The effect of raisins on biomarkers of endothelial function and oxidant damage; an open-label and randomized controlled intervention

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The effect of raisins on biomarkers of endothelial function and oxidant damage; an open-label and randomized controlled intervention Panagiotis T. Kanellosa, Andriana C. Kalioraa,⁎, Athanasios D. Protogeroub, Nikolaos Tentolourisc, Despina N. Perread, Vaios T. Karathanosa a

Department of Dietetics and Nutritional Science, School of Heath Science and Education, Harokopio University, Athens, Greece Cardiovascular Prevention and Research Unit, Department of Pathophysiology, National and Kapodistrian University of Athens Medical School, Athens, Greece c First Department of Propaedeutic and Internal Medicine, Laiko General Hospital, National and Kapodistrian University, Medical School, Athens, Greece d Laboratory of Experimental Surgery and Surgical Research N.S. Christeas, National and Kapodistrian University, Medical School, Athens, Greece b

A R T I C L E I N F O

A B S T R A C T

Keywords: Oxidative stress Endothelial function Raisins FMD PWV Antioxidants

Based on the existing data in grapes and wine, the aim of the present study was to investigate the probability that raisins improve clinical features and markers of oxidative stress, inflammation and arterial function in healthy smokers. Thirty-six apparently healthy smokers were recruited to an open-label and randomized, controlled, 4week prospective intervention. All participants were reported to consume less than the recommended amount of five servings fruits and vegetables daily. Participants in the intervention were instructed to consume raisins equal to five fruit servings (90 g/d). Anthropometric and blood pressure (BP) measurements, assessment of dietary intake, and fasting blood draws were conducted at baseline and at week 4. Biochemical (glucose, lipids, liver enzymes), inflammation [C-reactive protein (CRP), leptin], oxidative stress [Malondialdehyde (MDA), Advanced oxidation protein products (AOPPs)] and arterial function markers [Flow-mediated dilatation (FMD), Pulse wave velocity (PWV), Intercellular adhesion molecule-1 (ICAM-1), Nitric oxide (NO)] were assessed preand post-intervention. Baseline characteristics did not differ between the intervention and control arm. No effect of daily raisin consumption was observed on markers assessed between baseline and week 4 in either arm. Regarding vegetable consumption, no difference was observed in either group between baseline and post-intervention; however, as expected, a significant increase was reported in the intervention arm in fruit consumption between baseline and end point (p < 0.001) and between two arms post-intervention (p < 0.001). When analyzing according to age, ICAM-1 levels significantly decreased in subjects > 30 years (n = 8) in intervention arm (390.1 ± 17.6 to 302.2 ± 11 ng/mL, p = 0.004). After analysis of the data for sex, women in intervention (n = 5) decreased significantly diastolic BP (74.6 ± 4.2 to 67.4 ± 2.6 mg/dL, p = 0.043), total cholesterol (175.8 ± 7 to 166.6 ± 6.6 mg/dL, p < 0.001) and LDL-cholesterol (96.2 ± 9.6 to 89 ± 10.5 mg/dL, p = 0.012). However, due to the small sample size in the above, no safe conclusions can be exported.

1. Introduction

Health and Human Services, 2014). Mechanisms of the adverse effects of smoking have not been completely elucidated yet. Cigarettes contain numerous reactive oxygen and nitrogen species. At the same time, other oxidants are generated endogenously following exposure to tobacco smoking (Bartalis, Chan, & Wooten, 2007). The resulting oxidative stress consists a possible mechanism of the onset and/or progression of smoking-related pathologies (Bjørklund & Chirumbolo, 2017). Recently, the focus of epidemiologic research has shifted towards understanding the complex interaction of behavioral risk factors as

Smoking causes 80%–90% of all lung cancer deaths (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2004), while it increases risk for other cancers (Whiteman & Wilson, 2016) and for cardiovascular disease (Yanbaeva, Dentener, Creutzberg, Wesseling, & Wouters, 2007). Overall mortality among both male and female smokers in the United States is about three times higher than that among similar people who never smoked (U.S. Department of

Abbreviations: FMD, Flow-mediated dilatation; PWV, Pulse wave velocity; ICAM-1, Intercellular adhesion molecule-1; MDA, Malondialdehyde; AOOPs, Advanced oxidation protein products; NO, nitric oxide; CRP, C-reactive protein; LDL, Low density lipoprotein; BMI, Body mass index; BP, Blood pressure ⁎ Corresponding author. E-mail address: [email protected] (A.C. Kaliora). http://dx.doi.org/10.1016/j.foodres.2017.09.061 Received 9 June 2017; Received in revised form 19 September 2017; Accepted 21 September 2017 0963-9969/ © 2017 Published by Elsevier Ltd.

