Scholarly article on topic 'Dose and response to cocoa (DARC): A randomized double-blind controlled trial'

Dose and response to cocoa (DARC): A randomized double-blind controlled trial Academic research paper on "Clinical medicine"

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Abstract of research paper on Clinical medicine, author of scientific article — Valentine Yanchou Njike, Naomi Hamburg, Mark Kellogg, Amarnath Annapureddy, Joseph Vita

Abstract Background Habitual cocoa consumption has been shown to improve cardiometabolic risk. This study compared the effects of two doses of cocoa on blood pressure and other cardiometabolic risk factors in adults with stage 1 hypertension. Methods Randomized, controlled, modified Latin square parallel design to compare effects of two daily doses (i.e., 5 vs. 10g) of cocoa powder in cocoa-containing products for 8weeks on cardio-metabolic risk factors in 122 adults (average age 53.6years; 63 women and 59 men) with stage 1 hypertension on no more than one medication. Results Daily cocoa consumption did not improve (p >0.05) blood pressure, endothelial function, lipid profile, or insulin resistance in analyses of our entire sample. Daily consumption of cocoa, compared to placebo, reduced blood pressure in participants on ACE inhibitors (24-hour SBP: −3.2±9.3 vs. 3.6±8.5; p =0.038, 24-hour DBP: −2.0±5.0 vs. 2.3±5.6mmHg; p =0.023), beta blockers (−4.6±3.2 vs. 1.8±2.8mmHg; p =0.009) or diuretics (24-hour SBP 5.5±7.4 vs. −0.6±4.7; p =0.022); and improved endothelial function (3.1±2.3 vs. −3.4±7.4%; p =0.031) in participants on beta blockers. Dose-response on blood pressure was evident in participants on ACE inhibitors, with more benefit from the high dose compared to the low dose. Conclusions Including cocoa in the diet of patients with stage 1 hypertension seems to exert differential beneficial effects on cardiometabolic risk factors in certain sub-groups of patients.

Academic research paper on topic "Dose and response to cocoa (DARC): A randomized double-blind controlled trial"

clinical trials and regulatory science IN _

i~î- Cardiology ^ 11

ELSEVIER

Contents lists available at ScienceDirect

Clinical Trials and Regulatory Science in Cardiology

journal homepage: http://www.elsevier.com/locate/ctrsc

Dose and response to cocoa (DARC): A randomized double-blind controlled trials

Valentine Yanchou Njike, MD, MPHa'd'*, Naomi Hamburg, MDb, Mark Kellogg, PhDc, Amarnath Annapureddy, MDd, Joseph Vita, MDb

a Yale University Prevention Research Center, United States b Boston University Medical Center, Boston, MA, United States c Boston Children's Hospital, Harvard Medical School, Boston, MA, United States d Griffin Hospital, Derby, CT, United States

ARTICLE INFO

ABSTRACT

Article history: Received 17 August 2016 Accepted 19 November 2016 Available online 7 December 2016

Keywords: Cocoa

Dose-response Hypertension Blood pressure Endothelial function

Background: Habitual cocoa consumption has been shown to improve cardiometabolic risk. This study compared the effects of two doses of cocoa on blood pressure and other cardiometabolic risk factors in adults with stage 1 hypertension.

Methods: Randomized, controlled, modified Latin square parallel design to compare effects of two daily doses (i.e., 5 vs. 10 g) of cocoa powder in cocoa-containing products for 8 weeks on cardio-metabolic risk factors in 122 adults (average age 53.6 years; 63 women and 59 men) with stage 1 hypertension on no more than one medication.

Results: Daily cocoa consumption did not improve (p > 0.05) blood pressure, endothelial function, lipid profile, or insulin resistance in analyses of our entire sample. Daily consumption of cocoa, compared to placebo, reduced blood pressure in participants on ACE inhibitors (24-hour SBP: — 3.2 ± 9.3 vs. 3.6 ± 8.5; p = 0.038, 24-hour DBP: — 2.0 ± 5.0 vs. 2.3 ± 5.6 mm Hg; p = 0.023), beta blockers ( — 4.6 ± 3.2 vs. 1.8 ± 2.8 mm Hg; p = 0.009) or diuretics (24-hour SBP 5.5 ± 7.4 vs. — 0.6 ± 4.7; p = 0.022); and improved endothelial function (3.1 ± 2.3 vs. — 3.4 ± 7.4%; p = 0.031) in participants on beta blockers. Dose-response on blood pressure was evident in participants on ACE inhibitors, with more benefit from the high dose compared to the low dose. Conclusions: Including cocoa in the diet of patients with stage 1 hypertension seems to exert differential beneficial effects on cardiometabolic risk factors in certain sub-groups of patients.

© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Cardiovascular disease (CVD) is the leading cause of death in the United States [1]. Dietary intake, particularly a high consumption of fruits and vegetables, is among the recommended lifestyle modifications to reduce risk of cardiovascular disease and lower blood pressure [2]. Studies indicate that flavonoids, a major class of polyphenolic compounds in plant-based foods, mediate the favorable effects of cocoa products on cardiovascular health [3]. A prospective study showed that daily consumption of 6 g of chocolate was associated with a 39% reduced risk of myocardial infarction and stroke [4].

