A serving of blueberry (V. corymbosum) acutely improves peripheral arterial dysfunction in young smokers and non-smokers: two randomized, controlled, crossover pilot studies

Cristian Del Bo’ *a, Valeria Deon a, Jonica Campolo b, Claudia Lanti a, Marina Parolini b, Marisa Porrini a, Dorothy Klimis-Zacas c and Patrizia Riso a
aUniversità degli Studi di Milano, Department of Food, Environmental and Nutritional Sciences, Division of Human Nutrition, Milan, Italy. E-mail: cristian.delbo@unimi.it; Fax: +39 02503 16721; Tel: +39 02503 16727
bCNR Institute of Clinical Physiology, CardioThoracic and Vascular Department, ASST Great Metropolitan Hospital Niguarda, Milan, Italy
cSchool of Food and Agriculture, University of Maine, ME, USA

Received 12th June 2017 , Accepted 19th September 2017

First published on 20th September 2017


Several studies have documented the important role of polyphenol-rich foods in the modulation of vascular remodelling and function. This study aimed to evaluate the capacity of a single portion of blueberry (V. corymbosum) to acutely improve peripheral arterial dysfunction in a group of young volunteers. Twenty-four healthy males (12 non-smokers and 12 smokers) were recruited for two different randomized, controlled, crossover pilot acute studies. In the first study, non-smokers were exposed to a control treatment (C; 300 mL of water with sugar) and a blueberry treatment (BB; 300 g of blueberry). In the second study, smokers underwent 3 different protocols: (1) – smoking treatment (S); (2) – control treatment (CS; 300 mL of water with sugar + smoking); (3) – blueberry treatment (BS; 300 g of blueberry + smoking). Each treatment (1 day long) was separated by a one week washout period. Blood pressure, peripheral arterial function (reactive hyperemia index, RHI, a marker of endothelial function) and arterial stiffness (digital augmentation index, dAix and dAix normalized by considering a heart rate of 75 bpm, dAix@75) were measured before and after each treatment. In the first study, the consumption of blueberry and control treatment acutely increased peripheral arterial function in the group of non-smokers. The improvement in RHI was higher and significantly different after blueberry treatment compared to the control treatment (54.8 ± 8.4% BB vs. 28.2 ± 8.3% C; p = 0.01). No effects were observed for markers of arterial stiffness, blood pressure and heart rate. Acute cigarette smoke significantly increased blood pressure and heart rate, while no significant effect was registered in peripheral arterial function and stiffness. The intake of blueberry and control treatment before a cigarette did not counteract the increase in blood pressure and heart rate, while it significantly improved peripheral arterial function. In particular, a significant increase was observed following BS (35.2 ± 7.5% RHI; p = 0.02) and CS treatments (34.6 ± 11.9% RHI; p = 0.02) when compared to only smoking treatment. No difference between BS and CS was detected. In conclusion, the intake of blueberry and control treatments acutely improved peripheral arterial dysfunction both in smoker and in non-smoker subjects. Further studies should be performed to confirm the results obtained and reveal the potential mechanisms of blueberry in the improvement of endothelial function.


Introduction

Endothelial dysfunction is characterized by an imbalance between vasodilator and vasoconstrictor mediators resulting in altered vascular tone, organ blood flow and peripheral vascular resistance.1 The endothelium, in response to mechanical and hormonal stimuli, releases vasoconstrictor and vasodilator agents that regulate vasomotor function by inducing vasoconstriction or vasodilation.2 Nitric oxide (NO) is one of the most important molecules involved in the process of vasodilation. When endothelial damage occurs, the bioavailability of NO decreases causing vasoconstriction of the endothelium.2 Endothelial dysfunction represents the first step leading to the development of atherosclerosis.1 Convincing evidence supports the pivotal role of smoking, as a source of oxidative stress, in the development of ED.3 Cigarette smoke causes a temporal increase in blood pressure, heart rate and acute endothelial damage, and vascular and systemic inflammation.4

