Background: The 2015–2020 Dietary Guidelines for Americans recommend a Mediterranean-type diet as one of three healthful eating patterns. However, only one previous trial has evaluated the effects of a Mediterranean diet intervention in a US sample population. Methods: To address this gap, we conducted a pilot, non-blinded, 8-week randomized controlled trial on the comparative efficacy of consumption of a Mediterranean diet or a diet supplemented with fish oil, walnuts, and grape juice versus controls. Participants (overweight or obese US adults; 73% female and mean age 51 years) were randomly assigned to one of three groups: (1) Mediterranean diet; (2) habitual high-fat American-type diet supplemented with fish oil, walnuts, and grape juice; or (3) habitual high-fat American-type diet (controls). Intent-to-treat analysis of within-subject differences (Student’s paired t-test or Wilcoxon sign ranks test) and between-subject differences (mixed-effects models with a group-by-time interaction term, adjusted for baseline health outcome) was conducted. Results: Participants in the Mediterranean diet arm (n = 11) had significantly greater weight loss despite no significant change in total caloric intake, and lower plasma cystine, indicative of decreased oxidative stress, compared to controls (n = 9) at both 4 and 8 weeks. Compared to controls, they also had significantly lower total cholesterol and low- density lipoprotein cholesterol levels at 4 weeks. Participants in the supplement arm (n = 10) had significantly lower adiponectin levels compared to controls at 4 weeks. No significant improvements in endothelial function or inflammatory biomarkers were observed in either intervention group compared to controls. Conclusion: These results suggest that adopting a dietary pattern reflecting a Mediterranean diet improves weight and cardio-metabolic health among overweight or obese US adults, and may be more beneficial than supplementing habitual American diets with fish oil, walnuts, and grape juice. Trial registration: ClinicalTrials.gov NCT00166088. Registered 14 September 2005. Keywords: Mediterranean diet, Omega-3 fatty acids, Hyperlipidemia, Endothelial function, Oxidative stress * Correspondence: firstname.lastname@example.org The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. Department of Global Health and Population, Harvard T.H. Chan School of Public Health, 665 Huntington Ave, Building 1, Room 1211, Boston, MA 02115, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jaacks et al. BMC Nutrition (2018) 4:26 Page 2 of 8 Background conducted by trained study staff at the Emory University A Mediterranean diet supplemented with either olive oil Hospital Clinical Research Center (CRC), formerly the or nuts is the only dietary intervention that has been shown General Clinical Research Center, of the Atlanta Clinical to prevent major cardiovascular events in a randomized and Translational Science Institute. The study was ap- controlled trial . Consistent with this, several smaller proved by the Emory University Institutional Review Board trials of the Mediterranean diet conducted in Italy, Greece, and all participants provided written informed consent. Spain, France, and Finland, have variously reported pro- The inclusion criteria were as follows: aged 40–65 years, 2 2 tective effects against cardiovascular disease (CVD) risk body mass index (BMI) ≥28 kg/m and < 35 kg/m ,stable factors including adiposity, hypercholesterolemia, hypergly- body weight in the past 6 months (defined as weight cemia, insulin resistance, endothelial dysfunction, oxidative change < 2 kg), currently a non-smoker, currently con- stress, and/or inflammation [2–5]. suming a stable habitual diet as assessed by interview Despite new recommendations in the 2015–2020 with the research nutritionist, not consuming anti-oxi- Dietary Guidelines for Americans suggesting that a dants or vitamin-mineral preparations in the past Mediterranean-type diet is healthful , only one trial, 4 weeks, and 24-h diet recall showing saturated and to our knowledge, has evaluated the effects of a Mediter- trans fat intake of > 10% of total daily calories and total ranean diet intervention in a US population . In that cholesterol intake of > 300 mg/day. Participants meet- study of non-obese adult women in Michigan, guidance ing any of the following criteria were excluded: known on Mediterranean diet intake using a dietary component history of cardiovascular diseases including coronary exchange list was coupled with serial counseling by a artery disease, valvular heart disease, arrhythmias or dietitian for adherence based on food records at baseline, cardiomyopathies; renal or liver disease defined as cre- 3 months, and 6 months . Compared to the control atinine > 2.0 mg/dL and liver function tests > 3-times group following a habitual diet, no changes in blood lipids, upper limit of normal; history of diabetes or fasting except for an increase in plasma monounsaturated fatty plasma glucose (FPG) > 126 mg/dL; history of cancer acids, were noted in the Mediterranean diet group . other than skin cancer; systolic blood pressure > It is unclear from previous studies whether the Mediter- 180 mmHg and diastolic blood pressure > 110 mmHg; ranean diet as a whole is necessary to see improvements hematocrit < 30%; and other known acute or chronic ill- in health, or if habitual diets can be supplemented with ness, including psychiatric disorders, excessive chronic key components of the Mediterranean diet with similar alcohol consumption (> 2 alcoholic beverages/day), sta- benefit. It has been hypothesized that much of the benefit tin or other hypolipidemic therapy, and pregnant or of the Mediterranean diet stems from omega-3 polyunsat- lactating females. No imaging was performed for exclu- urated fatty acids, including eicosapentaenoic acid (EPA) sion of fatty liver disease. and docosahexaenoic acid (DHA) found in fish, and Following a screening visit, participants were randomly α-linolenic acid found in nuts [8, 9]. Moderate alcohol assigned to one of three groups: (1) Mediterranean diet; consumption, especially the phytochemicals found in (2) habitual high-fat American-type diet supplemented wine, is also thought to provide cardio-metabolic with fish oil, walnuts, and grape juice daily; or 3) habitual benefit [10, 11]. high-fat American-type diet (controls). Study measurements The rationale for this study is therefore two-fold: (1) were made at a baseline visit and again at 4 and 8 weeks to establish whether or not a Mediterranean-type diet is after the baseline visit. protective against CVD risk factors in a US population and (2) whether or not supplementing a habitual high-fat Mediterranean diet intervention American-type diet with several key components of the Participants randomized to the Mediterranean diet arm Mediterranean diet can produce similar reductions in received three meals with beverages and two snacks per CVD risk factors. Here we report results of a pilot, day as a prototypical Mediterranean diet prepared by the non-blinded, 8-week randomized controlled trial to test CRC metabolic kitchen for four consecutive weeks, whether a Mediterranean diet or a habitual high-fat coupled with verbal and written dietary instruction. American-type diet supplemented with several key com- During the first four-week period, meals and snacks ponents of the Mediterranean diet reduces CVD risk were picked up by participants at the CRC metabolic factors compared to a habitual high-fat American-type kitchen every three to four days and any issues with the control diet among overweight or obese adults. meal plan were discussed with the research nutritionist. The seven-day menu from the first week was rotated Methods on a day-to-day basis during the subsequent three weeks Sample population to avoid monotony. Verbal and written dietary instruction Participants were recruited from Emory Healthcare out- on the advantages and composition of the Mediterranean patient clinics and by advertisements. Assessments were diet were given by the CRC research nutritionist during Jaacks et al. BMC Nutrition (2018) 4:26 Page 3 of 8 the first four-week period. During the second four-week Outcome assessment period, participants received intensive verbal and detailed Anthropometric measurements (weight and waist circum- written dietary instruction and reinforcement to ensure ference) were assessed