Dr. John Folts is currently the Director of the Coronary Thrombosis Research Laboratory at The University of Wisconsin in Madison, Wisconsin.
The following material was transcribed from an audiocassette recording. A presentation at which ProvexCV was introduced during The American Dietetic Conference on October 22, 1998 by Dr. John Folts.
This material may be difficult for some people to follow and visualize if they do not have a medical background as Dr. Folts utilized numerous slides, graphs and overheads during his presentation. If you will take the time to read this transcript, Dr. Folts will help you understand the mechanisms that cause coronary heart disease and his current research that appears to play a key role in helping to prevent a stroke or a heart attack.
Dr. Folts’ current research is on in vitro platelet mediated coronary thrombosis and anti-oxidants. Dr. Folts developed the experimental model on Aspirin in 1974 and demonstrated by in vitro testing that aspirin can reduce arterial blood clots.
He is currently studying anti-platelet, anti-oxidant properties of poly phenol compounds found in red wine, beer, fruit juices and tea. Dr. Folts received his Bachelors of Science degree in electrical engineering, his masters in medical physiology and his Ph.D. degree in cardiovascular physiology and pathology from the University of Wisconsin. Today, in his lecture, he will describe the role of platelets in clot formation and his research on the ability of wine, fruit juice and tea to reduce or inhibit platelet mediated clot formation.
The American Dietetic Association presents Dr. John Folts, Ph.D.
October 22, 1998
Dr. Folts speech:
I want to talk about platelets, free radicals and LDL cholesterol and why we need something to protect against that in reducing coronary heart disease. Coronary artery disease is a major problem in the western world. We have made some progress, but we still have a lot left to do.
Epidemiological studies have shown an inverse relationship between dietary intake of flavonoids and the incidence of fatal and non-fatal myocardial infarction or heart attack. Flavonoids are micronutrients found in vegetables, fruits and their juices but not in any animal products. They have no nutritional value because they are not carbohydrates, fats or proteins. In vitro in the test tube and in tissue culture studies, many of them have shown to have both good anti-platelet and anti-oxidant properties.
Looking at a longitudinal section through a blood vessel when we are born, the endothelial cells lining the arteries are slippery smooth and they are very functional and they release substances called nitric oxide and other substances that chase blood cells away from sticking on the wall. Platelets are blood cells that get involved in clotting but we do not want them to stick on the walls of large arteries. The nitric oxide they release also relaxes the vascular smooth muscle cells and protects them from things we are going to see in a minute.
When these endothelial cells are damaged they are sloughed off and they are lost. This leaves a very clot-producing surface beneath them. The best way to damage the endothelial cells or have them sloughed off is by smoking cigarettes. Shown in the slide are several layers of platelets collecting on the vessel wall because endothelial cells are either damaged or gone and they release substances that tell vascular fluid muscles cells to multiply. It is really a smooth muscle cell tumour if you will. If they would stay down there in the media of the artery, which is part of the outer wall, they wouldn’t compromise the Lumina (opening) shown here. The platelets release substances also that say to these dividing fluid muscles cells to come up here where we are, so they migrate up and cause intimal thickening and this contributes to the narrowing of the artery.
So we would like to have something in our diet to restore endothelial function and also to inhibit the platelets, so they are less likely to contribute to this problem. Now here is another part of the vessel wall showing the endothelial cells and the vascular smooth muscle cells and here are the LDL cholesterol. We have to have cholesterol in our diets because the gonads use it to make sex hormones, the adrenal cortex uses it to make cortigo steroids, as well as, every new cell that is made in the body has to have cholesterol in its cell walls. The problem is when we have too much; there is no place to put it. The cholesterol diffuses down into the sub endothelial space, resides there for a while and then diffuses back out. Obviously, the more we have here the easier it is to diffuse in here, the harder it is to get it back out due to the concentration gradient. While cholesterol is in this space, oxidants and free radicals can be released by endothelial cells, vascular smooth muscle cells and many other cells. If the LDL cholesterol gets oxidized it is much more difficult for it to get out and it goes down the path of destruction. As it is oxidized, it moves down here in the sub endothelial space, thickening. It tells the endothelial cells, especially if they are sick, to release substances that cause white cells called monocytes to migrate in and then they get converted to macrophages. They then pick up cholesterol in an unregulated fashion and they become foam cells. These foam cells can be seen be seen in the arteries of children 10 –15 years old who have been killed in car accidents, when they autopsy them they find fatty streaks already developing in their coronary arteries, this is because of the presence of foam cells.
Now, here in this slide I have put everything together. Here we have the platelets simulating migration and proliferation of the smooth muscle cells down into here, we have the oxidized LDL’s migrating down into here (referring to the sub endothelial space), this process is really an anti-inflammatory reaction and the production of foam cells and finally, platelets of the cells initiates the clots that cause death. Initially there is very little fibrin in the clots; most of the clot is made of platelets.
So once again, we would like to have something in the diet that would improve the function of the endothelial cells, that would ‘turn down’ the activity of the platelets, that would protect the LDL cholesterol from oxidation, that would have some anti-inflammatory properties as well, and, that would inhibit a clot formation that cuts off the blood flow to the coronary arteries causing death.
The two main factors involved in coronary death are platelet activity and oxidized cholesterol; so far we have seen some mechanisms that can contribute to both the progressive narrowing of the arteries as well as the clot that kills.
