Protocols and study design

This study follows a randomized, controlled, cross-over design (single portion of blueberry versus a control product) in a sample of free-living older subjects (≥ 60y). The study involves two phases (Phase 1 and 2), both including two different appointments, each one spaced by a 1 week wash-out period, as illustrated in the SPIRIT schedule of enrollment, interventions, and assessments (Fig 1 and S1 Checklist).

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Fig 1. Schedule of enrollment, interventions, and assessment: Recommendations for interventional trials (SPIRIT).

https://doi.org/10.1371/journal.pone.0275132.g001

Phase 1 is aimed at evaluating the absorption of blueberry polyphenols and the effect of blueberries consumption on markers of vascular function (i.e., endothelin-1, NO, ICAM-1, VCAM-1 and VEGF), inflammation (i.e., IL-6, IL-8, and TNF-α), oxidative stress (i.e., levels of DNA damage), blood glucose and insulin response (Fig 2). Phase 2 is devoted to the evaluation of vascular functionality, using a non-invasive biosensor and the assessment of reactive hyperemia index (RHI), Framingham reactive hyperemia index (fRHI) and arterial stiffness (i.e., augmentation index (AI) and AI@75); in addition, blood pressure is recorded, as depicted in Fig 3. The approach, divided into two phases, is necessary to facilitate blood drawing (venous cannula) and the evaluation of vascular function (at the level of the brachial artery) avoiding potential sources of interferences between the two measurements. The day before the experiment, participants receive a list of instructions to follow. Following these instructions, subjects are asked to maintain their habitual lifestyle habits (also in terms of physical activities) and to refrain from consuming foods rich in (poly)phenols (e.g., berries, tea, coffee, chocolate, fruit juices). In addition, to limit the possible noise deriving from a long-fasting period the day of the experiment, subjects participating at the Phase 1 are invited to consume a standardized light breakfast. Specifically, volunteers can consume 200 mL of partially skimmed milk or 125 g of yogurt and 3 biscuits (i.e., shortbread) or 3 rusks. The meal has to be consumed at least 90 min before blood collection and at the same hour each test day. The adherence to instructions is evaluated through a food diary and a face-to-face interview that will be scheduled the day of the experiment. In addition, subjects are motivated to complete all the intervention by providing them with free meals the test days and with the access to all laboratory analysis at the end of the trial.

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Fig 2. Schematic representation of the study design for the evaluation of blueberry bioactive absorption and of different metabolic/functional markers (phase 1).

https://doi.org/10.1371/journal.pone.0275132.g002

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Fig 3. Schematic representation of the study design for the evaluation of the effects of intervention on vascular function markers (phase 2).

https://doi.org/10.1371/journal.pone.0275132.g003

Trial status

The trial has been prospectively registered (May 7, 2021; ISRCTN18262533) and is currently ongoing.

Location

The study is carried out at the Department of Food, Environmental and Nutritional Sciences (DeFENS), Università degli Studi di Milano (Milan, Italy) through its International Center for the Assessment of Nutritional Status (ICANS).

Participant enrollment

For the study, a group of 20 subjects (age ≥ 60y; 10 women and 10 men) are enrolled. The enrollment is carried out through advertisements by using emails, social networks, bulletin boards, and by word of mouth. The subjects who express their willingness to participate undergo a first general medical anamnesis through questions about their health status and their lifestyle (e.g., diseases, use of drugs and supplements, dietary habits, alcohol consumption, and physical activity) followed by an accurate clinical evaluation to confirm their eligibility to participate. Subjects interested in participating in the study sign an informed consent in which they agree with all the information on the dietary intervention, the analysis, and protocols that they are asked to follow. Volunteers are selected according to the inclusion and exclusion criteria reported below:

Inclusion criteria

• Age ≥ 60y.

Exclusion criteria

• Diabetes;
• Major condition related to cardiovascular system (e.g., a medical history of thrombosis or myocardial infarction);
• Presence of allergies (or other adverse reaction) to the ingestion of blueberry.

Changes in the volunteer status during the experimentation (e.g. occurrence of an exclusion criteria such as drug prescription, not evidenced at the enrolment) will be assessed to define the need of final exclusion from the study.

Blueberry and control product

Blueberry product consists of a mousse obtained by shredding a serving of blueberries (organic highbush blueberries, cultivar Legacy) able to provide at least 300 mg of anthocyanins (ACNs). This amount of ACNs is considered sufficient to induce an improvement of vascular function as previously reported [30, 31]. The control product consists of a drink prepared by suspending the same amount of fructose, glucose, and saccharose of blueberry (matching the blueberry product for energy) in water, as already published [30, 31].

Information on potential adverse effects

Even though no reports of adverse effects due to a blueberry consumption have been registered or reported in the literature, subjects are advised to annotate and communicate any adverse symptom perceived during the intervention period.

