Eur J Nutr DOI 10.1007/s00394-016-1185-1

ORIGINAL CONTRIBUTION

Acute intake of quercetin from onion skin extract does not influence postprandial blood pressure and endothelial function in overweight‑to‑obese adults with hypertension: a randomized, double‑blind, placebo‑controlled, crossover trial Verena Brüll1 · Constanze Burak1 · Birgit Stoffel‑Wagner2 · Siegfried Wolffram3 · Georg Nickenig4 · Cornelius Müller4 · Peter Langguth5 · Birgit Alteheld1 · Rolf Fimmers6 · Peter Stehle1 · Sarah Egert1  Received: 10 August 2015 / Accepted: 6 February 2016 © Springer-Verlag Berlin Heidelberg 2016

Abstract  Purpose  To determine whether postprandial metabolic and vascular responses induced by a high-fat and high-carbohydrate meal are attenuated by ingestion of the flavonol quercetin. Methods  Twenty-two overweight-to-obese hypertensive patients participated in a randomized, double-blind, controlled, crossover meal study. They consumed a test meal (challenge) rich in energy (4754 kJ), fat (61.6 g), saturated fatty acids (53 % of total fatty acids), and carbohydrates (113.3 g) with either placebo or 54 mg quercetin. Blood pressure, reactive hyperemia index (RHI), high-sensitive C-reactive protein (hs-CRP), soluble endothelial-derived adhesion molecules, parameters of lipid and glucose metabolism, and markers of antioxidant status were measured before the meal and at 2 and 4 h postprandially.

* Sarah Egert s.egert@uni‑bonn.de 1



Department of Nutrition and Food Sciences, Nutritional Physiology, University of Bonn, Endenicher Allee 11‑13, 53115 Bonn, Germany

2

Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany

3

Institute of Animal Nutrition and Physiology, Christian-Albrechts-University Kiel, Kiel, Germany



4

Department of Cardiology, Angiology and Pneumology, University Hospital Bonn, Bonn, Germany

5

Institute of Pharmacy and Biochemistry, Department of Biopharmaceutics and Pharmaceutical Technology, Johannes Gutenberg University, Mainz, Germany

6

Institute of Medical Biometry, Informatics and Epidemiology, University Hospital Bonn, Bonn, Germany





Results  Systolic and diastolic blood pressure increased significantly over time, but were not affected by treatment (placebo or quercetin). During both treatments, serum endothelin-1, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and plasma asymmetric dimethylarginine slightly decreased over time, whereas RHI increased. Serum triglycerides, total cholesterol, and insulin significantly increased, whereas HDL cholesterol and glucose significantly decreased over time, again with no effect of treatment. Plasma α-tocopherol significantly increased, and plasma Trolox equivalent antioxidative capacity decreased over time. Serum hs-CRP, plasma retinol, and β-carotene did not significantly change during the trial. Conclusion In hypertensive patients, a high-energy meal did not lead to postprandial impairment of vascular endothelial function. Postprandial metabolic responses induced by the challenge, such as lipemia and insulinemia, were not attenuated by the concomitant ingestion of quercetin. Clinical trial  This trial was registered at www.germanctr. de/ and http://apps.who.int/trialsearch/ as DRKS00000555. Keywords  Quercetin · Blood pressure · Postprandial metabolism · Cardiovascular diseases · Endothelial function Abbreviations ADMA Asymmetric dimethylarginine CVD Cardiovascular diseases FMD Flow-mediated dilatation hs-CRP High-sensitive C-reactive protein NO Nitric oxide PAT Peripheral arterial tonometry RHI Reactive hyperemia index

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RM-ANOVA Repeated-measures ANOVA sE-Selectin Soluble endothelial selectin sICAM-1 Soluble intercellular adhesion molecule-1 sVCAM-1 Soluble vascular cell adhesion molecule-1 TEAC Trolox equivalent antioxidative capacity

Introduction Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is one of the predominant flavonols. It is ubiquitously distributed in (edible) plants and is one of the most potent antioxidants of plant origin [1]. A recent comprehensive meta-analysis of 13 prospective cohort studies comprising 344,488 subjects showed that regular dietary intake of flavonols is inversely associated with the risk of cardiovascular diseases (CVD). The pooled RR comparing the highest and lowest categories of flavonol intake was 0.89 (95 % CI 0.84, 0.94; P for trend  = 0.001) [2]. Although the physiological mechanisms of this epidemiological benefit remain incompletely defined, animal studies and human intervention studies have identified many relevant effects of quercetin [3–5]. For example, studies in animal models suggest beneficial effects of quercetin on obesity-associated metabolic disorders including insulin resistance and dyslipidemia [6–8] as well as on hypertension and vascular dysfunction [9–11]. We recently showed that in patients with metabolic syndrome and/or hypertension, 6-week supplementation with supra-nutritional doses (150–162 mg/day) of quercetin reduced systolic blood pressure [12–14] and fasting plasma concentration of atherogenic oxidized low-density lipoproteins [12]. There is increasing evidence that the magnitude of stress that occurs in the postprandial state is an important contributing factor to CVD [15, 16]. The postprandial state is characterized by lipemia and glycemia/insulinemia and associated events, such as oxidative stress, inflammation, and impaired endothelial function [15]. The magnitude and duration of these postprandial responses are influenced by the quantity and quality of macronutrients in the consumed food [17]. Long-term repeated overshoot of postprandial metabolic stress may lead to chronic low-grade inflammation and impairment of the antioxidative defense, especially in individuals with insulin resistance syndrome. Recent data suggest that when flavonols are consumed with high-fat and readily available carbohydrate “pro-oxidant and pro-inflammatory” meals, they may counterbalance their negative postprandial effects [15]. Previous intervention studies in patients at increased CVD risk only measured quercetin effects in the fasting state with the last quercetin intake typically in the evening, 8–12 h before blood sampling. To the best of our knowledge, no studies have been published considering the acute