Now, if we are going to look at something that will turn down the platelet activity, we obviously have to have some methods of measuring platelet activity. Many of my clinical colleagues think of platelets only in terms of their numbers, example, if you have 50,000 you are going to bleed, if you have 300,000 you should be alright, if you have 900,000 or 1,000,000 you should be a clotter. Platelet activity can be turned up or down in a matter of 5-10 minutes and one way to turn it up in 5 minutes is by smoking of a cigarette.
So now we are going back to an animal model for studying platelet mediated clot formation and damaged and narrowed arteries, and then we are going to look at a method for studying platelet activity in the blood of human volunteers like myself. Dogs and pigs can be fully anesthetised, the chest opened, the heart is exposed and here is the circumflex coronary artery, one of the main branches of the coronary arteries. We put this flow probe devise around the coronary artery that measures blood flow through it without interfering with the blood flow in any way. These devises are commonly placed on the exposed arteries of patients at the time of surgery. In order to mimic the atherosclerotic plague, we clamp the artery damaging the endothelial cells, damaging vascular smooth muscle cells and exposing clot-producing surfaces. Then, to produce some vascular narrowing, I make these plastic cylinders with a range of internal diameters and one is placed around the outside of the artery so that it is narrowed down, now we can mimic the problem the patient has with coronary disease. Here is the model again, here is the flow probe, and here is the area of stenosis and vessel damage. Coronary blood flow starts out okay, it’s normal here, but, as the platelet plug accumulates in here, the coronary flow gradually declines until it’s fully occluded. If this animal were awake it would have chest pains because this part of the heart is getting no blood flow. These cyclic flow reductions; as we call them, are caused by periodic repeated platelet mediated clots that form and then embolize distally. This is thought to be the mechanism of unstable angina in patients with coronary artery disease.
Back in the early seventies, I discovered that if we gave them aspirin; and this is equivalent to one aspirin in you or I, these clots gradually went away and blood flow was restored and maintained. We were excited about this and I presented this at several meetings and the people there thought I was ‘crazy-as-a-loon’ because back in the seventies, aspirin was just for headaches and things like that. Now I am sure there are people in the audience that are taking their aspirin, as are those people who thought I was crazy. In the late seventies however, we discovered that if we raised the adrenaline or epinephrine, that the same thing, 75% -80% of the time clot formation comes back with aspirin and that’s a serious problem. That’s why we have spent the last 20 years looking for drugs and food substances that would inhibit the platelets and prevent the clot formation, but also, prevent the renewal of clot formation from elevated epinephrine.
Now, in our previous findings with the animal model, we have shown that red wine, moderate amounts equivalent to 2 glasses in you or I, purple grape juice; not the clear or white grape juice, tea, but not coffee, in this animal model reduced or inhibited platelet mediated clot formation and protected against potentially fatal coronary thrombosis. These flavonoids in red wine, purple grape juice and tea inhibit platelets about the same as aspirin but they have one unique feature. THEY PROTECT AGAINST ELEVATIONS OF ADRENALINE, WHICH ASPIRIN DOES NOT. That makes the flavonoids more desirable.
Now obviously, we would like to do this in humans, but not too many humans want to volunteer to have their chests opened and their arteries exposed and studied, so we have another way to do this. Here we have a device where I can take a blood sample from one of you, place it in this test tube shown here, put two electrodes here that are part of an ohmmeter. Many of us have volt ohmmeters in our cars and workshops at home; these ohmmeters measure the electrical resistance between these two electrodes, which is normally quite low, because blood has a lot of electrolytes in it that allows conduction of electricity. The ohmmeter displayed on this flip strip chart recorder, where this resistance and this is time. If I then put an agonist or something known to stimulate platelets to clump together into this test tube, they will start forming layers on these two electrodes and the more active they were when the samples were drawn, there will be more layers on these electrodes. It acts like a resistance and the resistance will go up. So this top tracing here (on the graph) is of a healthy person who has very healthy active platelets, this here shows how much their activity is represented. I then take that person and give them 1 aspirin, wait an hour, wash off the electrodes, get a fresh test tube with fresh blood, put the electrodes down, give the same amount of collagen, now we get this trace here. The difference here and here is how much aspirin has turned down platelet activity in that human being. If I then have them exercise and raise their adrenaline, or if I put some adrenaline in the test tube and look at a third sample, now platelet activity is restored back to near normal because adrenaline or epinephrine overcomes much of the inhibitory effect of aspirin.
Now this is a protocol for a study we did looking at the effects of a single dose of French red wine, white wine or purple grape juice. Because of the animal work we know that there is a variety of substances in our diet that would interfere with the study. So there is a 1 week ‘wash out’ where there is no alcoholic beverage allowed, no tea, no fruit juices and no aspirin products, grapes, oranges or grapefruit. A sample blood test is drawn and then we would have them drink the red wine, white wine or purple grape juice. This is about 2 glasses of wine for a person my size and the purple grape juice is about 4 ½ glasses, the reason why is because there are more flavonoids in red wine than there are in purple grape juice.
Now here is the results of the French red wine, here is the average of all the platelet activity before we did the red wine, here you can see that there is significant reduction in platelet activity in response to collagen and a response to a synergistic effect between adrenaline and epinephrine and adenosine diphosphate, which is another substance found in the blood that will stimulate platelets to aggregate. The white wine shows NO significant effect on collagen or adenosine diphosphate, and that’s because there is about 7 times the amount of flavonoids in red wine as there is in white wine and that is because of how the different coloured wines are made.
