Effects of Freeze-Dried Grape Powder on High-Density Lipoprotein Function in Adults with Metabolic Syndrome: A Randomized Controlled Pilot Study

Abstract

Background:

High-density lipoprotein (HDL) particles are protective against atherosclerosis. However, HDL function is impaired in metabolic syndrome (MetS) due to low-grade inflammation and dyslipidemia. Foods containing polyphenols, such as grapes, may prevent HDL dysfunction via antioxidant or anti-inflammatory effects. We evaluated the effects of grape powder ingestion on measures of HDL function in adults with MetS.

Methods:

Twenty adults (age: 32–70 years; body mass index: 25.3–45.4 kg/m²) consumed either 60 grams/day of freeze-dried grape powder (GRAPE) or a placebo for 4 weeks, separated by a 3-week washout period, in a randomized, double-blind crossover study. The primary outcome was serum paraoxonase-1 (PON1) arylesterase activity, a measure of HDL antioxidant function. Secondary outcomes included PON1 lactonase activity, plasma lipids, metabolic markers, cholesterol efflux capacity, and other HDL functional markers.

Results:

After 4 weeks, GRAPE did not alter the serum PON1 activity or other markers of HDL function compared with placebo. Measures of HDL function were positively correlated with each other and inversely with measures of insulin resistance and inflammation. GRAPE intake led to a significant reduction in fasting plasma triglycerides compared with placebo (P = 0.032). No other significant effects of GRAPE were observed for other plasma lipids, anthropometrics, or metabolic measures.

Conclusions:

Grape powder consumption did not impact HDL function in this cohort of adults with MetS. However, it was shown to improve fasting triglycerides, a risk factor for cardiovascular disease.

Introduction

High-density lipoprotein (HDL) particles have been demonstrated to be protective against atherosclerosis and cardiovascular disease (CVD).^1
–3 The protective functions of HDL are due to its role in reverse cholesterol transport (RCT) but also may be related to antioxidant and anti-inflammatory activities.^1,3,4 The functionality of HDL is often impaired under chronic inflammation, which is observed in metabolic syndrome (MetS), resulting in reductions in HDL cholesterol efflux capacity and antioxidant activity.^5

–8 Thus, HDL dysfunction may be a common feature of MetS, in addition to the established CVD risk factors of dyslipidemia, abdominal obesity, insulin resistance, and hypertension.

The biological activity of HDL is largely due to its transport of over 50 distinct proteins.⁹ Inflammatory conditions can remodel HDL to a proinflammatory particle with impaired antioxidant activity, characterized by an enrichment in serum amyloid A (SAA), an acute phase protein, and a depletion of paraoxonase-1 (PON1), an HDL-associated antioxidant enzyme.¹⁰ HDL-SAA enrichment has been shown to impair macrophage cholesterol efflux to HDL¹¹ and results in a more proinflammatory HDL particle,¹² whereas PON1 has been shown to have the opposite effect on HDL function.^13,14 These mediators of HDL function provide potential therapeutic targets beyond HDL-cholesterol (HDL-C) concentrations.¹⁵ With the failure of HDL-C-targeted drugs to reduce risk for CVD events,¹⁶ identifying compounds that impact HDL functionality is of particular interest.

Consumption of polyphenols has been shown to increase the serum PON1 activity in animal and human studies.^4,17,18 Grapes contain a variety of polyphenols, including anthocyanins. In human studies, mixed anthocyanin supplementation was shown to increase the serum PON1 activity and HDL cholesterol efflux capacity in dyslipidemic patients.^19,20 Furthermore, adults with low-grade inflammation have demonstrated improvements in plasma lipids and inflammatory markers with moderate dosages of anthocyanins (<100 mg/day).^21,22

In this study, we evaluated the effects of consuming freeze-dried grape powder (GRAPE), which is equivalent to 2.5 cups of fresh grapes daily, on HDL function in patients with MetS. Serum PON1-arylesterase activity was used as a primary endpoint to evaluate HDL antioxidant function. Secondary outcomes included plasma lipids, HDL subfractions and size, and other measures of HDL function. We hypothesized that GRAPE ingestion for 4 weeks would improve the PON1-arylesterase activity compared with a placebo. Additionally, grape powder intake was also expected to positively impact plasma lipids and other markers of HDL function.