Please cite this article as: Kanellos, P.T., Food Research International (2017), http://dx.doi.org/10.1016/j.foodres.2017.09.061

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chosen and the randomization sequence was computer generated. An independent statistician used a computer randomization software. After randomization, the statistician sent the randomization list to the trial principal investigator who completed a participant form for each subject, including the treatment and the subject trial number and put it in a sealed envelope. Blinding of the allocated treatment was maintained to data analysts and was exposed only after the assessment of outcomes. Participants in the intervention arm incorporated in their daily diet the consumption of 90 g of raisins equal to five fruit servings replacing snacks of alike nutritional value (low fat yogurt, mini crackers, or bread with low fat cheese). Raisins were provided in packages of 36 g each amount equal to two fruit servings, kindly donated by the Agricultural Cooperatives Union, Aegion, Greece.

determinants of health; this includes the interaction of tobacco consumption and dietary exposures (Shivappa et al., 2015). The assessment of nutritional habits in population studies has demonstrated that smokers and non-smokers differ in the kind of food they eat. Smokers tend to have higher intake of energy, total fat, saturated fat, cholesterol and alcohol (Northrop-Clewes & Thurnham, 2007), lower consumption of fruits and vegetables (Dauchet et al., 2010) and overall lower adherence to the Mediterranean dietary pattern (Hu et al., 2013) compared with non-smokers. This suggests that some of these differences may exacerbate the deleterious effects of components in tobacco smoke on risk of cancer and coronary heart disease. Since fruits and vegetables contain several nutrient and non-nutrient compounds, including phenolic compounds, vitamins, trace elements and fibers, smokers tend to have lower serum dietary antioxidant concentrations (Yanbaeva et al., 2007) compared with non-smokers. It is well known that fruit and vegetable consumption can protect against reactive oxygen species damage and improve antioxidant status and endothelial function (Gomes-Rochette et al., 2016). Corinthian raisins are small sun-dried fruits, produced almost exclusively in Greece, with significant phenolic content (Kanellos et al., 2013). Recently, the antiinflammatory and anti-oxidative properties of Corinthian raisins in diabetic patients have been reported, indicating a potential role in cardiovascular disease prevention (Kanellos et al., 2014). Focusing on improving the clinical features and markers of oxidative stress, inflammation and arterial function in healthy smokers, we designed a randomized controlled clinical trial to investigate the effects of diet supplementation with raisins in between meals.

2.3. Anthropometrics and blood pressure (BP) Anthropometric indices such as body weight (BW), height, waist and hip circumferences (WC, HC) and BMI were recorded both at baseline and after the end of the trial. BW was measured early in the morning in the fasting state with subjects in light clothing without shoes using a flat scale (Tanita WB-110MA, Japan) recorded to the nearest 0.1 kg and height was measured on a stadiometer (Seca Model 220, Germany) recorded to the nearest 0.1 cm. BMI was calculated as weight (in kg) divided by height2 (in m2). The WC was measured at the midpoint between the lower margin of the last palpable rib and the top of the iliac crest, using a stretch-resistant tape. HC was measured around the widest portion of the buttocks, with the tape parallel to the floor. All anthropometric measurements were recorded after a ≥12hour fast. Diastolic and systolic BP (DBP and SBP in mmHg, respectively) and heart frequency were evaluated at baseline and after the study completion using an electronic sphygmomanometer (OMRON HEM-907 XL, OMRON, Kyoto, Japan). Participants were asked to lie down and relax for a few minutes, after which, two consecutive BP measurements were recorded at an interval of 1–2 min. The recorded value was the mean of the two measurements.

2. Materials and methods 2.1. Study population Apparently healthy volunteers were invited to participate in the study through a University advertisement and word of mouth. Recruitment was based on the following inclusion criteria: age between 20 and 40 years, normal Body Mass Index (BMI) values (18.5–24.9 kg/ m2), smoking at least 10 cigarettes per day for at least 5 years, low adherence to the Mediterranean type diet indicated by i) MedDietScore values lower than 30 and ii) fruit and vegetable consumption lower than the recommendation of 5 servings daily. As assessed by a medical history questionnaire, physical examination and biochemical and hematological indices, all subjects included in the study were healthy. Exclusion criteria were alcohol abuse or drug use, any medication or vitamin/mineral supplementation or alternative diet (vegetarian, macrobiotic, etc.), pregnancy or lactation prior to the study. An additional exclusion criterion was pre-existence or clinical evidence of any gastrointestinal disease, such as inflammatory bowel disease, gastric ulcer and stomach or intestinal cancer. The primary end-point was deemed to be malondialdyhyde (MDA), as conventional measure of oxidative damage. The CV in published reports for MDA is 20% (Lai et al., 2005). A sample size of 33 was required to detect a 25% difference between any two treatments for MDA with 80% power assuming a CV of 50% and a two-sided α of 0.05. To allow for a 10% dropout rate over the 4 weeks, a total sample of no < 36 participants was required.