Blueberries are a rich source of polyphenolic compounds such as phenolic acids and anthocyanins involved in the modulation of several functional and metabolic pathways related to endothelial function.5–8 For example, anthocyanins (ACNs) have been reported to induce the expression and activity of numerous enzymes involved in NO metabolism.7 In particular, ACNs have been documented to stimulate endothelial nitric oxide synthase (eNOS) and to decrease endothelial NADPH oxidase activity and O2˙ levels as a result of haem oxygenase-induction.7 Furthermore, ACNs have been shown to reduce the expression of a plethora of pro-inflammatory agents involved in the adhesion of monocytes to endothelial cells by inhibiting the redox-sensitive transcription factor NF-κB and by eliciting cell adaptive responses involving the transcription factor Nrf2.7,9–11

The role of blueberries in the modulation of vascular function has been evaluated in different acute and chronic intervention studies.5,12–17 Stull et al.12 reported that a 6-week intervention with blueberry (22.5 g of freeze-dried blueberry powder, providing 290 mg of anthocyanins) smoothie (twice per day) improved endothelial function in subjects with metabolic syndrome. We previously documented that a 6-week consumption of 250 mL of a wild blueberry drink (25 g of freeze-dried blueberry powder, providing 375 mg of anthocyanins) failed to show an improvement of peripheral arterial function, a marker of endothelial function, in the whole group of subjects with cardiovascular risk factors.17 The beneficial effects were documented only in smokers and in those with endothelial dysfunction.17 Recently, we showed that a serving of 300 g of blueberry purée (providing about 300 mg of anthocyanins) was able to counteract the temporary impairment of endothelial function induced by acute cigarette smoke in a group of smoker volunteers with normal endothelial function.14 Based on our previous research, we evaluated whether the consumption of the same portion of blueberries could improve endothelial function also in subjects with a dysfunctional endothelium (single risk factor) and in those exposed to oxidative stress (smoking) and showing an impairment in peripheral arterial function (double risk factor). To this aim, two randomized, controlled, pilot studies (one in a group of smokers and the other one in non-smoker subjects) were performed in which the effect of a single blueberry portion on peripheral arterial function was evaluated.

Materials and methods

Subject selection

Twenty-four healthy male (12 non-smokers and 12 smokers) subjects with peripheral arterial dysfunction (RHI < 1.67) were recruited from the student population of the University of Milan according to the following criteria: 20–30 years of age, reactive hyperemia index, as a marker of peripheral arterial function (RHI < 1.67), moderate physical activity (up to 25–30 min day-1 of brisk walk or jog) and alcohol consumption (no more than 14 drinks of wine or beer per week). Smokers were homogeneous for smoking habits (about 15 cigarettes per day). Exclusion criteria were hypertension (systolic pressure >140 mmHg and diastolic pressure >90 mmHg), fasting hyperglycemia (>10 mmol L−1), hypercholesterolemia (high total serum cholesterol, >5.17 mmol L−1; high low density lipoprotein cholesterol, >3.36 mmol L−1), hypertriglyceridemia (>1.69 mmol L−1), and overweight/obese (BMI >25 kg m−2) based on American Heart Association guidelines.18 Subjects with normal endothelial function (RHI > 1.67) were automatically excluded from the study. Moreover, diabetic patients, subjects with renal insufficiency, constipation, diarrhea or gastrointestinal problem or diseases were not included. Subjects with traumas of the arms or hands, fingers, atopic dermatitis, thyroid disturbance, depression, anxiety, palpitations, asthma, and chronic backache were excluded. Other exclusion criteria were as follows: allergies, high (>5 portions per day) or low (<2 portions per day) intake of fruit and vegetables, specific diet (e.g. vegetarian, vegan or macrobiotic), specific aversion to blueberries or their products, and use of drugs, supplements, and medications during the last month. The study was performed in accordance to the ethical standards established in the 1964 Declaration of Helsinki and approved by the Ethics Committee of the University of Milan. All participants signed the informed consent form. The study was registered at http://www.isrctn.org as ISRCTN59129089.

