How Vitamin E works: An interview with Dr. Maret G. Traber
Part III: The function of the Tocopherol Transfer Protein
Richard A. Passwater, Ph.D.
Dr. Maret G. Traber of the University of California at Berkeley received the VERIS Award for her vitamin E research in 1993. She has made major contributions to our understanding on how vitamin E is absorbed and transported. Of special importance is her elucidation of the function of the alpha-tocopherol transfer protein (TTP). Understanding TTP and its functions clarifies much about why the alpha-tocopherol is the best vitamin E form for the body. This is not to say that other forms of vitamin E are of minor importance as they could make other significant contributions to health.
Part I of this series reviewed the various forms of the vitamin E family and presented the basic structures and nomenclature. Part II detailed many of the differences between natural vitamin E and synthetic vitamin E. Though both are good, the natural vitamin E is better. Throughout the earlier parts, we have made occasional reference to TTP and Dr. Marets research. Now, with the review of the basics completed, we can move ahead with a discussion of TTP and its significance.
Passwater: You indicated that vitamin E became of interest to you because of the problems in babies with cholestatic liver disease; this raised your curiosity about how vitamin E was transported. Tell us a little about the detective work that led from your curiosity to receiving the VERIS award for elucidating so much about the tocopherol transport protein. What was your first experiment along these lines?
Traber: Since it was known for some 20 years that lipoproteins carry vitamin E in the plasma, we thought that with our expertise in lipoproteins, we should be able to figure out just how lipoproteins transport vitamin E. I guess looking back it was rather a naive approach. We thought what we would start off with something easy: we would take some low-density lipoprotein (LDL) and feed it to cells having LDL receptors and other cells not having LDL receptors to see if those with LDL receptors took up the vitamin E. The answer was yes.
Passwater: Well, thats certainly part of the story. What did you do next?
Traber: We tried a couple of other simple things that suggested pretty much however lipid and lipoprotein metabolism worked -- thats how vitamin E got where it was going. We were all in the mindset that vitamin E was just going along for the ride with other fats. It turned out we were wrong. It was at that time that Dr. Gary Handelman published a report showing that if you fed vitamin E supplements to normal healthy people, the gamma tocopherol in their plasma ( the portion of blood minus red blood cells) went down. That just seemed so wrong to me.
Alpha-tocopherol is the most biologically available form of vitamin E and while gamma tocopherol is the most prevalent form of vitamin E in the diet. It was absolutely unthinkable that if you gave an alpha-tocopherol supplement that gamma-tocopherol levels in plasma should go down. None of the studies wed done so far had even suggested that would happen. Dr. Herb Kayden was so incensed that immediately he took some vitamin E. We drew his blood and then the next day we drew his blood again just to prove this couldnt happen. We were both absolutely wrong. Dr. Handelman was right: when you give someone vitamin E supplements, gamma-tocopherol in the plasma goes down. There was no way we could figure why this was true.
We had just finished a study in which we were infusing different forms of vitamin E into the stomachs of laboratory rats. We gave huge amounts of alpha tocopherol, collected the chylomicron fraction in the lymph and found there was no inhibition of the absorption of any other form. So Dr. Handelmans explanation that there had to be some sort of limitation in gamma absorption in competition between the forms of vitamin E wasnt right. This sent us off on a quest to figure out how could it be that you would get this discrimination in the plasma.
This is where Drs. Keith Ingold and Graham Burton come into the story. As it turned out, Drs. Ingold and Kayden were at a meeting in Japan when they were talking about this perplexing problem. Drs. Ingold and Burton had just synthesized some deuterated tocopherol and they saw discrimination also. We all decided all to join forces and study how is it that the body is able to discriminate between these various forms of vitamin E.
Passwater: How did you go about resolving this question?
Traber: Drs. Keith Ingold and Graham Burton had different stereoisomers of vitamin E labeled with deuterium, a heavy isotope of hydrogen. Maybe we should take a moment and discuss this interesting technique. I am sure our readers know about radioactive material, but perhaps they are not familiar with the technique of using stable isotopes, which are not radioactive, to trace the movements of compounds through the body.
Isotopes are forms of a chemical element having almost identical properties. We have discussed molecular isomers, but now we are speaking of elemental isotopes. Isotopes of elements have the same atomic number and the same number of protons in their atomic nucleus, but they differ in their atomic weights because they have different numbers of nuclear neutrons. Isotopes have the same chemistry, but slightly different physical properties.