2.4. Dietary history and analysis Food data were collected. Each participant was asked to keep a 3day food record (non-consecutive days, including one weekend day). Dietitians trained participants and reviewed unclear descriptions, errors, omissions, or doubtful entries in records and asked the participants to clarify them. The research dietitian supervisor checked all completed records for accuracy. The MedDietScore questionnaire was applied to estimate adherence to the Mediterranean dietary pattern (Panagiotakos, Pitsavos, Arvaniti, & Stefanadis, 2007). Furthermore, during the trial, dietary counseling was monitored by a mid-term nonscheduled phone call receiving a 24-hour dietary recall. To calculate energy intake and macronutrient breakdown (fat, protein, and carbohydrate) nutritional data were analyzed by Nutritionist Pro nutrient analysis software version 5.2.0 (Axxya Systems, Nutritionist Pro, Stafford, TX). 2.5. Clinical analyses Blood samples were drawn at baseline and at the end of the study (week 4) through a catheter in an antecubital vein after a 12 h overnight fast. Freshly drawn blood samples were used for the determination of glucose, lipid profile, liver enzymes, urea, uric acid, creatinine and total proteins using an automatic analyzer. Low density lipoprotein (LDL) cholesterol was calculated using the Friedewald formula. For assays to determine inflammation and oxidative stress biomarkers, serum and plasma samples were collected, separated by centrifugation at 1800g for 10 min at 4 °C, and stored at − 80 °C for subsequent analyses.

2.2. Study design All eligible subjects signed an informed consent form after a full review of the inclusion and exclusion criteria and an explanation of the risks and benefits of the study, which were approved by the Ethics Committee of Harokopio University, based on the Helsinki Declaration. In a two-armed, single center, randomized, controlled, 4-week prospective intervention trial, subjects were randomly assigned either to the control arm or to the intervention arm. The allocation of patients in the two arms was random. In this study simple randomization was 2

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2.6. Smoking status marker

Table 1 Demographic, anthropometric and clinical characteristics of participants at baseline (mean ± SEM).

Cotinine, which consists a metabolite of nicotine, has been used in research as a reliable marker for smoking status and smoking cessation studies due to its longer half-life than nicotine. Thus, a Cotinine ELISA Kit was used for the measurement of cotinine in serum samples (Αbnovva, Germany).

Age (y) Gender (n) Men Women Smoking years Number of cigarettes/day MedDietScore Body weight (kg) BMI (kg/m2) WC (cm) HC (cm) Systolic BP (mm Hg) Diastolic BP (mm Hg)a Pulse rate (min− 1) Cotinine (μΜ)

2.7. Inflammatory, adhesion and oxidative stress biomarkers Sandwich Enzyme Linked-Immunosorbent Assay (ELISA) kits were used to measure high sensitive C-reactive protein (hsCRP) (g/L) (IBL, Germany), leptin (pg/mL) (Invitrogen, UK), Intercellular Adhesion Molecule-1 (ICAM-1) (ng/mL) (Invitrogen, UK). MDA (μM) (Cell Biolabs, USA), Advanced Oxidation Protein Products (AOPPs) (μM) (Immunodiagnostik, Germany) and Nitric oxide (NO) (μmol/L) (R & DSystems, UK) were measured spectrophotometrically. 2.8. Arterial measurements

Control

Intervention

p

n = 14

n = 22

29.8 ± 1.4

30.8 ± 1.6

0.658

11 3 12.2 ± 1.4 19.4 ± 2.7 27.8 ± 1.4 77.5 ± 3.7 24.4 ± 0.8 91.2 ± 3.7 99.7 ± 1.9 118.3 ± 2.5 78.1 ± 2 68.0 ± 2.9 94.5 ± 3.2

16 6 12.4 ± 1.4 15.1 ± 1.3 29.1 ± 1.2 78.1 ± 3 24.4 ± 0.6 92.0 ± 2.6 100.8 ± 1.4 117.7 ± 2.4 74.9 ± 1.9 62.9 ± 2.1 86.1 ± 3.8

0.924 0.118 0.488 0.899 0.996 0.846 0.618 0.878 0.311 0.157 0.122

BMI: Body Mass Index; WC: Waist circumference; HP: Hip circumference; BP: Blood pressure. Statistical significance level p < 0.05. a p: Comparison of baseline characteristics between control and intervention group using Independent sample t-test or Mann-Whitney test.

Brachial BP was assessed with Macrolide BP Office device; the average of three consecutive recordings was used in the statistical analysis and the following Pulse wave velocity (PWV) calibration process. A B-Mode high-resolution ultrasoundimaging (Vivid 7 Pro, GE) was used for assessment of Flow-mediated dilatation (FMD), as previously described (Corretti et al., 2002), in order to evaluate endothelial function. Radial artery and femoralartery tonometry were used to assess aortic stiffness via the carotid to femoral PWV with Sphygmocor System (ActorMedical, Sydney, Australia). The methodology and the technical details for the measurement of PWV have been described previously (Laurent et al., 2006).