Food preparation and composition

Fresh blueberry (Vaccinium corymbosum L. “Brigitta”), from a single batch, was purchased and immediately stored at −20 °C until use. A portion (300 g) of frozen blueberry was thawed at +4 °C overnight and provided to the participants. It contained 27 g of total sugars, 856 mg of total phenolic acids, 309 mg of total anthocyanins, 30 mg of chlorogenic acid and 2.4 mg of ascorbic acid. The control treatment was prepared by suspending 16.4 g of fructose and 10.6 g of glucose (the same amount and type of sugars as contained in the blueberry) in 300 ml of water. No bioactive compounds were added.

Experimental design

Prior to the intervention, a 10-day run-in period was performed. Subjects were deprived of polyphenol-rich foods such as chocolate, berries, red wine and red to blue fruits, and green tea. Volunteers were asked to limit their intake of coffee to three cups per day, as well as caffeine-rich beverages (e.g. energy drinks), to reduce a potential effect on vascular function. The day before the experiment breakfast, lunch and dinner were standardized to provide adequate energy/macronutrient intake, taking into account Italian dietary habits.14 All participants refrained from physical activity the day before the experiment, while smokers were asked to maintain their smoking habits as reported in the questionnaire but to refrain from cigarettes the morning of the experiment.

Study 1

Twelve nonsmokers with peripheral arterial dysfunction were randomly divided into 2 groups of 6 subjects each: group 1 was assigned to the sequence BB treatment/wash-out/C treatment, whereas group 2 followed the sequence C/wash-out/BB treatment. The study consisted of a repeated measure 2-arm randomized-controlled trial (Fig. 1A). Each protocol was separated by a 7-day wash-out period. RHI levels were assessed in the morning after overnight fasting and following the consumption of 300 g blueberries or control treatment (BB or C, respectively). The protocol was designed to measure vascular function (peripheral arterial function and arterial stiffness) 120 min after blueberry/control intake by considering our previous observations on the specific time-point effect on endothelial function observed following the intake of the same portion of blueberry.14 The evaluation of peripheral arterial function and arterial stiffness was performed at baseline (T = 0) and after the intake of blueberry and control treatment (T = 120 min). Systolic (S) and diastolic (D) blood pressure (BP) and heart rate (HR) were measured in duplicate as follows: before BB and C intake (T = 0 min), after BB and C intake (T = 100 min), and following the measurements of endothelial function and arterial stiffness (T = 120 min).
image file: c7fo00861a-f1.tif
Fig. 1 Experimental designs. (A) Non-smoker study: Two arms, randomized controlled crossover design; (B) smoker study: three arms, randomized controlled crossover design.

Study 2

Twelve smokers with peripheral arterial dysfunction were randomly assigned to 3 different groups: S – smoking treatment; BS – blueberry treatment (300 g of blueberry) + smoking; and CS – control treatment (300 mL of water with sugar) + smoking. The study consisted of a repeated measure 3-arm randomized-controlled trial (Fig. 1B). Each protocol was separated by a-7 day wash-out period. On the day of the experiment, baseline RHI levels were assessed early in the morning after overnight fasting and without smoking. Successively, peripheral arterial function was assessed after smoking (S) or following the consumption of 300 g blueberries or control treatment and smoking (BS or CS, respectively). The protocol was designed to measure reactive peripheral arterial function and stiffness 120 min after blueberry/control intake (corresponding to 20 minutes after cigarette smoking) according to our previous publication.14 Systolic (S) and diastolic (D) blood pressure (BP) and heart rate (HR) were measured in duplicate at baseline (T = 0 min), before smoking (T = 100 min), 5 min after smoking one cigarette (T = 105 min), and following the measurements of endothelial function and arterial stiffness (T = 120 min).