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effects of quercetin on postprandial responses in hypertensive subjects. Thus, the aim of the present study was to systematically investigate whether postprandial metabolic and vascular responses induced by a high-fat and high-carbohydrate meal are attenuated by the concomitant ingestion of quercetin. Study variables included markers of endothelial function, blood pressure, parameters of lipid and glucose metabolism, and markers of antioxidant status. We hypothesized that, due to its strong antioxidant potential [4], quercetin would attenuate the oxidative stress-related inflammatory changes that result from the consumption of high-fat/carbohydrate meals. This trial was part of our recently published clinical intervention study examining the effects of 6-week quercetin supplementation on 24-h ambulatory blood pressure profile and fasting endothelial function in patients with prehypertension and stage I hypertension (“chronic study”). Our major finding was that quercetin reduced 24-h systolic ambulatory blood pressure in hypertensive patients, but did not affect fasting endothelial function [14]. The present postprandial trial was conducted at the end of the two supplementation periods of our previous chronic study after 6-week supplementation with quercetin or placebo.

Participants and methods Participants Details of the study design and patient recruitment, enrollment, and randomization have been previously described [14]. Interested volunteers aged 25–65 years, who were overweight or obese, attended a screening that included physical assessments (body height and weight, resting blood pressure, heart rate, and waist and hip circumference), clinical assessments [liver function, serum lipids and lipoproteins, glucose and uric acid, hematology, and highsensitive C-reactive protein (hs-CRP)], medical history, and a dietary questionnaire. The inclusion criteria were as follows: (1) central obesity (waist circumference ≥94 cm for men and ≥80 cm for women); (2) prehypertension (120–139 mmHg systolic and/or 80–89 mmHg diastolic) or stage 1 hypertension (≥140–159 mmHg systolic and/or ≥90–99 mmHg diastolic); (3) dyslipidemia (fasting serum triglyceride concentration ≥1.7 mmol/L and/or serum HDL cholesterol concentration <1.0 mmol/L for men and <1.3 mmol/L for women); and/or a pro-inflammatory state (hs-CRP ≥ 2 mg/L) [14]. Twenty-two subjects (11 male and 11 female) were included in this trial. The study protocol was explained in detail to all participants, who gave written informed consent at the beginning of the study. The study was registered at http://www.germanctr.de/ and http://apps.who.int/trialsearch/ as DRKS00000555.

Eur J Nutr

Test meal The test meal (challenge) consisted of 80 g croissant, 40 g bread roll, 20 g butter, 60 g cheese, 25 g jam, and 400 mL lemonade (Capri Sun, Eppelheim, Germany) and provided 4754 kJ, 61.6 g fat (50 % of the total energy), 113.3 g carbohydrates (41 % of the total energy), 24.1 g protein (9 % of the total energy), and 0 mg flavonoids. The nutrient composition of the test meal was calculated from the German Nutrient Database Bundeslebensmittelschlüssel (Max Rubner-Institute, Germany). The detailed composition is given in Table 1. With the meal, the subjects ingested a single capsule (placebo or 54 mg of quercetin, see below) with a glass of water. All subjects completely ingested the test meal within 15 min. Quercetin and placebo capsules Two types of hard gelatin capsules, quercetin and placebo, were manufactured at the Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Mainz, Germany. Quercetin capsules contained onion skin extract powder (132 mg per capsule), and placebo capsules contained mannitol (~170 mg per capsule). The quercetin content of the onion skin extract powder (Allium cepa L.; Rudolf Wild GmbH & Company KG, Heidelberg/Eppelheim, Germany) was 41.25 % (dry mass, 95.94 %). Therefore, each quercetin capsule contained 54 mg quercetin. Hard gelatin capsules (Coni-Snap®), size 0, were supplied by Capsugel, Belgium NV. Quercetin and placebo capsules were identical in shape and taste. Capsule filling was performed using a Dott