Table 2 Biochemical and arterial function markers of participants at baseline (mean ± SEM).

a

Fasting glucose (mg/dL) Total chol (mg/dL) HDL-chol (mg/dL) LDL-chol (mg/dL) Triglycerides (mg/dL)a Creatinine (mg/dL)a Urea (mg/dL)a Uric acid (mg/dL) ALP (U/L) γ-GT (U/L) SGOT (U/L)a SGPT (U/L)a Total proteins (g/dL)a Carotid-Radial PWV (m/s) Carotid-Femoral PWV (m/s) FMD (%)

2.9. Statistical analysis All analyses were conducted applying the Statistical Package for the Social Sciences (SPSS 21.0 for Windows, Chicago, IL, USA). Descriptive statistics were calculated for all parameters and the KolmogorovSmirnov test was applied to investigate if all measures were characterized by normal distribution. For variables with a normal distribution, the independent samples t-test was applied to compare the differences between the two arms pre- and post-intervention, while for variables without a normal distribution, the Mann-Whitney test was applied. Before the intervention this test served to ensure that the study population was characterized by homogeneity. For investigating possible intra-group differences, a paired sample t-test was applied for parametric variables and the Wilcoxon test for non-parametric ones. All data are expressed as mean values ( ± SEM) and statistical significance was set at p < 0.05.

Control

Intervention

p

91.7 ± 2.7 187.1 ± 6.8 51.6 ± 3.2 112.2 ± 6.5 115.7 ± 17.8 1 ± 0.0 31.9 ± 1.3 5.4 ± 0.5 69.8 ± 4.7 27.4 ± 2 29.4 ± 4.6 25.5 ± 3.9 7.3 ± 0.0 6.1 ± 0.3 6.6 ± 0.2 3.8 ± 0.6

89.7 ± 1.9 181.1 ± 7.8 58.2 ± 2 103.2 ± 6.9 98.1 ± 10.5 0.9 ± 0.0 29.8 ± 1.3 4.9 ± 0.2 66.9 ± 3.7 26.4 ± 2 20.7 ± 1.2 19.2 ± 1.6 7.4 ± 0.1 6 ± 0.2 6.4 ± 0.2 2.7 ± 0.4

0.561 0.591 0.072 0.376 0.454 0.077 0.249 0.268 0.632 0.723 0.210 0.235 0.987 0.879 0.558 0.104

ALP, alkaline phosphatase; γ-GT, γ-glutamyl transpeptidase; SGOT, serum glutamic oxalacetic transaminase; SGPT, serum glutamic pyruvic transaminase; Carotid-Radial PWV: Carotid-Radial pulse wave velocity; Carotid-Femoral PWV: Carotid-Femoral pulse wave velocity; FMD: Flow Mediated Dilatation. Statistical significance level p < 0.05. a p: Comparison of baseline markers between control and intervention group using Independent sample t-test or Mann-Whitney test.

3. Results

difference was observed in either group between baseline and post-intervention; however, as expected, a significant increase in fruit consumption was reported in the intervention arm between baseline and end point (p < 0.001) and between the two arms post-intervention (p < 0.001) (Table 4). As regards biochemical features between baseline and week 4 in either arm, we did not report any effect by daily raisin consumption (Table 5). Similarly, the incorporation of five fruit servings daily in the diet of healthy smokers did not exhibit any effect on either inflammatory or oxidative stress (Table 6) or endothelial function and arterial stiffness markers (Table 7). Although no effect was observed when analyzing all the participants, after analysis of the data for each gender separately, women in intervention (n = 5) decreased significantly diastolic BP (74.6 ± 4.2 to 67.4 ± 2.6 mg/dL, p = 0.043) (Fig. 1), total cholesterol

Among 36 healthy subjects who were eligible to participate, 33 were eligible for analysis (13 participants in the control and 20 in the intervention arm). None of the participants in the intervention arm reported any discomfort by daily consumption of raisins. Baseline characteristics, including age, BMI, and clinical features, did not differ between the intervention and control arm (Table 1). Similarly, biochemical, inflammatory, oxidative stress and arterial function markers of participants did not differ at baseline (Table 2). Anthropometric and clinical characteristics did not differ significantly between control and intervention arm at the end of the study (Table 3). Since cotinine levels did not differ between the two time points in either arm, smoking habits were considered unchanged in both arms between baseline and week 4 (Table 3). The daily energy and nutrient intakes did not differ between the two arms at baseline or at the end of the study (Table 4). Regarding vegetable consumption, no 3

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Table 3 Differences in anthropometric and clinical characteristics between control and intervention arm at the end of the study (mean ± SEM).

Table 5 Differences in biochemical markers between control and intervention arm at the end of the study (mean ± SEM).