When you look at the blood alcohol content (BAC) it was .06 here and was .06 on the other slides, so the alcohol content was the same. While there is a lot of controversy about which alcoholic beverages are better than other alcoholic beverages, we know that there are factors that contribute to the development and promotion of atherosclerosis and coronary thrombosis. So we would like to have something that is a good anti-oxidant, something that is a good anti-platelet substance, we would like to have something that improves the HDL/LDL ratio and we would like to have something that improves endothelial cell function.
Now, alcohol at moderate concentrations, a BAC of 1 or less has no anti-oxidant properties, does not inhibit platelets, it does increase HDL a little bit; and that’s a plus for alcohol, but it does not have any protective effect or improvement in endothelial function. The flavonoids in red wine and dark beer have been studied, they’re good anti-oxidants, they are good anti-platelet agents, and there is some evidence that they lower LDL cholesterol and we have evidence that they improve endothelial function. So this is why I say that it is the flavonoids in the beverages rather than the alcohol.
Now, here is the purple grape juice and here is the control for all of us, and here you can see that platelet activity is significantly turned down by the purple grape juice and the synergism between epinephrine and ADP is also turned down. So grape juice also has platelet inhibiting properties. Now, we did this second study for a couple of good reasons, it is suggested in the literature that flavonoids will bind to cell membranes and bind to plasma proteins. So the thought is, if you feed a human flavonoids for 7 days, there might be a tissue loading affect, and, you might produce a greater effect in giving a single dose. So we have chosen here to study grape juice given half the dose for 7 days compared to the previous study. Also, it is known that grapefruits and oranges have flavonoids in them but they are a different group of flavonoids than the ones found in grapes. So what we are going to test here is equal amounts of purple grape juice, orange juice, and grapefruit juice given for 7 days in twelve healthy human subjects. So once again we have a week ‘wash out’, no alcoholic beverages, no tea, no fruit juices, no aspirin products etc. Then they come in, have the blood sample drawn, platelets studied, and then they do the juice for 7 days. Then there is a ‘wash out’ period, we come and do the second group and then there is a week ‘wash out’ and we do the third group. The juices were randomized in the subjects.
Here is the control for all of us and here is the major inhibition of platelet activity from purple grape juice. Standard deviation is fairly wide and that’s because every body’s platelets are different and every body’s diet is different, but still, very significant reduction in platelet activity from purple grape juice and absolutely nothing from the orange or grapefruit juice. Now I don’t want to offend the citrus fruit growers but in this one specific test, clearly purple grape juice turns platelet activity, grapefruit and orange do not in equal amounts given.
In regards to anti-oxidant properties, this is a study published 2 years ago where they took off the shelf, purple grape juice, grapefruit, tomato, orange and apple with equal concentration and they tested the anti-oxidant capacity of the other juices, including the grapefruit, orange which we have studied. There is evidence to suggest that the flavonoids did inhibit platelet activity, they would also be good anti-oxidants so the data is supportive.
Now here on this slide is a slice of pizza that we might have for lunch, you can see pepperoni, sausage, tomato and cheese. Actually, this is a ruptured abdominal aorta, so in the patient ‘this is where the pizza is going to go right into the wall of the artery’ causing significant damage.
Now, you will see a lot of these advertisements these days, the implication is that you would have to eat 25 lbs of vegetables to equal the benefit of ‘2 of these capsules’. Now I did not study this product called Phytoplex (off the record: Phytoplex is a product sold by GeroVita Co.), now I want to repeat, I did not study this one. I use their advertisement because it is an impressive advertisement, unfortunately this company recommends taking 2-3 capsules a day, and you are supposed to get all these anti-oxidants properties and some anti-platelet benefits from it. What I found is that it would take 6-8 or 10 capsules to get any actual benefit from it in a living animal. None of these (flavonoid products) have been studied in animals or humans. If anything, there might be a test tube test but that is all. When I reported back to the company that it took 8 or 10 capsules they said, ‘thank you very much’ and continued to recommend the same 2 capsules per day. The reason is, it would cost too much and people would not buy it.
So here is the problem with commercial nutritional supplements, a variety of supplements containing flavonoids like grape seed, pycnogenol is one of these, grape skins, various berries like bilberry, a variety of ginkgo biloba extract and other plant sources are commonly sold in pharmacies and health food stores. The Federal Trade Commission estimated in 1996 that over 2 billion dollars was spent on these products.
When you work with your patients you will have to ask them specifically and question them a little bit to get them to tell you whether or not they are taking some of these supplements. The problem is that they have not been tested to show that a given dose produces a specific measured effect in either animals or humans. The reason they have not been tested is because they don’t have to. The FDA does not control nutritional supplements, the Federal Trade Commission does, but it only oversees them and does not require that you have hard evidence that they are efficacious and that they are safe.
Now this is one that I have looked at, called ProvexCV. It’s made by Melaleuca Inc. in Idaho Falls, Idaho and the company approached me and asked me to test their products. They provided grant money to the University of Wisconsin, not to me, but to do studies to find out if it really works. Here is what’s in it; there is a specific grape skin extract, grape seed extract, ginkgo biloba extract, bilberry, quercitin, a specific flavonoid and an enzyme blend to enhance bioavailability. A problem with some of these substances is they are not absorbed very well from the gut and don’t get into the blood where they are needed to do their thing. So, this is what I was going to test and many of these individually have shown in vitro to have either anti-oxidant, and/or anti-platelet properties so they are potentially useful. So the hypothesis is, do the flavonoid sources in ProvexCVT have anti-platelet properties in vitro, how can we demonstrate this and what’s more important, how much does one take?
