Effects of a hypoenergetic diet associated with açaí (Euterpe oleracea Mart.) pulp consumption on antioxidant status, oxidative stress and inflammatory biomarkers in overweight, dyslipidemic individuals

Clinical Nutrition xxx (xxxx) xxx

Contents lists available at ScienceDirect

Clinical Nutrition journal homepage: http://www.elsevier.com/locate/clnu

Original article

Effects of a hypoenergetic diet associated with açaí (Euterpe oleracea Mart.) pulp consumption on antioxidant status, oxidative stress and inflammatory biomarkers in overweight, dyslipidemic individuals Luciana Nicolau Aranha a, Mariana Gomes Silva a, Sofia Kimi Uehara a, Ronir Raggio Luiz c,
ucia Maria Moraes de Oliveira a, *
Firmino Nogueira Neto d, Glorimar Rosa a, b, Gla Jose a

Postgraduate Program in Medicine (Cardiology), Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil Department of Nutrition and Dietetics, Josu
e de Castro Nutrition Institute, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil Institute for Studies in Collective Health, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil d Lipids Laboratory-LabLip, Faculty of Medical Sciences, Rio de Janeiro State University, Rio de Janeiro, Brazil b c

a r t i c l e i n f o

s u m m a r y

Article history: Received 1 April 2019 Accepted 6 June 2019

Objective: To evaluate the effects of a hypoenergetic diet (HD)associated with açaí pulp consumption on oxidative stress, antioxidant status and inflammatory biomarkers in overweight, dyslipidemic individuals. Research methods & procedures: A randomized, double-blind, placebo-controlled clinical trial was conducted for 90 days. The study began with a 30-day run-in period, during which the intervention was exclusively a HD. Following this period, volunteers were randomized into 2 groups, and 200 g of either açaí pulp or placebo were added to the HD for 60 days. Anthropometric measurements, arterial pressure, oxidative stress and antioxidant status biomarkers, inflammatory and biochemical biomarkers were evaluated. Results: Sixty-nine volunteers completed the clinical trial, 30 of which were in the HD þ açaí group and 39 in HD þ placebo group. Plasma 8-isoprostane concentrations significantly reduced 60 days after the intervention in the açaí group (p ¼ 0.000), and there was a significant difference between the groups (açaí versus placebo; p ¼ 0.037). Regarding inflammatory status parameters, a significant reduction in IL6 was observed in the HD þ açaí group (p ¼ 0.042), and IFN-g decreased significantly in both groups, HD þ açaí (p ¼ 0.001) and HD þ placebo (p ¼ 0.008); there were, however, no differences between the groups. Lipid profile parameters and blood glucose levels did not show change, regardless of nutritional intervention. Conclusion: The addition of açaí to a HD, for 60 days, reduced oxidative stress and improved inflammation in overweight, dyslipidemic individuals. © 2019 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Keywords: Euterpe Acai Berry Oxidative stress Inflammation Dyslipidemia Obesity

1. Introduction The prevalence of obesity has increased worldwide. In the year 2017, elevated body mass index (BMI) ranked fourth among risk factors for mortality, mainly due to its effects on cardiovascular diseases (CVD) [1]. In Brazil, 2016 research data from the Telephonebased Surveillance of Chronic Diseases (VIGITEL) revealed that more

* Corresponding author. FESC, FACC, Universidade Federal do Rio de Janeiro, R.
ria, CEP Prof. Rodolpho P. Rocco, 255 e 8, Andar Sala 6, UFRJ, Cidade Universita 21941-913, RJ, Brazil. E-mail addresses: [email protected], [email protected] (G.M. Moraes de Oliveira).

than half of the population was overweight, with 18.9% of Brazilians being classified as obese [2]. Obesity influences the development of many risk factors associated with CVD, such as dyslipidemia, systemic arterial hypertension (SAH), insulin resistance, and type 2 diabetes mellitus [3]. Oxidative stress and inflammation also contribute to the development of these diseases [4]. The accumulation of adipose tissue promotes changes in adipokine secretion, as well as infiltration and activation of macrophages within the tissue [5]. There is a resulting increase in the expression of pro-inflammatory cytokines, such as interleukin 6 (IL6) and tumor necrosis factor alpha (TNF-a), and in the production of reactive oxygen species (ROS) by the infiltrated macrophages [5]. The main enzymatic antioxidants system includes superoxide

https://doi.org/10.1016/j.clnu.2019.06.008 0261-5614/© 2019 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Please cite this article as: Aranha LN et al., Effects of a hypoenergetic diet associated with açaí (Euterpe oleracea Mart.) pulp consumption on antioxidant status, oxidative stress and inflammatory biomarkers in overweight, dyslipidemic individuals, Clinical Nutrition, https://doi.org/ 10.1016/j.clnu.2019.06.008