Variable

Arm

Week 0

Week 4

p

pb

Variable

Arm

Week 0

Week 4

p

pb

Body weight (Kg)

Control Intervention Control Intervention Control Intervention Control Intervention Control

75.1 ± 3.9 79.8 ± 2.9 23.9 ± 0.9 24.7 ± 0.6 89.9 ± 3.8 93.3 ± 2.5 99.1 ± 1.9 101.8 ± 1.3 117.2 ± 2.8

74.6 ± 3.8 79.8 ± 2.8 23.8 ± 0.8 24.7 ± 0.6 90.1 ± 3.5 93.2 ± 2.5 99.2 ± 1.8 102 ± 1.2 117.5 ± 2.9

0.425 1.000 0.398 0.769 0.357 0.658 0.894 0.509 0.799

0.371

Fasting glucose (mg/dL)a

Control

90.8 ± 2.6

85.6 ± 2.7

0.082

0.128

Intervention Control

89.7 ± 2 186.7 ± 7.9

89.4 ± 2.1 178.8 ± 6.9

0.809 0.310

0.755

Intervention Control

117.2 ± 2.2 77.1 ± 2.2

115.7 ± 2.4 75.4 ± 2.9

0.543 0.432

Intervention Control Intervention Control Intervention Control

183.2 ± 7.9 52.8 ± 3.6 58.2 ± 2.1 110.8 ± 7.5 105.1 ± 7 115 ± 20.6

177.9 ± 7.3 51.2 ± 3.7 58 ± 2.1 102.2 ± 4.8 99.3 ± 7.1 121.2 ± 20

0.246 0.182 0.297 0.256 0.179 0.789

0.812

Intervention Control Intervention Control Intervention

74.7 68.1 63.8 94.7 85.1

73.3 69.5 64.3 93.9 83.4

0.380 0.613 0.816 0.764 0.597

Intervention Control

99.8 ± 10.9 1 ± 0.0

102.2 ± 10.6 1 ± 0.0

0.392 0.499

1.000

Intervention Control Intervention Control Intervention Control Intervention Control Intervention Control Intervention Control Intervention Control

0.9 ± 0.2 32 ± 1.5 30.3 ± 1.3 5.1 ± 0.5 5 ± 0.2 68.1 ± 5.2 67.3 ± 3.9 27.2 ± 2.2 26.8 ± 2 27.5 ± 3.9 21.1 ± 1.2 25.7 ± 4.3 19.2 ± 1.7 7.3 ± 0.1

0.9 ± 0.0 35.1 ± 2 32.1 ± 1.3 4.9 ± 0.4 5 ± 0.2 73.7 ± 5.8 68.6 ± 3.1 24.9 ± 2.5 28 ± 2 27 ± 3 21.2 ± 1.1 26.1 ± 4.4 18.4 ± 1.5 7.3 ± 0.1

0.115 0.325 0.134 0.584 0.968 0.093 0.649 0.114 0.298 0.906 0.877 0.919 0.467 0.688

Intervention

7.4 ± 0.1

7.3 ± 0.1

0.468

BMI (kg/m2) WC (cm) HC (cm) Systolic BP (mm Hg) Diastolic BP (mm Hg)a Pulse rate (min− 1) Cotinine (μΜ)a

± ± ± ± ±

1.9 3.4 2.2 3.6 3.9

± ± ± ± ±

1.9 2.3 2.1 3.6 5.2

0.294 0.210

Total chol (mg/ dL)

0.875

HDL-chol (mg/dL)

0.582

LDL-chol (mg/dL)

0.876

Triglycerides (mg/dL)a

0.767

Creatinine (mg/ dL)a

0.843

Urea (mg/dL)a Uric acid (mg/dL)

BMI: Body Mass Index; WC: Waist circumference; HP: Hip circumference; BP: Blood pressure. Statistical significance level p < 0.05. a p: Comparison of differences between baseline versus week 4 in variables in individual groups analyzed by paired sample t-test or Wilcoxon test. b p: Comparison of differences in variables between the control and the intervention group at week 4 analyzed by Independent sample t-test or Mann-Whitney test.

ALP (U/L) γ-GT (U/L) SGOT (mg/dL) SGPT (mg/dL)

(175.8 ± 7 to 166.6 ± 6.6 mg/dL, p < 0.001) (Fig. 2) and LDLcholesterol (96.2 ± 9.6 to 89 ± 10.5 mg/dL, p = 0.012) (Fig. 3) Additionally, when analyzing according to age, ICAM-1 levels significantly decreased in subjects above 30 years (n = 8) in intervention arm (390.1 ± 17.6 to 302.2 ± 11 ng/mL, p = 0.004) (Fig. 4). Furthermore, in a subgroup of ten heavy smokers (number of cigarettes ≥ 15 per day) in intervention arm, a trend in lower waist circumference was observed after the 4-week intervention compared to baseline (94.8 ± 3.7 vs 94.3 ± 3.6 cm, p = 0.054). However, no safe conclusions can be extrapolated as these results are unpowered.