Evaluation of blood pressure, heart rate, peripheral arterial function and arterial stiffness

Peripheral arterial function was measured by a non-invasive plethysmographic method (Endo-PAT 2000, Itamar Medical Ltd, Caesarea, Israel), as previously reported in detail.14 During the assessment, participants were in a supine position in a comfortable, dimly lit and temperature-controlled room. After application of the occlusion cuff to the dominant arm and fingertip probes to the index fingers of each hand, the study began with a 5 minute baseline, 5 minutes of occlusion, and last 5 minutes of post-occlusion measurements (hyperemic period). Occlusion of the brachial artery was performed on the dominant upper arm (at least 60 mmHg above the systolic blood pressure; minimally 200 mmHg and maximally 300 mmHg). The Endo-PAT system generates automatically a value of reactive hyperemia index (RHI) as an index of the endothelial-dependent flow-mediated dilation (FMD; the gold standard method for the evaluation of endothelial function).19,20 An RHI value less than 1.67 provides a sensitivity of 82% and a specificity of 77% for diagnosing endothelial dysfunction.21 The Endo-PAT device also provides dAix, strongly correlated to aortic Aix, calculated from the shape of the pulse wave recorded by the probes during baseline.22 Because Aix is influenced in an inverse and linear manner by heart rate, the dAix was automatically normalized by considering a heart rate of 75 bpm (dAix@75).

Statistical analysis

The sample size was calculated taking into account the expected variation of RHI as the primary endpoint considered. Based on our previous observation,14 twelve subjects were calculated to be sufficient to evaluate a difference of RHI of 0.30, after blueberry intake, (standard deviation 0.35), with alpha = 0.05 and a statistical power of 80%. In addition, the repeated measures design reduces the variance of estimates of treatment-effects allowing using fewer subjects. Finally, the number of subjects enrolled was comparable to that reported in other studies evaluating the role of cocoa, chocolate and mango in the modulation of endothelial function through PAT technology.23–26 Results for each treatment are reported as the percentage change (i.e. [after treatment − before treatment]/before treatment × 100). Mean changes are described as a mean with 95% CI. Variables were analyzed by one way ANOVA with time or treatment as dependent factors. Differences were considered significant at p ≤ 0.05; post-hoc analysis of differences between treatments was assessed by the Least Significant Difference (LSD) test with p ≤ 0.05 as the level of statistical significance. Data are reported as mean values and standard error of the mean (SEM). Statistical analysis was performed by means of the STATISTICA software (Statsoft Inc., Tulsa, OK, USA).

Results

Study 1

Baseline characteristic of non-smoker subjects. The anthropometric and clinical characteristics at baseline of the 12 healthy non-smoker subjects enrolled are reported in Table 1. All volunteers presented values of RHI lower than 1.67 (cut-off to discriminate an endothelial dysfunction). Blood pressure, heart rate and BMI were in the normal range.
Table 1 Characteristics of non-smoker subjects at baseline
Variables Mean ± SEMa
a N = 12. Data are reported as mean ± SEM; SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate; RHI, reactive hyperemia index; dAix, digital augmentation index; dAix@75, digital augmentation index standardized for a heart rate of 75 bpm; SEM, standard error of the mean
Age (years) 24.2 ± 1.2
Height (cm) 175.8 ± 1.4
Weight (kg) 70.5 ± 2.1
BMI (kg m−2) 22.5 ± 1.2
SBP (mmHg) 116.9 ± 3.2
DBP (mmHg) 75.3 ± 2.9
HR (beats per min) 61.8 ± 5.3
RHI 1.41 ± 0.07
dAix (%) −14.6 ± 2.7
dAix@75 (%) −20.0 ± 5.8


