Study population
Participants from the general population were recruited by means of news in the newspapers, social networks, and tableaux advertisements in the Hospital Universitari Sant Joan (HUSJ)-Eurecat, Reus, Spain, between January 2016 and June 2017. From 311 subjects assessed for eligibility, 159 (53 women and 106 men) pre- or stage-1 hypertensive individuals, according to current guidelines [21], were recruited. Inclusion criteria were as follows: age from 18 to 65, systolic blood pressure (SBP) ≥ 120 mmHg, no family history of cardiovascular disease or chronic disease, and willingness to provide informed consent before the initial screening visit. Exclusion criteria were: body mass index (BMI) ≥ 35 kg/m2, fasting glucose > 125 mg/dL, SBP ≥ 160 mmHg and diastolic blood pressure (DBP) > 100 mmHg or taking antihypertensive medications, hyperlipemia or antilipemic medication; smoking, pregnancy or intending to become pregnant, use of medications, antioxidants, vitamin supplements or adherence to a vegetarian diet, chronic alcoholism, physical activity > 5 h/week, intestinal disorders, anemia (hemoglobin ≤ 13 mg/dL in men and ≤ 12 mg/dL in women), consumption of a research product in the 30 days prior to inclusion in the study, or failure to follow the study guidelines. Participants signed informed consent prior to their participation in the study, which was approved by the Clinical Research Ethical Committee of HUSJ (14-12-18/12aclaassN1), Reus, Spain. The protocol and trial were conducted in accordance with the Helsinki Declaration and Good Clinical Practice Guidelines of the International Conference of Harmonization (GCP ICH) and were reported as CONSORT criteria. The trial was registered at Clinical-Trials.gov: NCT02479568.
Intervention products
Intervention beverages (supplied by the Florida Department of Citrus, USA) were control drink (CD), an OJ containing 690 mg/L of hesperidin (the natural hesperidin content), and an enriched orange juice (EOJ) containing 1200 mg/L of hesperidin. Ferrer HealthTech (Murcia, Spain) provided the Micronized 2S Hesperidin used in EOJ enrichment. The 2S form, the one present naturally in the OJ, is the most bioavailable [18]. Beverages were analyzed for hesperidin and narirutin content using chromatography–mass spectrometry (LC–MS/MS) (Supporting Information Table S1). Daily doses of 500 mL of CD, OJ and EOJ, provided 0 mg/day, 345 mg/day, and 600 mg/day of hesperidin, and 0 mg/day, 64 mg/day, and 77.5 mg/day of narirutin, respectively. Intervention drinks were similar in appearance and smell, and were differentiated only by a code assigned by an independent researcher not related to the study to guarantee blinding. Flavanone contents of the OJ and the EOJ were stable throughout the study.
Study design
A randomized, parallel, double-blind, placebo-controlled clinical trial was performed (Supporting Information Fig. S1). Participants were randomly assigned to one of the three intervention groups—CD, OJ, or EOJ—to consume 500 mL/day of the corresponding beverage for 12 weeks. Nested within the sustained consumption study were two dose–response studies, one at baseline and the other after 12 weeks of sustained consumption, where the 500 mL/dose was administered all at once and changes in the outcomes were recorded in the postprandial state. Participants were randomly allocated to the three intervention groups by a computerized random-number generator made by an independent statistician. PROC PLAN (SAS 9.2, Cary, NC: 83 SAS Institute Inc.) with a 1:1:1 allocation using random block sizes of 2, 4, and 6 was used. Participants, researchers and the statistician remained blinded to the type of product administered throughout the study.
After enrolment and following a 1-week run-in period with a control diet consisting of a maintained lifestyle and normal dietary habits based on nutritionist recommendations, the participants started the intervention trial. However, during the intervention period, the participants were instructed to also maintain their dietary habits, to completely refrain from consuming citrus-containing foods and to limit their total intake of flavonoid-rich foods (tea, coffee, cocoa, wine and other fruit juices) to reduce the possible masking effects that can exert these foods on BP [22, 23]. During the sustained study, participants attended seven visits (V) at the HUSJ-Eurecat. Dose–response postprandial studies, performed at V1 and V7, lasted from 08:00 a.m. to 02:00 p.m., and participants received a light meal before leaving. In addition to the baseline (0 h), blood samples were collected at 2 h, 4 h, and 6 h after the single dose of 500 mL. The adherence of the volunteers to their dietary habits throughout the study was assessed by a 3-day food record at V1, V3, V5, and V7. At each visit, subjects underwent a physical examination by a general practitioner and completed a Physical Activity Questionnaire Class AF [24], and anthropometric measurements were recorded. Samples were stored at -80ºC in the central laboratory’s Biobanc of HUSJ-Eurecat (biobanc.reus@iispv.cat) until required for batch analyses.
Compliance measures
The plasma levels of the following biomarkers of nutrient exposures were measured by LC–MS/MS in the plasma samples: hesperetin-7-O-β-d-glucuronide, hesperetin-3-O-β-d-glucuronide, hesperetin-7-O-sulfate, naringin-4-O-β-d-glucuronide, naringin-glucuronide and naringin sulfate. The extraction was carried out with a semi-automated process using Agilent Bravo Automated Liquid Handling Platform. Briefly, 20 μL of internal standard (Hesperetin d4) was mixed with 125 μL of plasma and 750 μL of methanol. The mixture was vortexed and centrifuged at 4700 rpm at 4 °C, and then 900 μL was evaporated in a Speed-Vac at room temperature. Residues were reconstituted in 25 μL of MeOH and 75 μL of H2O (1% of HFor) and injected in the LC–MS/MS, an Agilent 1200 series ultra-high-performance liquid chromatography (UHPLC) system coupled to a 6490 Triple Quad mass spectrometer, with electrospray source ionization (ESI) operating in negative mode.