Bonapace semi-automatic capsule-filling machine. Mannitol was obtained from Fagron, Barsbüttel, Germany. The primary investigators, all study personnel, and all participants were blinded to the treatments. The quercetin dose of 54 mg was selected to represent one-third of the total daily dose of quercetin (162 mg) that was shown to reduce systolic blood pressure after 6 weeks of supplementation [14]. Our previous plasma kinetic study showed that maximal plasma concentration of quercetin is reached 2 h after bolus intake of 50 mg of quercetin [18]. Study protocol This postprandial study was undertaken as a double-blinded, randomized, placebo-controlled crossover trial within a long-term supplementation study with 6-week treatment periods (162 mg/day quercetin or placebo) separated by a 6-week washout period. Each subject participated in two 4-h meal tests at the end of each of the 6-week intervention periods of the long-term study [14]. Subjects were assigned to quercetin or placebo according to a block-wise randomization scheme. The subjects were instructed to maintain their diet, body weight, body composition, and lifestyle during the study period, to abstain from alcohol on the day before the test, and to refrain from intensive physical activity for 12 h before the test. The tests were carried out in the morning after a 10- to 12-h overnight fast. Venous blood sampling, vascular testing, and measurements of blood pressure and heart rate were conducted before the test meal (0 h) and at 2 and 4 h after finishing the test meal (Fig. 1). Measurements

Table 1  Nutrient composition of the test meal per serving Test meal

Blood pressure and heart rate

Energy (kJ) Carbohydrates (g) Mono- and disaccharides (g) Polysaccharides (g) Ratio of polysaccharides to mono- and disaccharides Protein (g) Total fat (g) Saturated fatty acids (g) Monounsaturated fatty acids (g) N-6 polyunsaturated fatty acids (g) N-3 polyunsaturated fatty acids (g) Cholesterol (mg) Dietary fiber (g) Vitamin C (mg)

4754 113.3 61.4 51.6 0.84 24.1 61.6 32.9 19.4 4.0 2.0 104.2 3.5 49.3

Measurements of blood pressure and heart rate were taken with an automatic blood pressure measurement device (boso carat professional, Bosch + Sohn GmbH u. Co. KG, Jungingen, Germany) under standardized conditions according to the recommendations of the American Heart Association Council on High Blood Pressure Research [19]. Each participant sat quietly for 5–10 min, after which their arm was placed at heart level, and systolic and diastolic blood pressure was measured at least twice in 3- to 5-min intervals. If blood pressure measurements varied by ≥10 mmHg, an additional measurement was taken. The accumulated measurements were then averaged to determine overall systolic and diastolic BP.

Vitamin E (mg)a

5.4

One serving of the test meal consisted of 80 g croissant, 40 g bread roll, 20 g butter, 60 g cheese, 25 g jam, and 400 mL lemonade a

  α-Tocopherol equivalent

Vascular testing For vascular testing, we used noninvasive peripheral arterial tonometry (PAT) technology to assess the reactive

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Eur J Nutr

Fig. 1  Study design. Participants (n  = 22) received capsules (placebo or 54 mg of quercetin) and a test meal (challenge) and underwent vascular testing, venous blood sampling, and measurement of resting blood pressure and heart rate before the test meal and 2 and

4 h after finishing the test meal. This postprandial protocol was conducted at the end of the two supplementation periods of our previous chronic study [14] after 6-week supplementation with quercetin or placebo

hyperemia index (RHI). PAT was performed using the EndoPAT plethysmography device (Endo-PAT2000, Itamar Medical Ltd, Caesarea, Israel). Details are described elsewhere [20]. Briefly, the PAT signal measures the change in the peripheral arterial tone of peripheral arterial beds by recording the arterial pulsatile volume in the fingertip. For this purpose, we placed a plethysmography biosensor on each index finger and a blood pressure cuff on the upper arm of the study arm (left arm). The right arm served as a control arm (without a cuff). The recording of the PAT signal started after a resting period of at least 15 min with a 1-min standby-test period. If the signal recording was free from interference, the test was started. The test lasted 15 min and was split into three 5-min periods. The first period was a baseline recording of the pulse wave amplitude (0–5 min). Next, the blood pressure cuff was inflated to supra-systolic values for 5 min to induce ischemia (5–10 min). Finally, the cuff was deflated to induce reactive hyperemia (10–15 min). PAT signals were analyzed with automated software (Itamar Medical), and the RHI was calculated as the ratio of the average PAT signal amplitude over a 1-min period starting 1 min after cuff deflation divided by the average PAT signal amplitude over a 3.5-min period of the baseline recording. The RHI in the study arm was normalized to the RHI in the control arm. The RHI correlates with brachial artery flow-mediated dilatation (FMD) [21] and is significantly influenced by nitric oxide (NO) [22]. In subjects with coronary endothelial dysfunction, the RHI is lower than in subjects without coronary endothelial dysfunction [23] and it is lower in the presence of CVD risk factors [24].

