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Flavanones protect from arterial stiffness in postmenopausal women consuming grapefruit juice for 6 mo: a randomized, controlled, crossover trial

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Flavanones protect from arterial stiffness in postmenopausal women

consuming grapefruit juice for 6 mo: a randomized, controlled,

crossover trial

1

Véronique Habauzit,2–4Marie-Anne Verny,2,3Dragan Milenkovic,2,3Nicolas Barber-Chamoux,2,5Andrzej Mazur,2,3Claude Dubray,4and Christine Morand2,3*

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French National Institute for Agricultural Research (INRA), Joint Research Unit (UMR) 1019, Human Nutrition Unit (UNH), Research Center for Human Nutrition Auvergne (CRNH) Auvergne, Clermont-Ferrand, France;3Clermont Université, Université d’Auvergne, Unité de Nutrition Humaine, Clermont-Ferrand, France;4Institut National de la Santé et de la Recherche Médicale (INSERM), CIC 501, UMR 766, Clermont-Ferrand, France;

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Department of Cardiology, Gabriel Montpied Hospital, Clermont-Ferrand University Hospital, Clermont-Ferrand, France ABSTRACT

Background: The consumption of citrus fruits is associated with health benefits. However, clinical data regarding the effects of grape-fruit flavanone consumption on vascular function are lacking. Objective:The objective of the present study was to address the role of flavanones in the long-term effects induced by grapefruit juice (GFJ) consumption on vascular function in healthy postmenopausal women.

Design:Forty-eight healthy postmenopausal women aged 50–65 y within 3–10 y since menopause, a body mass index (in kg/m2) of 19–30, and a waist size.88 cm completed this double-blind, domized, controlled, crossover trial. These volunteers were ran-domly assigned to consume 340 mL GFJ/d, providing 210 mg naringenin glycosides, or a matched control drink without flavanones for 6 mo each, with a 2-mo washout between beverages. The primary endpoint was the assessment of endothelial function in the brachial artery by using flow-mediated dilation. Blood pressure, arterial stiff-ness, and endothelial function in the peripheral arterial bed were also evaluated as indicators of vascular function. These measurements and blood collection for clinical biochemical markers were per-formed in overnight-fasted subjects before and after the 6-mo treat-ment periods.

Results: The mean 6 SD carotid-femoral pulse wave velocity, which reflects central aortic stiffness, was statistically signifi-cantly lower after consumption of GFJ (7.36 6 1.15 m/s) than after consumption of the matched control drink without flavanones (7.706 1.36 m/s), with a P value of 0.019 for the treatment effect. Endothelial function in macro- and microcirculation, blood pres-sure, anthropometric measures, glucose metabolism, and biomarkers of inflammation and oxidative stress were not affected by the in-tervention.

Conclusions:Regular GFJ consumption by middle-aged, healthy post-menopausal women is beneficial for arterial stiffness. This effect may be related to flavanones present in grapefruit. This trial was registered at clinicaltrials.gov as NCT01272167. Am J Clin Nutr 2015;102:66–74.

Keywords: grapefruit juice, clinical trial, dietary flavanones, post-menopausal women, vascular function

INTRODUCTION

The incidence rates of cardiovascular diseases (CVDs)6rise sharply in the years after menopause. Many prospective cohort studies have provided consistent evidence that a higher intake of fruit and vegetables is associated with a lower risk of car-diovascular mortality (1). Plant-based foods are the exclusive sources of flavonoids, a large family of bioactive compounds (2). With a database of flavonoid contents in foods, the total intake of flavonoids has been estimated atw0.5 g/d in a French cohort (3). These compounds may help to promote the health benefits of plant-based foods; accumulating evidence from cohort studies has indicated that an increased intake of dietary flavonoids may reduce the risk of CVDs and type 2 diabetes (4, 5). This evidence is supported by studies conducted in animal models with nutritionally realistic doses of isolated flavonoids (2) and in humans consuming flavonoid-rich foods (6, 7). The most convincing clinical data are available for few flavonoid-rich products, including tea, cocoa, and soy, which are also the most studied (7, 8). These studies have reported beneficial effects of flavonoid-rich product consumption on some intermediate risk factors for CVD, such as LDL cholesterol, blood pressure (BP), and endothelial function. However, the trials with flavonoid-rich foods often cannot dissociate the specific effect of flavonoid compounds from that of the entire food due to the lack of appro-priate controls.

Although fruits have been recognized as major contributors to dietary flavonoid intake in humans, clinical studies focus-ing on fruit flavonoids remain scarce. Flavanones are almost

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Supported by a grant from the Florida Department of Citrus (to CM). The funders donated the study drinks.

*To whom correspondence should be addressed. E-mail: christine.morand@ clermont.inra.fr.

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Abbreviations used: BP, blood pressure; CD, control drink; CVD, cardio-vascular disease; FMD, flow-mediated dilation; GFJ, grapefruit juice; PAT, peripheral arterial tonometry; PWV, pulse wave velocity; RCT, randomized controlled trial.