Biological sampling

Blood samples (7 mL) are collected at baseline (t0) and after 1 h (t1), 1 h 30 ‘(t2), 2 h (t3) and 4 h (t4) from the consumption of blueberry or control product, as depicted in Fig 2. A cannula needle is used to facilitate blood collection procedures and minimize the discomfort of the volunteers. Tubes containing silicon for serum and tubes containing heparin as anticoagulants for peripheral blood mononuclear cells (PBMCs) are used. An aliquot of blood (500 μL) is immediately processed to obtain PBMCs, while the rest of the blood is maintained at room temperature (22°C) for 30 min before being processed by centrifugation at 1088 g for 15 min at 4°C. The serum obtained is collected, divided in aliquots, and stored at -80°C until analysis.

Outcome measurements

The primary selected outcome of the study is RHI as a vascular function marker, while other markers including vascular function (i.e., fRHI, AI, AI@75, Endothelin-1, NO, ICAM-1, VCAM-1 and VEGF), inflammation (i.e., IL-6, IL-8, and TNF-α) and oxidative stress markers (DNA damage), together with circulating levels of (poly)phenols, salicylates, glycaemia and insulin, are included as secondary outcomes to support and validate the study hypothesis.

Anthropometric measurements

Body weight and height are assessed at the beginning of the study by trained personnel following the international guidelines reported by Lohman et al. [32]. BMI calculation is obtained by dividing a person’s weight by the height to the power of 2 (BMI = weight (kg) / height (m)2).

Blood pressure

Volunteers are monitored during each intervention period measuring both systolic and diastolic blood pressure (SBP and DBP) obtained in a resting, seated position based on the validated JNC 7 guidelines [33].

Metabolic and functional markers

At enrollment, metabolic and functional parameters (i.e., glucose, insulin, lipid profile, liver, and renal function) are analyzed by a standardized validated protocol, using an automatic biochemical analyzer (YSI 2300 STAT Plus Glucose and Lactate Analyzer, Marshall Scientific, Hampton, VA, USA)). In details, non-high density lipoprotein cholesterol (non-HDL-C) is calculated by subtracting high density lipoprotein cholesterol (HDL-C) from total cholesterol (TC) while low density lipoprotein cholesterol (LDL-C) concentration is estimated using the Friedewald formula [34]. The levels of insulin sensitivity are assessed using the widely used homeostasis model assessment for insulin resistance (HOMA-IR), that is calculated considering fasting plasma concentrations of glucose and insulin, as indicated by Wallace et al. [35]. The blood samples are used for the evaluation of: i) kinetics of the absorption of the anthocyanins by gas chromatography–mass spectrometry analysis (GC–MS); ii) kinetics of the absorption of salicylates by means of ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS); iii) serum markers of vascular function (i.e. ET-1, NO, ICAM-1, VCAM-1 and VEGF) and inflammation (i.e. IL-6, IL-8, and TNF-α) evaluated through ELISA kit, while serum markers of oxidative stress (i.e. levels of DNA damage) evaluated through comet assay iv) blood glucose concentrations evaluated by oxidation with YSI 2300 STAT Plus Glucose and Lactate Analyzer and insulin levels determined by ELISA kit.

Vascular function and arterial stiffness

Endothelial-dependent vasodilation in the small finger arteries is assessed by a non-invasive plethysmographic method (Endo-PAT 2000, Itamar Medical Ltd, Caesarea, Israel) as described elsewhere [36, 37]. Briefly, the Endo-PAT equipment consists of two finger-mounted probes, which include a system of inflatable latex aircushions. The pulsatile volume changes of the fingertip are sensed by a pressure transducer, located at the end of each probe, and transferred to a personal computer where the signal is transduced, amplified, displayed, and stored. For the evaluation, subjects are requested to lie down in a supine position, with both hands on the same level, in a comfortable and thermoneutral environment. Then, a blood pressure cuff is placed on one upper arm (study arm), while the contralateral arm serves as a control arm. After a 10 min equilibration period, the blood pressure cuff on the study arm is inflated to 200–220 mmHg for 5 min. The cuff is then deflated to induce reactive hyperemia while the signals from both PAT channels (Probe 1 and Probe 2) are recorded by a computer. The RHI, an index of the endothelial-dependent flow-mediated dilation, is derived automatically in an operator independent manner. In fact, RHI is calculated as the ratio of the average pulse wave amplitude during hyperemia (60–120 s of the post-occlusion period) to the average pulse wave amplitude during baseline in the occluded hand, divided by the same values in the control hand and then multiplied by a baseline correction factor. According to manufacturing indications, an RHI value of 1.67 provides a sensitivity of 82% and a specificity of 77% for diagnosing endothelial dysfunction [38].