2

L.N. Aranha et al. / Clinical Nutrition xxx (xxxx) xxx

dismutase, catalase, glutathione peroxidase, and reduced glutathione; whereas dietary antioxidant compounds, such as vitamin E, vitamin C, carotenoids, uric acid, and polyphenols, are part of the non-enzymatic system. Together, they act through mechanisms to prevent and control ROS formation [5,6]. Dietary patterns characterized by higher consumption of fruits, vegetables, whole grains, low-fat dairy products, and lean meats are associated with a lower risk of all-cause morbidity and mortality [7]. Bioactive compounds, such as polyphenols found in fruits and vegetables, are also associated with beneficial effects on improvements in CVD risk factors, inhibiting inflammation and platelet aggregation, regulating lipid metabolism and intestinal microbiota, and eliminating free radicals [8]. Açaí (Euterpe oleracea Mart.), is a typical fruit from the Amazon Region of Brazil, which is rich in polyphenols, such as anthocyanins (cyanidin-3-glucoside and cyanidin 3-rutinoside), oleic acid, fibers, and phytosterols. It has attracted interest due to its antioxidant potential [9e11]. In vitro studies in experimental models have demonstrated that açaí has antioxidant [9,12], anti-inflammatory [13], and lipid-profile-improving effects [14]. Studies with humans, however, are still limited [15e17]. Although it is a highly caloric fruit, açaí may be part of a healthy balanced diet, and it may be useful in weight-loss programs. As previously shown, this fruit is rich in monounsaturated fatty acids and fibers which may act by stimulating metabolism, promoting satiety, and improving bowel function. Furthermore, the anthocyanins present in açaí may have antioxidant and anti-inflammatory effects and be beneficial to individuals who are overweight. Considering the presence of a high quantity and variety of compounds present in açaí and their possible effects on protecting against oxidative stress and inflammation, the objective of this study was to evaluate the effects of a hypoenergetic diet associated with açaí consumption on oxidative stress and antioxidant status and inflammatory biomarkers in overweight, dyslipidemic individuals. 2. Materials and methods 2.1. Population Two hundred and twenty-four volunteers were recruited at the Clinical Nutrition Research and Extension Center of the Clementino Fraga Filho University Hospital of the Federal University of Rio de Janeiro (Rio de Janeiro, RJ, Brazil), from October 2016 to February 2018. Of the 224 individuals screened, 131 were eligible to participate in the study. Selection criteria were: adults with BMI 25 kg/ m2 [18], both sexes, ages 20 to 59, and at least one lipid profile alteration according to the Brazilian Guidelines on Dyslipidemias and Prevention of Atherosclerosis [19]. Exclusion criteria were: allergy to açaí or food coloring; pregnant or breastfeeding; alcoholism or tobacco use; energy restriction diets; use of dietary supplements containing antioxidant vitamins or minerals; use of corticoids or lipid-lowering agents; autoimmune, infectious, or thyroid diseases; chronic renal failure; liver disease; cancer, and AIDS. This study was approved by the Research Ethics Committee of the Clementino Fraga Filho University Hospital of the Federal University of Rio de Janeiro (certificate number 1.436.233), and it was registered in the Brazilian Network of Clinical Trials (RBR-72dvqv). All participants signed Free and Informed Consent Forms.

OpenEpi [20], considering a confidence interval of 95% and a test power of 80%, with the parameter of reduced isoprostane levels obtained in a pilot study including 10 individuals, which resulted in a minimum sample size of 25 volunteers per group. The study began with a 30-day run-in period, during which volunteers received only balanced and individualized HD, with the aim of facilitating volunteer adaptation and thus minimizing loss of follow-up during the clinical trial. After this step, the individuals were randomized and allocated into 2 groups: HD þ açaí or HD þ placebo, adding either 200 g of frozen açaí or 200 g of placebo to the HD. Addition of frozen açaí pulp or placebo to the diet occurred for 2 months (60 days), with monthly follow-up. The study used simple randomization, in blocks of 10, based on a random number table, which was blinded to all researchers involved in the study. The volunteers were instructed to consume a package containing 200 g of açaí or placebo once daily, at breakfast. Diet calculation was individualized, based on total energy expenditure (TEE), using different formulas for men and women, according to weight, height, age, and physical activity coefficient [21]. In order to calculate the energy deficit, we used the method described by Wishnofsky [22] with the intention of promoting 3 kg of weight loss per month, thus prescribing a minimum caloric requirement which was not lower than the basal metabolic rate, using the formula: 7700  kg (to lose) ÷ 30. Macronutrient distribution was balanced, and dietary planning followed the Brazilian Guidelines on obesity, dyslipidemia, and hypertension [19,23]. The average energetic value of the complements was considered in the total energy value (TEV) for dietary planning, so that neither the açaí nor the placebo would exceed the daily energy value. Caloric intake was measured using a 3-day food register and complement consumption was monitored via dietary questionnaires (3-day food registers and 24-h recalls) and evaluation of leftovers for every participant during research follow-up. Every month, 3 extra complement containers were given, and we asked volunteers to give the leftover containers back every 30 days. General adverse effects such as dyspepsia, diarrhea, constipation, nausea, or allergic reactions to açaí or placebo were monitored via questionnaire throughout the study. 2.3. Nutritional value of açaí and placebo
m, Para
, Brazil, Frozen açaí pulp of the medium variety, from Bele was obtained from a commercial establishment in the city of Rio de Janeiro. Two hundred grams of açaí contained 154 kcal, 8.2 g of carbohydrates, 2.4 g of proteins, 12.4 g of total lipids (5.9 g oleic fatty acid), and 684 mg of gallic acid equivalents (GAE) of phenolic compounds. The placebo was made up of water, carboxymethylcellulose, sucralose, açaí flavoring, and soybean oil. Two hundred grams of placebo contained 55.4 kcal, 2.2 g of carbohydrates, 0.4 g of proteins, 5 g of total lipids, and 10 mg GAE/g of phenolic compounds. 2.4. Anthropometric parameters and arterial pressure Anthropometric evaluation was conducted monthly, including measurements of weight in kg, height in m, waist circumference in cm, neck circumference in cm, BMI in kg/m2, and waist-to-height ratio (WHtR). BMI was classified in accordance with the WHO [18], and arterial pressure (AP) was taken by the auscultatory method [23].