A meta-analysis of 24 randomized, controlled studies found cardioprotective effects of flavonoid-rich cocoa consumption on

☆ This study was conducted with funding from Hershey Company.

* Corresponding author at: Yale University Prevention Research Center, Griffin Hospital, 130 Division Street, Derby, CT 06418, United States.

E-mail addresses: valentine.njike@yalegriffinprc.org (V.Y. Njike), nhamburg@bu.edu (N. Hamburg).

flow-mediated dilatation (FMD), systolic blood pressure, and LDL and HDL cholesterol levels [5]. In another study, daily consumption of 37 g of dark chocolate along with a sugar-free cocoa beverage in overweight adults showed enhanced vasodilation in both conduit and resistance arteries, along with significant reductions in arterial stiffness in women [6]. Muniyappa et al. also found a significant improvement of 2.3% in endothelial function in hypertensive patients without drug therapy after consumption of cocoa beverage for 15 days [7].

Since CDV is both serious and prevalent [1], it is essential to determine the dietary patterns that are protective. Previously, our lab has shown that ingestion of 74 g of solid dark chocolate (providing 10 g of cocoa powder) acutely increases FMD compared to an equal amount of white chocolate [8] as does sustained ingestion of 10 g of cocoa beverage [9]. Although there are positive effects of chocolate and cocoa products, precautions do exist. Because chocolate is calorie-dense, high in fat, and sometimes high in added sugar, it is likely prudent for individuals to consume the smallest effective quantity. The purpose of this randomized, controlled, modified Latin square parallel design was to compare the effects of two doses of cocoa consumption on blood

http: //dx.doi.org/10.1016/j.ctrsc.2016.11.001

2405-5875/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

pressure, endothelial function and other cardiometabolic risk factors in 122 adults with stage 1 hypertension.

2. Methods

2.1. Design

This was a randomized, controlled, modified Latin square parallel design study with two treatment arms to examine variations in dose of cocoa consumption and effects on blood pressure, endothelial function and other cardiovascular risk factors over an 8-week period. The effects of two doses of cocoa consumption on blood pressure in 122 adults were compared. We hypothesized that a dose-response relationship would be found in which increasingly higher doses of cocoa-containing product consumption would demonstrate correspondingly greater beneficial effects on blood pressure and endothelial function over an 8-week period in individuals with stage 1 hypertension.

22. Participants

A total of 122 adults with stage 1 hypertension (i.e., 140-159/9099 mm Hg) on no more than one blood pressure medication were recruited. The study population was selected to represent the demographic profile of the two sites. Each site equally recruited 60 participants. The inclusion criteria included: men and women age 1875 years; stage 1 hypertension (i.e., 140-159/90-99 mm Hg) without anti-hypertensive medication or taking only one anti-hypertensive medication; body mass index <40 kg/m2; and willing to discontinue the use of chocolate/cocoa products at least 4 weeks prior to intervention. The exclusion criteria included: anticipated inability to complete or comply with study protocol; use of lipid-lowering medication or aspirin unless stable on medication for at least 1 month and willing to refrain from taking medication for 12 h prior to endothelial function scanning; severe hypertension (systolic blood pressure > 160 mm Hg or diastolic blood pressure > 100 mm Hg), or use more than one antihypertensive medications; allergy to cocoa products (chocolate or cocoa powder); regular use of vitamin C, vitamin E, fish oil, flax seed oil, omega-3 fatty acids, Coenzyme Q10, fiber supplements, garlic pills, arginine, red yeast rice, and/or any kind of antioxidant and unwilling to discontinue supplementation for at least 4 weeks prior to study initiation and for the study duration (use of a multi-vitamin containing no more than two times the recommended daily allowance for vitamins C and E was permissible); diagnosed eating disorder; on any specific diet, weight control diet, and/or vegan diet; substance abuse (chronic alcoholism and/or other chemical dependency); any unstable medical condition (e.g., cancer, AIDS, tuberculosis, psychotic disorder) that would limit the ability to participate fully in the trial; and/or current or impending pregnancy.

2.3. Recruitment and screening

This study was approved by the Griffin Hospital and Boston Medical Center Institutional Review Boards prior to the recruitment and screening. The study coordinators pre-screened potential participants for eligibility via a structured telephone interview using established inclusion criteria. Those who met preliminary eligibility criteria and agreed to participate were invited to undergo clinical eligibility screening, and were asked to sign a consent form approved by the Griffin Hospital/Boston Medical Center Institutional Review Boards. All participants were informed of the option of discontinuing participation at any time during the study. Participants were advised to discontinue the use of cocoa-containing products for at least 4 weeks prior to receiving intervention. The clinical screening physical examination consisted of weight, height, and blood pressure measures obtained by experienced study personnel using calibrated equipment. Blood pressure was measured (average of three measurements with five minutes between

measurements) with the participant sitting in a quiet room using an approved automated device (SunTech® 247™ Automated Blood Pressure Device). Participants underwent a fasting blood profile for lipids (total cholesterol, HDL, LDL, and triglycerides (TG)) and plasma glucose. All screening laboratory assays were performed at the Griffin Hospital/Boston Medical Center clinical laboratories. Participant eligibility was determined based on the clinical screening results. Participants flow through the trial are presented in Fig. 1.

