John D. Folts

Purple Grape Juice Improves Endothelial Function and Reduces the Susceptibility of LDL Cholesterol to Oxidation in Patients With Coronary Artery Disease

BACKGROUND In vitro, the flavonoid components of red wine and purple grape juice are powerful antioxidants that induce endothelium-dependent vasodilation of vascular rings derived from rat aortas and human coronary arteries. Although improved endothelial function and inhibition of LDL oxidation may be potential mechanisms by which red wine and flavonoids reduce cardiovascular risk, the in vivo effects of grape products on endothelial function and LDL oxidation have not been investigated. This study assessed the effects of ingesting purple grape juice on endothelial function and LDL susceptibility to oxidation in patients with coronary artery disease (CAD). METHODS AND RESULTS Fifteen adults with angiographically documented CAD ingested 7.7+/-1.2 mL. kg(-1). d(-1) of purple grape juice for 14 days. Flow-mediated vasodilation (FMD) was measured using high-resolution brachial artery ultrasonography. Susceptibility of LDL particles to oxidation was determined from the rate of conjugated diene formation after exposure to copper chloride. At baseline, FMD was impaired (2.2+/-2. 9%). After ingestion of grape juice, FMD increased to 6.4+/-4.7% (P=0.003). In a linear regression model that included age, artery diameter, lipid values, and use of lipid-lowering and antioxidant therapies, the effect of grape juice on FMD remained significant (mean change 4.2+/-4.4%, P<0.001). After ingestion of grape juice, lag time increased by 34.5% (P=0.015). CONCLUSIONS Short-term ingestion of purple grape juice improves FMD and reduces LDL susceptibility to oxidation in CAD patients. Improved endothelium-dependent vasodilation and prevention of LDL oxidation are potential mechanisms by which flavonoids in purple grape products may prevent cardiovascular events, independent of alcohol content.

Select Flavonoids and Whole Juice From Purple Grapes Inhibit Platelet Function and Enhance Nitric Oxide Release

Background—Moderate red wine consumption is inversely associated with coronary ischemia, and both red wine and purple grape juice (PGJ) contain flavonoids with antioxidant and antiplatelet properties believed to be protective against cardiovascular events. Acute cardiac events are also associated with decreased platelet-derived nitric oxide (NO) release. In this study, the effects of PGJ and PGJ-derived flavonoids on platelet function and platelet NO production were determined. Methods and Results—Incubation of platelets with dilute PGJ led to inhibition of aggregation, enhanced release of platelet-derived NO, and decreased superoxide production. To confirm the in vivo relevance of these findings, 20 healthy subjects consumed 7 mL · kg−1 · d−1 of PGJ for 14 days. Platelet aggregation was inhibited after PGJ supplementation, platelet-derived NO production increased from 3.5±1.2 to 6.0±1.5 pmol/108 platelets, and superoxide release decreased from 29.5±5.0 to 19.2±3.1 arbitrary units (P <0.007 and P <0.05, respectively). &agr;-Tocopherol levels increased significantly after PGJ consumption (from 15.6±0.7 to 17.6±0.9 &mgr;mol/L;P <0.009), and the plasma protein–independent antioxidant activity increased by 50.0% (P <0.05). Last, incubation of platelets with select flavonoid fractions isolated from PGJ consistently attenuated superoxide levels but had variable effects on whole-blood aggregation, platelet aggregation, and NO release. Conclusions—Both in vitro incubation and oral supplementation with PGJ decrease platelet aggregation, increase platelet-derived NO release, and decrease superoxide production. These findings may be a result of antioxidant-sparing and/or direct effects of select flavonoids found in PGJ. The suppression of platelet-mediated thrombosis represents a potential mechanism for the beneficial effects of purple grape products, independent of alcohol consumption, in cardiovascular disease.