2.2. Study design and dietary intervention 2.5. Biochemical parameters A randomized, double-blind, placebo-controlled clinical trial was conducted for 90 days, providing either açaí or placebo, in conjunction with a hypoenergetic diet (HD), which was calculated individually. Sample size was calculated using the software

Blood samples were collected in the morning, following a fasting period of 12e14 h. Blood was collected in tubes with gel to obtain serum or anticoagulant (EDTA) to obtain plasma. Thirty

Please cite this article as: Aranha LN et al., Effects of a hypoenergetic diet associated with açaí (Euterpe oleracea Mart.) pulp consumption on antioxidant status, oxidative stress and inflammatory biomarkers in overweight, dyslipidemic individuals, Clinical Nutrition, https://doi.org/ 10.1016/j.clnu.2019.06.008

L.N. Aranha et al. / Clinical Nutrition xxx (xxxx) xxx

minutes after collection, the tubes were centrifuged at 4000 rpm for 15 min using a Spinlab tabletop, in order to obtain serum and plasma. Serum concentrations of glucose, triglycerides (TG), total cholesterol (TC), and HDL-c were obtained in duplicate by an automated method (BioSystems A25 automated analyzer), using BioSystems commercial kits. LDL-c and VLDL-c were calculated using the Friedewald formula [24], which is only valid for triglyceride concentrations below 400 mg/dL. Plasma 8-isoprostane concentrations were obtained using the Enzyme Linked Immunosorbent Assay (ELISA) method, using an 8Isoprostane EIA KIT® (Cayman Chemical, USA). Results were expressed in pg/mL. Plasma vitamin A (retinol) and vitamin E (tocopherol) concentrations were obtained using the High Performance Liquid Chromatography (HPLC) method with UV detection, using a kit from Chromsystems Instruments & Chemicals GmbH® (Munich, Germany) in Waters 515 equipment, following the methodology described by Vuilleumier et al. [25]. Sample preparation consisted of a precipitation step, followed by supernatant analysis in the HPLC-UV system. This method uses a selected HPLC column and a mobile phase composed of methanol solution with a flow rate of 1.5 mL/min. Results were expressed in mmol/L. Inflammatory status biomarkers (INF-g, IL-6, IL-10, and TNF-a) were evaluated by the technology platform Luminex® xMAP® which involves an exclusive process that dyes latex microspheres with 2 fluorophores. The microspheres were processed in Luminex 200 equipment, using a HCYTOMAG-60K/Milliplex MAP Human Cytokine commercial kit (Merck Millipore, Darmstadt, Germany). Results were expressed in pg/mL. 2.6. Statistical analyses Statistical analyses were carried out using IBM® SPSS® Statistics software, version 21. Per-protocol (PP) statistical analysis was used, including only patients who completed treatment and did not violate protocol. Results were shown as frequency and mean ± standard deviation. The KolmogoroveSmirnov test was used for variable normality. Chi-squared was used to evaluate differences between categorical variables. Paired t-tests or Wilcoxon's test were used to evaluate intragroup effects, and effects between groups were evaluated by t-tests for independent samples or the ManneWhitney test, in accordance with variable distribution. Results with p-values < 0.05 were considered statistically significant. 3. Results Two hundred and twenty-four volunteers were recruited, 93 (41.5%) of which did not meet eligibility criteria. One hundred and twenty-five volunteers, with an average age of 39.8 ± 10.7, 76% (n ¼ 95) of which were women and 24% (n ¼ 30) of which were men, participated in the run-in period. Table 1 shows the anthropometric, arterial pressure, biochemical characteristics of the volunteers who participated in the run in. Significant reductions in body mass and BMI were observed. There was a reduction in biochemical parameters, which, however, was not statistically significant. During the 30-day run-in period, 16% (n ¼ 20) of the volunteers withdrew from this first step of the study, and 84% (n ¼ 105) were randomized to initiate the second step, during which the complement (açaí or placebo) was added to the HD. Sixty-nine volunteers completed all steps of the study, 30 in the HD þ açaí group and 39 in the HD þ placebo group. Their baseline characteristics may be observed in Table 2. An intergroup difference was observed in relation to WHtR (p ¼ 0.027). The other parameters were not divergent.

3

Table 1 - Anthropometric, arterial pressure, and biochemical characteristics during run in (n ¼ 105). Variables

Beginning of run in

End of run in

p-value

Body mass (kg) BMI (kg/m2) WC (cm) NC (cm) WHtR SAP (mmHg) DAP (mmHg) Blood glucose (mg/dL) TC (mg/dL) LDL (mg/dL) HDL (mg/dL) VLDL (mg/dL) TG (mg/dL)

96.1 ± 20.2 35.2 ± 6.0 107.8 ± 15.1 38.7 ± 4.4 0.7 ± 0.1 118.6 ± 14.0 78.4 ± 8.3 101.6 ± 46.2 206.6 ± 50.2 123.6 ± 41.2 50.0 ± 14.5 29.8 ± 12.8 163.3 ± 88.8

94.9 ± 19.9 34.8 ± 5.9 107.4 ± 15.2 38.5 ± 4.3 0.7 ± 0.1 117.6 ± 13.4 76.1 ± 7.3 98.8 ± 42.2 202.2 ± 46.4 119.9 ± 38.6 49.8 ± 14.2 28.6 ± 12.6 155.4 ± 85.4

0.000 0.000 0.301 0.072 0.312 0.373 0.338 0.701 0.224 0.231 0.701 0.308 0.230

Values shown as average ± standard deviation. Paired t-tests for variables with normal distribution and Wilcoxon's test for variables with non-parametric distribution. Values considered statistically significant: p < 0.05. BMI: body mass index; DAP: diastolic arterial pressure; HDL: high-density lipoprotein; LDL: low-density lipoprotein; NC: neck circumference; SAP: systolic arterial pressure; TC: total cholesterol; TG: triglycerides; VLDL: very low-density lipoprotein; WC: waist circumference; WHtR: waist-to-height ratio. Data in bold indicate significance.