Received December 3, 2014. Accepted for publication April 27, 2015. First published online May 27, 2015; doi: 10.3945/ajcn.114.104646.

66 Am J Clin Nutr 2015;102:66–74. Printed in USA.Ó 2015 American Society for Nutrition

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exclusively present at high concentrations in citrus fruits and in citrus fruit–derived food products. The major flavanones are naringenin and hesperetin, which are present as glycosides in grapefruit, orange, or clementine, respectively (9). Data from animal and cell studies indicated that flavanones might exhibit anti-inflammatory and anti-atherogenic properties, improve vascular reactivity, and exert antihypertensive and insulin-sensitizing effects when used for relevant nutritional condi-tions (9). A large prospective study examining the link between flavonoid intake and CVD mortality in postmenopausal women reported a statistically significant inverse association between flavanone intake and CVD (10). In this study, the intake of grapefruit was associated with a statistically significant reduction in CVD mortality (.1 serving/wk: RR: 0.85; P = 0.001). In an-other cohort of women, higher flavanone intake was associated with a lower relative risk of ischemic stroke (RR: 0.81; 95% CI: 0.66, 0.99; P = 0.04), and citrus fruit/juice consumption tended to be associated with a reduced risk of stroke (RR: 0.90; 95% CI: 0.77, 1.05) when comparing extreme quintiles (11). These epi-demiologic associations suggest that citrus flavanones may be cardioprotective. However, clinically based evidence remains scare (12, 13), and to our knowledge, the role of grapefruit fla-vanones in vascular protection has never been addressed through well-designed, randomized controlled trials (RCTs) in humans. Therefore, the present study was designed to investigate whether grapefruit flavanones could affect markers of vascular function in healthy postmenopausal women consuming grapefruit juice for 6 mo.

METHODS

Study population

Subjects were recruited in the Clermont-Ferrand region (France) through several media venues from February through October 2010. Nonsmoking Caucasian women who were 50–65 y old and 3–10 y after menopause were eligible for inclusion if these in-dividuals had a normal to overweight BMI (in kg/m2; 19–30), a waist circumference.88 cm, normal BP levels, normal blood analyses at screening, and normal baseline electrocardiogram examination with a corrected Q-T interval,420 ms. The exclu-sion criteria included a medical history of cancer or severe met-abolic diseases, medical history of major gastrointestinal surgery, therapy with blood lipid-lowering drugs or antihypertensive drugs, therapy with CYP3A4-metabolized drugs or other drugs that may interfere with grapefruit consumption (14), hormone replacement therapy in the previous 3 mo before entering the study, allergic reactions to citrus-containing foods, special dietary habits (e.g., vegetarians and vegans), use of dietary supplements (e.g., phytoestrogens and antioxidants), or physical activity level .5 h/wk. Subjects were also excluded if their usual consumption of one or more flavonoid-rich beverages, such as tea, coffee, wine, cocoa, or fruit juices, exceeded 500 mL/d, as estimated from a diet history interview at screening.

The 52 enrolled subjects provided written informed consent, and all subjects completed the study except for 4 who were lost to follow-up during the first 6-mo intervention period, as outlined in Figure 1. Ethical approval was obtained from the local human

FIGURE 1 Consolidated Standards of Reporting trials diagram showing flow of participants through the study. CD, control drink; GFJ, grapefruit juice; QTc, corrected Q-T interval.

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ethics committee (CPP Sud-Est VI, France). The clinical trial was performed at the Clinical Research Unit of the University Hospital of Clermont-Ferrand (France).

Characteristics of the intervention and randomization Concentrate blond grapefruit juice (GFJ) and the analysis of its constituents (Table 1) were provided by the Florida Department of Citrus. A control drink (CD) matched for the macro- and micro-nutrients of the juice but without naringenin glycosides (Table 1) was manufactured from a powder mix (PYC-DENA Laboratory, Eupen, Belgium). Ocean Spray produced and packed both GFJ and CD from the supplied grapefruit concentrate and powder mix, respectively. Throughout the intervention period, bottles of both beverages were blinded at the time of packaging.

Volunteers were required to consume daily one bottle containing 340 mL blond GFJ or the same volume of the isocaloric CD. This daily dose of GFJ corresponds to 2 servings of fruit juice, and it provided w210 mg naringenin glycosides (corresponding to 105 mg naringenin aglycone). The intake of each drink pro-vided 110 kcal/d. Subjects were asked to consume 170 mL in the morning at breakfast and 170 mL of beverage over lunch. The deliveries of bottles occurred every 1.5 mo, and volunteers were instructed to keep the bottles at home in a dark place and at a low temperature.