using standardized procedures consumption of a prototypical Mediterranean diet using by trained CRC staff. Blood lipids (total cholesterol, tri- their own home-cooked meals. The CRC research nutri- glycerides, high-density lipoprotein [HDL] cholesterol, tionist formally discussed and reinforced dietary principles and low-density lipoprotein [LDL] cholesterol) were with the participant weekly, either in person (baseline, measured using a CX7 chemistry analyzer (Beckman weeks 2, 4, and 6) or via telephone (weeks 1, 3, 5, and 7). Diagnostics, Fullerton, CA) by technicians at the Emory Appropriate adjustments were made, as needed, to indi- Healthcare Medical Laboratory. Inflammatory biomarkers vidual food items to ensure high compliance. and adipokines included IL-6, IL-8, C-reactive protein Meals and food plans were designed using ProNutra™ (CRP), and adiponectin. IL-6 and IL-8 were measured (Viocare, Inc., Princeton, NJ) to provide daily energy for with a Fluorokine MAP MultiAnalyte Profiling Human weight maintenance as determined by the Harris-Bene- Base Kit (R&D Systems, Inc., Minneapolis, MN) on a dict equation . Protein intake was provided at the Luminex – 200 platform. IL-6 values that were below the Recommended Dietary Allowance level of 0.8 g/kg/day; limit of detection were assigned a value of 0.01 pg/mL. saturated and trans fats at < 7% of total energy intake; CRP was measured using the Dade-Behring Nephelometry and cholesterol at < 200 mg/day. Using ProNutra™, meals System (BNII). One participant had a CRP level less than for individual participants were based on the diet scoring the limit of detection (LOD) and was assigned a value for Mediterranean diet adherence . Meals included of LOD/sqrt(2). Adiponectin was measured with the an abundance of plant food (fruits, vegetables, whole Quantikine Human Adiponectin Immunoassay solid-phase grains, nuts, and legumes); olive oil as the primary source ELISA (R&D Systems, Inc.,Minneapolis,MN).Markers of of fat; fish, poultry, and eggs in moderate to low amounts; hyperglycemia, including FPG and insulin were measured low consumption of red meats, saturated fats, and sweets; using a CX7 chemistry analyzer (Beckman Diagnostics, and consumption of either one to two ounce drinks per Fullerton, CA) by technicians at the Emory Healthcare day of wine (in those who habitually consumed wine) or Medical Laboratory. grape juice (in those who did not habitually consume Brachial artery reactivity was used to test flow-mediated alcohol). vasodilation (FMD) as a measure of endothelial function and more specifically of nitric oxide bioavailability. This Diet supplementation intervention outcome was chosen because endothelial dysfunction Participants assigned to their habitual high-fat American- integrates risk factor-mediated injury to the endothelial type diet supplemented with key components of the Medi- cells. It is an early marker of risk for development of terranean diet were given an eight-week supply of specific atherosclerosis and its adverse outcomes and thus provides dietary supplements by the CRC nutritionist and advised a sensitive, reproducible and non-invasive tool for investi- to consume them daily, in addition to their usual diet. gation of the effects of short-term dietary intervention (e.g., They were also advised to decrease caloric intake from an excellent experimental tool) [14, 15]. All measurements usual dietary constituents (e.g., not the dietary supple- were performed in a temperature-controlled laboratory ments) if they observed body weight gain of more than after an overnight fast. The brachial artery of the 0.5 kg during any given week. The supplements included: non-dominant arm was imaged using a high-resolution (1) two 1-g pills of Omacor® fish oil supplements (1.8 g of 10 MHz linear array ACUSON™ ultrasound transducer EPA/DHA) per day, (2) 1/3 cup shelled walnuts per day, (Siemens Medical Solutions USA, Inc., Malvern, PA) at and (3) 16 ounces (about 475 mL) Welch’s® 100% Concord baseline and continually for 120 s after producing 5-min grape juice per day. ischemia of the hand. Arterial diameter was measured as the distance from the leading edge of the intima-lumen Control group interface of