Table 2 Anthropometric, clinical, and laboratorial baseline characteristics of study volunteers. Variables

HD þ açaí (n ¼ 30)

HD þ Placebo (n ¼ 39)

p-value

Age (years) Women (n, %) Body mass (kg) BMI (kg/m2) WC (cm) NC (cm) WtHR SAP (mmHg) DAP (mmHg) Blood glucose (mg/dL) TC (mg/dL) LDL (mg/dL) VLDL (mg/dL) HDL (mg/dL) TG (mg/dL) 8-isoprostanes (pg/mL) Vitamin A (mmol/L) Vitamin E (mmol/L) IFN-g (pg/mL) TNF-a (pg/mL) IL-6 (pg/mL) IL-10 (pg/mL)

42.3 ± 9.1 18 (60%) 95.4 ± 18.9 34.2 ± 5.1 106.2 ± 13.4 39.7 ± 4.8 0.6 ± 0.1 119.3 ± 17.4 80.3 ± 10.7 105.6 ± 51.4 208.2 ± 42.9 126.8 ± 35.7 33.0 ± 13.6 47.1 ± 9.9 189.2 ± 102.0 17.0 ± 28.8 1.9 ± 0.5 28.7 ± 5.2 7.9 ± 26.8 12.1 ± 18.0 6.5 ± 18.6 9.6 ± 39.0

40.4 ± 10.2 28 (71.8%) 100.6 ± 23.9 37.1 ± 7.2 112.4 ± 17.4 39.6 ± 4.8 0.7 ± 0.1 120.3 ± 13.5 77.2 ± 7.9 103.9 ± 42.7 209.4 ± 62.1 126.6 ± 50.8 31.5 ± 14.0 49.3 ± 17.1 166.5 ± 89.7 13.2 ± 17.2 1.9 ± 0.6 28.0 ± 4.5 1.9 ± 0.9 8.1 ± 3.5 2.7 ± 1.9 2.9 ± 2.7

0.432 0.302 0.336 0.066 0.108 0.907 0.027 0.456 0.142 0.707 0.928 0.989 0.546 0.521 0.273 0.781 0.987 0.602 0.755 0.475 0.096 0.712

Values shown as average ± standard deviation or frequency (n, %). T-tests for independent samples with normal distribution and ManneWhitney test for variables with non-parametric distribution. Chi-squared test for categorical variables. Values considered statistically significant: p < 0.05. BMI: body mass index; DAP: diastolic arterial pressure; HD: hypoenergetic diet; HDL: high-density lipoprotein; IFN-g: interferon gamma; IL-6: interleukin 6; IL-10: interleukin 10; LDL: low-density lipoprotein; NC: neck circumference; SAP: systolic arterial pressure; TC: total cholesterol; TG: triglycerides; TNF-a: tumor necrosis factor alpha; VLDL: very lowdensity lipoprotein; WC: waist circumference; WHtR: waist-to-height ratio. Data in bold indicate significance.

Sixty days after the intervention, there was a significant reduction in body mass (p ¼ 0.022) and BMI (p ¼ 0.015) in the HD þ açaí group, as well as a reduction in body mass (p ¼ 0.010), BMI (p ¼ 0.006) and waist circumference (p ¼ 0.007) in the HD þ placebo group. Diastolic arterial pressure (DAP) significantly increased in the group that received placebo as an intervention. There was an intergroup difference in WHtR. Biochemical parameters, such as glucose, total cholesterol, LDL-c,

Please cite this article as: Aranha LN et al., Effects of a hypoenergetic diet associated with açaí (Euterpe oleracea Mart.) pulp consumption on antioxidant status, oxidative stress and inflammatory biomarkers in overweight, dyslipidemic individuals, Clinical Nutrition, https://doi.org/ 10.1016/j.clnu.2019.06.008

4

L.N. Aranha et al. / Clinical Nutrition xxx (xxxx) xxx

Table 3 Anthropometric, arterial pressure, and biochemical characteristics of volunteers, after 60-day intervention with açaí or placebo. Variables

Body mass (kg) BMI (kg/mb) WC (cm) NC (cm) WtHR SAP (mmHg) DAP (mmHg) Blood glucose (mg/dL) TC (mg/dL) LDL (mg/dL) VLDL (mg/dL) HDL (mg/dL) TG (mg/dL)

HD þ açaí (n ¼ 30)

pb

HD þ placebo (n ¼ 39)

T0

T60

DT60-T0

pa

T0

T60

DT60-T0

pa

93.6 ± 18.2 33.5 ± 4.9 105.4 ± 12.7 39.4 ± 4.9 0.6 ± 0.1 118.7 ± 14.5 77.3 ± 7.8 96.2 ± 30.8 203.9 ± 41.6 124.3 ± 33.5 32.7 ± 15.2 46.9 ± 14.3 163.7 ± 75.9