Lets look at the three isotopes of the element hydrogen. Hydrogen actually exists as the common hydrogen, and its heavier isotopes, deuterium and tritium. Hydrogen, along with oxygen, forms water. "Heavy" water has deuterium instead of hydrogen. The atomic weight of hydrogen is one, while the deuterium atom is two, and tritium is three; only the tritium isotope is an unstable isotope and gives off radioactive particles. Deuterium is a heavy isotope of hydrogen, but it is stable and is not radioactive so it doesnt have any harmful side effects. The nice part is it can be given to people as a special tracer of a molecule of interest.
Drs. Graham Burton and Keith Ingold used deuterium to make the vitamin E forms we used a little bit heavier than the natural forms. They then used a special detector, called a mass spectrometer, which measures how heavy the molecules are, to detect and identify the various forms of vitamin E in the samples of blood we obtained from our subjects.
As we have discussed earlier, stereoisomers are isomers that are mirror opposites, kind of like the differences between your right and your left hands. They look the same but you cant really put them on top of each other; your right hand is not the same as your left hand. Drs. Ingold and Burton chose to label two of the eight different stereoisomers present in synthetic vitamin E with deuterium. The two stereoisomers that they chose have opposite directions where the phytyl "tail" portion of the vitamin E molecule meets the chromane head portion. Basically the difference between RRR-alpha-tocopherol stereoisomer and the SRR-alpha-tocopherol stereoisomer is how the phytyl tail fits to the chromane head.
This is the most significant difference affecting in how the different forms of vitamin E fit into cell membranes. We fed to normal, healthy people equal amounts of these two stereoisomers of vitamin E, and what we found is that in the first six-to-nine hours there were equal concentrations of the two stereoisomers in the plasma. However, after twenty-four hours it was quite clear that there was a significant increase in the natural form of vitamin E, the RRR-alpha-tocopherol. The SRR-alpha-tocopherol disappeared rapidly from the plasma. 
Drs. Ingold and Burton were good enough to also label natural gamma-tocopherol with deuterium for us. Synthetic vitamin E contains only alpha-tocopherol, albeit eight isomers of alpha-tocopherol. As you pointed out in the introduction, gamma-tocopherol has one less methyl group on the chromane head portion of the molecule and this means that it has a free "ortho" position. Now it gets a little confusing because we have altered the chromane heads but the phytyl tail is in the same conformation as the natural RRR-alpha-tocopherol, but the gamma-tocopherol behaves the same as SRR-alpha-tocopherol, a synthetic form.
Passwater: Could you summarize the half-lives of the various forms in the body?
Traber: Well, "half-life" means how long does it take for half of the concentration of something to disappear. It is a term often used by pharmacologists to study drugs how long do they stay in the blood and are the concentrations high enough to be effective? In the case of vitamin E, because we had labels on them, we could distinguish the vitamin E we gave our subjects from the vitamin E that they already had present in their bodies. It turned out that the half-life for RRR-alpha-tocopherol was about 48 hours, while that of SRR-alpha-tocopherol was about 12 hours. That means that the SRR-alpha-tocopherol that is present only in synthetic vitamin E and not found in nature left the plasma four times faster than did the natural vitamin E. Gamma-tocopherol behaved like SRR-alpha-tocopherol. 
Passwater: How can something that is not exactly the same, just a little bit different from natural vitamin E, disappear so dramatically from the body?
Traber: We were truly perplexed. Others had studied gamma-tocopherol a lot, and have just concluded that the tissues lose it faster. None of these seemed like the right explanation. But we did find a report by Drs. G. L. Catignani and John Bieri of the National Institutes of Health from the late 1970's that said there was a tocopherol-binding protein in rat liver cytosol.  Almost four years later, Drs. D. J. Murphy and R. D. Mavis reported that rat liver cytosol could transfer vitamin E.  These reports made us think about the liver a little bit.
Near the same time, Dr. Ron Sokol at the University of Colorado Health Science Center had been looking at patients with cholestatic liver disease. He had a pretty good idea of what neurologic abnormalities due to vitamin E deficiency looked like. He and his friends in the neurologic clinic were looking for patients that had those symptoms. The symptoms are subtle. Its the inability to feel tingling of your toes or feel the vibrations from a tuning fork placed on your knee or your elbow. This loss of feeling or tingling sensation is the beginning of vitamin E deficiency symptoms. He found some patients who turned out to be vitamin E deficient without any other known cause. These findings were occurring about the same time.
Passwater: But, werent all of these new findings confusing?