Effect of blueberry and control treatments on blood pressure, heart rate, arterial function and arterial stiffness in non-smokers. The mean percentage changes pre- to post-treatment following the intake of blueberry and control are presented in Table 2. Both treatments did not have a significant impact on SBP, DPB and HR.
Table 2 Mean percentage variation (Δ) of systolic blood pressure (SBP), diastolic blood pressure (DPB) and heart rate (HR) in non-smokers following the intake of blueberry (BB) and control (C) treatmenta
  Δ% BB Δ% C p valueb
a N = 12. Data are expressed as means ± SEM. SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate; BB, blueberry treatment; C, control treatment; SEM, standard error of the mean. b Overall p value for t-TEST with STATISTICA software (Statsoft Inc., Tulsa, OK, USA).
SBP −0.89 ± 0.91 −2.93 ± 2.03 0.236
DBP −2.76 ± 2.33 −1.38 ± 1.04 0.431
HR −1.08 ± 1.84 0.56 ± 4.05 0.869


The effect of blueberry and control treatment on RHI (A), dAix (B) and dAix@75 (C) is reported in Fig. 2. Both treatments had a favorable effect on RHI showing an improvement compared to baseline (time effect). In particular, 10 out of 12 subjects (83%) reversed their RHI impairment following blueberry treatment, while 7 out of 12 subjects (58%) did similarly following the control treatment. The mean percentage change pre- to post-treatment following the BB treatment for RHI was +54.8% (95% CI: +37.9%, +71.7%) and +28.2% (95% CI: +11.5%, +44.9%) following the C treatment (Fig. 2A). This increase was higher and significantly different after the BB treatment when compared to C treatment (RHI, p = 0.011; treatment effect). In contrast, no significant effect was observed for dAix and dAix@75 as markers of arterial stiffness (Fig. 2B and C).


image file: c7fo00861a-f2.tif
Fig. 2 Mean percent variation of RHI (A), dAix (B), and dAix@75 (C) measured during blueberry (BB) and control (C) treatment. Data are expressed as mean ± SEM. C, control treatment; BB, blueberry treatment; RHI, reactive hyperemia index; dAix, digital augmentation index; dAix@75, digital augmentation index standardized for heart rate of 75 bpm. *Significantly different compared to control treatment (p ≤ 0.01).

Study 2

Baseline characteristics of smoker subjects. The anthropometric and clinical characteristics of the 12 smoker subjects enrolled are reported in Table 3. All volunteers were apparently healthy with blood pressure, heart rate and BMI in the normal range while the levels of peripheral arterial function were lower than 1.67, implying impaired peripheral arterial function.
Table 3 Characteristics of smoker subjects at baseline
Variables Mean ± SEMa
a N = 12. Data are reported as mean ± SEM; SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate; RHI, reactive hyperemia index; dAix, digital augmentation index; dAix@75, digital augmentation index standardized for a heart rate of 75 bpm; SEM, standard error of the mean.
Age (years) 24.5 ± 1.9
Height (cm) 180.1 ± 1.3
Weight (kg) 70.7 ± 1.2
BMI (kg m−2) 22.9 ± 1.1
Smoke (cigarettes per day) 15 ± 0.4
SBP (mmHg) 118.2 ± 2.9
DBP (mmHg) 75.7 ± 2.7
HR (beats per min) 64.9 ± 5.1
RHI 1.47 ± 0.05
dAix (%) −12.7 ± 2.5
dAix@75 (%) −18.2 ± 5.0