Methods

Study design

Men and women, aged 30–70 years, were recruited for this double-blind, crossover placebo-controlled study. Participants were qualified for this study if they met the NCEP ATP-III guidelines for MetS. Those who had other chronic ailments or were taking fibrates, anti-inflammatory, or glucose-lowering medications were excluded from the study since these may impact HDL functional measures. Informed consent was obtained from individuals before screening.

Twenty subjects completed both arms of the study and were included in data analysis. For the duration of the study, subjects were asked to adhere to their habitual diet and exercise patterns, as well as refrain from consuming foods rich in polyphenols. Subjects first entered a 2-week run-in period during which no powder was administered. Subjects were then randomly assigned to either 60 grams/day of a standardized freeze-dried grape powder (contributing 195 mg total polyphenols) or a calorically and nutritionally matched placebo powder, devoid of any polyphenols. Study sponsor-provided nutrient and phytochemical compositions of the GRAPE powder have been described previously.²³

For 4 weeks, subjects were asked to supplement their diet with two 30 grams packets of each respective powder daily. The powder was recommended to be reconstituted in an 8 fl oz. glass of water. As a measure of compliance, subjects were asked to bring empty packets to endpoint visits. Once the first arm was completed, subjects entered a 3-week washout period in which no powder was consumed and then underwent another 4-week intervention period with the alternate powder.

At the beginning and end of each period, venous blood was collected after a 12-hr overnight fast into EDTA-coated or serum collection tubes for plasma/serum isolation. Samples were frozen at −80°C until analysis. This protocol was approved by the University of Connecticut Institutional Review Board (#H15–218). This study is registered at clinicaltrials.gov, trial number NCT02542176.

Study outcomes

The primary outcome of this study was the effect of GRAPE on the PON1-arylesterase activity. The secondary outcomes were the effect of GRAPE on other metabolic variables, such as blood pressure, plasma lipids, measures of HDL profile, and cholesterol efflux capacity.

Anthropometrics, blood pressure, and dietary analysis

Body weight, waist circumference, and blood pressure were measured at baseline and the end of each experimental period. Subjects were asked to fill out 5-day diet records at the end of each intervention period. Dietary records were analyzed using the Nutrition Data System for Research software version 2013, developed by the Nutrition Coordinating Center, University of Minnesota, Minneapolis, MN.

Biochemical analyses

Total plasma cholesterol, HDL-C, triglycerides, and glucose were measured using enzymatic assays (Wako Diagnostics, Richmond, VA). Plasma alanine aminotransferase (ALT) was measured via spectrophotometric methods (Pointe Scientific, Inc., Canton, MI). Serum apoA-I (R&D Systems, Inc., Minneapolis, MN) and HDL-associated SAA (Invitrogen, Carlsbad, CA) were measured using commercially available sandwich ELISAs per manufacturer's instructions. SAA was measured in apolipoprotein B (apoB)-depleted serum, after using dextran sulfate Mg²⁺ precipitation methods (Stanbio Laboratory, Boerne, TX). Assays were measured using a UV-Vis microplate spectrophotometer (BioTek Instruments, Winooski, VT).