Traber: Yes, they were confusing, but they were also interesting. In one of the first patients we found almost no vitamin E in the plasma but there was some in the adipose (fat) tissue. I thought, "Gee thats funny, if there is none in the plasma, how could it get to the adipose tissue?"
In trying to put all these pieces of the puzzle together, I came up with a possible explanation. The vitamin E was absorbed and transported in the chylomicrons. Vitamin E could then be transferred from the chylomicrons along with the fat to the adipose tissue. Then those chylomicrons, after they are broken down, were delivered to the liver. Once they are in the liver, if the patients were missing this protein that Dr. John Bieri had described, they wouldnt be able to put vitamin E back in the plasma, and thus vitamin E would disappear from the plasma. I wondered if maybe this protein might recognize and bind natural alpha-tocopherol, but not the synthetic alpha- tocopherol nor gamma-tocopherol. It seemed like a plausible hypothesis.
So, we started isolating all kinds of lipoproteins and looking around the world for vitamin E deficient patients that might have a genetic abnormality in the vitamin E binding protein.
To make a long story short, there is in fact a vitamin E binding protein, which we now call tocopherol transfer protein (TTP), in human livers. Patients with this disorder that we then called "familial isolated vitamin E deficiency," (but is now called "ataxia with vitamin E deficiency) have a genetic defect in their TTP. Some of these patients are actually lacking the last third of the protein and they are absolutely unable to recognize and bind to any of the various forms of vitamin E. Vitamin E rapidly disappears from their plasma, and those patients by about age 10 or so, have severe neurologic abnormalities.
There are other patients whose defects in the protein are not quite as dramatic. They are able to transfer some of vitamin E from the liver into the lipoproteins, but it still leaves their plasma pretty fast.
This was followed up by Dr. T. Gotoda and colleagues, who found a patient on an isolated island in Japan. They studied more than 800 people and found twenty or so that were heterozygotes ( having two different "alternative" genes on the same locus of each pair of chromosomes) for the mutation found in the patient. These heterozygotes have one normal gene and they have one defective gene, and it turns out that they have half of normal plasma vitamin E concentrations. It all kind of fits nicely together. 
Passwater: Lets talk about some of the properties of this tocopherol transfer protein (TTP). When TTP is normal -- a complete molecule -- what does it transfer from what to what?
Traber: First of all, let me emphasize during absorption from the dietary or the supplemental vitamin E, you dont need a transfer protein. The chylomicrons contain all the various forms of vitamin E that are in the food or supplements. For some reason we havent figured out you dont need a transfer protein in your intestine. What happens when vitamin E gets to the liver, it is salvaged by the TTP.
When cave persons were walking around chasing the woolly mammoths and being a hunter/gatherer, there was relatively very little vitamin E in the diet. It was important for the body that whenever the diet did supply a decent amount of vitamin E, it needed to hang onto however much there was. We think that TTP is there to hang onto those traces of vitamin E.
This also explains why Dr. Max Horwitts had difficulty producing vitamin E deficiencies in his early studies of vitamin E deficiency in humans (See Whole Foods November, 1992). Dr. Horwitt had a terrible time; he had people on vitamin E - deficient diets for years and could never show any deficiency symptoms.  He could barely develop anemia in the volunteers because TTP hangs onto every little bit of vitamin E that flies past it and puts it back into the circulation. Weve actually calculated that it manages to replace the entire plasma pool of vitamin E every day. So it is working very hard.
Passwater: Thats remarkable, but it still doesnt explain where vitamin E goes and what it is doing.
Traber: We havent a clue. I have been working for the last two years with the worlds experts on lipoproteins, Drs. Richard Havel and Richard Hamilton at University of California at San Francisco. Dr. Havel wrote the original method how to isolate lipoproteins, so I thought they could help me figure out how vitamin E gets into lipoproteins as they are being assembled.  What we have found so far is that all the steps along the pathway for lipoprotein assembly are not the sites where vitamin E is added to the lipoproteins.
We dont know where the tocopherol transfer protein grabs the vitamin E as it is coming into the liver and we dont know where it is putting it to back out of the liver. I have done studies with Dr. Larry Rudel in North Carolina who has primate colonies. We have fed monkeys deuterated vitamin E, then perfused their livers and isolated their lipoproteins. We can demonstrate that once the very-low lipoprotein (VLDL) gets out of the liver, it is preferentially enriched with natural alpha tocopherol.  I am at a loss, because it seems to be the proverbial scientific " big black box." We know what we put in are equal amounts of whatever you just gave and we know what comes out is preferentially enriched in RRR-alpha-tocopherol. We know that people who have a defective hepatic TTP dont export the vitamin E properly from the liver but we dont know how it does it. Thats the big mystery. I am working diligently on the problem, but I need a brilliant inspiration.