a

a

Total proteins (g/ dL)a

0.128 0.714

0.587 0.560 0.349 0.064 0.841 0.771 0.441

ALP, alkaline phosphatase; γ-GT, γ-glutamyl transpeptidase; SGOT, serum glutamic oxalacetic transaminase; SGPT, serum glutamic pyruvic transaminase. Statistical significance level p < 0.05. a p: Comparison of differences between baseline versus week 4 in variables in individual groups analyzed by paired sample t-test or Wilcoxon test. b p: Comparison of differences in variables between the control and the intervention group at week 4 analyzed by Independent sample t-test or Mann-Whitney test.

supplementation with raisins on oxidant damage in healthy smokers with low adherence to the Mediterranean diet and low fruit/vegetable consumption. The mean fruit/vegetable intake was 2 servings/day. This was apparently below the 5–9 servings recommended as part of a

4. Discussion The main objective of the study was to estimate the effects of diet

Table 4 Differences in in energy and in nutrient intakes between control and intervention arm at the end of the study (mean ± SEM). Variable

Arm

Week 0

Week 4

p

pb

Energy (kcal)a

Control Intervention Control Intervention Control Intervention Control Intervention Control Intervention Control Intervention Control Intervention Control Intervention Control Intervention

2061.7 ± 165.7 1991.7 ± 121 225.3 ± 15.7 199 ± 18.9 84.6 ± 7.8 66 ± 8.3 71.4 ± 10.3 60.8 ± 8.6 28.5 ± 5 23.3 ± 4.3 25.1 ± 2.8 22 ± 2.5 11.8 ± 2.4 10.5 ± 1.6 0.7 ± 0.3 0.6 ± 0.2 1.1 ± 0.1 1.1 ± 0.1

2088.5 ± 212.7 1980.4 ± 117.7 223.8 ± 16 203.1 ± 19.1 89.6 ± 9.8 65.6 ± 7.9 76.5 ± 13.9 60 ± 8.3 32.7 ± 7.5 22.6 ± 4.1 26.1 ± 3.5 21.1 ± 2.7 11.7 ± 2.4 10.5 ± 1.7 0.8 ± 0.3 4.5 ± 0.4 1.1 ± 0.2 1.1 ± 0.2

0.333 0.778 0.575 0.494 0.878 0.748 0.799 0.533 0.799 0.286 0.517 0.232 0.333 0.231 0.798 < 0.001 1.000 0.804

0.668

Carbohydrates (g)a a

Protein (g)

Total fat (g)a Saturated (g)a Monounsaturated (g) Polyunsaturated (g)

a

Fruits (servings)a Vegetables (servings)a

Statistical significance level p < 0.05. a p: Comparison of differences between baseline versus week 4 in variables in individual groups analyzed by paired sample t-test or Wilcoxon test. b p: Comparison of differences in variables between the control and the intervention group at week 4 analyzed by Independent sample t-test or Mann-Whitney test.

4

0.333 0.804 0.735 0.769 0.668 0.875 < 0.001 0.882

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Table 6 Differences in inflammatory and oxidative stress markers between control and intervention arm at the end of the study (mean ± SEM). Variable

Arm

Week 0

Week 4

p

pb

CRP (μg/mL)a

Control Intervention Control Intervention Control Intervention Control Intervention

1.5 ± 0.6 1.9 ± 0.3 5.1 ± 0.6 4.3 ± 0.3 10.7 ± 1.9 13.2 ± 2.1 5.1 ± 0.4 4.6 ± 0.2

2 ± 0.8 2.7 ± 0.7 4.4 ± 0.3 4.5 ± 0.4 10.5 ± 1.5 13.5 ± 2 4.6 ± 0.2 4.5 ± 0.2

0.239 0.717 0.117 0.433 0.776 0.813 0.066 0.631

0.716

MDA (μΜ)a Leptin (pg/mL) AOPPs (μM)

0.113 0.753 0.276

CRP: C-reactive protein; MDA: Malondialdyhyde; AOPPs: Advanced Oxidation Protein Products. Statistical significance level p < 0.05. a p: Comparison of differences between baseline versus week 4 in variables in individual groups analyzed by paired sample t-test or Wilcoxon test. b p: Comparison of differences in variables between the control and the intervention group at week 4 analyzed by Independent sample t-test or Mann-Whitney test.

Fig. 2. Differences in total cholesterol (mean ± SEM) in men and women in intervention arm at baseline and at the end of the study. Statistical significance (p < 0.05) between two timepoints is indicated by *.