Here is the animal model again, here are the animals making clots on a very regular basis, which indicates the high level of platelet activity. We give the animals a regular dose of 10 mg per kilogram orally, we wait 3 hours for it to be absorbed, now we see the blood pressures varying a little bit here, so the flow varies a little bit, but no more clots, significant reduction in platelet activity and when we infuse the epinephrine or the adrenaline the animal is still protected. With aspirin they are not protected here (referring to the visual graphics on the slides). So the animal model would suggest at this dose, that ProvexCVT does inhibit platelet activity and better than aspirin.
Now, here is the same device that we looked at before, we can take a person’s blood, test it here, then give them ProvexCVT for a week, then draw another blood sample to see if it indeed is inhibitive.
Here is the protocol, we now have 14 healthy human subjects, eight men and six women, and once again with usual exclusions so we don’t confound the data, no alcoholic beverages, tea, these are aspirin containing compounds. Blood withdrawal for whole blood platelet aggregation studies with the subject fasting. Then the subjects consumed the ProvexCVT; it turns out that it takes 20 mg per kilogram in the human of ProvexCVT daily for seven days. Then a repeat blood sample was drawn for whole platelet aggregation studies on the seventh day. Here we can see the response to collagen. Collagen is important, because when the endothelial cells are sloughed off, there is collagen just beneath them and that’s a clot-promoting surface.
This is what we were surprised to find, ADP (adenosine diphosphate), was inhibited over 40% by the ProvexCV. There are two drugs, which I am sure you have probably heard of, one is Ticlodopomine or Ticlid and the other one is Clopidigrel or Plavex. They are prescribed specifically to inhibit adenosine diphosphate in humans. They carry some nasty side effects including suppression of the bone marrow and they are very expensive and those two drugs have no effect at all on collagen because they block specifically ADP.
Now, just a brief word about anti-oxidant properties, we can collect LDL’s from a person, put them in a test tube, and here on the ‘Y’ axis is a measure of production of products that occur when the LDL cholesterol gets oxidized, such as conjugated, dying or other things. So the ‘Y’ axis indicates oxidation of the LDL cholesterol. In a person with a poor diet, when you put their cholesterol in the test tube it starts the reaction, nothing happens for about fifty minutes. That means the person is protected from oxidation of LDL cholesterol for about fifty minutes, this is called the lag time. Then the oxidation takes off rapidly and becomes oxidized. A person with a better diet who eats more fruits and vegetables might be this person with a lag time prolonged out here for a hundred minutes, and then oxidation is a little bit slower than it was here. If you give someone a supplement, or maybe a vegetarian who eats lots of fruits and vegetables, their lag time maybe will be way out here to one hundred and fifty minutes, now they have protection for one hundred and fifty minutes before their cholesterol becomes oxidized.
Here is five different people’s LDL cholesterol; this is the control here, about fifty minutes. At a certain dose of vitamin E, an anti-oxidant, the protection is doubled. Equal amounts of ProvexCVT compared to vitamin E were up here, and that is partly because ProvexCVT has multiple flavonoids from multiple sources and that’s why I think it adds more protection.
If we go back to these figures here for just a minute, we said here that LDL cholesterol diffuses down in here and stays awhile (referring to the sub endothelial space) and comes back out. While it’s in here, you would like to have it protected from oxidation so it does not go down the pathway of destruction when there is a chance to get it back out.
So the longer the lag time, or the longer before the time that it starts to get oxidized theoretically, the better off you would be. So you would like to have anti-oxidants bound to the cholesterol to protect it while it is in this state.
So in summary with regard to ProvexCVT, ProvexCV significantly inhibits platelet activity in experimental coronary thrombosis in the animal models at a specific known dose. In fourteen healthy human subjects, ProvexCVT at 20 mgs per kilogram taken for seven days significantly inhibits human whole blood platelet aggregation and in preliminary studies the ProvexCVT also appears to have significant anti-oxidant properties as well.
With regard to the other beverages, the poly phenolic compounds, flavonoids being the major groups, in grape beverages like purple grape juices, red wine and tea; black and green, studied in the animal model, appeared to have significant anti-platelet properties in the animal model. They also have significant anti-oxidant properties when studied in a test tube. More work needs to be studied in vitro or ex vitro in human volunteers.
The flavonoids in grape beverages and in ProvexCVT appear to have anti-platelet and anti-oxidant properties in healthy human volunteers. We have not studied ProvexCVT in patients, but we have studied purple grape juice in patients with coronary disease and there are significant anti-platelet and anti-oxidant properties in the patients. More people need to be studied to confirm and expand these results. The key thing then is how much of something, whether it is a beverage, a fruit or a vegetable, is needed to inhibit platelet activity with a certain measurement i.e. compared to 1 aspirin tablet a day, or how much is needed to inhibit or to provide anti-oxidant protection comparable to say vitamin E, which is sort of the standard, and finally, how much is needed to improve endothelial function to protect that endothelial wall so the vessel is less likely to get involved in the atherosclerotic process.