the near wall to the leading edge of the These participants did not alter their diet, take supplements, lumen-intima interface of the far wall by an investigator alter usual activity, and were not given dietary advice. blinded to the treatment. FMD was calculated as the percent increase in brachial artery vasodilator response: Dietary intake assessment (post-hyperemia diameter-baseline diameter)/baseline Participants were asked to complete 3-day food re- diameter × 100. cords at baseline and again at 4 and 8 weeks after the Circulating pro-angiogenic cell activity (CFU-As) were baseline visit. Nutrient composition was determined measured using a colony forming assay from circulating using ProNutra™ (Viocare, Inc., Princeton, NJ). The mononuclear cells as described previously [16, 17]. Briefly, 3-day average nutrient intake at each time point was mononuclear cells were isolated by density-gradient cen- used in the final analysis. trifugation from a 20 ml sample of venous blood using Jaacks et al. BMC Nutrition (2018) 4:26 Page 4 of 8 CPT tubes (Becton, Dickinson and Company, Franklin intake, and a significant increase in monounsaturated, Lakes, NJ), and washed two times with PBS. The cells were omega-3, and omega-6 fatty acid intakes compared to suspended in growth medium (DMEM supplemented with controls at 4 weeks (Table 1). These differences were 20% fetal bovine serum and 6.5% endothelial cell growth attenuated and no longer statistically significant by supplement), and plated on human fibronectin-coated cell 8 weeks. Participants in the supplement arm had sig- culture dishes. To eliminate mature circulating endothelial nificantly higher total fat, monounsaturated, omega-3, cells, cells adherent after 24 h were discarded and nonad- and omega-6 fatty acid intakes compared to controls at herent cells were re-plated onto new fibronectin-coated both 4 and 8 weeks. plates at 1 million cells/well. Growth medium was changed every 2 days. After 7 days, CFU-As were counted manually Effects of Mediterranean diet andrecordedbyanobserverwho was blindedtothe clin- Among participants who were assigned to the Mediterra- ical data. Colonies were identified as central clusters of nean diet arm, there was a significant decrease from base- rounded cells with multiple flat cells emanating from the line to 8 weeks in body weight (− 2.2 [2.6] kg, p =0.03), central clusters. total cholesterol (− 24.9 [19.8] mg/dl, p = 0.003), LDL Biomarkers of oxidative stress included plasma cysteine cholesterol (− 24.9 [17.3] mg/dl, p = 0.003), and CFU-As (reduced form), cystine (oxidized form), and glutathione (− 40.3 [36.4] cfu, p = 0.01). There was also a non-signifi- (reduced form), and were measured using high-per- cant increase in FMD from baseline to 4 weeks (2.3 [4.0] formance liquid chromatography with fluorescence percent, p = 0.08). The average percent increase in FMD detection as described previously . relative to baseline among participants assigned to the Mediterranean diet arm was 66.1% at 4 weeks and 44.3% Statistical analysis at 8 weeks. Values presented are mean with standard deviation given When compared to changes in controls, participants in parentheses. Normality was tested using the Shapiro- in the Mediterranean diet arm had significantly greater Wilk test statistic and visually assessed using Q-Q plots. weight loss at both 4 and 8 weeks and greater decreases Differences in demographic, clinical, and dietary factors in total cholesterol and LDL cholesterol at 4 weeks between arms at baseline were assessed using Fisher’s (Table 1; Fig. 1). Moreover, compared to controls, partic- exact test for categorical variables and analysis of vari- ipants in the Mediterranean diet arm had significantly ance (ANOVA) for continuous variables. Within-subject greater decreases in CFU-As at 8 weeks, and in plasma differences in the health outcomes were analyzed using cystine (indicative of decreased oxidative stress) at both Student’s paired t-test for normally distributed variables 4 and 8 weeks. However, compared to changes in controls, and Wilcoxon sign ranks test for non-normally distrib- participants in the Mediterranean diet arm had no signifi- uted variables [19, 20]. Mixed-effects