2.4. Randomization

Participants were randomly assigned to one of the two different arms (i.e., 5 or 10 g of cocoa-containing products), and within each arm were further randomly assigned to two different sequence permutations: intervention/placebo or placebo/intervention. Participants underwent the intervention, control and washout phases during the duration of the study. In the intervention phase, participants consumed products containing cocoa powder for 8 weeks, and in the control phase they consumed the same amount of nutrient-matched products not containing cocoa powder for 8 weeks. A 4-week washout phase was incorporated between the two phases. The randomization was stratified by two sites.

2.5. Interventions

The two treatment arms were as follows: (1) cocoa products providing 10 g cocoa powder every day; (2) cocoa products providing 5 g cocoa powder every day. In the control phase, the participants consumed the same amount of nutrient-matched products not containing cocoa powder for 8 weeks. The cocoa-containing products were consumed as chocolate bars and cocoa beverages. Detailed information about the tested products is presented in Table 1.

2.6. Blinding

The investigators and study participants were blinded to treatment assignments. The test products were wrapped with different wrapper colors by the manufacturer. Only the manufacturer knew what test product corresponded to which treatment assignment. The cocoa-containing products and placebos were identical to appearance and taste. The manufacturer released the codes of the treatment assignments to the investigators after the completion of the statistical analysis of the data.

2.7. Outcome measures

2.7.1. Primary outcome measure

2.7.1.1. Ambulatory blood pressure monitoring (ABPM). Subjects underwent 24-hour ambulatory blood pressure monitoring using an FDA-approved device (Model 90207 with standard adult cuff or large cuff (depending on arm size), Spacelabs Medical, Inc.). Blood pressures were measured every 30 min throughout the 24-hour monitoring period. Mean systolic, mean diastolic, and average mean blood pressure (BP) were calculated from all valid blood pressure measurements. Daytime and nighttime (11:00 PM till 6:30 AM) BP were also assessed. Data were cleaned by: (1) deleting any readings taken > 24 h after the initial reading, (2) deleting the second of any two readings taken <20 min apart, and (3) deleting any readings with error codes per Spacelabs, Inc. default limits.

2.7.12. Office blood pressure. Systolic and diastolic BP were measured at each visit using an approved automated device (SunTech® 247™ Automated Blood Pressure Device). Blood pressures were measured (average of three measurements with five minutes between measurements) with the participant sitting in a quiet room.

Assessed for eligibility (YGPRC only) (n=1000)

• Did not meet inclusion criteria (n=835)

• Did not pass clinical screening (n=141)

• Subjects completed clinical screening (n=295)

• Enrolled after clinical screening (n=122)

Randomization

Placebo (n=31)

Dropped out (ri-4) -1 due to stomach discomfort -1 due to relocation -1 disliked test product -1 lost to follow up

Dropped out (n=2)

-Due to relocation

-Lost to follow up

4 weeks of washout

Dropped out

-1 due to busy

work schedule

-1 unable to use

BP monitor

Dropped out(n=3)

-1 Stomach upset

-1 lost to follow up

-1 unable to use BP

monitor

lOg cocoa powder (n=25)

Dropped out (n=5)-

1 Disliked test

product

-2 lost to follow-up

-1 change in BP med

-1 Syncopal episode

5g cocoa powder (n=Z4)

Completed the trial (n=101)

Note: Total number assessed for eligibility does not include total from Boston University Medical Center. However, all other values include totals from BUMC.

Fig. 1. Flow chart for participants participation.

2.7.2. Secondary outcome measures

2.7.2.1. Endothelial function. Flow-mediated dilatation (FMD) was measured as the percent change in brachial artery diameter from pre-cuff inflation to 60-second post-cuff release (upper arm cuff position). In addition to brachial diameter at 60 second post-cuff release, flow after cuff deflation within the first 15 s was used as an indicator of stimulus strength, hyperemic flow being the stimulus for endothelial reactivity. To account for potential variability in stimulus strength, a secondary analysis was performed in which FMD was divided by flow at 15 second post-cuff deflation to create a stimulus-adjusted response measure. This was compared to the unadjusted FMD measures.

2.7.2.2. Fasting serum lipids. Total cholesterol (Tchol), triglycerides (TG), and high-density lipoprotein (HDL) were obtained by direct measurements. Very-low-density lipoprotein (VLDL) and low-density-

lipoprotein (LDL) were obtained by calculation: VLDL = TG / 5; and LDL = Tchol - (VLDL + HDL). HDL:Tchol ratio was used to evaluate the impact of treatment assignments on lipid panel.