Plasma TNF-α and insulin were measured by Luminex magnetic bead-based assay using MAGPIX instrumentation (EMD Millipore, Billerica, MA). Insulin resistance was calculated using the homeostasis model assessment of insulin resistance (HOMA-IR) equation based on fasting plasma insulin and glucose measurements.²⁴

Lipoprotein particle size

NMR spectroscopy was conducted on plasma samples by LabCorp (Morrisville, NC). This method was used to determine lipoprotein particle diameter and concentrations, as previously described.²⁵

Paraoxonase-1 activity

PON1-arylesterase activity toward phenyl acetate (Sigma-Aldrich, St. Louis, MO) was measured in serum, as previously described.²⁶ PON1-lactonase activity toward delta-valerolactone (Sigma-Aldrich) was also determined using a modification of the method described by Khersonsky and Tawfik.²⁷

Cholesterol efflux

J774A.1 macrophages (ATCC, Manassas, VA) were maintained in RPMI media (Sigma-Aldrich) containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 100 U/mL penicillin, 100 μg/mL streptomycin (ThermoFisher Scientific, Waltham, MA), and 100 μg/mL Normocin (Invitrogen) at 37°C with 5% CO₂.

Cells were treated with antibiotic-free RPMI containing 1% FBS, [1,2-³H(N)]-cholesterol (2 μCi/mL; Perkin Elmer, Waltham, MA), and 2 μg/mL of the acyl-CoA:cholesterol acyltransferase inhibitor, CI 976 (Sigma-Aldrich), for 24 hrs to load cells with radiolabeled cholesterol. Cells were incubated for 16 hrs with antibiotic-free RPMI containing 0.2% (w:v) bovine serum albumin (BSA) (Sigma-Aldrich), 2 μg/mL CI 976, and 0.3 mM cpt-cAMP (Sigma-Aldrich), referred to as cell treatment media. ApoB-depleted serum was prepared by polyethylene glycol precipitation (Pointe Scientific, Inc.). Fresh cell treatment media with 2.8% (v:v) apoB-depleted serum from each participant was added to the cells for 4 hrs.

Cholesterol efflux capacity of HDL was then calculated as the percentage of radioactivity found in the media relative to the total radioactivity (media and cell lysate), after subtracting radioactivity released into RPMI/BSA media alone.

Statistical analysis

Sample size was estimated using GraphPad StatMate2 software to enable the detection of at least a 10% difference in the serum PON1-arylesterase activity at 80% power with a two-tailed level of significance at P = 0.05. Estimated data variability was based on PON1 activity measures in a previous cohort of adults with MetS.²⁵ Plasma lipids and anthropometric values were analyzed using two-way repeated-measures ANOVA, with significant values at P < 0.05. Dietary intake and HDL function-related values were analyzed by using a paired t test at the end of each intervention arm. Bivariate Pearson correlations were used to assess the relationships between HDL function-related variables. SPSS version 25 for Windows (IBM, Armonk, NY) was used for statistical analysis, and data are reported as mean ± SD.

Results

Participant characteristics

Twenty patients (12 men and 8 women) with MetS completed both arms of the intervention and were included in the final data analysis. Subject characteristics at baseline are shown in Table 1. Subjects had elevated blood lipids, blood glucose, and blood pressure, as well as a mean body mass index of 33.0 kg/m². Compliance to the daily consumption of powder was >95% for both GRAPE and placebo (data not shown).

Table 1.

Baseline Characteristics of Study Participants

Variable	All subjects (n = 20)
Age (years)	53.5 ± 10.1
Male, n (%)	12 (60)
BMI (kg/m²)	33.0 ± 4.77
Weight (kg)	93.9 ± 17.4
Waist circumference (cm)	109 ± 13.1
Total cholesterol (mg/dL)	193 ± 46.9
HDL-C (mg/dL)	43.9 ± 15.3
Non-HDL-C (mg/dL)	150 ± 48.4
Total cholesterol/HDL-C	4.9 ± 2.0
Triglycerides (mg/dL)	161 ± 68.2
Glucose (mg/dL)	99.4 ± 11.3
ALT (U/L)	32.2 ± 12.2
Systolic blood pressure (mmHg)	131 ± 11.3
Diastolic blood pressure (mmHg)	86.7 ± 7.51
Antihypertensive medication, n (%)	6 (30)
Statins, n (%)	2 (10)

Values are mean ± SD for all subjects (n = 20).

ALT, alanine aminotransferase; HDL, high-density lipoprotein.