There is no substitute for trying to do the five to seven servings of fruits and vegetables a day.
The end.
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The following material was transcribed from an audiocassette recording. A presentation at which ProvexCV was introduced during The American Dietetic Conference on October 22, 1998 by Dr. John Folts.
This material may be difficult for some people to follow and visualize if they do not have a medical background as Dr. Folts utilized numerous slides, graphs and overheads during his presentation. If you will take the time to read this transcript, Dr. Folts will help you understand the mechanisms that cause coronary heart disease and his current research that appears to play a key role in helping to prevent a stroke or a heart attack.
Dr. Folts’ current research is on in vitro platelet mediated coronary thrombosis and anti-oxidants. Dr. Folts developed the experimental model on Aspirin in 1974 and demonstrated by in vitro testing that aspirin can reduce arterial blood clots.
He is currently studying anti-platelet, anti-oxidant properties of poly phenol compounds found in red wine, beer, fruit juices and tea. Dr. Folts received his Bachelors of Science degree in electrical engineering, his masters in medical physiology and his Ph.D. degree in cardiovascular physiology and pathology from the University of Wisconsin. Today, in his lecture, he will describe the role of platelets in clot formation and his research on the ability of wine, fruit juice and tea to reduce or inhibit platelet mediated clot formation.
The American Dietetic Association presents Dr. John Folts, Ph.D.
October 22, 1998
Dr. Folts speech:
I want to talk about platelets, free radicals and LDL cholesterol and why we need something to protect against that in reducing coronary heart disease. Coronary artery disease is a major problem in the western world. We have made some progress, but we still have a lot left to do.
Epidemiological studies have shown an inverse relationship between dietary intake of flavonoids and the incidence of fatal and non-fatal myocardial infarction or heart attack. Flavonoids are micronutrients found in vegetables, fruits and their juices but not in any animal products. They have no nutritional value because they are not carbohydrates, fats or proteins. In vitro in the test tube and in tissue culture studies, many of them have shown to have both good anti-platelet and anti-oxidant properties.
Looking at a longitudinal section through a blood vessel when we are born, the endothelial cells lining the arteries are slippery smooth and they are very functional and they release substances called nitric oxide and other substances that chase blood cells away from sticking on the wall. Platelets are blood cells that get involved in clotting but we do not want them to stick on the walls of large arteries. The nitric oxide they release also relaxes the vascular smooth muscle cells and protects them from things we are going to see in a minute.
When these endothelial cells are damaged they are sloughed off and they are lost. This leaves a very clot-producing surface beneath them. The best way to damage the endothelial cells or have them sloughed off is by smoking cigarettes. Shown in the slide are several layers of platelets collecting on the vessel wall because endothelial cells are either damaged or gone and they release substances that tell vascular fluid muscles cells to multiply. It is really a smooth muscle cell tumour if you will. If they would stay down there in the media of the artery, which is part of the outer wall, they wouldn’t compromise the Lumina (opening) shown here. The platelets release substances also that say to these dividing fluid muscles cells to come up here where we are, so they migrate up and cause intimal thickening and this contributes to the narrowing of the artery.
So we would like to have something in our diet to restore endothelial function and also to inhibit the platelets, so they are less likely to contribute to this problem. Now here is another part of the vessel wall showing the endothelial cells and the vascular smooth muscle cells and here are the LDL cholesterol. We have to have cholesterol in our diets because the gonads use it to make sex hormones, the adrenal cortex uses it to make cortigo steroids, as well as, every new cell that is made in the body has to have cholesterol in its cell walls. The problem is when we have too much; there is no place to put it. The cholesterol diffuses down into the sub endothelial space, resides there for a while and then diffuses back out. Obviously, the more we have here the easier it is to diffuse in here, the harder it is to get it back out due to the concentration gradient. While cholesterol is in this space, oxidants and free radicals can be released by endothelial cells, vascular smooth muscle cells and many other cells. If the LDL cholesterol gets oxidized it is much more difficult for it to get out and it goes down the path of destruction. As it is oxidized, it moves down here in the sub endothelial space, thickening. It tells the endothelial cells, especially if they are sick, to release substances that cause white cells called monocytes to migrate in and then they get converted to macrophages. They then pick up cholesterol in an unregulated fashion and they become foam cells. These foam cells can be seen be seen in the arteries of children 10 –15 years old who have been killed in car accidents, when they autopsy them they find fatty streaks already developing in their coronary arteries, this is because of the presence of foam cells.
Now, here in this slide I have put everything together. Here we have the platelets simulating migration and proliferation of the smooth muscle cells down into here, we have the oxidized LDL’s migrating down into here (referring to the sub endothelial space), this process is really an anti-inflammatory reaction and the production of foam cells and finally, platelets of the cells initiates the clots that cause death. Initially there is very little fibrin in the clots; most of the clot is made of platelets.
So once again, we would like to have something in the diet that would improve the function of the endothelial cells, that would ‘turn down’ the activity of the platelets, that would protect the LDL cholesterol from oxidation, that would have some anti-inflammatory properties as well, and, that would inhibit a clot formation that cuts off the blood flow to the coronary arteries causing death.
The two main factors involved in coronary death are platelet activity and oxidized cholesterol; so far we have seen some mechanisms that can contribute to both the progressive narrowing of the arteries as well as the clot that kills.