models were used cant changes in any of the inflammatory biomarkers or to analyze between-subject differences in the health adipokines (IL-6, IL-8, CRP, and adiponectin), markers of outcomes . Models included a group-by-time inter- hyperglycemia (FPG and insulin), or FMD. In a subgroup action term. Baseline health outcome was adjusted in analysis of participants with FMD < 6% at baseline (n =8 all models. Statistical significance was considered for P < Mediterranean diet arm and n = 5 controls), there was a 0.05. All analyses were conducted using SAS version 9.4 non-significant increase in FMD (p = 0.07) among those in (SAS Institute, Cary, NC). the diet group compared to controls at 4 weeks. Results Effects of dietary supplementation with fish oil, walnuts, 48 individuals were screened for inclusion. Of these, 11 and grape juice (23%) were ineligible (n = 9 for BMI < 28 kg/m or ≥ Among participants who received supplements, there was 35 kg/m and n = 2 for cancer history) and 37 were ran- a significant decrease from baseline to 8 weeks in triglycer- domly assigned to control (n = 11), Mediterranean diet ides (− 17.4 [16.8] mg/dl, p = 0.01), but in none of the other (n = 11), or diet supplements (n = 15). Of these, n =7 measures. When compared to changes in controls, the (19%; n = 2 control and n = 5 supplement) were non- supplements resulted in significantly greater decreases in compliant with follow-up visits and excluded. The final adiponectin levels at 4 weeks, but no significant changes in sample was therefore 9 randomized to control, 11 to weight, waist circumference, lipid levels, inflammatory or Mediterranean diet, and 10 to supplements. Participants oxidative stress biomarkers, markers of hyperglycemia, were 51.4 (6.6) years old and 73.3% female. FMD, or CFU-As (Table 1;Fig. 1). Change in dietary intake Discussion Participants in the Mediterranean diet arm exhibited a Eight weeks of substitution of an American-type diet significant decrease in their saturated fat and cholesterol with a Mediterranean diet was found to reduce CVD risk Jaacks et al. BMC Nutrition (2018) 4:26 Page 5 of 8 a,b Table 1 Characteristics of participants at baseline and after 4 and 8 weeks of the dietary intervention Control n = 9 Mediterranean Diet n = 11 Supplements (Fish Oil, Walnuts, and Grape Juice) n =10 Baseline 4 weeks 8 weeks Baseline 4 weeks 8 weeks Baseline 4 weeks 8 weeks Dietary variables Energy, kcal 2159 ± 411 2047 ± 436 1964 ± 680 2346 ± 742 2368 ± 486 2192 ± 1110 1647 ± 494 2163 ± 775* 2266 ± 321* Tot fat, 40.3 ± 4.8 40.8 ± 5.7 32.1 ± 5.6 40.4 ± 5.3 35.1 ± 2.6 31.8 ± 10.2 34.4 ± 5.8 43.4 ± 5.8* 43.3 ± 4.3* %kcal Sat fat, 12.8 ± 2.9 14.2 ± 3.5 9.7 ± 2.2 12.5 ± 4.6 5.8 ± 1.5* 7.6 ± 2.7 11.0 ± 3.2 8.6 ± 3.2 8.7 ± 1.5 %kcal Carb, %kcal 44.3 ± 9.0 44.9 ± 7.3 50.5 ± 6.1 47.4 ± 6.2 52.0 ± 3.9 51.0 ± 7.6 45.0 ± 15.1 41.0 ± 9.0 42.4 ± 4.5 Protein, 17.1 ± 3.2 15.5 ± 3.4 18.1 ± 3.2 14.0 ± 3.9 15.9 ± 1.5 16.9 ± 3.1 19.4 ± 6.1 16.9 ± 3.4 15.0 ± 1.7* %kcal Fiber, g 18.1 ± 3.8 19.2 ± 9.5 21.2 ± 9.99 19.7 ± 9.0 29.6 ± 10.1 28.7 ± 10.0 20.6 ± 12.7 22.3 ± 9.8 19.2 ± 6.6 Chol, mg 292 ± 73 306 ± 128 282 ± 136 362 ± 216 223 ± 35.4* 334 ± 459 269 ± 120 301 ± 63.6 277 ± 47.4 MUFA, g 23.0 ± 6.8 17.3 ± 6.0 18.1 ± 10.6 16.9 ± 13.6 41.3 ± 10.0* 29.3 ± 20.1 14.4 ± 6.8 24.1 ± 9.7* 27.2 ± 4.6* Omega-3, g 0.81 ± 0.74 0.58 ± 0.22 0.91 ± 0.69 0.81 ± 0.96 2.25 ± 0.76* 1.97 ± 2.32 0.47 ± 0.28 5.92 ± 1.39* 5.62 ± 1.31* Omega-6, g 7.62 ± 3.65 4.54 ± 1.66 6.00 ± 3.13 7.09 ± 7.76 21.5 ± 6.89* 12.6 ± 12.5 4.25 ± 2.87 26.6 ± 10.0* 25.5 ± 2.61* Clinical variables Weight, kg 92.7 ± 7.2 96.2 ± 12.4 96.6 ± 11.1 93.4 ± 12.8 91.7 ± 12.5* 90.5 ± 13.0* 98.6 ± 16.8 99.5 ± 16.9 99.8 ± 16.8 WC, cm 105.7 ± 9.2 112.0 ± 12.3 107.4 ± 10.5 107.3 ± 6.7 107.9 ± 6.5 104.5 ± 5.9 114.1 ± 11.4 114.7 ± 11.5 112.8 ± 17.0 TC, mg/dl 189.6 ± 42.6 190.3 ± 30.9 179.3 ± 32.3 187.4 ± 38.4 163.3 ± 36.0* 164.2 ± 36.2 185.4 ± 19.9 178.3 ± 21.3 180.8 ± 19.4 TG, mg/dl 80.4 ± 37.3 84.5 ± 34.3 97.9 ± 51.7 141.9 ± 110.9 140.5 ± 100.6 109.3 ± 50.3 82.1 ± 26.9 66.0 ± 20.5 59.9 ± 19.7 HDL, mg/dl 46.2 ± 12.8 43.5 ± 11.9 40.9 ± 12.1 43.8 ± 14.1 40.2 ± 10.7 45.1 ± 12.5 43.6 ± 12.0 43.6 ± 13.2 47.3 ± 16.3 LDL, mg/dl 125.4 ± 35.5 134.0 ± 35.0 