Anthropometrics and plasma lipids

There were no significant differences in dietary intake, anthropometric measures, ALT, or glucose homeostasis measures (glucose, insulin, and HOMA-IR) between intervention arms for variables assessed (data not shown). GRAPE consumption did not significantly alter plasma total cholesterol, HDL-C, total cholesterol/HDL-C ratio, or non-HDL-C compared with placebo (Table 2). However, GRAPE consumption lowered fasting plasma triglycerides (−21%) over the course of the study, which was significant compared with placebo (P = 0.032).

Table 2.

Plasma Lipid Panel Before and After 4 Weeks of Grape or Placebo Intake

			P-value (RM-ANOVA)
Variable	Grape	Placebo	Time	Treatment	Interaction
Total cholesterol, mg/dL
Baseline	199 ± 46.1	198 ± 38.9	0.42	0.25	0.17
4 weeks	191 ± 46.9	201 ± 46.7	0.42	0.25	0.17
HDL-C, mg/dL
Baseline	45.5 ± 20.9	44.5 ± 14.9	0.97	0.65	0.57
4 weeks	45.1 ± 18.2	45.0 ± 17.0	0.97	0.65	0.57
Total cholesterol/HDL-C
Baseline	5.0 ± 2.2	4.9 ± 1.8	0.22	0.58	0.11
4 weeks	4.7 ± 1.7	5.0 ± 2.1	0.22	0.58	0.11
Non-HDL-C, mg/dL
Baseline	153 ± 49.1	153 ± 41.2	0.44	0.14	0.21
4 weeks	146 ± 46.8	156 ± 49.6	0.44	0.14	0.21
Triglycerides, mg/dL
Baseline	171 ± 75.4	135 ± 69.6	0.15	0.28	0.032
4 weeks	133 ± 59.0	146 ± 72.6	0.15	0.28	0.032

Values are mean ± SD for all subjects (n = 20). P-values are from two-way repeated-measures ANOVA (RM-ANOVA).

HDL particle profiles and markers of HDL function

Overall, GRAPE consumption did not significantly alter our primary outcome of the PON1-arylesterase activity (Table 3). Additionally, it did not affect HDL particle concentrations, HDL size, apoA-I, PON1-lactonase activity, or HDL cholesterol efflux capacity compared with placebo (Tables 3 and 4). However, GRAPE consumption resulted in a nonsignificant trend for higher PON1-lactonase activity/HDL-SAA ratio (P = 0.09), which was used as a marker of HDL anti-inflammatory potential. As expected, HDL functional measures were positively correlated with each other and inversely with measures of insulin resistance and inflammation across both intervention arms (Table 5).

Table 3.

Serum Markers of High-Density Lipoprotein Functionality in Study Participants After 4 Weeks of Grape or Placebo Intake

Variable	Grape	Placebo	Grape–placebo	P
PON1-arylesterase, kU/L	84.5 ± 17.4	86.3 ± 16.2	−1.8 ± 7.5	0.29
PON1-lactonase, kU/L	15.8 ± 3.2	15.6 ± 2.5	0.2 ± 1.8	0.60
ApoA-I, mg/dL	144 ± 60.8	144 ± 37.4	0 ± 61.7	0.97
HDL-SAA, μg/mL	4.0 ± 3.4	4.2 ± 3.4	−0.2 ± 2.0	0.74
HDL cholesterol efflux, %	15.1 ± 5.0	14.4 ± 5.5	0.7 ± 4.2	0.47
ApoA-I/SAA ratio	670 ± 565	526 ± 345	144 ± 387	0.13
PON1-arylesterase/SAA ratio, U/μg	38.7 ± 29.4	34.7 ± 25.1	4.0 ± 10.0	0.10
PON1-lactonase/SAA ratio, U/μg	7.1 ± 5.2	6.3 ± 4.6	0.8 ± 1.8	0.09

Values are mean ± SD for all subjects (n = 20). P-values from paired-samples t test.

Table 4.