Effect of smoking on blood pressure, heart rate, arterial function and arterial stiffness in smokers. Smoking induced a significant temporary increase in the levels of SBP (from 117.1 ± 4.40 mmHg to 129.6 ± 2.99 mmHg; p = 0.006), DBP (from 74.2 ± 4.00 to 83.2 ± 3.32; p = 0.03) and HR (from 65.2 ± 3.58 beats per min to 75.3 ± 5.07 beats per min; p = 0.04). The rise was registered after 5 min from smoking and the values dropped to baseline after 20 min (Fig. 3). Table 4 reports the levels of RHI, dAix and dAix@75 before and after smoking. No significant effect on markers of arterial function and stiffness was observed after acute cigarette smoking.
image file: c7fo00861a-f3.tif
Fig. 3 Variation of systolic and diastolic blood pressure (A) and heart rate (B) during acute cigarette smoking over time. Data are expressed as mean ± SEM. SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate. *Data are significantly different (p < 0.05).
Table 4 Arterial function and arterial stiffness before and 20 min after smoking a cigarette in smokers
  Before smoking 20 min after smoking p valuea
N = 12. Data are expressed as means ± SEM. RHI, reactive hyperemia index; dAix, digital augmentation index; dAix@75, digital augmentation index standardized for a heart rate of 75 bpm.a Overall p value for t-TEST with STATISTICA software (Statsoft Inc., Tulsa, OK, US).
RHI 1.47 ± 0.05 1.58 ± 0.07 0.324
dAix (%) −12.7 ± 2.5 −15.6 ± 3.5 0.455
dAix@75 (%) −18.2 ± 5.0 −18.8 ± 4.9 0.895


Effect of blueberry and control treatments on blood pressure, heart rate, arterial function and stiffness in smokers. The effect of BS and C treatment on blood pressure and heart rate in smoker volunteers is reported in Table 5. No significant effect in the mean percentage change pre- to post-treatment was observed on SBP, DPB and HR following the three interventions (S vs. CS vs. BS). Fig. 4 shows the effect of S, BS and CS treatment on RHI (A), dAix (B) and dAix@75 (C). ANOVA revealed a significant effect of the treatment for the variable RHI (p = 0.03; Fig. 4A). In particular, the mean percentage change pre- to post-treatment was +8.38% (95% CI: −4.57%, +21.3%) following the S treatment, +34.6% (95% CI: +10.6%, +58.7%) following the CS treatment and +35.2% (95% CI: +20.2%, +50.3%) following the BS treatment. Post-hoc analysis (LSD test) showed that the consumption of a single serving of blueberry and control significantly reversed the impairment of RHI (Fig. 4B) when compared to the S treatment (BS vs. S, p = 0.022 and CS vs. S, p = 0.023).
image file: c7fo00861a-f4.tif
Fig. 4 Mean percent variation of RHI (A), dAix (B), and dAix@75 (C) measured during each treatment. aData are expressed as mean ± SEM. S, smoking treatment; CS, control-drink + smoking treatment; BS, blueberry intake + smoking treatment; RHI, reactive hyperemia index; dAix, digital augmentation index; dAix@75, digital augmentation index standardized for a heart rate of 75 bpm. *Significantly different compared to smoking treatment (p ≤ 0.01).
Table 5 Mean percentage variation (Δ) of systolic blood pressure (SBP), diastolic blood pressure (DPB) and heart rate (HR) in smokers measured during each treatmenta
  Δ% S Δ% BS Δ% CS p valueb
a N = 12. Data are expressed as mean ± SEM. S, smoking treatment; CS, control-drink + smoking treatment; BS, blueberry intake + smoking treatment. SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate. b Overall p value for ANOVA using STATISTICA software (Statsoft Inc., Tulsa, OK, USA).
SBP 11.3 ± 3.5 4.13 ± 1.27 5.38 ± 2.52 0.174
DBP 13.6 ± 5.5 4.09 ± 3.05 2.35 ± 2.34 0.130
HR 15.5 ± 3.7 9.80 ± 4.50 10.8 ± 5.1 0.961


However, the BS and CS treatments did not differ in their effect (p = 0.954). No significant variation was detected for dAix and dAix@75 following the three treatments (Fig. 4C).