Participants were assigned to one of the 2 treatment sequences (GFJ then CD or CD then GFJ) after a blocked randomization procedure by using a random block of 4 participants. An independent person working at the clinical research unit generated a computerized random list. The allocation sequence was concealed from the re-searchers in opaque sealed envelopes. The envelopes were handed to persons responsible for packing bottles of drinks in opaque bags numbered for each woman according to the random schedule.

Study design

The objective of the present study was to examine the role of flavanones in the chronic effects of GFJ consumption on vascular

function in healthy postmenopausal women. This study was a double-blind, crossover RCT that included two 6-mo periods of intervention and a 2-mo washout period.

The primary outcome was the change in endothelium-dependent flow-mediated dilation (FMD) measured from the brachial artery after the intervention period. Secondary outcomes included the effect on BP, carotid-femoral pulse wave velocity (PWV), and digital peripheral arterial tonometry (PAT signal), as well as body composition and metabolic and inflammatory markers. These markers included glycemia, insulinemia, systemic endothelial function biomarkers (endothelin 1, soluble intercellular cell ad-hesion molecule 1, soluble vascular cell adad-hesion molecule 1, von Willebrand factor), inflammation (high-sensitivity C-reactive protein, IL-6), and antioxidant status (ferric reducing ability of plasma, oxygen radical absorbance capacity). All measurements were performed in fasted subjects at baseline and at the end of each experimental period.

During the entire study period, participants were requested to maintain their dietary habits and to minimize their intake of citrus foods and their consumption of flavonoid-rich beverages (coffee, tea, cocoa, fruit juices, and wine) to 250 mL/d. At screening, in-formation regarding the habitual food intake of the participants was obtained from a diet history interview. At the beginning and at the end of each experimental period, anthropometric measures were made and nutritional intake was determined by using an in-house developed 3-d estimated dietary record. It has been previously demonstrated that a 3-d dietary record was valid to estimate dietary intakes in adults without cognitive impairments (15). The partic-ipants were instructed to record their entire food intake on 3 consecutive days directly after consumption. Each subject was trained in the level of detail required to adequately describe the foods and amounts consumed, including the name of the food, preparation methods, recipes for food mixtures, and portion sizes. For completeness and reliability, a trained dietitian screened all the 3-d food intake questionnaires to clarify entries, to probe for for-gotten foods, and to check the portion sizes with the subjects. Participants were assisted with the use of an instruction manual for coding food portions that included validated photographs of.250 foods represented in 3 different portion sizes (16). Nutrient intakes were estimated by using the French food-composition database originally developed for the Supplementation en Vitamines et Mineraux Antioxydants (SU.VI.MAX) study (17).

Adherence to the intervention was assessed by counting the nonconsumed study products (bottles) brought back at the end of each period and by comparing the plasma concentrations of vitamin C at the beginning and end of each period.

The safety and tolerance of the intervention were monitored during the study period by recording clinical and laboratory adverse events. During the intervention, blood was sampled every 3 mo to determine liver markers, including alanine aminotransferase, as-partate aminotransferase, g-glutamyl transferase, and alkaline phosphatase. A control electrocardiogram was performed 10 d af-ter the beginning of each inaf-tervention period and at each visit to avoid any risk of a prolonged corrected Q-T interval in response to grapefruit consumption (18).

Body composition analysis

Volunteers underwent a dual-energy X-ray absorptiometry examination at the beginning and at the end of each experimental

TABLE 1

Compositional analysis of 1 serving (340 mL) of each study drink1 Grapefruit juice Control drink

Total carbohydrates, g 27.3 27.3

Total organic acids, g 3.57 3.57

Citrus pectins, mg 174.1 174.1 Minerals, mg Potassium 637.5 637.5 Magnesium 26.9 26.9 Calcium 21.4 21.4 Sodium 11.6 11.6 Phosphorus 55.2 55.2 Vitamins Vitamin C, mg 141.3 141.3 Vitamin B-9, mg 9.5 9.5 b-Carotene, mg 13 13 Naringenin glycosides, mg 212.9 — Energy, kcal 110 110 1

Carbohydrates include sucrose (33.3%), glucose (33.3%), and fructose (33.3%). Organic acids include citric acid (98%) and malic acid (2%). Nar-ingenin glycosides include two-thirds naringin and one-third narirutin.

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period. Dual-energy X-ray absorptiometry scanning is accepted as an accurate and convenient method for measuring both re-gional and total body composition (19). All scans were performed at the University Hospital of Clermont-Ferrand on a Hologic QDR 4500 A (S/N 45339) densitometer. During the study period, daily phantom measurements were performed as required for quality control. Subjects were scanned by using standard imaging and positioning protocols. Technical investigators blinded to the allocation of treatments performed data collection and man-agement.

Noninvasive assessments of vascular function

Vascular function measurements were performed in the morning after an overnight fast in a quiet temperature-controlled room (22–258C). The subjects laid quietly for several minutes before the measurements were performed. During the study period, the same trained operator who was blinded to the allocation of treatments performed each vascular measurement.