Plasma High-Density Lipoprotein Particle Concentrations in Study Participants After 4 Weeks of Grape or Placebo Intake

Variable	Grape	Placebo	Grape–placebo	P
Total HDL-P, μM	31.9 ± 5.1	31.9 ± 4.8	0.0 ± 3.0	0.93
Large HDL-P, μM	6.7 ± 4.1	6.4 ± 3.4	0.3 ± 1.6	0.36
Medium HDL-P, μM	11.1 ± 7.6	11.1 ± 5.9	0.0 ± 4.2	0.95
Small HDL-P, μM	14.1 ± 7.6	14.3 ± 6.3	−0.2 ± 4.3	0.83
HDL diameter, nm	9.3 ± 0.6	9.3 ± 0.5	0.0 ± 0.3	0.73

Values are mean ± SD for all subjects (n = 20). P-values from paired-samples t test. Concentrations of total HDL-P (7.3–13 nm diameter), small HDL-P (7.3–8.2 nm diameter), medium HDL-P (8.2–8.8 nm diameter), and large HDL-P (8.8–13 nm diameter) are shown.

HDL-P, HDL particles.

Table 5.

Pearson Correlations of High-Density Lipoprotein-Related Markers in Study Participants After 4 Weeks of Grape or Placebo Intake

	HDL-C	ApoA-I	PON1-ARE	PON1-Lac	Large HDL-P	Medium HDL-P	Small HDL-P	HOMA-IR	TNF-α	HDL-SAA	PON1-Lac/HDL-SAA
Cholesterol efflux	0.19	0.18	0.18	0.24	−0.06	0.35 ^*	−0.21	−0.34 ^*	−0.16	−0.39 ^*	0.41 ^**
HDL-C	—	0.46 ^**	0.53 ^***	0.59 ^***	0.92 ^***	0.41 ^**	−0.57 ^**	−0.40 ^*	−0.43 ^**	0.15	−0.16
ApoA-I		—	0.34 ^*	0.27	0.47 ^**	0.01	−0.13	−0.13	−0.16	0.21	−0.15
PON1-ARE			—	0.88 ^***	0.59 ^***	0.15	−0.29	−0.40 ^*	−0.44 ^**	0.08	0.06
PON1-Lac				—	0.59 ^***	0.25	−0.39 ^*	−0.38 ^*	−0.42 ^**	0.09	0.05
Large HDL-P					—	0.28	−0.41 ^**	−0.31	0.53 ^***	0.19	−0.16
Medium HDL-P						—	0.81 ^***	−0.21	0.00	−0.14	0.06
Small HDL-P							—	0.35 ^*	0.06	−0.11	0.18
HOMA-IR								—	0.37 ^*	0.27	−0.15
TNF-α									—	0.16	−0.02
HDL-SAA										—	−0.68 ^***

Values are Pearson correlation coefficients. Bold values are significant, ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001.

ApoA-I, serum apoA-I concentration; cholesterol efflux, HDL cholesterol efflux capacity; HDL-C, plasma HDL-cholesterol; HDL-SAA, HDL serum amyloid A concentration; HDL-P, HDL particle concentration; HOMA-IR, homeostasis model assessment of insulin resistance; PON1-ARE, PON1-arylesterase activity; PON1-Lac, PON1-lactonase activity; TNF-α, plasma tumor necrosis factor-alpha concentration.

Discussion

Overall, GRAPE consumption did not significantly affect our primary outcome of the PON1-arylesterase activity, a measure of HDL antioxidant activity, or modify other measures of HDL function in this cohort of adults with MetS. However, GRAPE consumption improved fasting triglyceride concentrations compared with placebo. Thus, grape powder intake at this dosage had no impact on the HDL functionality measures assessed but may reduce the modest hypertriglyceridemia seen in this condition.