frozen and stored in cryovials at −80 °C until analysis. Fasting and postprandial lipids, insulin, glucose, and hsCRP were assayed from fresh samples within 4 h of sampling at the central laboratory of the Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Germany. All laboratory measurements were taken without knowledge of the treatment. Serum concentration of total cholesterol was measured using polychromatic endpoint measurement. Serum concentrations of low-density lipoprotein cholesterol, HDL cholesterol, and triglycerides, and plasma concentration of glucose were measured using bichromatic endpoint measurement with a Dimension Vista 1500 analyzer (Siemens Healthcare Diagnostics GmbH). Serum concentration of hs-CRP was determined using nephelometric methods with a Dimension Vista 1500 analyzer (Siemens Healthcare Diagnostics GmbH). Serum insulin concentration was measured using a chemiluminescent immunometric assay with the Immulite 2000 analyzer (Siemens Healthcare Diagnostics GmbH). Plasma concentration of asymmetric dimethylarginine (ADMA; Immundiagnostik AG, Bensheim, Germany), serum endothelin-1 and serum soluble adhesion molecules E-selectin (sE-Selectin), soluble intercellular adhesion molecule-1 (sICAM-1), and soluble vascular cell adhesion molecule-1 (sVCAM-1) (R&D systems, Inc., Minneapolis, USA) were determined in duplicate using commercially available enzyme-linked immunoassay kits according to manufacturer instructions and quality controls. Plasma concentration of l-arginine was determined using reversedphase HPLC as described previously [25]. Analyses of plasma concentrations of quercetin and its monomethylated derivatives tamarixetin (4′-O-methyl quercetin) and isorhamnetin (3′-O-methyl quercetin) as well as kaempferol were performed using HPLC with fluorescence detection as described previously [26]. All samples were treated enzymatically with β-glucuronidase/ sulfatase-type H-2 (crude enzyme extract from Helix pomatia; Sigma-Aldrich AG, Taufkirchen, Germany) prior to the extraction of the flavonols.

Blood sample processing and analysis Fasting and postprandial blood was drawn into tubes containing EDTA, lithium heparin and fluoride, or a coagulation activator (Sarstedt, Nümbrecht, Germany). Plasma and serum were obtained by centrifugation at 3000g for 15 min at 8 °C. Aliquots of plasma and serum for determination of flavonols and hemodynamic biomarkers were immediately

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The antioxidant capacity of plasma was measured according to Miller et al. [27] as Trolox equivalent antioxidative capacity (TEAC). For determination of β-carotene and vitamins A and E, the plasma samples were deproteinized by addition of ethanol (containing apocarotenal as the internal standard, 5 µmol/L). Fat-soluble vitamins were extracted with n-hexane and analyzed using normal-phase HPLC (column, Nucleosil 100-5 CN, 250 × 4.0 nm, Macherey-Nagel, Düren, Germany) and ultraviolet detection (292 nm). Statistical analyses All statistical analyses were performed using the IBM SPSS statistical software package (SPSS version 20, IBM Corporation, Somers, USA). Baseline characteristics of the gender groups (Table 2) were compared using independent samples t tests or Mann–Whitney U tests. Blood pressure, heart rate, EndoPAT, and blood parameters were compared using repeated-measures ANOVA (RM-ANOVA) with treatment (two levels: quercetin and placebo) and time of measurement (three levels: before the test meal and 2 and 4 h after finishing the test meal) as fixed factors. Comparisons are presented for differences between treatments, time points, and treatment × time point interaction. RM-ANOVAs were conducted with log-transformed variables if the residuals were not normally distributed, which was the case for hs-CRP, serum insulin, plasma quercetin, and plasma total flavonols. In all cases, P  ≤ 0.05 (two-sided) was Table 2  Baseline characteristics of participants Age (years) Body height (cm) Body weight (kg) BMI (kg/m2) Waist circumference (cm) Waist-to-hip ratio Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Heart rate (min−1) Serum triglycerides (mmol/L) Serum total cholesterol (mmol/L) Serum HDL cholesterol (mmol/L) Serum LDL cholesterol (mmol/L) Plasma glucose (mmol/L) Serum insulin (pmol/L) Serum hs-CRP (mg/L)

taken to indicate significance. Unless indicated otherwise, descriptive data are presented as arithmetic mean ± SD.

Results Baseline characteristics Baseline characteristics of the subjects are presented in Table  2. All subjects were overweight (54.5 %) or obese (45.5 %), had a visceral fat distribution, and were prehypertensive or stage 1 hypertensive. Height, waist circumference, waist-to-hip ratio, and fasting serum concentration of HDL cholesterol were different between men and women (Table 2). Blood pressure, heart rate, and endothelial function Fasting and postprandial blood pressure, heart rate, and biomarkers of endothelial function are shown in Table 3. Systolic blood pressure, diastolic blood pressure, and heart rate significantly increased over time. Serum endothelin-1, sICAM-1, sVCAM-1, and plasma ADMA decreased over time, whereas serum sE-Selectin and RHI increased over time. Serum hs-CRP did not change over time. There was no effect of treatment on blood pressure, heart rate, or any biomarker of endothelial function (Table 3).