Passwater: These are observations in humans, right?
Passwater: Does TTP exist in other mammals?
Traber: Yes, TTP was first found in rat liver. It has been isolated from human liver. And, we have been doing studies in mice and find it in mouse liver. Interestingly, there are horses that become vitamin E-deficient one possibility is that they have a defect in TTP. 
Passwater: You indicated that lipoprotein is "preferentially enriched" by natural RRR-alpha-tocopherol. Does it take any of the other isomers; the gamma, beta, delta? Also, does it take any of the synthetic"S" variations?
Traber: Actually there was a paper that came out in June 1997 FEBS Letters from a group in Japan who have isolated the protein.  They have tested purified TTP in an in vitro (laboratory glassware) assay. They find some transfer of other forms. Alpha-tocopherol is 100 percent transfer; gamma-tocopherol is only 9 percent transfer; delta-tocopherol, which is a pretty exotic form, is 2 percent transfer. TTP doesnt move tocopheryl acetate at all, and it doesnt recognize compounds like cholesterol. The synthetic form, SRR-alpha-tocopherol, is not transferred very effectively.
The point of their paper is that the biologic activity of vitamin E has always been very controversial. This is because the various forms of vitamin E which dont have huge chemical structural differences between them, all act as antioxidants, however, there are huge differences in their biologic activity. It seems as if the action of TTP determines why those biologic differences occur. That is, TTP prefers the RRR-alpha-tocopherol form of vitamin E and keeps this in circulation.
Passwater: So synthetic vitamin E is dumped more quickly from the membranes because of the kinks in the phytyl tail due to the "S" configurations, and it does not get re-circulated from the liver because TTP dont transfer it. Very, very interesting.
You mentioned that Japanese researchers isolated and purified TTP. Didnt they also determine the amino acid sequence?
Traber: Professor Hiroyuki Arai and his colleagues have determined the amino acid sequence of both rat and human TTP. And, importantly, they have demonstrated that patients with vitamin E deficiency have a genetic defect in TTP. About 20 mutations have been discovered in vitamin E deficient patients so far.
They have very interesting data that suggests that TTP is not only located in the liver, but may play a role in regulating vitamin E in the brain. I think that we have entered a new age and will find many tissues express vitamin E binding/transfer proteins.
I have just returned from a scientific meeting in Japan, and was fortunate enough to again meet with Professor Arai. We had met before at an International Symposium on Vitamin E in 1991.
At that time, I presented our data demonstrating that there were humans who were unable to effectively maintain plasma vitamin E concentrations and that this was due to their impaired ability to incorporate vitamin E into VLDL. I suggested that there must be a liver protein, perhaps that described by Drs. Bieri and Catignani as an alpha-tocopherol binding protein, that functions to put vitamin E into lipoproteins and that these patients perhaps had a genetic defect in this protein.
At the reception at the end of the day, I met Professor Arai, who had just purified the rat liver tocopherol binding protein and demonstrated that it transferred vitamin E between liposomes and membranes. They were searching for a physiological role for this protein. It was a very happy moment for me. 
Passwater: After receiving the VERIS award in 1993, you mentioned that you might some day be able to answer the question about our requirement for vitamin E and the maximum amount that can be transferred by TTP and anything more than that just couldnt be handled or transferred. Could you tell us a little bit about what is known about dosages maximum amounts that can be absorbed and utilized?
Traber: There are several lines of evidence that shed light on this question. We can glean information from the studies that support the evidence that vitamin E is protective against heart disease. Dr. Kenny Jialal at the University of Texas Southwestern has given various dosages of vitamin E to determine how much is needed to protect LDL from oxidation. He has found 400 IU produces statistical significance in LDL oxidation prevention.
If you look at epidemiological evidence, the reports such as those by the Harvard group in the New England Journal of Medicine in May 1993, indicate that more than 100 IU for more than two years may be required before you receive the full benefit from vitamin E.
The latest study, the CHAOS Study, was published in The Lancet.  In this study, some subjects were given 400 IU and others 800 IU, and at most, they managed to double or triple the plasma concentration of vitamin E in the subjects. Were talking 2,000 people, so its quite clear to me that if you give huge doses of vitamin E, you are not raising the plasma concentration tremendously. But, in this group of heart attack victims, the supplemental vitamin E was found to decrease the risk of a second non-fatal heart attack by more than 75 percent.