122.0 ± 26.5 119.4 ± 36.5 98.8 ± 31.7* 97.1 ± 31.8 125.4 ± 19.6 121.5 ± 21.3 121.5 ± 21.0 IL-6, pg/ml 1.87 ± 2.11 2.08 ± 2.98 2.03 ± 2.37 1.83 ± 2.14 1.62 ± 1.47 1.30 ± 1.23 2.26 ± 1.79 2.38 ± 2.48 2.44 ± 1.34 IL-8, pg/ml 3.29 ± 1.11 3.30 ± 1.41 44.6 ± 116.1 10.4 ± 21.1 131.2 ± 358.2 4.22 ± 3.36 2.51 ± 1.28 3.46 ± 3.30 3.01 ± 1.94 CRP, mg/l 5.52 ± 6.13 4.50 ± 5.83 5.22 ± 5.73 4.46 ± 8.02 4.18 ± 8.02 1.76 ± 1.72 6.51 ± 6.19 5.64 ± 6.22 7.77 ± 6.77 APN, μg/l 7732 ± 4166 8021 ± 4796 8634 ± 4458 5846 ± 3762 5530 ± 4205 5575 ± 3215 6403 ± 4439 5975 ± 4490* 6532 ± 4586 FPG, mg/dl 83.1 ± 13.7 80.7 ± 5.22 79.0 ± 6.11 89.6 ± 13.4 85.0 ± 7.15 93.8 ± 18.1 89.8 ± 11.0 87.1 ± 9.80 89.6 ± 9.90 Insulin, 7.19 ± 3.00 6.29 ± 3.47 6.80 ± 5.04 9.73 ± 6.87 10.1 ± 11.2 14.9 ± 27.8 11.3 ± 4.21 10.7 ± 6.49 12.0 ± 6.37 μU/ml FMD, % 6.16 ± 5.30 6.94 ± 5.48 5.28 ± 4.92 5.63 ± 3.35 7.97 ± 3.83 6.76 ± 3.61 6.90 ± 4.22 8.67 ± 4.48 9.61 ± 6.75 CFU-As 51.6 ± 25.9 41.8 ± 30.0 61.0 ± 42.7 89.4 ± 42.2 60.4 ± 30. 6 44.3 ± 22.3* 78.1 ± 48.1 53.3 ± 36.1 55.4 ± 30.0 Cysteine, 13.3 ± 3.53 11.0 ± 1.40 12.8 ± 2.06 12.1 ± 4.20 12.1 ± 3.35 9.93 ± 1.73 13.4 ± 3.18 13.3 ± 5.02 12.2 ± 2.80 μmol/l Cystine, 95.3 ± 18.9 103.6 ± 22.1 106.4 ± 30.6 95.1 ± 16.7 84.6 ± 18.3* 87.2 ± 11.0* 88.8 ± 18.2 91.5 ± 18.7 96.1 ± 18.5 μmol/l Glutathione, 1.98 ± 1.04 1.89 ± 0.48 1.87 ± 0.42 2.05 ± 1.24 2.09 ± 0.75 1.54 ± 0.52 2.38 ± 1.18 1.74 ± 0.59 1.89 ± 1.02 μmol/l Values are mean ± SD Means were calculated for all participants with non-missing data at the specified time point (baseline, 4 weeks, and 8 weeks) *P < 0.05 for group-by-time interaction term where control and baseline are referent from mixed-effects models Abbreviations: APN adiponectin, FMD flow-mediated vasodilation, MUFA monounsaturated fatty acids, CFU-As circulating pro-angiogenic cell activity, TC total cholesterol, TG triglycerides, WC waist circumference factors, specifically body weight and cholesterol levels, in a Supplementation of an American-type diet with key pilot randomized controlled trial in overweight or obese foods/nutrients thought to be responsible for the posi- US adults. These changes with the Mediterranean diet tive health effects (fish oil, walnuts, and grape juice) were accompanied by significant improvement in systemic was not associated with significant changes in CVD risk oxidative stress, measured as a decrease in cystine levels factors, potentially due to the lack of weight loss in this and a reduction in circulating pro-angiogenic cell activity. group. Jaacks et al. BMC Nutrition (2018) 4:26 Page 6 of 8 randomized to follow a modified Mediterranean diet using an exchange list compared to controls following their usual diet . There are several potential explana- tions for the discrepancy between our findings and the findings of that study, including the fact that our study was conducted among overweight or obese adults and dietary interventions may be more effective among those at increased risk. In addition, we provided foods to partici- pants for 4 weeks whereas the previous study provided only telephone counseling. However, given that we ob- served sustained effects on weight loss even after foods were no longer provided suggests that intensive counsel- ing about how to follow a Mediterranean-type diet may be effective for CVD prevention among overweight or obese US adults. We observed significant improvements in blood lipids, including total cholesterol and LDL cholesterol, among participants assigned to the Mediterranean diet arm compared to controls at 4 weeks. This is consistent with the observation that these participants had declines in saturated fat and cholesterol intake and increases in monounsaturated, omega-3, and omega-6 fatty acid in- takes at 4 weeks. It is also consistent with several previ- ous studies conducted in Europe and Brazil [5, 22–25]. Of note, the improvements in nutrient intake among Mediterranean diet arm participants were attenuated by 8 weeks, as participants were no longer provided prepared meals, but simply given counseling and reinforcement by the research nutritionist. This suggests that sustaining changes to an American-type diet that are consistent with a Mediterranean-type diet may