BP was measured on the right upper arm with a validated and automated sphygmomanometer device (SureSigns V3; Philips). The subjects rested 5–10 min in a seated or lying position before each BP assessment. Three consecutive BP readings were re-corded at 1-min intervals. The mean of the 3 consecutive readings was considered for statistical analysis.

FMD of the left brachial artery was measured in the lon-gitudinal plane above the antecubital fossa by using a high-resolution ultrasound system with a 7- to 12-MHz linear array transducer (Vivid S5; GE Healthcare). A mechanical arm device (Vascular Imaging), which was equipped with micrometer screws that allowed precise movements in the 3 dimensions, was used to obtain correct and stereotactic positioning of the transducer. After optimally positioning the transducer, images were recorded at baseline for 10 s and for 3 min after the release of forearm is-chemia, which was caused by inflating a sphygmomanometer cuff to 220 mm Hg for 5 min (occlusion). Images were coded and recorded on a videotape and then digitized for subsequent blinded analysis by using automated edge detection software (Hemodyn 3M apparatus; Dinap SRL). FMD is expressed as the percentage increase in the brachial artery diameter from baseline to maximal dilation, which occurs 30–90 s after the cuff is released.

Endothelial function in the peripheral arterial beds was assessed by using PAT (EndoPAT 2000; Itamar Medical Ltd.). The measurements of the endothelium-mediated changes in vascular tone were made in the fingertips by using a pair of unique plethysmographic biosensors. The PAT signal was measured from the fingertip by recording the finger arterial pulsatile volume changes before and after a 5-min occlusion of the brachial artery (identical to that applied for FMD). The measurement from the contralateral arm was used as a control for non–endothelial-dependent changes in vascular tone. Therefore, subjects served as their own control. The results are expressed as PAT ratios, which correspond to the mean pulse wave amplitude during hyperemia (60–120 s of the postocclusion period) to the mean pulse wave amplitude during baseline in the occluded hand, divided by the same values in the control hand, and then mul-tiplied by a baseline correction factor. The EndoPAT device also generates the augmentation index, which is a measure of pe-ripheral microvascular stiffness calculated from the shape of the pulse wave recorded by the probes during baseline. To correct

for the independent effect of heart rate, we adjusted the aug-mentation index to a heart rate of 75 beats/min.

The carotid-femoral PWV is an established index of aortic stiffness. Pulse, which travels at a higher velocity in stiff arterial vessels, is calculated from measurements of pulse transit time and the distance traveled between the 2 considered recording sites (i.e., carotid artery and femoral artery). The measurements were performed by using a validated noninvasive device (SphygmoCor; AtCor Medical Pty. Ltd.) that allows online pulse wave recording (requiring an acquisition ofw20 sequential waveforms) and au-tomatic PWV calculation [PWV = distance (m)/transit time (s)]. The R-wave of a simultaneously recorded electrocardiogram was used as a reference frame. The system software calculates 2 variables concerning the variability of the recorded waves, thus providing internal quality control for the recording and minimizing operator bias.

Biochemical analysis

Fasting blood was collected before and after each intervention period. Venous blood was collected into evacuated tubes containing EDTA or Na-heparin. The plasma samples were immediately iso-lated, processed, and stored at2808C until analysis. The plasma samples for vitamin C analysis were immediately transferred into Eppendorf tubes containing 2 volumes of meta-phosphoric acid (5%) and stored in the dark at2808C. The laboratory technicians in charge of blood sample analysis were not aware of the allocation of treatments.

Plasma glucose concentrations were measured according to a standard laboratory procedure, and plasma high-sensitivity C-reactive protein concentrations were measured according to a standard immunoturbidimetric method at the university hospital laboratory. Plasma insulin, inflammatory cytokine, and endo-thelial biomarker concentrations were assayed by ELISA with kits from Eurobio AbCys for insulin, IL-6, von Willebrand factor, soluble intercellular cell adhesion molecule 1, and soluble vas-cular cell adhesion molecule 1, as well as a kit from R&D Systems Europe Ltd. for endothelin 1. Ferric reducing ability of plasma was measured by spectrophotometry according to the method of Benzie and Strain (20). Oxygen radical absorbance capacity was determined by fluorimetry according to the method as previously described (21). The total nitric oxide pool as-sessment in plasma was based on the conversion of nitrate to nitrite in the presence of nitrate reductase, followed by the fluorimetric determination of nitrite (Cayman kit; Interchim). Vitamin C was quantified in deproteinized plasma by HPLC with a fluorescent detector (excitation wavelength, 360 nm; emission wavelength, 440 nm) as described previously (22).

HOMA-IR was calculated from fasting plasma glucose and insulin values by using HOMA calculator software developed by the University of Oxford (https://www.dtu.ox.ac.uk/homacalculator), which estimates steady-state b-cell function and insulin sensitivity as percentages of a normal reference population.