Administration of Wine and Grape Juice Inhibits In Vivo Platelet Activity and Thrombosis in Stenosed Canine Coronary Arteries

BACKGROUND Moderate daily consumption of alcoholic beverages is a negative risk factor for the development of atherosclerosis and coronary artery disease (CAD), especially in France and other Mediterranean areas where red wine is regularly consumed with meals. Platelets contribute to the development of atherosclerosis, CAD, and acute arterial thrombus formation. METHODS AND RESULTS Anesthetized dogs were prepared with the Folts model of mechanically stenosed coronary arteries and intimal damage. Periodic acute platelet-mediated thrombus formation occurred, causing cyclic flow reductions (CFRs) in coronary blood flow. The CFRs were eliminated by the administration of 1.62 +/- 1.12 mL/kg red wine intravenously (IV) and 4.0 mL/kg intragastrically (IG). The CFRs were abolished by 2.04 +/- 1.42 mL/kg of grape juice IV and 10 mL/kg IG. White wine did not have significant results in eliminating the CFRs, either IV (2.0 mL/kg) or IG (4.0 mL/kg), decreasing the slopes of the CFRs only slightly. CONCLUSIONS Pure ethanol has been shown to inhibit platelet aggregation in vitro, ex vivo, and in vivo, although a blood alcohol content (BAC) of > or = 0.2 g/dL is usually required. The BAC of dogs administered the red wine-saline solution intravenously was 0.028 g/dL, much less than is usually necessary for platelet inhibition with pure ethanol. Because red wine and grape juice, but not white wine, abolished the CFRs, this suggests there are compounds present in red wine and grape juice that are not present or are present in a lower concentration in white wine. Wine and grape juice contain a wide variety of naturally occurring compounds, including fungicides, tannins, anthocyanins, and phenolic flavonoids (including flavonols and flavones). These compounds have shown platelet inhibition in vitro by a variety of proposed mechanisms. Perhaps the biological activity of these compounds can explain the platelet-inhibitory properties of red wine and grape juice that are observed without high levels of ethanol.

Blood flow reductions in stenosed canine coronary arteries: vasospasm or platelet aggregation?

In 67 dogs with a 60-80% coronary stenosis produced by an external constricting plastic ring, blood flow measured with an electromagnetic flowmeter showed cyclical flow reductions of varying magnitude and duration, and then an abrupt return to control flow. In 45 dogs, heparin did not prevent these flow reductions, but ibuprofen (Motrin) or indomethacin abolished them. With incremental doses of each of these drugs, the cyclical flow reductions and the platelet function in vitro were diminished proportionately. In 10 more dogs, during low flow, pinching or poking the narrowed vessel suddenly restored normal flow. Topical application of papaverine and nitroglycerin proximal to the stenosis did not abolish the cyclic flow reduction, although a transient fall in systemic pressure indicated that they had been absorbed. Seven dogs had the constricting cylinder and flow probe chronically implanted for 4-6 weeks. A single oral dose of aspirin, 20 mg/kg, abolished their cyclic flow reductions for 2-4 days. In five dogs with 70% stenosis in the circumflex coronary artery, coronary arteriography was performed before coronary flow reduction and when coronary blood flow was low. This showed that there was a considerable additional reduction in the size of the mechanically constricted lumen during spontaneous flow reduction. In one dog, a nonopacified mass was dislodged from the area of constriction in 67 msec and this restored the lumen to its control diameter. Similar rapid clearing was filmed in two more dogs. In no case was vasospasm observed. These results suggest that obstruction from platelets aggregated in the narrowed lumen caused the cyclic flow reductions.

Changes in tissue levels of carnitine and other metabolites during myocardial ischemia and anoxia

Abstract The induction of ischemia in the open chest dog, or anoxia in the perfused rat heart, causes dramatic changes in the tissue levels of free acyl carnitine and related metabolites. During the early phase of ischemia or anoxia the tissue levels of free carnitine decline, while acetyl carnitine rapidly increases. These changes are accompanied by elevation in long-chain acyl carnitine, long-chain acyl CoA, and lactate and by decreases in acetyl CoA, CoA, ATP, and creatine phosphate. As the degree of ischemia becomes more severe, carnitine appears to be lost from the myocardium. A scheme is presented which relates carnitine-linked mitochondrial metabolism to the activity of carnitine acyl transferase, ANT, carnitine/acyl carnitine translocase, creatine phosphokinase, and pyruvate dehydrogenase. It is suggested that the conversion of carnitine to acyl carnitine during the onset of ischemia may play an important role, by virtue of its effect on these enzymes, in the regulation of metabolism during the early or reversible phase of ischemia.