The Endo-PAT tool also provides the digital augmentation index (AI), a measure of pulse wave reflection and a surrogate marker for arterial stiffness. AI derives from digital pulse volume waveforms and it has been reported to be strongly correlated with aortic AI. Peripheral AI is calculated from the shape of the pulse wave recorded during baseline [39, 40]. Since digital AI is affected in an inverse and linear manner by heart rate (HR) [41], the AI was automatically normalized by considering a HR of 75 bpm (AI@75).

Vascular and inflammatory markers

The concentrations of several markers related to vascular and inflammatory processes are quantified at least in duplicate using specific ELISA or colorimetric assay kits according to the instructions of each producer (Boster Bio, Valley Ave, Pleasanton, CA; Cayman Chemical Company, Ann Arbor, MI, USA). The following kits are used on serum samples obtained at each time points during the intervention periods: IL-6: BSR-EK0410—Human IL-6 PicoKine ELISA Kit, IL-8: BSR-EK0413—Human IL-8 PicoKine ELISA Kit, TNF-α: BSR-EK0525—Human TNF alpha PicoKine ELISA Kit, Endothelin-1: BSR-EK0945—Human Endothelin PicoKine ELISA Kit, ICAM-1: BSR-EK0370—Human ICAM-1 PicoKine ELISA Kit, VCAM-1: BSR-EK0537—Human VCAM-1 PicoKine ELISA Kit, VEGF: BSR-EK0539—Human VEGF PicoKine ELISA Kit, NO: CAY-780001–2x96—Nitrate/Nitrite Colorimetric Assay Kit.

Oxidative stress markers

The levels of oxidatively-induced DNA damage, as markers of oxidative stress, are assessed in PBMCs by the comet assay on an aliquot of fresh blood, immediately after its withdrawal. Oxidatively-induced DNA damage is measured by treating the cells with hydrogen peroxide and by evaluating the capacity of cells to counteract this oxidative insult. This procedure is performed according to Del Bo’ et al., [42]. Briefly, cell resistance against oxidatively induced DNA damage is measured by treating the isolated PBMCs with hydrogen peroxide (H2O2). Two slides with two gels each are prepared for each sample. One slide is treated with H2O2 (500 μmol/L in PBS) for 5 min in the dark, while the other slide is treated with PBS without H2O2, as it is used as a control. Following oxidative treatment, the slides are placed in lysis buffer (2.5M NaCl, 0.1M Na2 EDTA, 10mM Tris, 1% Triton X-100, 1% DMSO, and 1% N-lauroylsarcosine sodium salt, pH 10) for 1h at 4°C in the dark. After lysis, the gels are washed in buffer (40nM HEPES, 0.1M KCl, 0.5mM EDTA and 0.2mg/ml bovine serum albumin, pH 8) and then transferred to electrophoresis buffer (0.3M NaOH and 1mM Na2 EDTA) and incubated for 40min at 4°C in the dark. Electrophoresis is performed at 1.1V/cm2 for 20min. Slides are successively neutralized (0.4M Tris–HCl, pH 7.5) for 15min at 4°C in the dark, stained with ethidium bromide (2 μg/ml), washed in PBS, drained and coverslipped to permit the subsequent quantification of DNA damage through an epifluorescence microscope attached to a high sensitivity CCD video camera and to a computer equipped with an image analysis system (QCapture 2.9.13, Irvine, CA, USA; DNA COMET1.50.00, University of Milan, Italy). The levels of DNA damage are calculated as the percentage of DNA in the tail.

Serum anthocyanins

Serum samples are used to determine the kinetics of absorption of blueberry ACNs and salicylates. The extraction and analysis of ACNs from serum are performed through a microelution solid phase extraction (SPE) and ultra-HPLC methods, according to Martí et al., [43]. Briefly, prior to injection in the UPLC-MS/MS, ACNs are extracted using μElution Plates 30 mm conditioned with 250 mL of methanol and 250 mL/mL of 0.2%acetic acid. Then 350 μL of plasma is mixed with 350 μL of phosphoric acid 4%, loaded into the plate. The loaded plates are washed with 200 μL of Milli-Q water and 200 μL of 0.2%acetic acid. Then, the retained ACNs are eluted with 2 x 50 μL of acetone/Milli-Q water/acetic acid solution (70:29.5:0.5, v/v/v) and injected. Injection volume is 2.5 μL. UPLC-ESI-MS/MS is constituted by a stationary phase of 100% silica particles, particles while the mobile phase by 10% acetic acid (eluent A) and acetonitrile (eluent B). The flow rate is set at 0.4 mL/min.