Discussion

Several studies have emphasized the role of polyphenol-rich foods in the modulation of vascular function. Encouraging results have been obtained following the consumption of a serving of coffee,27,28 tea,29,30 dark chocolate and cocoa powder,25,31–33 and grape,34 while few and conflicting are those results reported after the intake of different types of berries.15,16,35–38 For example, Dohadwala and colleagues documented a significant increase in vascular function 4 h after the consumption of cranberry juice (480 ml, providing 835 mg total polyphenols and 94 mg anthocyanins) in a group of subjects with coronary artery disease.36 Rodriguez-Mateos et al.15 showed that the consumption of blueberry baked products (containing a total of 34 g of wild blueberry powder, equivalent to 240 g of fresh blueberry) improved endothelial function at 1, 2 and 6 h in healthy volunteers. In contrast, we previously failed to observe a beneficial effect on vascular function 1 h after the intake of a portion (300 g) of blueberry (providing 300 mg of anthocyanins) in young, healthy subjects with normal endothelial function.37 Also, Jin et al.38 reported that vascular reactivity was not affected 2 h from the intake of 250 ml blackcurrant juice drink in a group of healthy volunteers. In the present pilot studies, we documented that the administration of 300 g of blueberry acutely improved peripheral arterial function both in smoker and in non-smoker subjects 2 h after consumption. Discrepancies among studies may be dependent on the type and the dose of berry (e.g. cranberry vs. blackcurrant vs. blueberry), the experimental design, and/or the subject characteristics (e.g. healthy vs. subjects with cardiovascular risk factors/endothelial dysfunction), the length of time between berry intake and the evaluation of the endothelial function (e.g. 1 h vs. 2, 4 or 6 h), and the mode of measuring endothelial function (e.g. flow mediated dilation vs. peripheral arterial function). Another important variable to be considered may be the peak time of absorption of bioactives/metabolites and their blood concentration. Rodriguez-Mateos et al.16 reported that the administration of different doses of blueberry polyphenols (766, 1278, and 1791 mg total blueberry polyphenols, equivalent to 240, 400, and 560 g fresh blueberries, respectively) increased endothelial function at 1–2 h and 6 h in a group of healthy subjects and these beneficial effects were closely linked to the increase of the circulating levels of polyphenol metabolites in a time- and dose-dependent manner. In our study, we did not measure the absorption of blueberry bioactives and their metabolic products; however, we previously documented that the intake of the same blueberry portion increased anthocyanin absorption at 1 h, reaching the maximum peak at 1.5–2 h from consumption.39 These findings could explain the modulation of endothelial function observed at 2 h in our subjects.

It is widely recognized that cigarette smoking causes acute and chronic vascular damage. We reported that the smoke of one cigarette temporarily induces vascular dysfunction in a group of young smokers.40 In the present study, acute cigarette smoke did not further induce detrimental effects on vascular function in smokers with established endothelial dysfunction. On a positive note, the intake of blueberries reversed this condition, in agreement with our previous publication.14 Similar results were also reported by Schwarz and colleagues documenting that the pre-consumption of red wine prevented most of the negative vascular effects of acute smoking in a group of young healthy non-smokers.41 In our study, the beneficial effects observed following the consumption of blueberry were not significantly different compared to those observed after the intake of the control drink. The lack of a significant difference in RHI between blueberry and control treatment, together with the apparent increase observed also in the non-smoker group following the intake of the control drink, could be attributed to different factors. For example, it has been reported that the amount and type of sugar may affect endothelial function.42,43 The consumption of both blueberry and control drinks, providing the same amount of glucose and fructose, brought blood glucose to a comparable elevation within the first 15 min from intake and dropped to baseline after 1 h, as documented in a subgroup of subjects (data not shown). Since the evaluation of arterial function was performed 2 h from the intake of blueberry and control drinks, we can exclude a direct contribution of sugars for the above observation. However, different studies reported a possible involvement of insulin and glucagon, two hormones secreted in response to blood sugar levels, in the modulation of vascular function especially at the microvascular level.44–46 The secretion of insulin may induce changes in microcirculatory tone, activate the eNOS pathway and consequently lead to vasodilation.44,45 On the other hand, glucagon can trigger the formation of cAMP, which induces the formation of NO playing a pivotal role in vasorelaxation.46 Since PAT technology measures endothelial function at the microvascular level, and subjects did not consume food apart from the blueberry and control drinks during the entire duration of the experiment, the involvement of insulin or glucagon cannot be ruled out.