92.9 ± 18.8 33.3 ± 5.1 104.4 ± 13.8 39.2 ± 4.4 0.6 ± 0.1 118.0 ± 9.6 77.7 ± 6.2 100.5 ± 41.4 203.7 ± 41.4 122.1 ± 37.1 32.7 ± 13.6 47.9 ± 14.1 180.0 ± 113.3

0.7 ± 1.7 0.2 ± 0.6 1.0 ± 2.8 0.2 ± 1.5 0.0 ± 0.0 0.7 ± 12.6 0.3 ± 8.9 4.2 ± 30.1 0.2 ± 32.8 2.2 ± 38.8 1.1 ± 17.8 1.0 ± 16.9 16.3 ± 78.8

0.022 0.015 0.059 0.411 0.056 0.878 0.854 0.767 0.974 0.723 0.520 0.748 0.267

99.3 ± 23.7 36.6 ± 7.2 112.1 ± 17.3 39.4 ± 4.5 0.7 ± 0.1 118.2 ± 13.1 75.4 ± 8.2 103.2 ± 53.9 201.4 ± 53.7 117.2 ± 39.5 30.5 ± 12.1 48.5 ± 13.1 163.3 ± 90.9

98.6 ± 23.8 36.3 ± 7.3 110.7 ± 16.9 39.3 ± 4.7 0.7 ± 0.1 120.0 ± 12.8 78.5 ± 7.1 101.4 ± 46.2 196.1 ± 50.9 117.3 ± 42.9 32.1 ± 14.7 46.8 ± 14.8 159.5 ± 73.2

0.7 ± 1.7 0.3 ± 0.6 1.5 ± 3.2 0.1 ± 1.1 0.0 ± 0.0 1.8 ± 11.9 3.1 ± 9.2 1.8 ± 16.8 5.3 ± 33.5 3.1 ± 44.4 2.4 ± 13.6 1.7 ± 11.2 3.8 ± 53.1

0.010 0.006 0.007 0.409 0.060 0.397 0.044 0.280 0.327 0.419 0.658 0.336 0.653

0.287 0.054 0.105 0.944 0.028 0.717 0.601 0.676 0.508 0.628 0.848 0.748 0.365

Values shown as average ± standard deviation. D ¼ change after 60-day intervention (T60 e T0). BMI: body mass index; DAP: diastolic arterial pressure; HD: hypoenergetic diet; HDL: high-density lipoprotein; LDL: low-density lipoprotein; NC: neck circumference; SAP: systolic arterial pressure; TC: total cholesterol; TG: triglycerides; VLDL: very low-density lipoprotein; WC: waist circumference; WHtR: waist-to-height ratio. Data in bold indicate significance. a p-value e comparison of intragroup averages. Paired t-tests for variables with normal distribution and Wilcoxon's test for variables with non-parametric distribution. b p-value e comparison of averages between groups studied (açaí versus placebo): t-tests for independent samples with normal distribution and ManneWhitney test for variables with non-parametric distribution. Values considered statistically significant: p < 0.05.

HDL-c, and TG did not show change, regardless of nutritional intervention (Table 3). There was a significant reduction in plasma 8-isoprostane concentrations levels (p ¼ 0.000), IL-6 (P ¼ 0.042), and IFN- g (p ¼ 0.001) 60 days after the intervention in the HD þ açaí group. In the HD þ placebo group, there was a significant increase in plasma vitamin A concentrations (p ¼ 0.045) and a reduction in vitamin E (p ¼ 0.020) and IFN-g (p ¼ 0.008) levels. When comparing the means between the groups (açaí versus placebo), we observed that there was a significant difference in plasma levels of isoprostanes between the groups studied (p ¼ 0.037). We did not, however, find differences in the other biomarkers of the antioxidant status and inflammatory status between the groups studied (Table 4). 4. Discussion With the aim of reducing loss of follow-up during the clinical trial and adapting volunteers to the study, a 30-day run in period was conducted, during which volunteers received HD only. Notwithstanding the implementation of this period, we were unable to avoid a 34.3% loss of follow-up. High withdrawal rates have similarly been observed in dietary intervention studies. In a

retrospective clinical trial, Inelmen et al. [26] observed a 1-yeardrop-out rate of 77.3% in a sample of 383 overweight and obese patients receiving ambulatory treatment. Of the 296 patients who dropped out, 69.2% did so during the first 3 months of follow-up (approximately 80% of which abandoned treatment at the first visit); 20.6% between the 3rd and 6th months, and 10.1% during the final 6 months of the study. During the intervention period, we observed that açaí had no effects that were additional to the HD regarding anthropometric, arterial pressure, and lipid profile data. Similar results were observed in other studies which used açaí as an intervention. Pala et al. [17] evaluated the effects of consuming 200 g of açaí for 4 weeks in 40 healthy women, observing no alterations in TC, LDL, and HDL. They did, however, observe an increase in apolipoprotein A1, which is the primary protein component of HDL, as well as an increase in transfer of lipids to HDL, which is important to the metabolism of this lipoprotein. Barbosa et al. [16] also found no changes in anthropometric and lipid profile parameters following daily ingestion of 200 g of açaí pulp during 4 weeks, in 35 healthy women. In contrast, Udani et al. [15] conducted a pilot study with 10 healthy volunteers, offering 200 g of açaí daily during 1 month, and they observed a reduction in fasting glucose levels, TC, and LDL in

Table 4 Plasma levels of oxidative stress, antioxidant status, and inflammatory status biomarkers, after 60-day intervention with açaí or placebo. Variable