Blood salicylates

The analysis of salicylates in serum samples is performed using stable isotope dilution and gas chromatography–mass spectrometry analysis (GC–MS), as described previously [44]. Briefly, following adjustment for the pH to about 1.0 with hydrochloric acid, the organic material is extracted twice with ethyl ether and ethyl acetate (2 mL), and then is collected for derivatization prior to GC–MS analysis. Analysis is then carried out using a gas chromatograph interfaced with a single-quadrupole mass spectrometer equipped with a chamber provided with an electron ionization source and using helium as carrier gas. Finally, the identification of derivative salicylic acid is achieved by comparing the gas chromatographic retention times and the mass spectra of the serum samples with those of the standards.

Data management

Data management includes a strict control of data quality and check of accuracy of data entry. To ensure data protection, data will be saved in a password-protected Excel file on an electronic device equipped with an antivirus system locked up in a secure location when not in use. Once completed final data will be stored in an electronic database (Dataverse). Identifiable data are not recorded in the database or other documents, and participants are identified by a unique trial ID only. Hard copies of data sheets linking the participant identification number to the person’s contact details are kept securely within the University premises that are accessible only to dedicated research team members. Participant files and other source data (including copies of protocols, questionnaires, and original reports of test results, correspondence, records of informed consent and other documents pertaining to the conduct of the study) are kept for the maximum period of time permitted by the institution.

Sample size, randomization, and statistical analysis

The sample size is determined based on previous studies conducted in our laboratories (30–31) and other research groups [26, 28, 45] to test the efficacy of a blueberry consumption on markers of vascular function. Twenty volunteers are enrolled for the study. This number is considered sufficient (alpha = 0.05, 80% power) to determine an increase of 0.30 in the RHI parameter (endothelial function marker) after blueberry intake. This calculation also considers a possible drop out of 25% of the volunteers. Subjects are randomly divided using a computer random number generator. The randomisation and allocation is performed by a person not involved in the trial and blinded to the investigators and researchers involved in samples analysis. The statistical analysis is performed by means of STATISTICA software (Statsoft Inc., Tulsa, OK, US). Only complete data obtained will be analysed. The Shapiro–Wilk test is applied to verify the normal distribution of the variables under study. In particular, the following statistical elaborations are performed to identify significant differences between treatments: (i) the analysis of variance (ANOVA) with repeated measures, (ii) Wilcoxon paired data test, (iii) Linear Mixed Model (LMM) analysis. Furthermore, regression and correlation analyses (Spearman and Kendall test) are carried out to highlight potential associations between vascular activity, and markers of inflammation, vascular function, oxidative stress, other than biochemical and physiological markers. Corrections for potential confounding factors (i.e., age, gender, drugs/medications) are performed. When appropriate, a post-hoc p-value adjustment is performed using the Hochberg-Benjamin correction. Significance is set at p<0.05; significance in the range 0.05 < p < 0.10 is indicated as trend.

Monitoring

Given the limited objectives and the short-term nature of the intervention, this trial is monitored by the dedicated research team without the use of a formal data monitoring committee and will be performed independently from the sponsor. Data access is restricted to trained staff with unique password protected accounts. Eventual adverse events such as unfavorable and unintended signs, abnormal laboratory findings, and symptoms temporarily associated with the intervention are collected from the time of intervention until the end, whether or not considered related to the intervention study. All adverse events are considered until they are resolved, but adverse effects are unlikely to occur.

Ethics and dissemination

The study is conducted in accordance with the Declaration of Helsinki and the Data Protection Act. Trial personnel obtain informed consent from all participants prior to inclusion to the study, including consent for the use of data and biological specimens in other ancillary studies. The principal investigator stores informed consent. All subjects must agree to participate voluntarily and are free to withdraw from the study at any time. The study (S1 File) received ethical approval by the Ethics Committee of the University of Milan on the 12 December 2020 (S2 File). The Research Ethics Committee (REC) approval includes the trial protocol, information sheet and consent form. The trial is registered at the International Standard Randomized Controlled Trial Number (ISRCTN18262533, date of registration: 07 May 2021). Any amendment to the protocol and information provided to participants are submitted to the REC for approval prior to implementation. Substantial amendments may only be implemented after written Ethics Committees approval has been obtained, whereas non-substantial amendments can be implemented without written approval from the Ethics Committee. The Principal Investigator (PI) has to ensure that the participant’s privacy is maintained. Data and source documents are stored in such a way that they can be accessed later for the purposes of monitoring or inspection by the Ethics Committee. Public access to data and other materials could be requested to the PI at the end of the study after completion of elaboration and publication of data. At the end of the study, participants can receive a copy of the results of the study from the PI. The results from the trial are submitted for publication in a peer-reviewed journal irrespective of the outcome. Authorship of presentations and reports related to the study are in the name of the collaborative group and the list will be based on the extent of contribution. Results will be also communicated through other dissemination means including relevant scientific conferences.