Now, if we are going to look at something that will turn down the platelet activity, we obviously have to have some methods of measuring platelet activity. Many of my clinical colleagues think of platelets only in terms of their numbers, example, if you have 50,000 you are going to bleed, if you have 300,000 you should be alright, if you have 900,000 or 1,000,000 you should be a clotter. Platelet activity can be turned up or down in a matter of 5-10 minutes and one way to turn it up in 5 minutes is by smoking of a cigarette.
So now we are going back to an animal model for studying platelet mediated clot formation and damaged and narrowed arteries, and then we are going to look at a method for studying platelet activity in the blood of human volunteers like myself. Dogs and pigs can be fully anesthetised, the chest opened, the heart is exposed and here is the circumflex coronary artery, one of the main branches of the coronary arteries. We put this flow probe devise around the coronary artery that measures blood flow through it without interfering with the blood flow in any way. These devises are commonly placed on the exposed arteries of patients at the time of surgery. In order to mimic the atherosclerotic plague, we clamp the artery damaging the endothelial cells, damaging vascular smooth muscle cells and exposing clot-producing surfaces. Then, to produce some vascular narrowing, I make these plastic cylinders with a range of internal diameters and one is placed around the outside of the artery so that it is narrowed down, now we can mimic the problem the patient has with coronary disease. Here is the model again, here is the flow probe, and here is the area of stenosis and vessel damage. Coronary blood flow starts out okay, it’s normal here, but, as the platelet plug accumulates in here, the coronary flow gradually declines until it’s fully occluded. If this animal were awake it would have chest pains because this part of the heart is getting no blood flow. These cyclic flow reductions; as we call them, are caused by periodic repeated platelet mediated clots that form and then embolize distally. This is thought to be the mechanism of unstable angina in patients with coronary artery disease.
Back in the early seventies, I discovered that if we gave them aspirin; and this is equivalent to one aspirin in you or I, these clots gradually went away and blood flow was restored and maintained. We were excited about this and I presented this at several meetings and the people there thought I was ‘crazy-as-a-loon’ because back in the seventies, aspirin was just for headaches and things like that. Now I am sure there are people in the audience that are taking their aspirin, as are those people who thought I was crazy. In the late seventies however, we discovered that if we raised the adrenaline or epinephrine, that the same thing, 75% -80% of the time clot formation comes back with aspirin and that’s a serious problem. That’s why we have spent the last 20 years looking for drugs and food substances that would inhibit the platelets and prevent the clot formation, but also, prevent the renewal of clot formation from elevated epinephrine.
Now, in our previous findings with the animal model, we have shown that red wine, moderate amounts equivalent to 2 glasses in you or I, purple grape juice; not the clear or white grape juice, tea, but not coffee, in this animal model reduced or inhibited platelet mediated clot formation and protected against potentially fatal coronary thrombosis. These flavonoids in red wine, purple grape juice and tea inhibit platelets about the same as aspirin but they have one unique feature. THEY PROTECT AGAINST ELEVATIONS OF ADRENALINE, WHICH ASPIRIN DOES NOT. That makes the flavonoids more desirable.
Now obviously, we would like to do this in humans, but not too many humans want to volunteer to have their chests opened and their arteries exposed and studied, so we have another way to do this. Here we have a device where I can take a blood sample from one of you, place it in this test tube shown here, put two electrodes here that are part of an ohmmeter. Many of us have volt ohmmeters in our cars and workshops at home; these ohmmeters measure the electrical resistance between these two electrodes, which is normally quite low, because blood has a lot of electrolytes in it that allows conduction of electricity. The ohmmeter displayed on this flip strip chart recorder, where this resistance and this is time. If I then put an agonist or something known to stimulate platelets to clump together into this test tube, they will start forming layers on these two electrodes and the more active they were when the samples were drawn, there will be more layers on these electrodes. It acts like a resistance and the resistance will go up. So this top tracing here (on the graph) is of a healthy person who has very healthy active platelets, this here shows how much their activity is represented. I then take that person and give them 1 aspirin, wait an hour, wash off the electrodes, get a fresh test tube with fresh blood, put the electrodes down, give the same amount of collagen, now we get this trace here. The difference here and here is how much aspirin has turned down platelet activity in that human being. If I then have them exercise and raise their adrenaline, or if I put some adrenaline in the test tube and look at a third sample, now platelet activity is restored back to near normal because adrenaline or epinephrine overcomes much of the inhibitory effect of aspirin.
Now this is a protocol for a study we did looking at the effects of a single dose of French red wine, white wine or purple grape juice. Because of the animal work we know that there is a variety of substances in our diet that would interfere with the study. So there is a 1 week ‘wash out’ where there is no alcoholic beverage allowed, no tea, no fruit juices and no aspirin products, grapes, oranges or grapefruit. A sample blood test is drawn and then we would have them drink the red wine, white wine or purple grape juice. This is about 2 glasses of wine for a person my size and the purple grape juice is about 4 ½ glasses, the reason why is because there are more flavonoids in red wine than there are in purple grape juice.
Now here is the results of the French red wine, here is the average of all the platelet activity before we did the red wine, here you can see that there is significant reduction in platelet activity in response to collagen and a response to a synergistic effect between adrenaline and epinephrine and adenosine diphosphate, which is another substance found in the blood that will stimulate platelets to aggregate. The white wine shows NO significant effect on collagen or adenosine diphosphate, and that’s because there is about 7 times the amount of flavonoids in red wine as there is in white wine and that is because of how the different coloured wines are made.