Passwater: Have you found evidence to support the theory that 400 or 800 IU of vitamin E supplements increase the tissues stores of vitamin E?
Traber: Yes. We have fed deuterated alpha-tocopherol to two terminally ill volunteers in a study we did with Drs. Graham Burton and Robert Acuff in Tennessee to measure the vitamin E uptake and distribution.  We used two doses; one was a 30 milligram dose of deuterated alpha- tocopherol, as 15 milligrams of natural RRR-alpha-tocopheryl acetate and 15 milligrams of synthetic, all racemic alpha-tocopheryl acetate. As we discussed near the beginning of this section, this is the same kind of vitamin E that you could get from your store shelf except it has been labeled with a stable isotope so that we could follow its pathway in the body.
One volunteer got 30 milligrams, the other person got ten times as much, but the same forms. We reasoned that 30 milligrams was roughly what one could get if they were really good about eating vitamin E-containing foods. The 300 milligrams was in between the 100 IU and the 400 IU capsule, the most common level of supplementation that people are taking in the United States.
What we found that I think is absolutely startling --this has been accepted for publication in the American Journal of Clinical Nutrition we found that with the small dose (30 milligrams per day) that only about 6 percent of the vitamin E in the tissues was labeled. Now this person took vitamin E for almost a year. This dose might be even equivalent to the amount of vitamin E that is in a multi-vitamin tablet. The other terminally ill person the one who took the large dose -- took that 300 milligrams for two years before death occurred. These subjects donated their tissues for our study for which we are very grateful.
What we found is that virtually all the tissues had double the tocopherol content in the person taking taking the 300 IU vitamin E supplement. Meanwhile, the regular unlabeled form was about the same in the two people. In essence, what we found was that the total deuterated tocopherol virtually doubled in the person taking the larger dose. Of the total amount there, 60 percent was labeled with deuterium from the supplement we gave. These data tell me that those people who are taking vitamin supplements are actually loading all their tissues with twice as much vitamin E as they normally would have. It is interesting that the doubling of tissue vitamin E is about the same as the doubling we saw in the plasma.
Passwater: Now the question is which came first, the chicken or the egg? It kind of looks like you absorb vitamin E; this then gets transported in the plasma, and from there it is moved to all the lipoproteins during chylomicron catabolism. Once it gets to the liver, the liver can only put so much vitamin E back into circulation.
Traber: There is a limitation on that. The lipoproteins are always returning to the liver so that their job is to be like little trucks moving dietary fat out of the liver to the tissues where it can be used. These "trucks" keep going out and coming back in again. And when they come in, I think there is only so much vitamin E that can be loaded into them and put back in the circulation. I think the limiting factor in plasma vitamin E is TTP. But I havent been able to show it. Weve been working on this for three years and, even though we dont have the answer, I think we are getting closer.
Passwater: We have pretty much been describing the tocopherol transfer protein and its involvement with the delivery of vitamin E to tissues. What do we know about the uptake of vitamin E by tissues?
Traber: Tissues express (increase their production of ) the LDL receptor when they need cholesterol. The LDL receptor binds LDL that is in the plasma. LDL is the so-called "bad cholesterol" that everybody is always trying to reduce, but in fact it is a major delivery system for cholesterol needed by tissues. At the same time LDL delivers cholesterol it delivers vitamin E because vitamin E is part of the LDL particle. When LDL is taken into the cell by the LDL receptor, the particle is catabolized (broken down) and vitamin E is released.
There is also another mechanism for delivery of vitamin E to tissues that involves lipoprotein lipase. This is an enzyme that is involved in breaking up dietary fat, which is in the form of triglycerides, and releasing fatty acids so that they can be taken up by adipose tissues. The adipose tissue gets its fat from the diet, and in fact, in the same gigantic lipoproteins (the chylomicrons) that carry around the dietary fat also carry vitamin E. It turns out that lipoprotein lipase actually delivers vitamin E along with fats to adipose tissue.
Then just to make life really interesting, vitamin E can be delivered to tissues by one other non-specific mechanism. Vitamin E is a molecule that can move between membranes and so in the circulation, lipoproteins can give their vitamin E to tissue membranes, and thus, deliver vitamin E to the tissue. All of those kind of mechanisms are non-specific for the vitamin E form
Dr. Dutta-Roys group has been working hard on a tocopherol binding protein that is smaller than the one that is present in the liver. [45, 46] We dont know what that binding protein does. It is about 15,000 molecular weight; the one in the liver is about 32,000 molecular weight and the smaller tocopherol binding protein could possibly be regulating vitamin E concentrations in tissues. But, really, it is pretty much unknown what regulates tissue concentrations. That continues to be one of the things on which we are working very hard.