Total (n = 22)

Women (n = 11)

Men (n = 11)

P value women versus men

48.1 ± 10.9 174.5 ± 8.2 94.4 ± 15.1 30.9 ± 3.6 103.9 ± 8.3 0.94 ± 0.09 144.0 ± 10.5 93.0 ± 8.3 73.8 ± 10.5 2.21 ± 1.03 5.63 ± 0.95 1.37 ± 0.27 3.54 ± 0.82 5.13 ± 0.39 80.4 ± 51.7

47.7 ± 10.8 169.6 ± 8.2 91.3 ± 18.9 31.5 ± 4.4 99.8 ± 8.0 0.86 ± 0.04 142.1 ± 11.7 93.8 ± 8.4 74.6 ± 9.0 1.92 ± 1.11 5.48 ± 1.14 1.49 ± 0.26 3.41 ± 0.96 5.00 ± 0.41 76.3 ± 29.9

48.5 ± 11.5 179.4 ± 4.5 97.6 ± 9.9 30.3 ± 2.7 108.1 ± 6.6 1.01 ± 0.04 145.9 ± 9.3 92.2 ± 8.5 73.1 ± 12.2 2.51 ± 0.91 5.77 ± 0.74 1.26 ± 0.23 3.67 ± 0.66 5.27 ± 0.32 83.7 ± 65.8

1.000 0.002 0.204 0.661 0.019 <0.0001 0.430 0.838 0.848 0.136 0.492 0.015 0.472 0.147 0.392

2.82 ± 3.84

4.35 ± 5.03

1.29 ± 0.69

0.083

Shown is mean ± SD The comparison of men and women was performed using an unpaired Student’s t test or a Mann–Whitney U test hs-CRP high-sensitive C-reactive protein

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Table 3  Fasting and postprandial blood pressure, heart rate, and parameters of endothelial function Fasting

2 h postprandial

4 h postprandial

P values from RM-ANOVA Time

Treatment

Time × treatment

0.036

0.121

0.653

0.019

0.495

0.618

<0.001

0.874

0.907

0.014

0.320

0.696

0.736

0.093

0.469

<0.0001

0.453

0.839

<0.0001

0.235

0.074

0.005

0.176

0.377

0.007

0.864

0.174

0.008

0.820

0.483

0.020

0.451

0.298

Systolic blood pressure (mmHg)  Quercetin (n = 22)

149.3 ± 20.4

148.9 ± 18.4

154.1 ± 20.4

 Placebo (n = 22)

146.7 ± 20.3

146.4 ± 18.0

148.9 ± 18.2

 Quercetin (n = 22)

97.2 ± 9.8

94.9 ± 13.5

98.7 ± 10.8

 Placebo (n = 22) Heart rate (min−1)  Quercetin (n = 22)

96.3 ± 12.5

95.0 ± 11.6

96.8 ± 11.7

65.0 ± 8.3

70.2 ± 11.9

67.3 ± 9.6

 Placebo (n = 22)

64.9 ± 7.6

69.7 ± 11.9

67.3 ± 10.0

Diastolic blood pressure (mmHg)

RHI (EndoPAT)  Quercetin (n = 22)

1.90 ± 0.64

2.18 ± 0.52

2.17 ± 0.50

 Placebo (n = 22)

1.87 ± 0.56

2.05 ± 0.59

2.03 ± 0.49

Serum hs-CRP (mg/L)  Quercetin (n = 22)

2.28 ± 2.40

2.43 ± 2.79

2.37 ± 2.57

 Placebo (n = 22)

4.04 ± 5.45

4.00 ± 5.26

3.97 ± 5.32

Serum endothelin-1 (pg/mL)  Quercetin (n = 22)

2.15 ± 0.63

1.72 ± 0.56

2.07 ± 0.65

 Placebo (n = 22)

2.20 ± 0.71

1.81 ± 0.66

2.12 ± 0.67

 Quercetin (n = 22)

209.6 ± 36.3

207.2 ± 37.6

204.7 ± 39.2

 Placebo (n = 22)

218.7 ± 40.5

212.6 ± 39.0

204.9 ± 34.7

Serum sICAM-1 (ng/mL)

Serum sVCAM-1 (ng/mL)  Quercetin (n = 22)

571.7 ± 131.6

535.5 ± 120.0

543.5 ± 112.0

 Placebo (n = 22)

591.0 ± 137.1

575.5 ± 127.8

569.9 ± 126.8

 Quercetin (n = 22)

42.5 ± 14.6

42.2 ± 14.6

44.2 ± 16.2

 Placebo (n = 22)

42.8 ± 15.5

42.3 ± 15.6

43.2 ± 16.1

Serum sE-Selectin (ng/mL)

Plasma ADMA (µmol/L)  Quercetin (n = 22)  Placebo (n = 22) l-arginine/ADMA ratio

0.57 ± 0.14

0.52 ± 0.16

0.46 ± 0.06

0.55 ± 0.17

0.51 ± 0.14

0.49 ± 0.20

 Quercetin (n = 22)

144.0 ± 47.0

162.1 ± 46.7

178.0 ± 54.4

 Placebo (n = 22)