Table 7 Differences in endothelial function and arterial stiffness markers between control and intervention arm at the end of the study (mean ± SEM). Variable

Arm

Week 0

Week

p

pb

Carotid-Radial PWV (m/s)

Control

6.2 ± 0.3

5.8 ± 0.3

0.072

0.051

Intervention Control

6 ± 0.2 6.6 ± 0.3

6.1 ± 0.2 6.8 ± 0.2

0.444 0.588

0.555

Intervention Control Intervention Control

6.4 ± 0.2 3.8 ± 0.6 2.7 ± 0.4 387.6 ± 38.1

6.4 ± 0.2 3.9 ± 0.7 2.9 ± 0.5 414.3 ± 44.1

0.855 0.925 0.602 0.347

0.117

Intervention Control

353.9 ± 15.8 21.1 ± 2.3

327.5 ± 12.9 19.8 ± 2.5

0.232 0.688

0.497

Intervention

27.4 ± 2.5

23.4 ± 2.2

0.130

Carotid-Femoral PWV (m/s) FMD (%) ICAM-1 (ng/ mL)a Nitric oxide (μmol/L)

0.907

Carotid-Radial PWV: Carotid-Radial pulse wave velocity; Carotid-Femoral PWV: CarotidFemoral pulse wave velocity; FMD: Flow Mediated Dilatation; ICAM-1: Intercellular Adhesion Molecule-1. Statistical significance level p < 0.05. a p: Comparison of differences between baseline versus week 4 in variables in individual groups analyzed by paired sample t-test or Wilcoxon test. b p: Comparison of differences in variables between the control and the intervention group at week 4 analyzed by Independent sample t-test or Mann-Whitney test.

Fig. 3. Differences in LDL-cholesterol (mean ± SEM) in men and women in intervention arm at baseline and at the end of the study. Statistical significance (p < 0.05) between two timepoints is indicated by *.

Fig. 4. Differences in ICAM-1levels (mean ± SEM) between control and intervention arm in participants aged above 30 years. Statistical significance (p < 0.05) between two timepoints is indicated by *.

Fig. 1. Differences in diastolic blood pressure (mean ± SEM) in men and women in intervention arm at baseline and at the end of the study. Statistical significance (p < 0.05) between two timepoints is indicated by *.

5

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criteria. This is the first study evaluating the consumption of a dried fruit in healthy smokers. However, the short duration of the intervention and the photometric evaluation of MDA could be limitations of the study. Since previous studies have shown that smokers have different response compared to non-smokers, probably due to their increased need for antioxidants, an intervention with a higher amount of raisins could elucidate results.

healthy diet. Although, smokers increased fruit intake with incorporation of 5 servings of raisins in daily diet, this was not combined with differences in either levels of inflammatory and oxidative stress markers or levels of endothelial function and arterial stiffness markers. In the study of Alvarez-Parrilla et al. (2010), the effect of daily consumption of pear, apple and orange juice for 26 days on antioxidant capacity and lipid profile was evaluated in smokers and non-smokers. While fruit consumption increased antioxidant capacity in non-smokers, this was not observed in smokers, supporting the idea that total antioxidants supplied daily by apple, pear and 200 mL orange juice were not sufficient to neutralize reactive oxygen species produced by smoking, in the studied population. This is probably an explanation for results herein. Regarding the consumption of fruit or fruit juices and their effect on vascular function indices, some recent studies have been reported. The study of Dohadwala et al. (2011) examined the effect of cranberry juice enriched with polyphenols on vascular function in patients with coronary artery disease and found a reduction of PWV in the juice group vs placebo. However, the juice applied was enriched with phenolic compounds, in contrast to the present study where naturally dried black grapes were administered (equivalent to 5 fruit servings) based on the USDA recommendations. In another recent study (Johnson et al., 2015), researchers examined the effect of daily consumption of frozen blueberries powder for 8 weeks in postmenopausal women with prehypertension and hypertension stage 1. At the end of intervention, a significant decrease in systolic (p < 0.05) and diastolic BP (p < 0.01) and PWV was reported compared to baseline. NO levels increased in intervention arm. However, in a previous study, 3-week administration of 250 g blueberries in healthy smokers had no effect on BP and oxidative stress markers (McAnulty et al., 2005). Overall, results on the effect of polyphenol-rich foods in endothelial function are controversial and several methodological limitations in clinical trials occur. Vlachopoulos et al. (2005) studied the effect of dark chocolate rich in flavonoids (100 g) on endothelial function, arterial stiffness, wave reflection indices andoxidant status. Results showed improvement in FMD during the first 60 min of the study and reduction of the pulse wave reflections during the 3 h study but no effect on arterial stiffness and antioxidant capacity. Higher cocoa intake was found as an independent determinant of low arterial stiffness and wave reflection indexes was independently associated with lower central pulse pressure (Vlachopoulos et al., 2007). Nevertheless, cocoa intake was assessed by a cocoa-frequency self-administered questionnaire rather than by measurement of phenolics in serum. Similarly, in an unpowered study of healthy non-smoking men, consumption of 400 mL beer reduced arterial stiffness compared with vodka or beer without alcohol (Karatzi et al., 2013). The pulse wave reflections decreased significantly in all categories, suggesting a synergistic action of alcohol with beer-containing antioxidants. Another study (Riso et al., 2014) evaluated the effects of 10-day broccoli (250 g/day) intake on dietary markers and markers of inflammations in young male smokers. Plasma CRP decreased by 48% (post-hoc analysis, p < 0.05) following broccoli diet, consistent with epidemiologic observations that fruit and vegetable intake is associated with lower circulating CRP concentrations. A more recent study was investigated the effect of dietary supplementation with a standardized maqui berry (Aristotelia chilensis) extract, rich in anthocyanins, on products of lipid peroxidation. A decrease in ox-LDL and urinary F2-isoprostanes was observed at 4 weeks versus baseline implying that the maqui berry extract may improve oxidative status in healthy adults, overweight adults, and adult smokers (Davinelli, Bertoglio, Zarrelli, Pina, & Scapagnini, 2015). One of the major strengths of the present study is that the amount of raisins consumed was realistic, selected according to the daily recommendations for fruits and vegetables. Furthermore, all subjects (both males and females) were selected according to cautiously defined