Statistical analysis

The variables are presented as means 6 SDs. A power analysis based on FMD measurements performed in the labo-ratory, with an SD of the mean of 2.96 to observe a difference of

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25%, indicated that 42 of the 52 subjects were required to complete the study (a = 0.05, power of 80%).

All statistical analyses were conducted in a blind manner. Usual descriptive statistics and robust measures were computed. The data for outcome variables were tested for normality and log-normality by using the Shapiro-Wilk test.

According to the statistical guidelines for the analysis of crossover trials (23), the effects of the treatment on the outcome variables were assessed by using mixed models. Treatment (GFJ or CD), period (first or second period), and group/sequence (the order in which the subjects received the treatment) were used as fixed effects. The effects of subjects were included as random effects to account for correlations between the observations made on the same subject. The baseline values (for each period) were also used as covariates. Because hypotheses were prespecified a priori in this study, no adjustment for multiple comparisons was made (24). The treatment effect was considered statistically significant if the P value was,5%. All statistical analyses were performed with SAS version 9.1.3 software (SAS Institute).

Comparisons of baseline values between the 2 treatment pe-riods were performed by using paired t tests to determine whether any carryover effects were present. This procedure failed to reveal any statistically significant differences, in-dicating the absence of carryover effects. Therefore, the baseline values presented for all the measured variables were the means of the 2 periods.

RESULTS

Baseline characteristics of the study population, safety, and compliance

The baseline characteristics of the study population are summa-rized in Table 2. The enrolled subjects were healthy postmenopausal Caucasian women with a mean age of 57.8 6 3.7 y and with a mean time since menopause of 6.36 2 y. At screening, the en-rolled subjects were slightly overweight (BMI ranging from 21.1 to 29.9) and had a mean waist circumference of 95.76 5.3 cm. The subjects were normoglycemic, had typical laboratory test results (hematologic, renal, liver, and inflammatory markers), and were clinically normal (data not shown). The volunteers ranged from normal to slightly hyperlipidemic, as shown by the screening

values for plasma total cholesterol and LDL cholesterol concen-trations (Table 2). The recruited subjects exhibited normal to high-normal BP values. Using the Framingham risk equation, the 10-y risk of CVD for the study population was estimated at 8.56 3.9%, which corresponded to a moderate risk compared with the refer-ence value (5.9%) for a population of middle-aged women (25).

The 6-mo consumption of each beverage did not induce any hepatic toxicity, as reflected by the absence of changes in plasma liver enzyme activities for the duration of the study period (data not shown). At the end of the 2 experimental periods, the plasma concentrations of vitamin C were statistically signifi-cantly higher compared with baseline values: 74.76 19.7 mmol/ L and 71.13 6 15.2 mmol/L for GFJ and CD, respectively, compared with 61.6 6 18.4 mmol/L at baseline (P , 0.001). This result reflected the good compliance of the volunteers for the protocol and was consistent with an estimated level of compliance of.95% based on the counting of distributed and unused bottles. Anthropometric measures, body composition, and dietary intakes

The two 6-mo intervention periods with each beverage did not affect the body weight of volunteers or the distribution of fat mass between the abdominal region and whole body (Table 3). The intake of the 2 study drinks provided the subjects with an extra daily energy intake of 110 kcal but did not induce any statisti-cally significant changes in their total energy intake or in their dietary intake of individual macronutrients (Table 3).

Vascular function

As shown in Figure 2, after daily GFJ consumption for 6 mo, the PWV between carotid and femoral arteries was statistically significantly lower (7.36 6 1.15 m/s) than that measured after CD consumption (7.706 1.36 m/s), with a P value of 0.019 for the diet effect according to the mixed-model analysis adjusted from baseline values. The corresponding difference in PWV values between the 2 drink periods was estimated at20.524 m/s by using the mixed model. No statistically significant treatment effect was found on the augmentation index at 75 beats/min, which reflected the peripheral arterial stiffness (27.776 18.39% and 26.136 18.76% for GFJ and CD, respectively; P = 0.735). As presented in Table 4, the mixed-model analysis did not reveal any statistically significant dietary treatment effect (GFJ vs. CD) on endothelial function when assessed in the brachial artery (FMD) and in the peripheral arterial beds (PAT ratio), with P values of 0.739 and 0.208, respectively. The values for diastolic, systolic, and pulse blood pressures were not signifi-cantly different between GFJ and CD treatments, with P values estimated at 0.429, 0.729, and 0.248, respectively (Table 4). No differences in plasma biomarkers of hemodynamic function, nitric oxide, and endothelin 1 were observed between volunteers after the 2 interventions (Table 4).

Glucose metabolism, biomarkers of inflammation, endothelial function, and antioxidant status

As presented in Table 4, the fasting plasma glucose and insulin concentrations, as well as values of HOMA-IR reflecting insulin resistance, were not significantly different between the dietary interventions with GFJ and a matched CD without flavanones.