HD þ açaí (n ¼ 30) T0

Oxidative stress biomarker 8-isoprostanes (pg/ml) 36.3 ± 27.1 Antioxidant status biomarkers Vitamin A (mmol/L) 1.9 ± 0.6 Vitamin E (mmol/L) 31.8 ± 8.2 Inflammatory status biomarkers IFN-g (pg/mL) 6.7 ± 16.8 TNF-a (pg/mL) 11.2 ± 15.7 IL-6 (pg/mL) 7.9 ± 32.2 IL-10(pg/mL) 11.4 ± 53.4

pb

HD þ placebo (n ¼ 39)

T60

DT60-T0

pa

T0

T60

DT60-T0

pa

15.3 ± 25.3

21.0 ± 17.7

0.000

23.7 ± 20.1

28.3 ± 40.9

4.5 ± 45.6

0.474

0.037

2.0 ± 0.6 29.4 ± 6.2

0.1 ± 0.6 2.4 ± 7.5

0.524 0.093

1.8 ± 0.5 29.8 ± 5.7

2.0 ± 0.8 27.5 ± 4.5

0.2 ± 0.7 2.3 ± 5.8

0.045 0.020

0.492 0.153

3.6 1.8 3.6 6.0

0.001 0.371 0.042 0.864

3.6 8.4 2.3 2.1

± ± ± ±

1.1 ± 5.5 0.9 ± 5.3 0.4 ± 1.2 0.1 ± 1.3

0.008 0.306 0.159 0.553

0.738 0.414 0.081 0.716

3.1 9.3 4.4 5.4

± ± ± ±

4.5 9.0 10.9 15.9

± ± ± ±

12.5 8.1 21.8 38.5

± ± ± ±

4.9 5.0 1.6 3.1

2.5 7.5 2.6 2.0

2.7 4.4 1.8 2.1

Values shown as average ± standard deviation. D ¼ change after 60-day intervention (T60 e T0). HD: hypoenergetic diet; IFN-g: interferon gamma; IL-6: interleukin 6; IL-10: interleukin 10; TNF-a: tumor necrosis factor alpha. Data in bold indicate significance. a p-value e comparison of intragroup averages. Paired t-tests for variables with normal distribution and Wilcoxon's test for variables with non-parametric distribution. b p-value e comparison of averages between groups studied (açaí versus placebo): t-tests for independent samples for parametric variables and ManneWhitney test for non-parametric variables. Values considered statistically significant: p < 0.05.

Please cite this article as: Aranha LN et al., Effects of a hypoenergetic diet associated with açaí (Euterpe oleracea Mart.) pulp consumption on antioxidant status, oxidative stress and inflammatory biomarkers in overweight, dyslipidemic individuals, Clinical Nutrition, https://doi.org/ 10.1016/j.clnu.2019.06.008

L.N. Aranha et al. / Clinical Nutrition xxx (xxxx) xxx

comparison with baseline values. However, these researchers did not use a placebo and they instructed volunteers to avoid nitraterich foods (such as bacon and hot dogs). It is, thus, not possible to affirm that these positive results were due to açaí ingestion. In experimental models of rats with hypercholesterolemia, açaí supplementation was demonstrated to be capable of improving the animals' antioxidant status by reducing serum levels of carbonyl proteins and total, free, protein sulfhydryl groups and superoxide dismutase activity and increasing paraoxonase activity, suggesting that the flavonoids in açaí pulp may function to reduce stress caused by a hypercholesterolemic diet [12]. Barbosa et al. [16] also observed an increase in catalase activity and total antioxidant capacity and a reduction in ROS production, following supplementation of 200 g per day of açaí during 4 weeks in healthy women. Açaí has a high phenolic compound content, especially the anthocyanins cyanidin 3-rutinoside and cyanidin 3-glucoside, which are responsible for a significant part of its antioxidant capacity [9,27], and which may contribute to oxidative stress reduction in these individuals. To evaluate oxidative stress, we used plasma concentrations of 8-isoprostanes, which are specific lipid peroxidation markers. Additionally, as they are stable, they may be found in all biological fluids and tissues. Our results support the hypothesis that açaí may exercise beneficial effects in relation to improvements in oxidative stress, given that, 60 days after the intervention, we observed a significant reduction in plasma 8-isoprostane concentrations in the group that consumed HD associated with açaí and that this difference was observed between groups. Kim et al. [28] also found reduced urine levels of 8-isoprostanes in the açaí group in comparison with the placebo group in individuals with metabolic syndrome. As previously mentioned, açaí is rich in anthocyanins, and these compounds have a high antioxidant capability and act to impede ROS formation via enzyme inhibition or by sequestration of trace elements involved in free radical production, in addition to inhibiting lipid peroxidation [29]. Studies in humans which evaluate the effects of açaí on inflammatory markers are still limited. Recently, Kim et al. [28] evaluated plasma levels of C-reactive protein (CRP), TNF-a, IFNg, and IL-6, and they observed that consumption of an açaí beverage for 12 weeks significantly reduced plasma levels of IFNg. In our study, we observed a significant reduction in IFN-g in both groups; IL-6, however, was reduced only in the açaí group. The mechanisms by which anthocyanins may reduce inflammation are not yet clear. It has, however, been suggested that their anti-inflammatory action may be attributed to antioxidant activities that result in negative regulation of the Nuclear factor-kappa B signaling pathway, as well as the as well as the mitogenactivated protein kinase pathway [30]. There are some limitations to this study which may have influenced the results, such as selective loss of follow-up during the trial, the small number of volunteers, and the fact that the quantity of açaí provided during the study may not have been sufficient to observe the effects that were additional to the HD on lipid profile parameters. This study, however, may contribute to the scientific literature as a basis for future studies, given that the patients were controlled via diet, that we used a placebo, and that we obtained a higher number of volunteers in comparison with other studies that used açaí as an intervention.