2.7.2.3. C-reactive protein (CRP). Serum CRP values were determined using a high-sensitivity nephelometric method at Children's Hospital Boston [10].

2.7.2.4. Fasting glucose and insulin. Glucose and insulin were measured at each time point. Homeostasis model assessment of insulin resistance (HOMA-IR) values were calculated (HOMA calculator version 2.2.1) from fasting serum glucose and serum insulin levels.

2.7.2.5. Body weight. Body weight was measured for all study participants during each visit. Body weight was measured to the nearest 0.5 lb using a balance-type medical scale. Subjects were measured in the morning (fasting), unclothed with the exception of undergarments.

Table 1

Composition of the tested products.

High dose Low dose

Chocolate Cocoa beverage Placebo chocolate Placebo beverage Chocolate Cocoa beverage Placebo chocolate Placebo beverage

Variable (20 g) (40 g) (20 g) (34 g) (10 g) (20 g) (10 g) (17 g)

Energy (kcal) 94.2 140 105.2 100 47.1 70 52.6 50

Energy from fat (kcal) 58.6 0 52.2 0 29.3 0 26.1 0

Fat(g) 7 0 6 0 3.5 0 3 0

Sat fat (g) 4.4 0 3.6 0 2.2 0 1.8 0

Trans fat (g) 0 0 0 0 0 0 0 0

Cholesterol (mg) 1.6 10 3.8 5 0.8 5 1.9 2.5

Sodium (mg) 1.2 360 26.2 300 0.6 180 13.1 150

Carbohydrates (g) 10.6 20 11.8 20 5.3 10 5.9 10

Dietary fiber (g) 2 2 0 4 1 1 0 2

Sugar(g) 7.4 16 9.8 14 3.7 8 4.9 7

Protein (g) 1.6 12 1.6 10 0.8 6 0.8 5

Vitamin A (IU) 6.2 0 10.8 0 3.1 0 5.4 0

Vitamin C (mg) 0 0 0.4 0 0 0 0.2 0

Calcium (mg) 9 400 58.4 300 4.5 200 29.2 150

Iron (mg) 0.8 0 0 0 0.4 0 0 0

Caffeine (mg) 13.1 8.6 0 0.2 6.6 4.3 0 0.1

Theobromine (mg) 129.2 100 0 0.2 64.6 50 0 0.1

Flavanols (mg, PAC1-10) 178.8 83 bd bd 89.4 41.5 bd bd

Flavanols (mg, DMAC) 310 204 bd bd 155 102 bd bd

Epicatechin (mg) 26 19.8 bd bd 13 9.9 bd bd

2.7.2.6. Waist circumference. Waist circumference was measured using the U.S. government standard protocol. Waist circumference was measured around the narrowest point between ribs and hips when viewed from the front after exhaling.

2.7.2.7. Urinary theobromine. Theobromine was analyzed in urine by liquid chromatography-mass spectrometry using the method of Ptolemy et al. [11]. Basically 500 ^Lof sample was spiked with stable isotope labeled internal standard, passed thru a 10 kDa molecular weight cut off filter, diluted to remove matrix interference and injected onto a C18-BEH column for separation and infusion into the mass spectrometer for quantification. The method has a limit of quantification of 2.25 ^mol/L for caffeine and theobromine and a between day precision of 10% for theobromine and 5% for caffeine.

2.7.2.8. Compliance measure. The theobromine level in urine was measured to assess the compliance. Compliance was also assessed by collecting the returned consumption log or cocoa packages. Good compliance was defined as > 80% use of treatment.

2.8. Statistical analysis

Generalized linear models (GLM) were used to analyze these data. In addition to the effect of time on the outcome measures, other factors were incorporated into the GLM regressions to adjust for potential confounding factors (i.e., covariate imbalance between the treatment groups) such as individual characteristics (i.e., age, gender, BMI and baseline BP) and specific nutrient intake; as well as controlling for sequence in randomized treatment assignments. Descriptive and exploratory analyses of all measured outcomes were carried out before embarking on modeling or hypothesis testing procedures. Distributions ofvariables were expected to meet criteria for analysis with parametric statistics, but distributions were assessed prior to analyses. As indicated, log transformation of data or nonparametric analytic techniques was employed. All analyses of our outcome measures were based on intention-to-treat principle. In all analyses, a two-tailed a of <0.05 (adjusted for 3 pair-wise comparisons) was considered statistically significant. SAS software for Windows version 9.3 was used to carry out all statistical analyses.

The sample size was estimated to allow for 20% attrition and non-compliance and to provide > 80% power to detect a minimal difference of 3.5 mm Hg in DBP between cocoa-containing products and placebo

with maximum allowable type I error of 5% adjusted for 3 pair-wise comparisons.