TABLE 2

Baseline characteristics of the study population (n = 48) at screening Mean6 SD (range)

Age, y 586 4 (50–65)

Time since menopause, y 6.36 2 (3–10)

BMI, kg/m2 25.76 2.3 (21.1–29.9)

Waist circumference, cm 966 5 (88–111)

Corrected Q-T interval, ms 4036 15 (364–420)

Systolic blood pressure, mm Hg 1356 18 (99–167) Diastolic blood pressure, mm Hg 746 11 (53–93)

Fasting glucose, mmol/L 4.856 0.42 (4.2–5.7)

Triglycerides, mmol/L 1.226 0.54 (0.6–3.2)

Total cholesterol, mmol/L 6.186 0.98 (4.2–8.2)

HDL cholesterol, mmol/L 1.716 0.44 (1.06–3.66)

LDL cholesterol, mmol/L 3.976 0.92 (2.36–6.20)

Framingham Risk Score, % 8.56 3.9 (2.1–17.8)

Creatinine, mmol/L 656 9 (45–86)

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The plasma concentrations of high-sensitivity C-reactive protein, IL-6, vWF, soluble intercellular cell adhesion molecule 1, and soluble vascular cell adhesion molecule 1 did not statistically significantly differ between the 2 experimental periods (Table 4). The total plasma antioxidant status, as reflected by ferric reducing ability of plasma and oxygen radical absorbance capacity values, was also similar after 6 mo of regular GFJ or CD consumption (Table 4).

DISCUSSION

The main finding from this RCT is that GFJ consumption sta-tistically significantly lowered arterial stiffness compared with a matched CD without flavanones. Regardless of the significant effect of grapefruit flavanones on PWV, we did not detect statis-tically significant changes in endothelial function assessed by FMD and PAT ratio as well as in peripheral systolic and diastolic BP.

The consistency of clinical evidence regarding the relation between the consumption of fruit juices rich in polyphenols and improvement in vascular function has been recently reviewed (26, 27). These reviews suggested that improved endothelial function and reduced diastolic BP could be reliable mechanisms by which fruit juice–derived polyphenols can reduce CVD risk. Further-more, the beneficial effect of flavonoids on arterial stiffness is emerging. Indeed, a recent review has compiled data from studies that have evaluated the association between flavonoid consumption and arterial stiffness (28). These data support an improvement in arterial stiffness related to the intake of iso-flavones, anthocyanins, and, to a lesser extent, flavanols. Arterial stiffness is generally related to blood pressure (29). However, several chronic flavonoid-based intervention studies have re-ported a statistically significant reduction of arterial stiffness independently from blood pressure changes. Several studies performed in normotensive subjects (healthy volunteers and patients with type 2 diabetes or coronaropathy) showed im-provement in PWV without any change in peripheral BP (30– 32). In overweight men and postmenopausal women, a decrease in both PWV and BP was observed after an isoflavone-based intervention, but changes in these measures were independent (33). Therefore, results from these trials are consistent with those from our study showing a decrease in aortic stiffness without a concomitant decrease in BP in normotensive post-menopausal women.

The PWV measured between the carotid and femoral arteries is considered the gold-standard measurement of central arterial stiffness (34) and strongly correlates with cardiovascular events and all-cause mortality (35). The magnitude of the difference in PWV values between GFJ and CD (20.524 m/s) after the 6-mo consumption period could correspond to an absolutew5% re-duction in the global CVD risk (35). Although arterial stiffness is a consequence of vascular aging, this condition has been shown to be a reversible process, and several interventions, in-cluding physical activity and dietary changes, have been pro-posed to improve arterial stiffness (34). Arterial stiffness is influenced by vascular tone and the release of vasoactive en-dothelial mediators, as well as structural alterations that may cause deterioration in vessel elasticity (36, 37). In healthy people, the age-related stiffening of large arteries is estimated at w0.096 m/s per year (C Heiss, University of Du¨sseldorf,

TABLE 3

Effect of the intervention on anthropometric measures, body composition, and dietary intakes1

Baseline GFJ CD GFJ-CD P value2

Weight, kg 67.46 6.6 68.36 7.0 67.86 6.6 20.241 0.624

Body lean mass, g 427696 4076 430346 4100 427576 4150 252.6 0.229

Body fat mass, g 258176 4259 262116 4592 260536 4117 2296.1 0.422

% Body fat 36.46 3.7 36.66 4.0 36.76 3.6 20.456 0.128

% Abdominal fat 35.26 4.2 35.66 4.6 35.66 4.2 20.580 0.152

Energy intake, kcal 15546 330 15746 415 15496 408 9.065 0.895

Carbohydrate intake, g/d 171.86 45.9 166.256 54.49 162.26 54.2 21.956 0.830

Protein intake, g/d 73.86 17.0 75.26 14.5 73.76 13.2 1.477 0.484

Fat intake, g/d 59.16 18.5 63.476 23.40 62.626 21.27 1.183 0.736

1

All values are means6 SDs, n = 48. Mixed models are adjusted for baseline value. Dietary intake was determined by using a self-administrated 3-d estimated dietary record. CD, control drink; GFJ, grapefruit juice.