Numerous animal studies have shown that ingestion of grapes²⁸ and grape products^29,30 reduce risk of atherosclerosis development. Moreover, the consumption of freeze-dried grape powder over a period of 1 month has been shown to reduce blood pressure and improve endothelial function in men with MetS (46 grams/day)²³ and reduce oxidative stress in healthy women (36 grams/day).³¹ However, the overall effects of grape and grape products on traditional plasma lipids in human trials (including HDL-C) have been mixed (as reviewed in Ref³²).

Freeze-dried grapes showed no changes in plasma lipids in a study that included men with MetS at a dose of 46 grams/day.²³ Zunino et al.³³ observed significant reductions in the concentrations of large LDL particles and large LDL-cholesterol in obese adults with the consumption of 46 grams/day of freeze-dried grape powder for 3 weeks compared with placebo. The plasma triglyceride change observed with GRAPE, while modest, is an important finding due to elevated fasting triglycerides being a well-established biomarker for CVD risk.³⁴

The ability of HDL to accept cholesterol from macrophages, termed HDL cholesterol efflux capacity, has been shown in cohort studies to be a significant predictor of CVD, independent of HDL-C, apoA-I, and HDL particle concentrations.^35,36 Recently, it has been reported that serum cholesterol efflux capacity was progressively lower as the number of MetS criteria increased.⁸ We found that HDL cholesterol efflux capacity in this model system was inversely related to HOMA-IR and positively associated with medium HDL particle concentrations (Table 5). This finding is consistent with others who have found that most HDL-mediated macrophage cholesterol efflux is due to medium-sized HDL particles.³⁷

Although we did not observe significant effects of GRAPE with the current study, data from animal studies suggest that cyanidin glycosides, an anthocyanin commonly found in grapes, preserve HDL function under inflammatory conditions. Dietary cyanidin 3-glucoside administration to apoE^−/− mice for 4 weeks increased RCT and protected against atherosclerosis.³⁸ Cyanidin appears to promote HDL formation and function via liver X receptor activation and by regulating transcript stability of lipid transporters critical for cholesterol efflux.^38
–40

As previously mentioned, the inflammatory state of the HDL particle is critical to its functionality. SAA can impair HDL protective functions, such as cholesterol efflux from macrophages¹¹ and anti-inflammatory capacity.¹² Overall, the concentrations of HDL-SAA were unchanged with GRAPE in the current study. However, we found that HDL-SAA concentrations were inversely associated with HDL cholesterol efflux capacity, whereas PON1-lactonase/HDL-SAA ratio was positively associated (Table 5). Interestingly, GRAPE tended to improve both PON1 activity/HDL-SAA ratios; however, the effects did not reach significance.

A longer study duration or increased dosage of grape powder may have a more substantial effect on metrics of HDL function. PON1 is an HDL-associated enzyme that has been reported to protect against LDL oxidation and contributes to the anti-inflammatory and cholesterol-accepting properties of HDL.^13,14 Human PON1 activity is an important determinant of CVD risk.^41,42 Due to their reciprocal regulation and counteracting effects on HDL function,¹⁰ paired measurements of both PON1 and SAA have been suggested as useful disease markers.⁴³ This again alludes to the suggestion that metrics of HDL function, beyond HDL-C, may be helpful in assessing CVD risk.

Conclusions

We evaluated the effects of consuming freeze-dried grape powder on HDL functional measures in a group of adults with MetS. Although the primary outcome of this study, PON1-arylesterase activity, and other HDL measures were not altered, we found that GRAPE lowered fasting plasma triglycerides, a known risk factor for CVD. MetS is a heterogeneous condition, and certain individuals may be more sensitive to changes in HDL function with diet. Studies of longer duration and of larger size are needed to better understand the effects of polyphenols on HDL function in humans.

Footnotes

Acknowledgments

This work was supported by an award to C.N.B. from the California Table Grape Commission. This trial is registered at clinicaltrials.gov, trial number NCT02542176.

Author Disclosure Statement

C.N.B. and M.L.F. have received funding from the California Table Grape Commission. C.L.M., Q.D., C.G., G.H.N., B.S.L., and D.M.D. declare no competing financial interests exists.

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