3. Results

3.1. Study participants

Most of the study participants were white. Baseline values of all outcome measures were somewhat comparable in the active treatment groups as compared to the placebo group. The average age of the participants who received the high-dose treatment assignment was about 54 years, while the average age for those who received the low-dose treatment was about 53 years. The study participants were overweight with their body mass index averaging about 30 kg/m2. The average office systolic blood pressure (SBP) was within the stage 1 hypertension range while the average office diastolic blood pressure (DBP) was within the pre-hypertensive range. The average 24-hour BP of the study participants was within normal BP range. Other participants' baseline data are presented in Table 2.

3.2. Efficacy

3.2.1. All study participants

Daily consumption of 5 or 10 g of cocoa products for an 8-week period as compared to placebo did not lower the 24-hour BP or office BP. Daily consumption of cocoa products for 8 weeks did not show any dose-response on the office or 24-hour blood pressures. Daily consumption of 5 or 10 g of cocoa-containing products for 8 weeks as compared to placebo did not improve endothelial function, serum lipids, CRP level, body weight, waist circumference, glucose level, or insulin level (see Table 3). Also, theobromine urine level did not increase after daily ingestion of cocoa-containing products for 8 weeks as compared to placebo.

3.3. Sub-group analyses

3.3.1. Participants on ACE inhibitors

Daily consumption of 10 g of cocoa-containing products for 8 weeks as compared to placebo significantly lowered the 24-hour ambulatory systolic and diastolic BP (24-hour ambulatory SBP: — 3.2 ± 9.3 vs. 3.6 ±8.5; p = 0.038, 24-hour ambulatory DBP: — 2.0 ± 5.0 vs. 2.3 ± 5.6 mm Hg; p = 0.023) in participants who were on ACE inhibitors.

Table 2

Baseline characteristics of study participants.

Variable High dose cocoa placebo High dose cocoa products p-Value' Low dose cocoa placebo Low dose cocoa products p-Value¥

Age (years) 54.2 ± 10.1 54.2 ± 10.1 1.0000 53.0 ± 10.6 53.0 ± 10.6 1.0000

Gender

Female 16 (26.2%) 14 (23.0%) 0.5233 16 (26.2%) 17 (27.9%) 0.9061

Male 14 (23.0%) 17 (27.9%) 14 (23.0%) 14 (23.0%)

African Americans 12 (19.7%) 14 (23.0%) 0.7351 11 (18.0%) 10 (16.4%) 0.7423

Caucasians 17 (27.9%) 15 (24.6%) 17 (27.9%) 20 (32.8%)

Other 1 (1.6%) 2 (3.3%) 2 (3.3%) 1 (1.6%)

Weight (lbs) 191.1 ± 34.8 192.9 ± 35.2 0.7993 195.2 ± 36.7 197.4 ± 38.5 0.7481

Body mass index (kg/m2) 29.9 ± 4.2 30.3 ± 4.2 0.6750 30.5 ± 4.9 30.7 ± 5.0 0.8378

Office SBP (mm Hg) 141.3 ± 11.7 138.7 ± 12.1 0.2689 141.8 ± 12.1 142.4 ± 13.7 0.8171

Office DBP (mm Hg) 86.2 ± 7.9 83.9 ± 9.3 0.1636 85.0 ± 8.5 84.7 ± 8.7 0.8490

24-h SBP (mm Hg) 126.9 ± 9.2 129.3 ± 10.3 0.2456 130.8 ± 12.3 130.4 ± 11.2 0.8708

24-h DBP (mm Hg) 77.7 ± 7.7 78.1 ± 8.1 0.8099 79.3 ± 8.0 78.9 ± 7.6 0.8179

Wake SBP (mm Hg) 131.4 ± 10.0 134.3 ± 10.0 0.1591 135.1 ± 11.6 134.6 ± 11.0 0.8110

Wake DBP (mm Hg) 81.5 ± 7.9 81.9 ± 8.4 0.8101 82.6 ± 8.8 82.6 ± 8.8 0.9784

Sleep SBP (mm Hg) 118.0 ± 10.5 120.2 ± 13.3 0.4137 122.4 ± 17.0 122.1 ± 16.2 0.9117

Sleep DBP (mm Hg) 70.3 ± 9.1 71.2 ± 9.6 0.6298 72.7 ± 9.3 72.0 ± 8.7 0.6877

Fasting blood glucose (mg/dL) 96.2 ± 17.3 99.5 ± 39.4 0.5622 99.5 ± 22.0 102.9 ± 35.0 0.5509

Insulin, serum (mIU/mL) 13.2 ± 8.8 13.8 ± 8.8 0.7321 13.7 ± 10.6 14.1 ± 11.7 0.8656

HOMAIR 3.3 ± 2.6 3.6 ± 2.7 0.6437 3.7 ± 3.6 3.9 ± 4.1 0.7596

Waist circumference (cm) 100.8 ± 13.2 100.7 ± 12.8 0.9579 102.0 ± 12.1 102.5 ± 12.3 0.8171