163.0 ± 63.6

171.1 ± 53.0

173.1 ± 65.5

Shown is mean ± SD ADMA asymmetric dimethylarginine, hs-CRP high-sensitive C-reactive protein, sE-Selectin soluble endothelial selectin, sICAM-1 soluble intercellular adhesion molecule-1, RHI reactive hyperemia index, RM-ANOVA repeated-measures ANOVA, sVCAM-1 soluble vascular cell adhesion molecule-1

Serum lipids, lipoproteins, glucose, insulin, and plasma flavonols Fasting and postprandial parameters of lipid and glucose metabolism are shown in Table 4. Serum triglycerides, total cholesterol, and insulin significantly increased over time, and there was no effect of treatment on these parameters. Serum HDL cholesterol and plasma glucose significantly

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decreased over time, and there was no effect of treatment on these parameters. There was a significant effect of treatment and a significant time × treatment interaction on plasma quercetin and total flavonols (Table 4). Since this postprandial study was conducted at the end of the 6-week supplementation periods of our previously published trial [14], fasting quercetin concentration was higher during quercetin treatment than during placebo treatment (Table 4).

Eur J Nutr Table 4  Fasting and postprandial serum lipids, lipoproteins, insulin, plasma glucose, and plasma flavonols Fasting

2 h postprandial

4 h postprandial

P values from RM-ANOVA Time

Treatment

Time × treatment

Serum triglycerides (mmol/L)  Quercetin (n = 22)

1.91 ± 1.09

2.77 ± 1.30

3.26 ± 1.49

 Placebo (n = 22)

1.94 ± 0.94

2.77 ± 1.36

3.18 ± 1.25

 Quercetin (n = 22)

5.24 ± 0.79

5.36 ± 0.88

5.37 ± 0.86

 Placebo (n = 22)

5.32 ± 0.80

5.42 ± 0.78

5.41 ± 0.83

<0.0001

0.927

0.832

0.027

0.641

0.884

0.012

0.600

0.656

0.394

0.777

0.765

0.004

0.892

0.628

<0.0001

0.683

0.405

0.882

<0.0001

0.002

0.695

<0.0001

0.003

Serum total cholesterol (mmol/L)

Serum HDL cholesterol (mmol/L)  Quercetin (n = 22)

1.34 ± 0.40

1.32 ± 0.37

1.28 ± 0.36

 Placebo (n = 22)

1.32 ± 0.37

1.32 ± 0.38

1.25 ± 0.35

Serum LDL cholesterol (mmol/L)  Quercetin (n = 22)

3.29 ± 0.71

3.33 ± 0.70

3.35 ± 0.72

 Placebo (n = 22)

3.32 ± 0.68

3.37 ± 0.71

3.35 ± 0.70

Plasma glucose (mmol/L)  Quercetin (n = 22)

5.07 ± 0.47

4.43 ± 1.44

4.49 ± 0.45

 Placebo (n = 22)

5.17 ± 0.46

4.36 ± 1.09

4.52 ± 0.48

 Quercetin (n = 22)

58.9 ± 45.3

340.1 ± 339.6

114.6 ± 79.4

 Placebo (n = 22)

53.5 ± 46.7

320.3 ± 282.8

136.9 ± 131.2

481.3 ± 205.4

543.8 ± 202.4

560.5 ± 190.0

63.7 ± 46.1

51.3 ± 29.9

46.5 ± 27.1

419.0 ± 191.4

477.9 ± 192.7

496.6 ± 183.2

43.4 ± 67.7

34.3 ± 24.5

30.0 ± 22.4

Serum insulin (pmol/L)

Plasma total flavonolsa (nmol/L)  Quercetin (n = 22)  Placebo (n = 22) Plasma quercetin (nmol/L)  Quercetin (n = 22)  Placebo (n = 22) Shown is mean ± SD

RM-ANOVA repeated-measures ANOVA a

 Total plasma flavonols were calculated as: total flavonols (nmol/L) = quercetin (nmol/L) plus kaempferol (nmol/L) plus isorhamnetin (nmol/L) plus tamarixetin (nmol/L)

Plasma α‑tocopherol, retinol, β‑carotene, and TEAC Plasma α-tocopherol concentration significantly increased over time, but was not affected by treatment (Table 5). Plasma retinol and β-carotene were not affected by time or treatment (Table 5). Plasma TEAC decreased over time, but was not affected by treatment (Table 5). Fasting plasma concentrations of vitamin E, vitamin A, and β-carotene were within normal concentration ranges in all subjects (α-tocopherol, 12–48 µmol/L; retinol, 0.7–2.8 µmol/L; β-carotene, 0.28–2.3 µmol/L).