When you look at the blood alcohol content (BAC) it was .06 here and was .06 on the other slides, so the alcohol content was the same. While there is a lot of controversy about which alcoholic beverages are better than other alcoholic beverages, we know that there are factors that contribute to the development and promotion of atherosclerosis and coronary thrombosis. So we would like to have something that is a good anti-oxidant, something that is a good anti-platelet substance, we would like to have something that improves the HDL/LDL ratio and we would like to have something that improves endothelial cell function.
Now, alcohol at moderate concentrations, a BAC of 1 or less has no anti-oxidant properties, does not inhibit platelets, it does increase HDL a little bit; and that’s a plus for alcohol, but it does not have any protective effect or improvement in endothelial function. The flavonoids in red wine and dark beer have been studied, they’re good anti-oxidants, they are good anti-platelet agents, and there is some evidence that they lower LDL cholesterol and we have evidence that they improve endothelial function. So this is why I say that it is the flavonoids in the beverages rather than the alcohol.
Now, here is the purple grape juice and here is the control for all of us, and here you can see that platelet activity is significantly turned down by the purple grape juice and the synergism between epinephrine and ADP is also turned down. So grape juice also has platelet inhibiting properties. Now, we did this second study for a couple of good reasons, it is suggested in the literature that flavonoids will bind to cell membranes and bind to plasma proteins. So the thought is, if you feed a human flavonoids for 7 days, there might be a tissue loading affect, and, you might produce a greater effect in giving a single dose. So we have chosen here to study grape juice given half the dose for 7 days compared to the previous study. Also, it is known that grapefruits and oranges have flavonoids in them but they are a different group of flavonoids than the ones found in grapes. So what we are going to test here is equal amounts of purple grape juice, orange juice, and grapefruit juice given for 7 days in twelve healthy human subjects. So once again we have a week ‘wash out’, no alcoholic beverages, no tea, no fruit juices, no aspirin products etc. Then they come in, have the blood sample drawn, platelets studied, and then they do the juice for 7 days. Then there is a ‘wash out’ period, we come and do the second group and then there is a week ‘wash out’ and we do the third group. The juices were randomized in the subjects.
Here is the control for all of us and here is the major inhibition of platelet activity from purple grape juice. Standard deviation is fairly wide and that’s because every body’s platelets are different and every body’s diet is different, but still, very significant reduction in platelet activity from purple grape juice and absolutely nothing from the orange or grapefruit juice. Now I don’t want to offend the citrus fruit growers but in this one specific test, clearly purple grape juice turns platelet activity, grapefruit and orange do not in equal amounts given.
In regards to anti-oxidant properties, this is a study published 2 years ago where they took off the shelf, purple grape juice, grapefruit, tomato, orange and apple with equal concentration and they tested the anti-oxidant capacity of the other juices, including the grapefruit, orange which we have studied. There is evidence to suggest that the flavonoids did inhibit platelet activity, they would also be good anti-oxidants so the data is supportive.
Now here on this slide is a slice of pizza that we might have for lunch, you can see pepperoni, sausage, tomato and cheese. Actually, this is a ruptured abdominal aorta, so in the patient ‘this is where the pizza is going to go right into the wall of the artery’ causing significant damage.
Now, you will see a lot of these advertisements these days, the implication is that you would have to eat 25 lbs of vegetables to equal the benefit of ‘2 of these capsules’. Now I did not study this product called Phytoplex (off the record: Phytoplex is a product sold by GeroVita Co.), now I want to repeat, I did not study this one. I use their advertisement because it is an impressive advertisement, unfortunately this company recommends taking 2-3 capsules a day, and you are supposed to get all these anti-oxidants properties and some anti-platelet benefits from it. What I found is that it would take 6-8 or 10 capsules to get any actual benefit from it in a living animal. None of these (flavonoid products) have been studied in animals or humans. If anything, there might be a test tube test but that is all. When I reported back to the company that it took 8 or 10 capsules they said, ‘thank you very much’ and continued to recommend the same 2 capsules per day. The reason is, it would cost too much and people would not buy it.
So here is the problem with commercial nutritional supplements, a variety of supplements containing flavonoids like grape seed, pycnogenol is one of these, grape skins, various berries like bilberry, a variety of ginkgo biloba extract and other plant sources are commonly sold in pharmacies and health food stores. The Federal Trade Commission estimated in 1996 that over 2 billion dollars was spent on these products.
When you work with your patients you will have to ask them specifically and question them a little bit to get them to tell you whether or not they are taking some of these supplements. The problem is that they have not been tested to show that a given dose produces a specific measured effect in either animals or humans. The reason they have not been tested is because they don’t have to. The FDA does not control nutritional supplements, the Federal Trade Commission does, but it only oversees them and does not require that you have hard evidence that they are efficacious and that they are safe.
Now this is one that I have looked at, called ProvexCV. It’s made by Melaleuca Inc. in Idaho Falls, Idaho and the company approached me and asked me to test their products. They provided grant money to the University of Wisconsin, not to me, but to do studies to find out if it really works. Here is what’s in it; there is a specific grape skin extract, grape seed extract, ginkgo biloba extract, bilberry, quercitin, a specific flavonoid and an enzyme blend to enhance bioavailability. A problem with some of these substances is they are not absorbed very well from the gut and don’t get into the blood where they are needed to do their thing. So, this is what I was going to test and many of these individually have shown in vitro to have either anti-oxidant, and/or anti-platelet properties so they are potentially useful. So the hypothesis is, do the flavonoid sources in ProvexCVT have anti-platelet properties in vitro, how can we demonstrate this and what’s more important, how much does one take?