Flow mediated dilatation (%) 8.2 ± 3.9 8.2 ± 3.7 0.9932 9.3 ± 5.3 8.9 ± 4.4 0.6307

NTGFMD 10.7 ± 6.0 12.0 ± 6.9 0.3913 10.4 ± 6.2 12.2 ± 5.7 0.2542

Post FMD SBP (mm Hg) 140.1 ± 12.1 139.5 ± 10.1 0.8083 140.9 ± 8.3 140.1 ± 10.8 0.7735

Post FMD DBP (mm Hg) 83.3 ± 8.0 84.7 ± 7.6 0.4755 85.3 ± 7.3 83.8 ± 6.3 0.4426

Total cholesterol (mg/dL) 198.3 ± 40.6 188.9 ± 39.1 0.2115 190.8 ± 43.1 196.5 ± 34.1 0.4539

Triglycerides (mg/dL) 133.1 ± 69.0 143.7 ± 122.3 0.6709 155.3 ± 182.0 148.2 ± 126.4 0.7753

High density lipoprotein (mg/dL) 52.4 ± 17.9 50.4 ± 14.4 0.5147 51.8 ± 15.5 51.4 ± 15.1 0.9029

Low density lipoprotein (mg/dL) 119.9 ± 37.3 110.4 ± 34.5 0.1732 110.2 ± 39.1 115.7 ± 34.7 0.4421

Total cholesterol/HDL 4.1 ± 1.2 4.0 ± 1.2 0.6778 4.0 ± 1.5 4.1 ± 1.2 0.8116

High-sensitivity C-reactive protein (ng/mL) 4.1 ± 7.3 2.9 ± 3.8 0.3765 5.6 ± 9.6 4.5 ± 4.8 0.3780

Values are mean ± standard deviation except otherwise stated. ' p-Values obtained comparing high dose placebo versus high dose cocoa products. ¥ p-Values obtained comparing low dose placebo versus low dose cocoa products.

There was a dose response on 24-hour BP with the higher dose (i.e., 10 g) of cocoa powder in cocoa-containing product consumption, showing greater beneficial effects on BP as compared to the low dose (i.e., 5 g of cocoa powder). Daily consumption of 10 g of cocoa-containing

products for 8 weeks as compared to placebo significantly reduced HOMA-IR and triglyceride levels (HOMA-IR: -1.4 ± 2.2 vs. 0.7 ± 1.8; p = 0.034, triglycerides: - 63.5 ± 151.6 vs. 76.4 ± 198.6 mg/dL; p = 0.012) in participants on ACE inhibitors.

Table 3

Change in outcome measures from baseline after 8 weeks of treatment assignment.

Variable High dose cocoa placebo High dose cocoa products p-Value1 Low dose cocoa placebo Low dose cocoa products p-Value

Weight (lbs) 1.3 ± 4.8T 0.6 ± 3.5 0.3629 -0.1 ± 3.8 -0.5 ± 4.1 0.5788

Body mass index (kg/m2) 0.2 ± 0.8 0.1 ± 0.6 0.4327 -0.0 ± 0.6 -0.1 ± 0.6 0.6247

Office SBP (mm Hg) -2.1 ± 9.0 0.6 ± 8.5 0.1371 -1.2 ± 10.2 -0.4 ± 11.4 0.6457

Office DBP (mm Hg) -1.6 ± 5.8T 0.1 ± 5.8 0.1285 -1.0 ± 5.8 -0.8 ± 7.0 0.8359

24-h SBP (mm Hg) 0.3 ± 8.9 -1.3 ± 10.0 0.2981 -1.5 ± 7.5 -1.1 ± 6.5 0.7779

24-h DBP (mm Hg) 0.3 ± 5.5 - 0.2 ± 5.7 0.5898 -0.6 ± 3.9 -0.8 ± 4.2 0.8379

Wake SBP (mm Hg) 0.9 ± 10.2 -1.3 ± 10.3 0.1897 -1.7 ± 8.3 -1.0 ± 7.1 0.6687

Wake DBP (mm Hg) 0.6 ± 6.8 - 0.6 ± 5.8 0.2446 0.0 ±5.7 -1.1 ± 4.4 0.2992

Sleep SBP (mm Hg) -0.3 ± 9.4 -1.0 ± 12.1 0.8019 -1.5 ± 9.9 -1.0 ± 10.2 0.7099

Sleep DBP (mm Hg) 0.1 ± 6.1 - 0.2 ± 7.0 0.7409 - 0.8 ± 5.4 - 0.3 ± 6.6 0.6979

Fasting blood glucose (mg/dL) 0.1 ± 13.1 -2.5 ± 30.5 0.5291 2.7 ± 13.7 2.5 ± 29.6 0.9659