5. Conclusion This study showed that the addition of 200 g of açaí to a hypoenergetic diet, reduced oxidative stress and improved inflammatory status in overweight individuals with dyslipidemia.

5

Author contributions
ucia Maria Moraes de Oliveira and Glorimar Rosa were Gla responsible for study design, analyzed and interpretation of the results and writing of the manuscript. Luciana Nicolau Aranha was responsible for recruiting and care of patients, laboratory analysis, analyzed and interpretation of the results and the writing of the manuscript. Mariana Gomes Silva and Sofia Kimi Uehara assisted in recruiting and care of patients. Ronir Raggio Luiz assisted interpre
Firmino Nogueira Neto was responsible for tation of the results. Jose laboratorial analysis. All authors read and approved the final manuscript. The authors have no conflicts of interest to declare. Conflicts of interest The authors declare no conflict of interest. Acknowledgements This study was supported by the Coordination for the Improvement of Higher Education Personnel (CAPES). References [1] GBD 2017 Risk Factor Collaborators. Global, regional, and national comparative risk assessment of 84 behavioral, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 19902017: a systematic analysis for the Global Burden on Disease Study. Lancet 2018;392:1923e94. https://doi.org/10.1016/S0140-6736(18)32225-6.
rio da Saúde. Vigitel Brasil 2016: vigila ^ncia de fatores de risco e [2] Ministe ^ nicas por inque
rito telefo ^ nico dos 26 estados braproteç~ ao para doenças cro
rio da Saúde, Secretaria de sileiros e no Distrito Federal em 2016/Ministe ^ncia em Saúde, Departamento de Vigila ^ncia de Doenças e Agravos n~ Vigila ao
rio da Saúde; 2017. Transmissíveis e Promoç~ ao da Saúde. Brasília: Ministe
ME, Poirier P, Lemieux I, Despre
s JP. Overview of epidemiology and [3] Piche contribution of obesity and body fat distribution to cardiovascular disease: an update. Prog Cardiovasc Dis 2018;61:103e13. https://doi.org/10.1016/j.pcad. 2018.06.004. [4] Matsudo M, Shimomura I. Increased oxidative stress in obesity: implications for metabolic syndrome, diabetes, hypertension, dyslipidemia, atherosclerosis, ^ncer. Obes Res Clin Pract 2013;7:e330e41. https://doi.org/10.1016/j. and ca orcp.2013.05.004. [5] de Mello AH, Costa AB, Engel JDG, Rezin GT. Mitochondrial dysfunction in obesity. Life Sci 2018;192:26e32. https://doi.org/10.1016/j.lfs.2017.11.019. [6] Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O. Oxidative stress and antioxidant defense. World Allergy Organ J 2012;5:9e19. https://doi.org/10. 1097/WOX.0b013e3182439613. [7] Harmon BE, Boushey CJ, Shvetsov YB, Ettienne R, Reedy J, Wilkens LR, et al. Associations of key diet-quality indexes with mortality in the multiethnic cohort: the dietary patterns methods project. Am J Clin Nutr 2015;101: 587e97. https://doi.org/10.3945/ajcn.114.090688. [8] Chiva-Blanch G, Visioli F. Polyphenols and health: moving beyond antioxidants. J Berry Res 2012;2:63e71. https://doi.org/10.3233/JBR-2012-028. https://content.iospress.com/articles/journal-of-berry-research/jbr028. [9] Schauss AG, Wu X, Prior RL, Ou B, Patel D, Huang D, et al. Phytochemical and nutrient composition of the freeze-dried amazonian palm berry, Euterpe oleraceae Mart. (acai). J Agric Food Chem 2006;54:8598e603. https://doi.org/ 10.1021/jf060976g. [10] Pacheco-Palencia LA, Duncan CE, Talcott ST. Phytochemical composition and thermal stability of two commercial acai species, Euterpe oleracea and
ria. Food Chem 2009;115:1199e205. https://doi.org/10.1016/ Euterpe precato j.foodchem.2009.01.034. [11] Heinrich M, Dhanji T, Casselman I. Açai (Euterpe oleracea Mart): a phytochemical and pharmacological assessment of the species' health claims. Phytochem Lett 2011;4:10e21. https://doi.org/10.1016/j.phytol.2010.11.005. [12] de Souza MO, Silva M, Silva ME, Oliveira Rde P, Pedrosa ML. Diet supplementation with acai (Euterpe oleracea Mart.) pulp improves biomarkers of oxidative stress and the serum lipid profile in rats. Nutrition 2010;26:804e10. https://doi.org/10.1016/j.nut.2009.09.007. [13] Xie C, Kang J, Li Z, Schauss AG, Badger TM, Nagarajan S, et al. The açaí flavonoid velutin is a potent anti-inflammatory agent: blockade of LPS
and IL-6 production through inhibiting NF-e ^B activation mediated TNF-a and MAPK pathway. J Nutr Biochem 2012;23:1184e91. https://doi.org/10. 1016/j.jnutbio.2011.06.013. [14] Feio CA, Izar MC, Ihara SS, Kasmas SH, Martins CM, Feio MN, et al. Euterpe oleracea (açai) modifies sterol metabolism and attenuates experimentally-