Discussion As expected, we found significant alterations in the parameters of lipid, glucose, and insulin metabolism after ingestion of the meal. Moreover, these metabolic changes were

accompanied by an increase in blood pressure and heart rate. However, we were unable to detect any meal-induced impairment in endothelial function. In addition, we found no acute effects of quercetin on markers of endothelial function. In contrast to our hypotheses, all meal-induced changes in markers of endothelial function (decrease in endothelial-derived adhesion molecules and ADMA, and increase in RHI) showed a slight improvement across the 4-h time frame. This result is somewhat unexpected, given previous reports that meals rich in fat and energy lead to meal-induced impairment of endothelial function [28–30]. Plasma glucose and serum insulin concentrations at baseline and at 2 and 4 h postprandial indicate that the ingestion of the test meal induced a high hypoglycemic response. For example, serum insulin concentration increased by 500 % from baseline (fasting) to 2 h postprandially (Table 4). This high insulin response caused a rapid decline in glucose concentration, with plasma glucose

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Table 5  Fasting and postprandial plasma α-tocopherol, retinol, β-carotene, and TEAC Fasting

2 h postprandial

4 h postprandial

P values from RM-ANOVA Time

Treatment

Time × treatment

0.004

0.623

0.562

0.388

0.494

0.666

0.912

0.837

0.578

<0.0001

0.431

0.099

Plasma α-tocopherol (µmol/L)  Quercetin (n = 22)

46.19 ± 11.18

44.93 ± 8.82

46.46 ± 8.78

 Placebo (n = 22)

45.68 ± 10.13

45.35 ± 10.83

47.92 ± 12.49

 Quercetin (n = 22)

2.12 ± 0.58

2.15 ± 0.54

2.15 ± 0.47

 Placebo (n = 22)

2.07 ± 0.77

2.08 ± 0.61

2.12 ± 0.75

Plasma retinol (µmol/L)

Plasma β-carotene (µmol/L) 0.86 ± 0.55

0.86 ± 0.57

0.86 ± 0.56

0.85 ± 0.66

0.86 ± 0.66

0.86 ± 0.64

 Quercetin (n = 22)

1.50 ± 0.19

1.44 ± 0.18

1.38 ± 0.15

 Placebo (n = 22)

1.47 ± 0.17

1.45 ± 0.17

1.44 ± 0.19

 Quercetin (n = 22)  Placebo (n = 22) Plasma TEAC2 (mmol/L)

Shown is mean ± SD RM-ANOVA repeated-measures ANOVA, TEAC Trolox equivalent antioxidative capacity a

  Plasma TEAC in mmol of Trolox equivalents/L

concentration lower at 2 and 4 h postprandially than at baseline (fasting). These responses were not attenuated when quercetin was consumed with the test meal, confirming one previous postprandial observation in healthy men [31]. Data from further human quercetin trials are limited. Few human studies have examined the effects of fruit phenolic compounds on postprandial glycemia, and there is no evidence to support a glucose-lowering effect of consuming polyphenol-rich fruits or beverages with meals [reviewed in 15]. In accordance with previous postprandial studies [30, 32, 33], ingestion of a high-fat meal induced a strong and long-lasting increase in serum triglycerides (Table 4). In contrast to our expectation, quercetin did not attenuate the postprandial increase in triglyceride concentrations. However, quercetin reduced fatty acid and triglyceride synthesis in the rat liver [34], and Pfeuffer et al. [31] found decreased postprandial triglyceride increase after chronic quercetin treatment compared to placebo in healthy men. Triglyceride concentrations are usually inversely related to HDL cholesterol, and this may explain the small postprandial decrease in HDL cholesterol in the present study (Table 4). The postprandial state is a pro-oxidant state [15]. It is assumed that postprandial hyperlipidemia and hyperglycemia induce a relative oxidative stress that is exaggerated and prolonged in individuals who are obese or diabetic [15]. Postprandial oxidative stress is typically accompanied by postprandial inflammation and impaired endothelial function [15]. Thus, it is surprising that the strong and long-lasting hyperlipidemia and hyperglycemia/hyperinsulinemia induced by our test meal did not translate into

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impaired endothelial function in our overweight and obese subjects with hypertension. In fact, our results indicate that there was a transient increase in endothelial-dependent vasodilation during the postprandial state. Although this finding was unexpected, it supports some findings from the literature using the same (RHI, endothelial adhesion molecules) or other related measures of vascular function (e.g., FMD). For example, Liu et al. [35] showed that in apparently healthy males, fat-rich mixed snacks result in a transient improvement in peripheral vascular response at 2 h after snack intake. In two other studies of healthy adults, a high-fat meal induced a significant increase in forearm blood flow over a period of 6 h, but did not affect FMD [36, 37]. Our data differ from those of some previous studies which found that RHI [33, 38] or FMD [39–41] was impaired after short-term consumption of a high-fat meal with similar or lower energy level to the test meal used in the current study. Thus, our findings cannot be explained by an insufficient fat and/or energetic challenge. Our meal was rich in fat and saturated fatty acids and designed to reflect the typical fat consumption of a Western diet. A postprandial decline in vascular reactivity may have occurred at a time point different to those that were chosen for testing in our trial. However, in previous studies, significant impairments in vascular reactivity were observed at 2 and/or 4 h postprandially [38, 39, 41]. Thus, we consider the time points for potential changes in vascular reactivity suitable. The slight postprandial improvement in endothelial function observed in the present trial may be explained by diurnal fluctuations because the study was conducted in the morning and lasted for 4–5 h. Endothelial function exhibits