Main outcome measures
SBP and DBP were measured twice after 2–5 min of respite, with the patient in a seated position, with 1-min interval between, using an automatic sphygmomanometer (OMRON HEM-907; Peroxfarma, Barcelona, Spain). The mean values were used for statistical analyses. Office PP, which represents the force that the heart generates each time it contracts, was determined by the difference between SBP and DBP [25]. The main outcomes were measured in both dose–response and sustained consumption studies.
Secondary outcomes
Homocysteine in serum samples was determined by LC–MS/MS. F2α isoprostanes were determined by a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) (Caymanchem, MI, USA) in 24-h urine. Soluble Intercellular Adhesion Molecule-1 (ICAM-1) and Soluble Vascular Cell Adhesion Molecule-1 (VCAM-1) were determined in serum by the Luminex™xMAP technology with the EPX010-40,232-901 kit eBioscience (Thermo Fisher Scientific, Waltham, Massachusetts, USA), in the the Bio-Plex™ 200 instrument (Bio-Rad, Hercules, California, USA). Uric acid was measured by standardized methods on an autoanalyzer 182 (Beckman Coulter-Synchron, Galway, Ireland) in serum samples. All biological biomarkers were measured in the sustained consumption study. Homocysteine was additionally measured after the single 500-mL dose of the corresponding intervention product in both dose–response studies.
Transcriptomic analyses
Gene expression was assessed in peripheral blood mononuclear cells (PBMCs) with an Agilent Microarray Platform (Agilent Technologies, Santa Clara, California, USA) in a subsample (n = 37) of participants (11, 15, and 11, in CD, OJ, and EOJ groups, respectively) at baseline and after 12 weeks. PBMC RNA was isolated using Ficoll gradient separation GE Healthcare Bio Sciences, Barcelona, Spain), RNA yield was quantified with a Nanodrop UV–VIS Spectrophotometer and integrity was measured with an Agilent 2100 Bioanalyzer using the Total RNA Nano kit and the Eukaryote Total RNA Nano (Agilent Technologies, Santa Clara, California, USA). Total RNA from the PBMCs was labeled with one color (Cy3) (ref: 5190-2305, Agilent Technologies, Santa Clara, California, USA) and hybridized using a Gene Expression Hybridization Kit (ref: 5188-5242, Agilent Technologies, Santa Clara, California, USA). Image scanning was performed with an Agilent Microarray Scanner System with SureScan High Resolution Technology (Agilent Technologies, Santa Clara, California, USA). Differentially expressed genes were subjected to functional and biochemical pathway analysis using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.genome.jp/kegg) and PANTHER (protein annotation through evolutionary relationship classification system (https://www.pantherdb.org/) [26] biochemical pathway databases. The analysis was performed using GeneCodis (https://www.genecodis.dacya.ucm.es [27] software.
Selected genes related to hypertension were validated by PCR. Briefly, to analyze the expression of the genes and validate the DNA array results, cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4 Barcelona, Spain) and MyGene Series Peltier Thermal Cycler (LongGene Scientific, Zhejiang, China) and used for reverse transcription. The cDNA was subjected to quantitative reverse transcription-polymerase chain reaction amplification using LightCycler 480 SYBR Green I Master (Roche Diagnostic, Sant Cugat del Vallès, Barcelona, Spain) and a LightCycler 480 II system (Roche Diagnostic, Sant Cugat del Vallès, Barcelona, Spain).
Sample size and power analyses
A sample size of 159 individuals was calculated assuming an expected dropout rate of 20% and a type I error of 0.005 (two sided), which allows at least 80% power for the detection of statistically significant differences in the SBP of 4 mmHg among the groups. The population standard deviation of the SBP was estimated to equal 6 mmHg [28].
Statistical analyses
Descriptive data were expressed as the mean 95% confidence interval (CI). The normality of variables was assessed by the Kolmogorov–Smirnov test. Non-parametric variables were log transformed. ANOVA was used to determine differences in baseline characteristics. Analyses were made by intention-to-treat. Multiple imputation was made by linear regression analysis. Intra-treatment comparisons were performed by means of a general linear model with Bonferroni correction and age and sex as covariables. Inter-treatment comparisons were carried out by analysis of covariance (ANCOVA) model adjusted for age and sex. For transcriptomic analyses, quality control was performed through principal component analyses. Statistical comparisons were performed by Student’s t test or Welch’s t test if proceeded. Multiple testing correction was performed using the Benjamini–Hochberg False Discovery Rate (FDR) control procedure. Probes were assumed to be differentially expressed if they presented a P value < 0.05 and a fold change ≤ −0.58 or ≥ 0.58 in log2 scale (corresponding to 1.5-fold difference in natural scale). Calculations were performed using the R statistical language. Comparisons among treatments were carried out by an ANCOVA model adjusted by age and sex and baseline values. Statistical significance was defined as a P value ≤ 0.05 for a two-sided test. Analyses were performed using SPSS for Windows, version 21 (IBM corp., Armonk, NY, USA). All data were analyzed according to the pre-specified protocol.