Please cite this article as: Aranha LN et al., Effects of a hypoenergetic diet associated with açaí (Euterpe oleracea Mart.) pulp consumption on antioxidant status, oxidative stress and inflammatory biomarkers in overweight, dyslipidemic individuals, Clinical Nutrition, https://doi.org/ 10.1016/j.clnu.2019.06.008

6

L.N. Aranha et al. / Clinical Nutrition xxx (xxxx) xxx

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

induced atherosclerosis. J Atheroscler Thromb 2012;19:237e45. https://doi. org/10.5551/jat.11205. Udani JK, Singh BB, Singh VJ, Barrett ML. Effects of Açai (Euterpe oleracea Mart.) berry preparation on metabolic parameters in a healthy overweight population: a pilot study. Nutr J 2011;10:45. https://doi.org/10.1186/14752891-10-45. Barbosa PO, Pala D, Silva CT, de Souza MO, do Amaral JF, Vieira RA, et al. Açai (Euterpe Oleracea Mart.) pulp dietary intake improves cellular antioxidant enzymes and biomarkers of serum in healthy women. Nutrition 2016;32: 674e80. https://doi.org/10.1016/j.nut.2015.12.030. Pala D, Barbosa PO, Silva CT, de Souza MO, Freitas FR, Volp ACP, et al. Açai (Euterpe oleracea Mart.) dietary intake affects plasma lipids, apolipoproteins, cholesteryl ester transfer to high-density lipoproteinand redox metabolism: a prospective study in women. Clin Nutr 2018;37:618e23. https://doi.org/10. 1016/j.clnu.2017.02.001. World Health Organization. Obesity. Preventing and managing the global epidemic: report of a WHO Consultation. Geneva: WHO; 2000. WHO Technical Report Series 894. Faludi AA, Izar MCO, Saraiva JFK, Chacra APM, Bianco HT, Afiune Neto A, et al. ~o da Diretriz Brasileira de Dislipidemias e Prevença ~o da AteroAtualizaça sclerose e 2017. Arq Bras Cardiol 2017;109:1e76. Dean AG, Sullivan KM, Soe MM. OpenEpi: open source epidemiologic Statistics for public health. http://openepi.com/Menu/OE_Menu.htm. [Accessed 5 May 2019]. Institute of Medicine of the National Academies (IOM). Dietary reference intakes for energy, carbohydrate, fiber, fat, fatty acids, cholesterol, protein, and amino acids. Part 1. Washington: The National Academy Press; 2005. Wishnofsky M. Caloric equivalentes of gained or lost weight. Am J Clin Nutr 1958;6:542e6. https://doi.org/10.1093/ajcn/6.5.542.

~o AA, [23] Malachias MVB, Souza WKSB, Plavnik FL, Rodrigues CIS, Branda Neves MFT, et al. 7º Diretriz Brasileira de Hipertens~ ao arterial. Arq Bras Cardiol 2017;107:30e4. [24] Friedewald WT, Levy RI, Fredrickson DS. Estimation of concentration of lowdensity lipoprotein cholesterol in plasma, without use of preparative ultracentrifuge. Clin Chem 1972;18:499e502. http://www.ncbi.nlm.nih.gov/ pubmed/4337382. [25] Vuilleumier JP, Keller HE, Gysel D, Hunziker F. Clinical chemical methods for the routine assessment of the vitamin status in human populations. Part I: the fat-soluble A and E, and beta-carotene. Int J Vitam Nutr Res 1983;53:265e72. https://www.ncbi.nlm.nih.gov/pubmed/6685115. [26] Inelmen EM, Toffanello ED, Enzi G, Gasparini G, Miotto F, Sergi G, et al. Predictores of drop-out in overweight and obese outpatients. Int J Obes (Lond) 2005;29:122e8. https://doi.org/10.1038/sj.ijo.0802846. [27] Del Pozo-Insfran D, Brenes CH, Talcott ST. Phytochemical composition and pigment stability of açaí (Euterpe oleracea Mart.). J Agric Food Chem 2004;52: 1539e45. https://doi.org/10.1021/jf035189n. [28] Kim H, Simbo SY, Fang C, McAlister L, Roque A, Banerjee N. Açaí (Euterpe oleracea Mart.) consumption improves biomarkers for inflammation but not glucose- or lipid-metabolism in individuals with metabolic syndrome in a randomized, double-blinded, placebo-controlled clinical trial. Food Funct 2018;9:3097e103. https://doi.org/10.1039/c8fo00595h. [29] Reis JF, Monteiro VV, de Souza Gomes R, do Carmo MM, da Costa GV, Ribera PC, et al. Action mechanism and cardiovascular effect of anthocyanins: a systematic review of animal and human studies. J Transl Med 2016;14:315. https://doi.org/10.1186/s12967-016-1076-5. [30] Vendrame S, Klimis-Zacas D. Anti-inflammatory effect of anthocyanins via modulation of nuclear factor-kb and mitogen-activated protein kinase signaling cascades. Nutr Rev 2015;73:348e58. https://doi.org/10.1093/nutrit/ nuu066.

Please cite this article as: Aranha LN et al., Effects of a hypoenergetic diet associated with açaí (Euterpe oleracea Mart.) pulp consumption on antioxidant status, oxidative stress and inflammatory biomarkers in overweight, dyslipidemic individuals, Clinical Nutrition, https://doi.org/ 10.1016/j.clnu.2019.06.008