Effect of ingestion of purple grape juice on endothelial function in patients with coronary heart disease

T “French Paradox” refers to the observation that the coronary heart disease mortality rate is lower in France than in other industrialized countries with similar prevalences of coronary risk factors.1,2 This paradox has been attributed to frequent consumption of alcohol-containing beverages, which increase highdensity lipoprotein (HDL) cholesterol levels and inhibit platelet function.1–7 Several epidemiologic studies suggest that ingestion of red wine, which contains several hundred different types of flavonoids, is more cardioprotective than beer or spirits.1–4 Indeed, the flavonoids found in red wine and purple grape juice (GJ) also inhibit platelet aggregation, and in 1 study, were shown to be powerful antioxidants that improved endothelial function.8–10 In that study, however, most subjects were taking vitamin E, so it is unclear if the observed results were due solely to the flavonoids in GJ or a combination of vitamin E and GJ.10 Furthermore, a high dose of GJ was administered (approximately 8 ml/kg/day) for only 2 weeks. The purpose of this study was to assess the endothelial function and antioxidant effects of 2 doses of purple GJ alone and in combination with vitamin E for 8 weeks. • • • The institutional review board of the University of Wisconsin Medical School approved this study. All subjects provided informed consent before participation. Twenty-two adults with angiographically documented coronary artery disease were recruited for this study. Subjects were not allowed to take vitamin supplements for 4 weeks before enrollment or during this study, except as prescribed by the research protocol. Subjects also were prohibited from consuming fruit products, tea, or alcoholic beverages during this study. Medications could not be changed during the study. All subjects ingested purple GJ (Welch’s 100% Concord Grape, Concord, Massachusetts) for 56 days. The first 11 subjects were instructed to drink 8.0 ml/kg of GJ, twice daily. For an average 80-kg person, this was approximately 640 ml/day (21 ounces) of GJ, which contained approximately 112 g of carbohydrate. The next 11 subjects were instructed to drink 4.0 ml/kg of purple GJ, once daily (low-dose group). After 28 days, subjects added vitamin E (d,l-a-tocopheryl) 400 IU to their daily intake of GJ. Subjects’ diaries indicated .90% compliance with GJ and vitamin E. Endothelial function was evaluated by measuring flow-mediated vasodilation (FMD) of the brachial artery using B-mode ultrasound. Studies were performed at baseline and at subsequent visits on the morning of phlebotomy, after a 12-hour fast. Subjects were instructed to drink their daily dose of GJ at least 2 hours before testing. Brachial artery diameters and blood flows were measured with a 7.5-MHz linear array vascular ultrasound transducer and an Agilent Technologies 5500 Sonos ultrasound system (Palo Alto, California). Increased forearm blood flow was induced by inflating a blood pressure tourniquet around the widest part of the forearm to a systolic blood pressure of 250 mm Hg for 4.5 minutes. Repeat brachial artery diameter and blood flow scans were obtained immediately and 1 minute after deflation of the tourniquet. Resting brachial artery diameter and blood flow scans were repeated 15 minutes later. Sublingual nitroglycerin (400 mg) was administered and final scans were performed after 3 minutes. The brachial artery was imaged in longitudinal sections 2 to 15 cm above the elbow. Images were recorded using the digital storage and retrieval software of the ultrasound system. Vessel diameters were measured in triplicate using digital calipers (Freeland Systems, Westfield, Indiana). Measurements were performed and interpreted by investigators who were blinded to subject information and study date. The brachial artery diameter was measured at end-diastole, using intima-media interfaces, or if they could not be visualized, media-adventitia interfaces, as landmarks. FMD was calculated as the ratio of the brachial artery diameter after reactive hyperemia to the baseline diameter, expressed as a percent change. Nitroglycerinmediated vasodilation was calculated in an analogous fashion. In this laboratory, intraobserver reliability for measurement of the brachial artery diameter is 0.987, reflecting an interclass correlation coefficient across all readings and conditions.10 Lipid and glucose levels were measured using enzymatic techniques on a Hitachi 747 analyzer (Tokyo, Japan) using standard reagents (Roche, Mannheim, Germany). Insulin levels were measured by radioimmunoassay. Low-density lipoprotein (LDL) particles were isolated from serum by sequential density ultracentrifugation between densities of 1.006 and 1.063 g/ml using a Beckman Optima ultracentrifuge (Fullerton, California) at 100,000 rpm (.400,000 g). The LDL-containing fraction was desalted with a 2-ml column of preswollen 12% cellulose and 0.1 mol/L From the University of Wisconsin Medical School, Madison, Wisconsin. This study was supported by an unrestricted grant from Welch’s Foods, Inc., Concord, Massachusetts. Dr. Stein’s address is: Section of Cardiovascular Medicine, University of Wisconsin Medical School, 600 Highland Avenue, H6/315 CSC (MC 3248), Madison, Wisconsin 53792. E-mail: jhs@medicine.wisc.edu. Manuscript received February 21, 2001; revised manuscript received and accepted April 10, 2001.