Eur J Nutr

a circadian variability with an attenuation in the early morning [42]. This is in accordance with the peak incidence of cardiovascular events in the early hours after waking [43]. In other studies, FMD was lowest in the early morning and increased in the hours to noon [43, 44]. Thus, it is possible that our participants underlay the circadian variability with an improvement of the endothelial function from morning to noon. The mechanisms behind the impairment of endothelial function in the morning are still unclear. Proposed mechanisms include decreased NO production and increased NO degeneration or inactivation [44], decreased l-arginine availability [44], the circadian variation in endothelial NO synthase activity [42, 45], the circadian rhythm of glucocorticoids, catecholamines, and angiotensin II [42], and activation of the sympathetic nervous system and the sensitivity of adrenergic receptors [43]. The slight postprandial increase in RHI observed in the present study may also be explained by insulin-induced sympathetic nervous system activity and insulin-induced vasodilation [46, 47]. In the present study, plasma antioxidant capacity measured by TEAC slightly decreased over time, whereas plasma α-tocopherol concentration increased (Table 5). Based on these two changes, we cannot conclude that the fat-rich challenge increased postprandial oxidative stress as observed in previous studies [30, 41]. Although the optimum method for assessing postprandial oxidative stress has not yet been defined, previous studies largely demonstrated a significant positive correlation between lipemia and oxidative stress measured using free radical status, leukocyte superoxide anion production, nitrotyrosine, TBARS, and 8-PGF2α [30]. We found no effects of quercetin intake on vascular endothelial function in our hypertensive subjects (Table 4). In contrast, a study by Kukongviriyapan et al. [48] in mice has indicated that quercetin has a vascular protective effect associated with endothelial nitric oxide synthase (eNOS) upregulation, blood glutathione (GSH) redox ratio, and the reduction of oxidative stress. In addition, Calabriso et al. [49] recently studied the role of flavonols including quercetin on endothelial inflammatory gene expression. The flavonols reduced intracellular ROS and inhibited the endothelial expression of adhesion molecules. The decrease in endothelial inflammatory gene expression was related to the inhibition of NF-κB and AP-1 activation. Thus, the lack of effects of quercetin intake on endothelial function in our specific patients could be explained by a lack of effect of quercetin on antioxidant activity and oxidative stress. A major strength of this study is its double-blind, placebo-controlled, crossover design, in addition to the strictly controlled postprandial protocol and the examination of a wide range of metabolic and hemodynamic parameters. All subjects served as their own controls, so the betweenregimen variability was minimized. In addition, our study

design was able to detect statistically significant changes in metabolic and vascular parameters over time. However, this study also has a few potential limitations. First, the three time points at which we measured endpoints may not be representative of the entire postprandial period. In future studies, measurements should be extended over a longer interval. Also, blood samples should be drawn every hour. To further explore the mechanisms responsible for elevations in postprandial endothelial function, additional markers of antioxidant capacity and oxidative stress, vascular inflammation as well as glucocorticoids and catecholamines should be measured. Second, this postprandial study was conducted at the end of the 6-week supplementation periods of our chronic trial [14] and, therefore, fasting baseline quercetin concentrations were higher during quercetin bolus intake than during placebo (Table 4). Thus, strictly, our study design combines two questions, (i) the effects of an increased quercetin status on postprandial metabolism and also (ii) the effects of an acute quercetin intake. In conclusion, in hypertensive patients, a test meal rich in energy, fat, saturated fatty acids, and refined carbohydrates did not lead to the frequently reported postprandial impairment of vascular endothelial function. Postprandial metabolic responses induced by the challenge such as lipemia, glycemia, and insulinemia were not attenuated by the concomitant ingestion of quercetin. Acknowledgments  The authors are indebted to our volunteers for their interest and participation in our study; to Rudolf Wild GmbH & Company KG (Matthias Saß) for the supply of the onion skin extract; to Petra Pickert, Margret Schüller, Christel Bierschbach, Adelheid Schuch, Anke Ernst, Petra Schulz, Anke Carstensen, and Ute Hartung for excellent technical assistance; and to Sarah Krönung, Elvis Kolobara, Claudia Pagliarucci, Lisa Albrecht, Ramona Napp, and Michael Napp for performing the venipunctures. This study was supported by Grant No. EG292/3-1 of the German Research Foundation (to SE). Compliance with ethical standards  Conflict of interest  The authors declare that they have no conflict of interest. Ethical standard  The study was conducted according to the guidelines laid down in the 1964 Declaration of Helsinki, and its later amendments and all procedures involving human participants were approved by the ethical committee of the Medical Faculty of the Rheinische Friedrich-Wilhelms-Universität Bonn, Germany. Written informed consent was obtained from all participants.

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