Passwater: Is this smaller tocopherol binding protein specific for RRR-alpha-tocopherol just like TTP? And, whats its short designation?
Traber: The tocopherol binding protein has not been completely characterized nor does it have a jazzy abbreviation. It binds alpha-tocopherol better than gamma-tocopherol, but we dont know its amino acid structure nor its nucleotide sequence. So details on this protein are just in their infancy.
Passwater: When LDL carries tocopherol into the cell interior via LDL receptors and then releases the tocopherol after catabolization how does the tocopherol find its way into both sides of the bi-membrane and distribute itself?
Traber: Gee, you ask such easy questions! Alpha-tocopherol is a fat-like molecule, so it shouldnt move away from membranes very easily. Thats why I think every tissue must have a tocopherol transferring protein. Only trouble is, no one has found any proteins, except TTP and the other 14.2 kDa protein we just discussed.
Passwater: Do various types of tissues or various organs have differing needs for taking up vitamin E?
Traber: I think so. We have done a couple of studies that help to answer this question. We did a long complicated study looking at the vitamin E content of adult beagle dogs that were fed a vitamin E-deficient diet. What we found is it took a really long time for the brain to give up its vitamin E. Other tissues gave up vitamin E really readily but virtually every tissue has a different concentration and so it is not clear at all what determines the vitamin E content.
Recently, we measured vitamin E forms in hairless mice, and found that the brain has only alpha-tocopherol, while the skin has about 15 percent tocotrienols. This was surprising because the mice were fed regular laboratory mouse chow, not a special diet. We are still trying to figure out what these various forms do. 
Passwater: In part I, we talked a little about the tocotrienols possible being better antioxidants in the membranes if they could get there. You cautioned that although some tocotrienols might reach some tissues soon after they were absorbed but before they reach the liver, the liver would filter most of the tocotrienols out and the TTP would not efficiently load the tocotrienols from the liver back into lipoproteins for transport to tissues. Would you elaborate?
Traber: TTP doesnt seem to recognize the tocotrienols to put them into the lipoproteins very effectively. Meanwhile, if you take a supplement of vitamin E you know that all of the different forms are being absorbed. This goes along with studies showing that the tocotrienols are absorbed; they are going to be passed by the chylomicrons to essentially all of the tissues during chylomicron catabolism, during that one pass through the circulation.
So it is possible that we are delivering tocotrienols to tissues, but when you measure the plasma you dont see them again because they are leaving quickly. So no one has done this study of feeding tocotrienols to people to look at what is in the tissues. We dont even know what is in the skin. I think there have been studies done where large amounts of tocotrienols were fed to animals and then the researchers did find some tocotrienols in some tissues.
This type of research requires very sensitive and specific analytical methods to do the chemical analyses. I think it is still an open question as to whether tocotrienols in vivo are more effective antioxidants. What I do think is clear is that they dont have as high a level of biologic activity as alpha-tocopherol. But it is not clear to me that biologic activity and antioxidant activity are the same. It is not clear, for example, which is more important for protection against heart disease. This would require us to make a judgement on which of the following is more important -- what is in the plasma or what is in the tissue?
We always end up with questions. Thats the trouble with thinking you know the answer.
We dont know a lot about the tocotrienols because they are not that popular an item in the American diet so they have not been looked at extensively here. There has been a lot of work done by the Malaysians where because they eat palm oil and thus have a lot more in their diet. Basically, I personally havent measured the levels of plasma tocotrienols in people who eat a lot of palm oil.
Dr. K. C. Hayes and colleagues tried to do a study looking at plasma concentrations of tocotrienols and basically what they found is that when you feed the tocotrienols and then measure during the post absorption about the first six to ten hours --you can find tocotrienols in the plasma.  After that, they seem to disappear from the plasma.