2P value for dietary treatment effect (PROC MIXED; SAS Institute).

FIGURE 2 Effect of GPJ naringenin on PWV, index of central arterial stiffness. This study was a crossover study, and all participants (n = 48) had values for GFJ and CD. Values are means6 SDs. Mixed-model analysis adjusted for baseline value. CD, control drink; GPJ, grapefruit juice; PWV, pulse wave velocity.

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Germany, personal communication, 2014). Therefore, the ob-served difference in PWV between GFJ and the matched CD without flavanones is equivalent to 5 y of age-related stiffen-ing. The present study does not identify the nature or origin of the changes in arterial stiffness induced by grapefruit flava-nones. However, previous studies using different categories of flavonoids, including soy isoflavones and wine flavanols, have demonstrated their ability to modulate calcium handling in human and rat arteries (38, 39). This effect is consistent with clinical data showing the action of flavonoids on arterial stiffness because this action is directly related to vascular tone, which is itself dependent on intracellular free calcium. Further studies are needed to determine the mechanism involved in the reduction of central aortic stiffness by grapefruit flavanones.

FMD was initially considered the primary endpoint of this study. However, we failed to observe any effect of the in-tervention on FMD measured in overnight fasted subjects. FMD is largely dependent on nitric oxide bioavailability, which is acutely influenced by dietary flavonoids (40), and therefore can return to baseline levels in fasted subjects. The plasma con-centrations of flavanones peaked between 5 and 6 h after citrus juice intake, with a subsequent decline to baseline levels over the next 12 h (41, 42). An improved endothelium-dependent vaso-dilation was observed in the postprandial state but not in fasted subjects after a 1-mo period of intervention in a previous clinical trial with hesperidin from orange juice (12). In this former study, the observed changes in endothelial reactivity positively corre-lated with the plasma concentration of hesperetin after the intake of orange juice or of pure hesperidin. Because FMD was not measured postprandially in our study, the potential ability of

grapefruit flavanones to induce transient improvements in FMD when present at micromolar concentrations in the plasma has not been assessed. This issue should be addressed in further in-tervention studies with flavanones. In contrast to FMD, changes in the arterial wall and consequently PWV may conceivably persist for a longer period, thus being detectable after a 12-h fasting period with direct measurements of arterial stiffness (27). Be-sides, consistent with our results, a previous clinical trial aiming to study whether the chronic consumption of cranberry juice rich in flavonoids may improve vascular function also showed a re-duction in PWV in fasted subjects without any changes in FMD (31).

In this study performed in free-living subjects, the daily con-sumption of 2 servings of GFJ over 6 mo did not alter daily intake of macronutrients, body weight, or body fat distribution. Similarly, no adverse effect on glucose metabolism was observed. This result is consistent with a recent meta-analysis of RCTs evaluating the effects of a variety of fruit juices on glucose control and insulin sensitivity that showed that fruit juice consumption did not sig-nificantly affect fasting glucose and insulin concentrations (43).

The present RCT provides the first clinical evidence of the vasculoprotective property of flavanones in subjects who regu-larly consume GFJ. It should be noted that this effect was ob-served for an intake of flavanones equivalent tow40% of the estimated daily intake of flavonoids (3). This finding is consis-tent with recent epidemiologic studies reporting an inverse as-sociation between citrus flavanone intake and the risk of CVDs (10, 11). Previous in vitro and animal studies also support our results suggesting a positive effect of flavanones on vascular function (9).

TABLE 4

Effect of a 6-mo consumption of grapefruit flavanones on vascular function, glucose metabolism, biomarkers of inflammation, endothelial function, and antioxidant status1

Baseline GFJ CD GFJ-CD P value2 Blood pressure, mm Hg Systolic 126.046 13.99 125.896 17.08 125.286 14.36 1.413 0.429 Diastolic 70.756 9.02 69.396 8.66 70.066 8.87 20.283 0.729 Pulse pressure 55.36 9.5 56.56 12.4 55.26 10.6 1.5487 0.248 Endothelial function FMD dilation, % 4.216 2.33 3.976 2.37 4.326 2.54 20.123 0.739