Insulin, serum (mIU/mL) 0.7 ± 5.1 -0.8 ± 6.2 0.2181 2.3 ± 8.2T 0.7 ± 8.3 0.2064

HOMA IR 0.1 ± 1.5 - 0.3 ± 1.9 0.3921 0.7 ± 3.2 0.3 ± 3.4 0.3639

Waist circumference (cm) 0.2 ± 3.7 - 0.5 ± 4.2 0.3133 0.2 ±3.5 -0.8 ± 3.7 0.1241

Flow mediated dilatation (%) 0.4 ± 2.9 0.6 ± 3.2 0.6920 -0.1 ± 3.6 0.6 ± 3.0 0.2689

NTGFMD -0.7 ± 5.5 0.7 ± 4.8 0.3758 0.3 ± 5.6 1.5 ± 4.5 0.5044

Post FMD SBP (mm Hg) 1.9 ± 12.7 0.8 ± 11.2 0.7399 0.0 ± 11.1 2.6 ± 13.0 0.4041

Post FMD DBP (mm Hg) 0.5 ± 6.7 0.8 ± 7.6 0.8915 0.1 ± 7.2 1.6 ± 8.5 0.4333

Total cholesterol (mg/dL) 1.0 ± 20.2 1.5 ± 23.6 0.8984 2.4 ± 19.8 1.5 ± 21.1 0.8149

Triglycerides (mg/dL) 15.5 ± 105.8 -18.0 ± 79.9 0.0515 - 2.5 ± 79.8 35.5 ± 108.9T 0.0274

High density lipoprotein (mg/dL) 0.4 ± 6.1 2.2 ± 7.9T 0.1238 -1.3 ± 6.4 0.1 ± 5.1 0.2230

Low density lipoprotein (mg/dL) -3.0 ± 19.7 2.5 ± 20.2 0.1080 4.4 ± 15.9T -0.7 ± 18.7 0.1361

Total cholesterol/HDL -0.0 ± 0.6 - 0.1 ± 0.5 0.7774 0.1 ± 0.8 0.0 ± 0.6 0.5080

High-sensitivity C-reactive protein (ng/mL) -1.1 ± 6.3 - 0.2 ± 1.7 0.3938 -0.5 ± 8.5 -0.0 ± 3.2 0.6561

Values are mean ± standard deviation. T Indicate significant change from baseline.

' p-Values obtained comparing high dose placebo versus high dose cocoa products. ¥ p-Values obtained comparing low dose placebo versus low dose cocoa products.

3.3.2. Participants on beta blockers

Daily consumption of 5 g of cocoa-containing products for 8 weeks as compared to placebo significantly reduced SBP ( — 4.6 ± 3.2 vs. 1.8 ± 2.8 mm Hg; p = 0.009). Daily consumption of 5 g of cocoa-containing products for 8 weeks as compared to placebo significantly improved endothelial function (3.1 ± 2.3 vs. — 3.4 ± 7.4%; p = 0.031).

3.3.3. Participants on calcium channel blockers

Daily consumption of 10 g of cocoa-containing products as compared to placebo significantly increased total, HDL and LDL cholesterol (total cholesterol: 22.3 ± 28.3 vs. —12.2 ± 8.1; p = 0.015, HDL cholesterol: 7.7 ± 9.1 vs. —1.3 ± 5.3; p = 0.031, LDL cholesterol: 15.8 ± 17.2 vs. — 4.7 ± 9.0; p = 0.044).

3.3.4. Participants on diuretics

Daily consumption of cocoa-containing products for 8 weeks as compared to placebo significantly increased 24-hour ambulatory SBP (5.5 ± 7.4 vs. — 0.6 ± 4.7; p = 0.022).

4. Discussion

When data of all study participants were analyzed, daily consumption of cocoa-containing products as compared to placebo for 8 weeks did not lower 24-hour ambulatory or office BP in participants with stage 1 hypertension. There was no dose response on BP measures when comparing the beneficial effects of the higher dose (i.e., 10 g of cocoa powder) of the cocoa-containing products to the low dose (i.e., 5 g of cocoa powder) in these participants with stage 1 hypertension. Daily consumption of cocoa-containing products for 8 weeks as compared to placebo did not improve endothelial function, serum lipids, CRP level, body weight, waist circumference, glucose, or insulin.

However, in sub-group analyses, daily consumption of cocoa-containing products lowered 24-hour ambulatory BP in participants on ACE inhibitors and also showed a dose-response. Daily consumption of 10 g of cocoa-containing products also reduced insulin resistance and lowered triglycerides in participants on ACE inhibitors. Daily consumption of 5 g of cocoa-containing products reduced SBP and improved endothelial function. Daily consumption of 5 g of cocoa-containing products reduced 24-hour ambulatory BP in participants on diuretics. Consumption of cocoa-containing products increased HDL and LDL cholesterol in participants on calcium channel blockers.