Here is the animal model again, here are the animals making clots on a very regular basis, which indicates the high level of platelet activity. We give the animals a regular dose of 10 mg per kilogram orally, we wait 3 hours for it to be absorbed, now we see the blood pressures varying a little bit here, so the flow varies a little bit, but no more clots, significant reduction in platelet activity and when we infuse the epinephrine or the adrenaline the animal is still protected. With aspirin they are not protected here (referring to the visual graphics on the slides). So the animal model would suggest at this dose, that ProvexCVT does inhibit platelet activity and better than aspirin.
Now, here is the same device that we looked at before, we can take a person’s blood, test it here, then give them ProvexCVT for a week, then draw another blood sample to see if it indeed is inhibitive.
Here is the protocol, we now have 14 healthy human subjects, eight men and six women, and once again with usual exclusions so we don’t confound the data, no alcoholic beverages, tea, these are aspirin containing compounds. Blood withdrawal for whole blood platelet aggregation studies with the subject fasting. Then the subjects consumed the ProvexCVT; it turns out that it takes 20 mg per kilogram in the human of ProvexCVT daily for seven days. Then a repeat blood sample was drawn for whole platelet aggregation studies on the seventh day. Here we can see the response to collagen. Collagen is important, because when the endothelial cells are sloughed off, there is collagen just beneath them and that’s a clot-promoting surface.
This is what we were surprised to find, ADP (adenosine diphosphate), was inhibited over 40% by the ProvexCV. There are two drugs, which I am sure you have probably heard of, one is Ticlodopomine or Ticlid and the other one is Clopidigrel or Plavex. They are prescribed specifically to inhibit adenosine diphosphate in humans. They carry some nasty side effects including suppression of the bone marrow and they are very expensive and those two drugs have no effect at all on collagen because they block specifically ADP.
Now, just a brief word about anti-oxidant properties, we can collect LDL’s from a person, put them in a test tube, and here on the ‘Y’ axis is a measure of production of products that occur when the LDL cholesterol gets oxidized, such as conjugated, dying or other things. So the ‘Y’ axis indicates oxidation of the LDL cholesterol. In a person with a poor diet, when you put their cholesterol in the test tube it starts the reaction, nothing happens for about fifty minutes. That means the person is protected from oxidation of LDL cholesterol for about fifty minutes, this is called the lag time. Then the oxidation takes off rapidly and becomes oxidized. A person with a better diet who eats more fruits and vegetables might be this person with a lag time prolonged out here for a hundred minutes, and then oxidation is a little bit slower than it was here. If you give someone a supplement, or maybe a vegetarian who eats lots of fruits and vegetables, their lag time maybe will be way out here to one hundred and fifty minutes, now they have protection for one hundred and fifty minutes before their cholesterol becomes oxidized.
Here is five different people’s LDL cholesterol; this is the control here, about fifty minutes. At a certain dose of vitamin E, an anti-oxidant, the protection is doubled. Equal amounts of ProvexCVT compared to vitamin E were up here, and that is partly because ProvexCVT has multiple flavonoids from multiple sources and that’s why I think it adds more protection.
If we go back to these figures here for just a minute, we said here that LDL cholesterol diffuses down in here and stays awhile (referring to the sub endothelial space) and comes back out. While it’s in here, you would like to have it protected from oxidation so it does not go down the pathway of destruction when there is a chance to get it back out.
So the longer the lag time, or the longer before the time that it starts to get oxidized theoretically, the better off you would be. So you would like to have anti-oxidants bound to the cholesterol to protect it while it is in this state.
So in summary with regard to ProvexCVT, ProvexCV significantly inhibits platelet activity in experimental coronary thrombosis in the animal models at a specific known dose. In fourteen healthy human subjects, ProvexCVT at 20 mgs per kilogram taken for seven days significantly inhibits human whole blood platelet aggregation and in preliminary studies the ProvexCVT also appears to have significant anti-oxidant properties as well.
With regard to the other beverages, the poly phenolic compounds, flavonoids being the major groups, in grape beverages like purple grape juices, red wine and tea; black and green, studied in the animal model, appeared to have significant anti-platelet properties in the animal model. They also have significant anti-oxidant properties when studied in a test tube. More work needs to be studied in vitro or ex vitro in human volunteers.
The flavonoids in grape beverages and in ProvexCVT appear to have anti-platelet and anti-oxidant properties in healthy human volunteers. We have not studied ProvexCVT in patients, but we have studied purple grape juice in patients with coronary disease and there are significant anti-platelet and anti-oxidant properties in the patients. More people need to be studied to confirm and expand these results. The key thing then is how much of something, whether it is a beverage, a fruit or a vegetable, is needed to inhibit platelet activity with a certain measurement i.e. compared to 1 aspirin tablet a day, or how much is needed to inhibit or to provide anti-oxidant protection comparable to say vitamin E, which is sort of the standard, and finally, how much is needed to improve endothelial function to protect that endothelial wall so the vessel is less likely to get involved in the atherosclerotic process.
There is no substitute for trying to do the five to seven servings of fruits and vegetables a day.
The end.
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