5. Conclusions Conclusively, the results of the present study suggest that raisin consumption could be a means of increasing the low fruit and vegetable intake. Although herein favorable effect in arterial function or biochemical profile was reported in older or female participants solely, however raisins could be an alternative to fresh fruits and help people reach the recommendations, being rich in antioxidants and easy to store and carry simultaneously. The amount of raisins was realistic however insufficient to evoke a significant effect on the markers studied in a population of smokers. Funding The study was supported by IKY (SPhD/11186/13β) Fellowships of Excellence for Postgraduate Studies in Greece-Siemens Program. Conflict of interest All the authors declare that they have no conflict of interest. References Alvarez-Parrilla, E., De La Rosa, L. A., Legarreta, P., Saenz, L., Rodrigo-García, J., & González-Aguilar, G. A. (2010). Daily consumption of apple, pear and orange juice differently affects plasma lipids and antioxidant capacity of smoking and nonsmoking adults. International Journal of Food Sciences and Nutrition, 61(4), 369–380. Bartalis, J., Chan, W. G., & Wooten, J. B. (2007). A new look at radicals in cigarette smoke. Analytical Chemistry, 79(13), 5103–5106. Bjørklund, G., & Chirumbolo, S. (2017). Role of oxidative stress and antioxidants in daily nutrition and human health. Nutrition, 33, 311–321. Corretti, M. C., Anderson, T. J., Benjamin, E. J., Celermajer, D., Charbonneau, F., Creager, M. A., ... International Brachial Artery Reactivity Task Force (2002). Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: A report of the International brachial artery reactivity task force. Journal of the American College of Cardiology, 39, 257–265. Dauchet, L., Montaye, M., Ruidavets, J. B., Arveiler, D., Kee, F., Bingham, A., ... Dallongeville, J. (2010). Association between the frequency of fruit and vegetable consumption and cardiovascular disease in male smokers and non-smokers. European Journal of Clinical Nutrition, 64(6), 578–586. Davinelli, S., Bertoglio, J. C., Zarrelli, A., Pina, R., & Scapagnini, G. (2015). A randomized clinical trial evaluating the efficacy of an anthocyanin-maqui berry extract (Delphinol®) on oxidative stress biomarkers. The Journal of the American College of Nutrition, 34(Suppl. 1), 28–33. Dohadwala, M. M., Holbrook, M., Hamburg, N. M., Shenouda, S. M., Chung, W. B., Titas, M., & …Vita, J.A. (2011). Effects of cranberry juice consumption on vascular function in patients with coronary artery disease. The American Journal of Clinical Nutrition, 93, 934–940. Gomes-Rochette, N. F., Da Silveira Vasconcelos, M., Nabavi, S. M., Mota, E. F., NunesPinheiro, D. C., Daglia, M., & De Melo, D. F. (2016). Fruit as potent natural antioxidants and their biological effects. Current Pharmaceutical Biotechnology, 17(11), 986–993. Hu, E. A., Toledo, E., Diez-Espino, J., Estruch, R., Corella, D., Salas-Salvado, J., ... Martinez-Gonzalez, M. A. (2013). Lifestyles and risk factors associated with adherence to the Mediterranean diet: A baseline assessment of the PREDIMED trial. PLoS One, 8(4), e60166. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans (2004). Tobacco smoke and involuntary smoking. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, 83, 1–1438. Johnson, S. A., Figueroa, A., Navaei, N., Wong, A., Kalfon, R., Ormsbee, L. T., & …Arjmandi, B.H. (2015). Daily blueberry consumption improves blood pressure and arterial stiffness in postmenopausal women with pre- and stage 1-hypertension: a randomized, double-blind, placebo-controlled clinical trial. Journal of the Academy of Nutrition and Dietetics, 115(3), 369–377. Kanellos, P. T., Kaliora, A. C., Gioxari, A., Christopoulou, G. O., Kalogeropoulos, N., & Karathanos, V. T. (2013). Absorption and bioavailability of antioxidant phytochemicals and increase of serum oxidation resistance in healthy subjects following supplementation with raisins. Plant Foods for Human Nutrition, 68(4), 411–415.

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