Baseline brachial diameter, mm 3.566 0.46 3.566 0.43 3.576 0.46 20.068 0.228

PAT ratio 2.66 0.56 2.56 0.59 2.66 0.48 20.131 0.208 Hemodynamics NO, mmol/L 98.596 36.55 96.136 40.03 93.876 43.75 0.761 0.912 Endothelin 1, pg/mL 1.266 0.34 1.416 0.47 1.326 0.42 0.094 0.324 Plasma Glucose, mmol/L 4.86 0.52 4.926 0.78 4.816 0.4 0.113 0.312 Insulin, mU/mL 8.26 2.93 7.896 2.61 7.836 2.46 0.372 0.253 HOMA-IR 1.056 0.37 1.026 0.47 1.016 0.32 0.051 0.254 hs-CRP, mg/L 3.246 2.11 3.746 1.67 3.396 1.49 0.427 0.154 IL-6, pg/mL 0.216 0.28 0.256 0.22 0.286 0.44 20.087 0.393 vWF, mUnit/mL 1440.936 668.3 1514.816 748.1 1622.096 887.9 2139.766 0.345 sICAM-1, ng/mL 595.486 121.23 615.236 114.79 600.356 138.61 4.150 0.847 sVCAM-1, ng/mL 759.96 207.77 814.516 203.97 840.426 205.06 236.757 0.302 FRAP, mmol Fe2+/mL 718.636 110.44 730.236 122.71 749.456 126.53 218.459 0.376 ORAC, mmol/L 151886 2616 155446 2628 151296 2141 507.2 0.289 1

All values are means6 SDs, n = 48. Mixed models are adjusted for baseline value. CD, control drink; FMD, flow-mediated dilation; FRAP, ferric reducing ability of plasma; GFJ, grapefruit juice; hs-CRP, high-sensitivity C-reactive protein; NO, nitric oxide; ORAC, oxygen radical absorbance capacity; PAT, peripheral arterial tonometry; sICAM-1, soluble intercellular adhesion molecule 1; sVCAM, soluble vascular cell adhesion molecule 1; vWF, von Willebrand factor.

2

P value for dietary treatment effect (PROC MIXED; SAS Institute).

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Several limitations should be acknowledged in the present study. Special attention was paid to match the CD to GFJ as closely as possible for a range of components, but we cannot fully exclude the possibility that other known or unknown bioactives present in GFJ might also be causally related to the biological outcomes beyond flavanones. A way to strengthen the contribution of flavanones would have been to look at the correlation between the observed effect on PWV and plasma concentrations of flavanones. However, due to our study design, it is irrelevant to seek such a correlation because all flavanone metabolites disappeared from the circulation after an overnight fasting. Given the number of endpoint measures considered in our study, we cannot exclude some inflation of a risk due to multiple comparisons. To address this issue, a specific study focused on PWV is warranted. Despite these limitations, our trial was the first long-term study examining the impact of GFJ con-sumption on vascular function in healthy postmenopausal women. This trial provides evidence of an improvement in arterial stiffness over a long time frame of grapefruit consumption as part of a normal diet. It also highlights a possible role of flavanones in mediating this beneficial effect.

In conclusion, our results support that some bioactive com-pounds abundant in grapefruit, particularly flavanones, may prevent arterial stiffening. These results have potential implications for public health, but dietary recommendations regarding the intake of grapefruit products for the whole population cannot yet be pro-vided. Indeed, grapefruits also contain furanocoumarins, which can interact with the metabolism of a variety of drugs (44). To overcome this difficulty, the citrus industry is making great efforts to evolve toward grapefruit products low in these phytochemicals by altering grapefruit varieties, agronomic practices, postharvest treatments, processing, and storage.

We are grateful to the medical team of the clinical research unit (Christian Dualé, Gise`le Pickering, Gilles Ducheix, Sylvie Eschalier, Sylvia Boulliau, Nicolas Macian, Vincent Bigay, Delphine Roux, Dominique Morand, and Stéphanie Bagur) for their technical assistance in conducting the clini-cal aspects of this study. We also thank Dominique Bayle, Mathieu Rambeau, and Séverine Thien for their technical assistance with the sample analysis and Aurélie Maurer for monitoring and analyzing the food intake records. We give special thanks to Damian Craiem for his technical support with the vascular measurements. We thank Dr Christian Heiss (University Du¨sseldorf, Medical Faculty, Division of Cardiology, Pulmo-nology, and Vascular Medicine, Moorenstrasse 5, 40225 Du¨sseldorf, Germany) for personal communication regarding age-related changes in arterial stiffness.

The authors’ responsibilities were as follows—VH: conducted clinical study implementation and monitoring and performed statistical analysis; VH and M-AV: performed data acquisition or analysis; VH, DM, NB-C, AM, CD, and CM: prepared or revised the manuscript; M-AV: implemented protocol; DM, AM, CD, and CM: designed study; DM, NB-C, AM, CD, and CM: interpreted data; and CD: performed volunteer recruitment and clinical study management. None of the authors reported any conflicts of interest concerning the research described in this article. The funders had no role in the study design, data collection and analysis, publication decision, or man-uscript preparation.

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Figure

FIGURE 1 Consolidated Standards of Reporting trials diagram showing flow of participants through the study
FIGURE 2 Effect of GPJ naringenin on PWV, index of central arterial stiffness. This study was a crossover study, and all participants (n = 48) had values for GFJ and CD

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