require more intensive support. The significant decrease in plasma cystine level and hence systemic oxidative stress in the Mediterranean diet arm suggests that this may be an important under- lying mechanism explaining the protective effects of a Mediterranean diet on major cardiovascular events ob- served in the PREDIMED trial . We have recently shown that higher levels of cystine are associated with mortality in patients with coronary artery disease . Fig. 1 Mean change from baseline to 4 weeks (black bars) and 8 weeks Three secondary analyses of PREDIMED have focused (gray bars) in a body weight, b total cholesterol, and c LDL cholesterol. on oxidative stress [27–29]. In a randomly selected sub- Mean change was calculated for all participants with non-missing data sample of 75 PREDIMED participants with metabolic at both baseline and 4 weeks (black bars) or both baseline and 8 weeks (gray bars), thus may differ slightly from means presented in Table 1 syndrome, the plasma activity of superoxide dismutase (means calculated for all participants with non-missing data at a single and catalase was significantly increased and the activity time point). *Indicates p < 0.05 for group-by-time interaction term of xanthine oxidase significantly decreased in both inter- where control and baseline are referent from mixed-effects models vention groups (Mediterranean diet plus olive oil and Mediterranean diet plus nuts) compared to controls . To our knowledge, this is one of the first random- However, there were no significant differences across ized controlled trials to test the effects of a groups in plasma biomarkers of oxidative damage (nitrite Mediterranean-type diet in a US sample population. levels, nitrotyrosine index, or carbonylated proteins) with A previous study conducted among 69 healthy, non-obese the exception of nitrate levels, which were significantly women in the United States found no significant effect higher in the two intervention groups compared to on blood lipids, insulin, glucose, or CRP among those controls after 5 years of follow up . The other two Jaacks et al. BMC Nutrition (2018) 4:26 Page 7 of 8 secondary analyses of the PREDIMED trial found sig- cardio-metabolic health and may be more beneficial nificantly increased plasma total antioxidant capacity than supplementing habitual American diets with fish measured by colorimetric test in both intervention oil, walnuts, and grape juice. Future research might groups compared to controls after 3 years of follow up focus on conducting larger trials of verbal and written , and a non-significant (p = 0.059) decrease in counseling on the preparation and health benefits of a F2-isoprostane levels among women with metabolic Mediterranean diet among a more diverse group of syndrome in the intervention groups compared to con- Americans, and also on the potential for supplementing trols after 1 year of follow up . Improved antioxidant with other key components of the Mediterranean diet capacity with a Mediterranean diet compared to control (e.g., olive oil) on improving health in this population. diet has also been reported in several other small trials in Abbreviations Chile (n = 42 healthy male students)  and Greece ANOVA: Analysis of variance; APN: Adiponectin; BMI: Body mass index; (n = 90 adults with abdominal obesity) . CFU-A: Circulating pro-angiogenic cell activity; CRC: Clinical research center; CRP: C-reactive protein; CVD: Cardiovascular disease; DHA: Docosahexaenoic acid; Participants in the Mediterranean diet arm had signifi- EPA: Eicosapentaenoic acid; FMD: Flow-mediated vasodilation; FPG: Fasting cantly greater weight loss over the eight-week interven- plasma glucose; LDL: Low-density lipoprotein; LOD: Limit of detection; tion period compared to controls despite having similar MUFA: Monounsaturated fatty acids; TC: Total cholesterol; TG: Triglycerides; WC: Waist circumference total caloric intakes (this was not designed to be a weight loss trial). This observation is consistent with a pre-specified Acknowledgements secondary outcomes analysis of the PREDIMED trial that We would like to acknowledge the help of research staff at the Atlanta Clinical and Translational Science Institute at the Emory University Hospital found a significant difference in weight loss in the Mediter- clinical research site with conducting the study protocol. In particular, we ranean diet with olive oil group compared to controls . would like to thank Margaret Petersen, RD, MS, for design and preparation of Together, these results lend further support to the hypoth- the research diets. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the esis that it is not just diet quantity, but also diet quality, that Centers for Disease Control and Prevention. matters for weight management. In contrast, in the supple- ment arm, a significant increase in caloric intake was ob- Funding Funding was provided by the National Center for Advancing Translational served at both 4 and 8 weeks. This may at least partially Sciences of the National Institutes of Health (grant number UL1 TR000454), explain the lack of significantweightlossinthisgroup. the National Institute of Diabetes and Digestive and Kidney Diseases of We have previously shown that higher levels of circulat- the National Institutes of Health (grant number K24 DK096574), and the National Heart, Lung, and Blood Institute of the National Institutes of Health ing pro-angiogenic cell activity, measured as CFU-As, (grant numbers 5P20HL113451–01, 5P01HL101398–02, 1R56HL126558–01, were associated with increased CVD risk [16, 17]. Most 1U10HL110302–01, and U01 HL-079156). The funders had no role in the design, importantly, higher CFU-As were associated with an in- analysis, or writing of this article. creased risk of cardiovascular events in patients with cor- Availability of data and materials onary artery disease [16, 17]. Thus, a higher number of The dataset analyzed during the current study is available from the CFU-As are potentially a reflection of a stimulated state of corresponding author on reasonable request. the individual’s endogenous stem cell-dependent repara- Authors’ contributions tive or regenerative system. Herein, we demonstrate that a SS, MP, WRA, DPJ, VV, TRZ, and AAQ conceived and executed the trial. CDS Mediterranean diet intervention reduces the elevated and DPJ assessed the biomarkers. LMJ performed the statistical analysis and drafted the manuscript. All authors critically reviewed the manuscript and CFU-A count, implying significant reduction of the stimu- approved the final version. lated state of the endogenous reparative system, and of re- duced long-term risk of adverse cardiovascular outcomes. Ethics approval and consent to participate The study was approved by the Emory University Institutional Review Board This was a well-designed, carefully conducted study includ- and all participants provided written informed consent. ing thorough characterization of dietary intake and CVD risk. The lack of improvement in endothelial function and Competing interests some of the biomarkers of inflammation could be due to the The authors declare that they have no competing interests. limited sample size, relatively short period of intervention, and the fact that some of these outcomes were not abnormal Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in at baseline in a number of the subjects studied. For example, published maps and institutional affiliations. there was a trend towards improvement in endothelial func- tion in the subset of participants with baseline abnormalities Author details Department of Global Health and Population, Harvard T.H. Chan School of in FMD after the Mediterranean diet intervention. Findings Public Health, 665 Huntington Ave, Building 1, Room 1211, Boston, MA need to be confirmed in a larger study. 2 02115, USA. Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30307, USA. Hemostasis Laboratory Branch, Division of Blood Disorders, National Center on Birth Conclusions Defects and Developmental Disabilities, Centers for Disease Control and These results suggest that adopting a dietary pattern 4 Prevention, Atlanta, GA 30329, USA. Gwinnett Medical Center, Lawrenceville, reflecting a Mediterranean diet improves weight and GA 30046, USA. Division of Pulmonary, Allergy, Critical Care and Sleep Jaacks et al. 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Published: May 31, 2018