Arterial stiffness represents a significant determinant of pulse pressure and elasticity of the blood vessels.47 Numerous studies found that chronic smoking increases arterial stiffness.48 The loss of elasticity of the artery walls reduces its compensatory ability to absorb the pulsatile energy and the wave propagation effects that influence peripheral wave reflection. This inability for compensatory response results in the gradual increase in blood pressure with age, leading to the development of isolated systolic hypertension and to an increase of cardiovascular risk. The Endo-PAT system provides the value of the digital augmentation index (dAix) and the value of dAix, standardized for heart rate (dAix@75), as markers of arterial stiffness. Digital Aix reflects changes in vessel diameter, blood pressure and heart rate.49 In our experimental conditions, we documented that the subjects involved did not show impairment in arterial stiffness, probably due to their young age. Acute cigarette smoke, as well as the intake of a portion of blueberry, seem to be insufficient to alter arterial stiffness in accordance with previous observations.14 However, other studies reported a significant effect following medium-/long-term intervention with blueberries.50,51 Johnson et al.50 showed that 8 weeks of blueberry consumption (22 g freeze-dried blueberry powder, providing about 844 mg phenolics and 470 mg anthocyanins) reduced arterial stiffness in postmenopausal women with pre- and stage 1-hypertension. McAnulty and colleagues reported that 6 weeks consumption of 25 g of whole blueberry powder (equivalent to 250 g fresh berries, not characterized for phenolic compounds) improved arterial stiffness in sedentary men and women.51

Recent research emphasized the role of blueberries in the control of blood pressure in subjects with pre-hypertension, hypertension and/or metabolic syndrome.50–53 We previously reported that the intake of a portion of blueberry was unable to affect blood pressure in a group of healthy subjects,37 while we documented its capability to counteract the increase in systolic blood pressure induced by acute cigarette smoke.14 In the present study, we confirmed the effect of smoking on blood pressure and heart rate in accordance with the literature, but we failed to demonstrate the ability of blueberries to counterbalance this impairment, probably due to the small number of subjects enrolled. Similar results have been reported by McAnulty and colleagues that documented no significant effect on blood pressure following an acute and chronic intervention with blueberries in smokers.54

Possible study limitations are the small number of subjects and the lack of information regarding the circulating levels of insulin, glucagon and anthocyanins as potential mechanisms underpinning the improvement of endothelial function. Finally, another factor may be the absence of a real placebo as control treatment.

In conclusion, these studies documented that one portion of blueberries (300 g) can acutely improve endothelial function in young smokers and non-smokers with endothelial dysfunction. Additionally, acute and chronic intervention studies should be performed to confirm the results obtained and reveal the potential mechanisms of action through which blueberries can affect endothelial function. Moreover, since sugars have been shown to positively influence the endothelial response, the role of insulin and glucagon must be evaluated in future. Even though this pilot study concludes that blueberries can overcome the endothelial dysfunction associated with cigarette smoking, the authors do encourage people to stop smoking in order to reduce all risks associated with this habit.

Author contribution

Cristian Del Bo’ performed the study and drafted the manuscript; Valeria Deon and Claudia Lanti enrolled the subjects and contributed to performing the study; and Marisa Porrini and Patrizia Riso designed the study and provided funding. Jonica Campolo, Marina Parolini and Dorothy Klimis Zacas contributed to the study concept and design. All the authors critically revised the manuscript.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

This study was supported by intramural fundings and by a grant (Rif. Pratica 2010.2303) from the Cariplo Foundation. We are grateful to all our volunteers for their time and commitment.

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