The effects of cocoa consumption on blood pressure have been conflicting. In a recent study by Mastroiacovo et al. [12], daily consumption of cocoa beverages for 8 weeks lowered the SBP and DBP in elderly individuals. In another study by Grassi et al. [13], daily consumption of cocoa with different levels of flavonoid content, lowered office and 24-hour ambulatory BPs in healthy volunteers. In a meta-analysis of 20 studies by Ried et al. [14], short-term cocoa and chocolate product consumption lowered BP However, in a study by Dower et al. [15], flavo-noid-rich foods (i.e., cocoa and tea) did not lower office and 24-hour ambulatory BP. In our study, we did not see improvement in BP when we analyzed data for all study participants. However, in sub-group analyses, daily consumption of cocoa-containing products lowered BP in participants who were on ACE inhibitors, diuretics or beta blockers. The high flavanol content in cocoa powder could explain the beneficial effects of cocoa on BP. Flavanols increase the production of nitric oxide in the body, which relaxes the blood vessels and in turn lowers BP. Flavanols also inhibit the angiotensin-converting enzyme activity, which may contribute to the reduction of BP [16].

In this study, we did not see improvement in endothelial function after daily consumption of cocoa-containing products when we analyzed data in all our study participants. However, in a sub-group analysis, we observed improvement in endothelial function in participants on beta blockers. Numerous studies have shown that consumption of cocoa improved endothelial function in different populations [13,17-19]. The flavanols present in the cocoa powder stimulate nitric oxide synthase

which increase the production of nitric oxide, which in turn causes the blood vessels walls to relax and widen, thereby improving the endothelial function.

We did not see improvement in serum lipids or increase in CRP in this study when we analyzed data for all our study participants. In sub-group analysis, cocoa consumption lowered triglycerides cholesterol in participants on ACE inhibitors and raised HDL cholesterol in participants on calcium channel blockers. Interestingly, cocoa consumption also increased LDL cholesterol in a sub-group of our study participants who were on calcium channel blockers. The effects of cocoa on serum lipid in previous studies have been mixed. In a meta-analysis of 10 studies by Tokede et al. [20], short-term consumption of cocoa products lowered LDL and total cholesterol, but had no effects on triglycerides cholesterol and HDL cholesterol. The beneficial effects seen on consumption of cocoa on lipid profile could be attributed to the high content of flavanols in the cocoa.

Our data showed improvement in insulin resistance after consumption of cocoa-containing products only in study participants who were on ACE inhibitors. Stote et al. [21] did not see reduction in insulin resistance after short-term consumption of flavanol-rich foods in obese adults at risk for insulin resistance. Conversely, Grassi et al. [22] showed reduction in insulin resistance in hypertensive patients after cocoa consumption. A meta-analysis by Hooper et al. [19] found reduction in insulin resistance after short-term consumption of cocoa. The beneficial effects that have been observed on insulin resistance after cocoa consumption may be due to the high flavanol content in the cocoa products used.

Interestingly, daily consumption of cocoa-containing products as compared to placebo did not increase theobromine urine level in our study participants. The lack of increase in theobromine urine level may be due to inadequate amount of cocoa-containing products consumed or was confounded by the consumption of other foods (e.g., tea leaves) rich in this compound.

4.1. Limitations

The study is limited by the fact that the study population was predominantly Caucasian; therefore, this limits the generalizability of our findings. Study participants were a small sub-set of the sampling frame. The limitation of small study size was overcome by crossing over the population to different treatment assignments, thereby improving the power of the study. However, we were underpowered to conduct sub-group analyses. This study relied on self-report by the participants of their dietary intake and compliance, which can introduce measurement and recall biases. Another limitation of the study was that participants were not monitored on a daily basis and were not administered a supervised diet. However, this can also be viewed as a strength of the study, because it provides a more realistic scenario and potentially increases external validity.

5. Conclusions

The addition of cocoa-containing products in the habitual diet of patients with stage 1 hypertension taking ACE inhibitors, diuretics or beta blockers lowered their blood pressure. A dose-response impact on blood pressure was evident when comparing the beneficial effects observed by adding higher dose of the cocoa-containing products in the habitual diet to the low dose in patients on ACE inhibitors with stage 1 hypertension. The addition of cocoa-containing products in the habitual diet of patients with stage 1 hypertension improved endothelial function in patients on beta blockers, reduced triglycerides and insulin resistance in patients on ACE inhibitors, and increased HDL cholesterol in patients on calcium channel blockers. In addition, the addition of cocoa-containing products in the habitual diet of patients with stage 1 hypertension did not adversely affect their weight or waist circumference. Further studies are warranted to explore the benefits of the

addition of cocoa-containing products in the habitual diet among different sub-groups of patients with stage 1 hypertension.

Disclosure statement

The authors have nothing to disclose. Authors' contribution

The authors' responsibilities are as follows:

VYN and JV served as the Principal Investigators and were responsible for oversight of all study related activities. VYN was also responsible for the protocol development, data analysis, interpretation, and wrote manuscript.

NH was responsible for study management at the Boston site, analyzing the ultrasound readings and critical review of manuscript.

MK analyzed the chemistry data and also contributed to manuscript preparation.

AA contributed to manuscript preparation.

All authors declare that they have no conflicts of interest.

Acknowledgement

The authors wish to acknowledge the technical assistance of Susan Acheychek and Rockiy G. Ayettey.

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