We did a study looking at tocotrienols applied to the skin of hairless mice and we developed a whole fancy method for measuring tocopherols and tocotrienols in a single run with an HPLC with electric chemical detection. What we found much to our surprise in the animals that didnt have any tocotrienols applied to the skin we found tocotrienols. You can imagine I was screaming in the lab, "Whats going on; you guys are so messy, come on, clean up, behave here, lets try again. I dont want any tocotrienols anywhere near these mice. Lets measure these skins again." We found in animals that had never even seen the tocotrienols, that yes they had tocotrienols in the skin. Then we analyzed the diet and we found that the chow that was being fed to the mice in fact was probably made with palm oil. That was enough to put measurable amounts of tocotrienols in the diet of the lab animals. The most surprising part was that the skin of these animals had about 15 percent of the vitamin E was tocotrienols. This gets back to that question of what about tissue concentrations because we found that the brain only has alpha tocopherol. The skin had 15 percent tocotrienols. The other tissues, like the liver or the heart, or the lungs they had a smaller amount, a couple of percent. So, it really looks like different tissues collect different forms of vitamin E. We are still trying to figure out exactly how it gets to the skin and whats going on. I think it is an interesting thing that maybe if we had more tocotrienols in our diets, we would have nicer looking skin. Thats always a thought. The Malaysians have such beautiful complexions.
Passwater: Recently, Dr. Marvin Bierenbaum and colleagues from the Jordan Heart Institute in New Jersey gave a poster presentation showing that supplementation for three years with a good amount of tocotrienols regressed carotid atherosclerosis to a great extent.  Tocotrienols are going to become of great importance in the years to come as this information gets out. Hes published his findings at regular intervals -- six months, one year, two years, and now three years -- and with the passage of time, the regression (measured ultrasonically) has continued. I think that this is very exciting development.
Dr. Bierenbaum and his colleagues are giving about 324 milligrams of mixed tocotrienols and 96 milligrams of RRR-alpha-tocopherol. This may or may not be related to the action of vitamin E. It may be an independent action of the tocotrienols. Have you been following this?
Traber: I followed it some and I am familiar with the other research he published. I think that this is an effect of the antioxidant function of vitamin E. As I have pointed out in Part II, RRR-alpha-tocotrienol appears to have the greater antioxidant activity in vivo, especially in membranes.
There are several possibilities for why tocotrienols are effective in treating atherosclerosis. One might be that it is the antioxidant activity of the form of vitamin E -- whether it is alpha-tocopherol, alpha-tocotrienol, or the gamma-tocotrienol is involved as it protects the membrane. Then there is the possibility that alpha-tocopherol is having some effect on protein kinase C and that is causing less inflammation in these tissues. The third possibility is that gamma-tocotrienol inhibits cholesterol synthesis. It is not clear how much this is affecting the atherosclerotic lesion. Most of the cholesterol in the body in fact doesnt come from the diet, it comes from new synthesis. So these patients may be benefitting from what may be a "triple whammy" of different mechanisms of vitamin E.
Passwater: Wouldnt these three mechanisms just halt the progression of plaque formation, but what would explain the removal of the plaque material?
Traber: One possible mechanism could involve macrophages. Their job is to remove garbage. Macrophages are sitting there in the atherosclerotic lesion. You can see that if you stop hitting them over the head with more cholesterol, they might then have a chance to get rid of the cholesterol that is there.
This is just my speculation. I think what the Bierenbaum group has done is show ultrasonically that the lumen of the arteries are wider and more blood is flowing through them. I dont think anybody has shown yet that cholesterol is being removed from the lesions.
This is just my speculation. I think what the Bierenbaum group has done is show ultrasonically that the lumen of the arteries are wider and that more blood is flowing through them. I dont think anybody has shown yet that cholesterol is being removed from the lesions.
Passwater: How about the involvement of cellular adhesion molecules such as VCAM and ICAM?
Traber: VCAM and ICAM are adhesion molecules that are on the capillary wall and the surface of white cells. They cause white cells to stick to capillaries. Specific signals, such as in response to wounds and infection, cause the expression (production) and activation of certain adhesion molecules. The expression of these molecules is dependent on the redox (oxidation-reduction status) state of the cells. Thus, cells with lots of vitamin E or other antioxidants are less likely to make adhesion molecules. This is a new area and I havent done any research in this area myself.
What I would like to point out here is this really fits with the CHAOS study. In the CHAOS study, they fed vitamin E to people who had proven heart disease and who had actually had heart attacks already. Those people who received vitamin E had 75 percent less risk of having a second heart attack compared to the people who received placebos. I think the data is accumulating that vitamin E is really protective in heart disease.
Passwater: The FDA invited those of us involved with antioxidant research to a conference held at the National Academy of Sciences in November 1993. The purpose was to provide the FDA with scientific input on antioxidant vitamins and the prevention of heart disease and cancer as mandated by Congress.