Intravenous nitroglycerin infusion inhibits cyclic blood flow responses caused by periodic platelet thrombus formation in stenosed canine coronary arteries

BackgroundNitroglycerin and other clinically relevant organic nitrate derivatives have been shown to inhibit platelet aggregation in vitro. This antithrombotic effect of nitrates is potentiated by reduced thiol. To determine the potential relevance of this mechanism of action in vivo, we examined the effect of intravenous nitroglycerin infusion on periodic platelet thrombus formation in a canine model of coronary artery stenosis. Methods and ResultsWe used a canine model of coronary artery stenosis associated with cyclic reductions in coronary blood flow that have been shown to depend on periodic platelet thrombus formation. We quantified the frequency of cycles per 40-minute observation period and monitored the effect of a continuous infusion of intravenous nitroglycerin on the cycle frequency. The administration of 10-15 μg/kg/min nitroglycerin reduced cyclic platelet thrombus formation significantly and did so without a significant change in coronary artery blood flow. Pretreatment with the reduced thiol, N-acetylcysteine (100 mg/kg during 30 minutes), led to inhibition of cyclic platelet thrombus formation by intravenous nitroglycerin at doses that alone had no such effect (5 μg/kg/min). ConclusionThese data suggest that one mechanism by which intravenous nitroglycerin improves ischemia in acute coronary artery disease syndromes may be by inhibition of platelet thrombus formation and may highlight the potential importance of reduced thiol in this mechanism. (Circulation 1991;83:2122—2127)

Protection of the ischemic dog myocardium with carnitine

Abstract In 25 open chest anestheslzed dogs, left anterior descending coronary arterial blood flow was measured with an electromagnetic flowmeter while aortic blood pressure and epicardlal electrocardiograms were recorded. Ischemia was produced in the left anterior descending arterial bed by decreasing mean flow to one third of control levels for a 5 minute period with a micrometer snare device. This produced an increase in S-T segment deviation greater than 4 mv in the ischemic bed. Control and ischemic left anterior descending arterial bed tissue samples were obtained by drill biopsy and were analyzed for adenosine triphosphate (ATP) and creatine phosphate levels and adenlne nucleotide translocase activity. The ATP levels decreased from 5.6 ± 1.2 to 3.6 ± 1.4 μmoles/g, and creatine phosphate decreased from 15.3 ± 4.6 to 5.8 ± 3.8 μmoles/liter. The adenine nucleotide translocase activity decreased from an average control value of 42,957 ± 9,480 to 29,100 ± 6,609 disintegrations per minute (dpm)/mg during the 5 minute period of ischemia. With the ischemia maintained, 100 mg/cc of L-carnitine was infused into the ischemic left anterior descending arterial bed at a rate of 1 cc/min for 5 minutes (17 dogs), and 80 mg/kg of D-L carnltine was given intravenously in 8 dogs. The epicardial S-T segment deviation decreased to approximately 2 mv after the carnltine infusion, with ischemia maintained. A third biopsy sample of the ischemic bed showed that the ATP level had increased to 5.2 ± 1.1 and the creatine phosphate to 10.8 ± 4.8 moles/g; the adenine nucleotide translocase activity had increased to 37,800 ± 7,210 dpm/mg. In 9 dogs ventricular fibrillation developed at this level of ischemia before infusion of carnitine, whereas only one dog had fibrillation at comparable levels of ischemia after infusion. These results support the hypothesis that infusion of carnitine may benefit the ischemic myocardium by maintaining tissue levels of free carnitine, reversing inhibition of adenine nucleotide translocase by long chain acyl coenzyme A esters and in this manner restoring mitochondrial function.

