive days at the beginning (baseline) of the study and 3 months after enrollment. On day 1, vital signs (temperature, blood pressure, pulse, and respiratory rate), weight, height, and waist and hip circumferences were measured. Subjects were asked to discontinue anti-inflammatory medicines including aspirin or anti-histamines 72-h before each blood collection until 48-h after DTH implantation. On day one, blood was collected after a 12-h fast for blood chemistry, lipid profile, complete blood count (CBC) differential, fatty acids, and immunological tests described below. On day 2, a second blood sample was drawn for repeated ex vivo immune tests. Then, three (3) recall antigens (detailed below) and saline were implanted in the forearm of each subject for the DTH skin test, as an in vivo test of cell-mediated immune function.[49] On day 3 and 4, participants visited the MRU for an evaluation of their DTH skin response at 24-h and 48-h post-administration, respectively.

Diet Intervention Visits and Assessment of Dietary Intake

Dietitians at the MRU at HNRCA provided participants with instructions for substituting fats used in their diets with study oils and spreads. Dietary counseling was provided every two weeks or more often as needed to ensure that all subjects were following the instructions, were using the right oil and adhering to the appropriate diet. Compliance with consuming study oils was evaluated by the dietitians using a "checklist of study oils consumed per week" as indicated by participants when they returned used containers for a new supply of study oils and spreads as well as by plasma fatty acid analysis (see below). To assess dietary intake, participants completed a 3-day dietary record (3DDR) at both baseline and month 3, which were reviewed and analyzed by a dietitian using the Minnesota Nutrient Data System (Nutrition Coordinating Center, University of Minnesota, Minneapolis, MN), version 2010.

CBC Differential, Blood Chemistry, and Blood Lipid Profile

Fasting blood was used for evaluation of CBC-differential,[50] blood glucose,[51] and blood lipid profile [triglycerides,[52] total cholesterol,[53] low density lipoprotein cholesterol (LDL-C), and high density lipoprotein cholesterol (HDL-C)[54] at baseline and month 3 by the Nutritional Evaluation Laboratory (NEL) at the HNRCA.

Fatty Acid Analysis

Compliance of study oil intake was determined by measuring fatty acid composition in the plasma of participants by gas chromatography (GC) method.[55]

Immunological Assessment

Lymphocyte Phenotype. To determine lymphocyte phenotype, whole blood surface staining of different white blood cell markers was done using fluorescent antibodies (all from eBioscience, San Diego, CA) for total T cells (CD3+), helper T cells (CD4+), cytotoxic T cells (CD8+), naïve and memory subpopulations within CD4 and CD8 T cells (determined by their combined expression of CD45RA, CD45RO, and CD62L), B cells (CD19+), and natural killer T cells (CD3 + NK+). Isotype staining was also done as the negative control. Flow cytometric measurement was conducted using an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA). Data were analyzed using Flowjo 7.6 software (TreeStar Inc., Ashland, OR).

Lymphocyte Proliferation. T cell proliferation was measured by [3H]-thymidine incorporation after stimulation of whole blood culture with different stimuli described below. To account for the day-to-day variability in ex vivo immunological measurements, blood was collected two times (day 1 and day 2), and the average of the two measurements was used in statistical analysis. Heparinized blood was diluted 1:10 (final v: v) with RPMI 1640 supplemented with 100 kU/L penicillin, 100 mg/L streptomycin, 2 mmol/L L-glutamine, and 25 mmol/L HEPES (Gibco Laboratories, Grand Island, NY). Diluted blood was stimulated with T cell mitogen concanavalin A (Con A, Sigma, St. Louis, MO) at 5, 25, and 50 mg/L, or immobilized anti-CD3 (eBioscience) at 1, 5, and 10 mg/L and soluble anti-CD28 (1 mg/L), and incubated in 96-well round-bottom cell culture plates for 72-h. Cultures were pulsed with 0.5 μCi [3H]-thymidine during the last 4-h of incubation. Cells were harvested onto glass fiber mats using a Perkin Elmer cell harvester (model No. C961961, Waltham, MA). Cell proliferation was quantified as the amount of [3H]-thymidine incorporation into DNA, which is determined by liquid scintillation counting in a Perkin Elmer counter (model No. 2450–0060). T cell proliferation is expressed as count per minute (cpm).

Cytokine and PGE2 Production. Heparinized whole blood was diluted 1:4 (final v: v) with RPMI 1640. To determine T cell cytokine IL-2 and IFN-γ production, diluted whole blood was stimulated with Con A (25 mg/L) or immobilized anti-CD3 (5 mg/L) and soluble anti-CD28 (1 mg/L) in 24-well plate for 48-h. In addition, we also assessed the impact of olive oil on inflammatory response. Diluted whole blood was stimulated with lipopolysaccharide (LPS) (1 mg/L) in 24-well plates for 24-h to measure the production of pro-inflammatory cytokines IL-6 and TNF-α as well as lipid inflammatory mediator PGE 2 using ELISA.

For all cytokines including IL-2, IFN-γ, IL-6, and TNF-α, reagents were from BD Bioscience. For PGE 2, high sensitivity enzyme immune assay (EIA) kit (ENZO Life Science Inc, PA, USA) was used. Plates were read in a micro-plate reader (BioTek, model No. EL808) and analyzed by Gene5 software.

Delayed-Type Hypersensitivity (DTH) Skin Test. Three recall skin test antigens, Candida Albicans (Candin, Allermed Laboratories Inc, San Diego, CA), Tetanus Toxoid (Toxoid Adsorbed, Sanofi Pasture, CA), and Trichophyton Mentagrophytes (Trichophyton, Allermed Laboratories Inc, San Diego, CA) were injected intradermally at separate sites on the volar surface of the forearm. Saline (0.9 %) was used as the negative control. Candida, Trichophyton and saline were injected in a standard volume of 0.1 ml, and tetanus toxoid was injected in a volume of 0.025 ml. Skin tests were read 24 and 48-h after antigens were applied; a test was considered positive if the skin induration was ≥ 5 mm.

Statistical Analysis

The demographic characteristics and other variables of interest of the participants in the CON versus OO group were compared at baseline by using Student's t-test or Chi-square test. Paired t-test or nonparametric test was performed to compare the measures at baseline and month 3 within each group. Differences in outcome measures of interest between the groups were determined using Analysis of Covariance (ANCOVA) controlling for their corresponding baseline levels, sex and age or using non-parametric test. We used log transformation for the data that were not normally distributed. Two-sided significance at P < 0.05 was considered to be significant. Analyses were performed using IBM SPSS Statistics version 21.