The oxidized-LDL research was of quite a bit of interest. Every speaker in one session had presented evidence supporting the oxidized-LDL theory of atherosclerosis. Then a scientist from the audience was asked by the FDA to present new information. She was Dr. Lenore Kohlmeier from the University of North Carolina. She followed everybody else, each person giving glowing reports about their studies showing significant correlation between vitamin E and prevention of heart disease, and she reported on a study in which the research team took biopsies from patients in emergency wards. They compared the vitamin E content of the adipose tissue from the gluteus maximus with the incidence of those patients who had heart disease rather than other complaints.
Although she told us that they couldnt find any correlation, the study as it was published about a month later did show correlation with taking vitamin E supplements and reduced incidence of heart attacks. She said, "to summarize it, no independent association between alpha-tocopherol levels measured in gluteal fat biopsies taken within 24 hours of an infarction and myocardial infarction risk, was seen as comparing them with controls largely drawn from the native local population in question. We have no significant relationship between alpha-tocopherol concentrations and myocardial infarction risk in these nine countries."
When she was introduced, she said she had evidence that would change the picture of the other findings. I vividly remember her smug smile when she stepped down from the podium and remarked something like, "sorry to spoil your fun, but it doesnt look to be true." Yet, the paper as published  said, "the findings for alpha-tocopherol are compatible with previous observations of reduced risk among vitamin E supplement users only."
Is the vitamin E content of adipose tissue a reliable indicator of vitamin E intake?
Traber: Yes. This is also in agreement with the study we were just discussing  Many avenues of research are coming together when looking at vitamin E and heart disease. Epidemiological studies such as Dr. Fred Geys and those from Harvard.
Passwater: And my 1976 study of 18,000 persons which few scientists seem to know about.  I only mention that because it impacts on the Harvard studies. Since I published the details in many books and articles, it has encouraged many people to take vitamin E supplements through the years. Thus, they were protected for decades, and as a result, this has shown up in the Harvard studies. The Harvard studies would have nothing to show if there wasnt a large number of people taking vitamin E supplements for the to compare against those not taking the vitamin E supplements. They found that only those who took vitamin E supplements above 100 IU for two years or more had the reduction in heart disease incidence. I kid Drs. Charles Hennekens and Julie Buring about that a lot. It amuses me that scientists can study groups like that and not wonder why the people decided to take the supplements. Certainly they had good reason to invest their time and money in the chore of taking vitamin E supplements over decades.
Traber: Interesting. Well. In addition to these epidemiological studies, we now have mechanistic studies such as those showing that vitamin E reduces the oxidation of lipoproteins. There may be many factors in heart disease risk, and vitamin E may be involved in controlling several or even most of these factors. There isnt just one answer. I think fat is important, cholesterol is important, but it is quite clear there is an inflammatory component that can be redox - regulated.
Passwater: Yes, and lets also note the fact that alpha-tocopherol, and especially gamma-tocopherol, traps the peroxynitrite formed from the nitric oxide released by macrophages during inflammation; this is another mechanism by which vitamin E prevents heart disease. Also important are the roles of folic acid, vitamin B-6 and vitamin B-12 in reducing homocysteine build-up which can initiate arterial damage and produce free radicals in the artery wall which in turn can oxidize LDL.
Well, its clear that the good news about vitamin E reducing the risk of heart disease has moved from the research journals to the heart specialty journals, and now, to the general medical journals. Its official now; the New England Journal of Medicine published a review article in August. 
Thanks to your research, we better understand why vitamin E supplements help protect us. Dr. Traber, thank you for your chatting with us about your research and also taking the time to cover some of the basics about this exciting vitamin.
NOTE: In Part II of this series, Dr. Traber and I discussed our preference of alpha-tocopherol, rather than tocopheryl esters for topical applications such as ointments and cosmetics. Since this was a side issue, we didnt provide references to the literature. However, since that time there seems to be a proliferation of mass-market products being advertised that use tocopheryl acetate (usually synthetic at that). I would like to refer cosmetic chemists to reference 53 which reports that there is no conversion of tocopheryl acetate to free tocopherol in the skin and that under some conditions, tocopheryl acetate enhances skin cancer development whereas free alpha-tocopherol significantly reduces experimental UVB carcinogenesis. I dont imply that this report represents the totality of the evidence nor that it is the last word, but it does support our discussion in Part II. My advice is that until we have more information, consumers should play it safe and use free alpha-tocopherol in their cosmetics and sunscreens.
© 1998 Reprinted with permission of the copyright owner, Whole Foods magazine, Whole Foods, Inc.