Grape seed and skin extracts inhibit platelet function and release of reactive oxygen intermediates.

Red wine and purple grape juice contain polymeric flavonoids with antioxidant properties believed to be protective against cardiovascular events but the alcohol and sugar content of these beverages has curtailed their medicinal use. Acute cardiac events are also associated with enhanced inflammation and thrombosis. In this study, the extracts from grape skins or seeds were examined for their anti-inflammatory properties and effect on platelet release of reactive oxygen intermediates. Incubation of platelets with seed or skin extract led to a decrease in platelet aggregation from 68.8 ± 19.8% to 45 ± 3.6% for seeds and to 27 ± 7.2% for skin, respectively (P < 0.05). Platelet incubation with grape skin or seed extracts led to a marked decrease in superoxide release from 73 ± 6.2 to 2 ± 3.4 for grape seeds and to 0.33 ± 0.57 for grape skin (chemilum. units; P < 0.05) as well as a significant increase in radical-scavenging activity, decrease in reactive oxygen species release by confocal microscopy, and enhanced platelet NO was measured using an NO-sensitive microelectrode. These effects were dose dependent for both grape extracts. Coincubation with seeds and skins led to additive inhibition of platelet aggregation, enhanced NO release, and prevented superoxide production. Incubation with seed or skin extracts led to an immediate attenuation of release of the inflammatory mediator, soluble CD40 ligand. Thus, the extracts from purple grape skins and seeds inhibit platelet function and platelet-dependent inflammatory responses at pharmacologically relevant concentrations. These findings suggest potentially beneficial platelet-dependent antithrombotic and anti-inflammatory properties of purple grape-derived flavonoids.

Potential health benefits from the flavonoids in grape products on vascular disease.

In the dog, monkey, a nd human we have shown that 5 ml/kg of red wine or 5-10 ml/kg of purple grape juice but not orange or grapefruit juice inhibits platelet activity, and protects against epinephrine activation of platelets. Red wine and purple grape juice enhances platelet and endothelial production of nitric oxide (Fitzpatrick et al., 1993, Parker et al., 2000). This is thought to be one of the mechanisms whereby purple grape juice significantly improved endothelial function in 15 patients with coronary artery disease. The consumption of purple grape juice by the patients also offered increased protection against LDL cholesterol oxidation, even though all the patients were also taking another antioxidant vitamin E, 400 IU/day. The number of people and animals in these studies was small; however, each one acted as their own control as measurements were made in each before, and then after consumption of red wine or purple grape juice. Thus these studies are thought to be significant. We feel that the results of these studies are encouraging and justify further research on larger numbers of subjects. This suggests that the flavonoids in purple grape juice and red wine may inhibit the initiation of atherosclerosis by one or more of the mechanisms described above. It will take years to fully characterize the potential benefits of daily consumption of red wine or purple grape juice for maintaining a healthy heart. Based on the existing evidence of antiplatelet and antioxidant benefits and improved endothelial function from red wine and purple grape juice, it seems reasonable to suggest that moderate amounts of red wine or purple grape juice be included among the 5-7 daily servings of fruits and vegetables per day as recommended by the American Heart Association to help reduce the risk of developing cardiovascular disease.