Hermann Esterbauer, Ph.D. is a professor of biochemistry and the Head of the Institute of Biochemistry of the Karl-Franzens-University of Graz in Austria. He serves on the Editorial Boards of Free Radical Research Communications, Biochemical Journal, Amino Acids, Free Radical Biology and Medicine, and Journal of Biotechnology.

Dr. Esterbauer's major fields of research are free radical reactions and lipid peroxidation in health and disease, particularly atherosclerosis, and the roles of antioxidants in health and in preventing disease.

The interest in the role of vitamin E and carotenoids in the reduction of heart disease by their preventing oxidation of low density lipoproteins continues to grow. This is virtually the title the ground-breaking report in the Annals of the New York Academy of Sciences in 1989 by Dr. Hermann Esterbauer.[1] Many scientists are now following up on this relationship. I have been interviewing the major scientists involved in this important research over the past several months. I covered the background of vitamin E and free radicals, but now I will begin to tie everything together in the next two interviews.

This month I have the pleasure of bringing Dr. Esterbauer's research to your attention, and next month, the research that started it all, that of Dr. Daniel Steinberg's group.

Passwater: Dr. Esterbauer, when did you become interested in vitamin E research?

Esterbauer: A long time ago. In the early 1960s I did my Ph.D. thesis on autoxidation of fatty acids contained in plant oils. Naturally, this evoked my interest in vitamin E as an antioxidative factor preventing rancidity. Later on, when we studied lipid peroxidation induced by xenobiotics in liver and liver cells, we realized, as many other investigators did, that vitamin E is perhaps one of the most important components in cell membranes protecting membrane lipids against oxidative damage by free radicals.

Passwater: What attracted your interest to this field of research?

Esterbauer: The University of Graz has a long tradition in lipid and lipoprotein research, and Prof. Erwin Schauenstein, the supervisor of my Ph.D. thesis, had the idea that perhaps some of the lipid oxidation products formed endogenously or ingested with food have a biological or pathological importance. More than 30 years ago, I isolated compounds from oxidized linoleic acid. Amongst many other substances I found 4-hydroxynonenal (HNE), a substance which is now, 30 years later, investigated in many laboratories throughout the world as marker of oxidative stress, and as a second "toxic messenger" of free radical damage.

Passwater: In 1987, you reported on the relationship between vitamin E and the oxidation of the cholesterol carrier, low-density lipoprotein (LDL). What piqued your curiosity to look at this possible relationship?

Esterbauer: In the early 1980s, the groups of Dr. Daniel Steinberg in La Jolla and Dr. Guy Chisolm in Cleveland published some remarkable papers on implications of the oxidation of LDL in atherosclerosis. [2-5] We were interested in whether we could identify some substances in oxidized LDL which we had isolated a long time ago from oxidized linoleic acid.

Together with my colleague, Dr. Gunther Jurgens, of the Institute of Medical Biochemistry, who had worked on lipoproteins for years, we set up experiments to oxidize LDL in vitro. Much to my surprise, the LDL, although containing a high content of linoleic acid, was very resistant to oxidation. In an article, which we published in 1987 in the Journal of Lipid Research, we commented, "For one of us (H.E.), who has in the past studied lipid peroxidation in many biological systems, the most surprising result was the high resistance of the polyunsaturated fatty acids in LDL against oxidation." [6]

We learned from these studies that nature protects LDL with vitamin E, carotenoids and perhaps other not yet identified antioxidants.

Passwater: We have discussed LDL in many of my columns and interviews over the past few years, but lipoproteins are still new subjects to the general public. Since the understanding of how vitamin E protects against heart disease requires a rudimentary knowledge of the role of lipoproteins, I am still looking for help in describing them to my readers. You describe have an excellent description. So let me ask you, what are low density lipoproteins?

Esterbauer: Cholesterol is a lipid, i. e., a fat-soluble compound. Therefore, cholesterol is not soluble in blood, which is a water-based fluid. To overcome this incompatibility, the body has designed a means to transport this fat-soluble compound inside water compatible particles called lipoproteins. There are several lipoproteins, but the two with which we are most interested are the low density lipoproteins (LDL and the high-density lipoproteins (HDL). In lay terms, LDL is associated with "bad" cholesterol and HDL is associated with "good" cholesterol.

LDL primarily carries cholesterol from where it is manufactured in the liver to various cells that need cholesterol. HDL primarily carries excess cholesterol back to the liver. LDL is the main carrier of cholesterol in our blood stream. In persons having normal cholesterol and other blood fats, typically about 60% of the total blood cholesterol is contained in LDL. Many epidemiological studies and case control studies have shown that increased levels of LDL are associated with an increased risk of atherosclerosis.

LDL is a very large spherical particle with a molecular weight of about 2.5 million, consisting of an oily central core of about l,600 molecules of esterified cholesterol and several hundred molecules of triglycerides. This core is surrounded by a shell of phospholipids, the polar head groups of these molecules face the outside and make the particle soluble in blood despite the high cholesterol and fat content.

It is important to keep in mind that LDL is not only rich in cholesterol but also in polyunsaturated fatty acids, mainly linoleic acid, arachidonic acid, which are - if not protected - highly susceptible to oxidation. On average, about l.500 molecules of PUFAs are present in an LDL particle. They clearly need protection by antioxidants. The major ones are vitamin E and carotenoids. Vitamin E is contained in the shell, whereas B- carotene is in the core.

Passwater: LDL is a carrier of cholesterol, but the cholesterol has to get inside of the cell to be used. Tell us a little about the receptors that recognize LDL, latch on to it and bring the contents into the cell interior.

Esterbauer: Embedded in the LDL shell is also a large protein termed apolipoprotein B. The Nobel Price winners, Dr. Joseph Goldstein and Dr. Michael Brown, discovered that a specific receptor (termed LDL-receptor) that can recognize apolipoprotein B of LDL is present at the surface of most cells in our body. When LDL binds to such receptors it is quickly taken up by the cell and the LDL particle is degraded intracellularly into its constituents. Most of them are reused again as building blocks for membranes and new lipoproteins.

The liver is particularly effective in removing LDL from the circulation. On average, an LDL particle circulates in the blood for about 2 days before it is cleared by this receptor-mediated uptake.

Passwater: For years, many researchers thought that LDL was the main culprit in initiating the cholesterol deposits in arteries. You mentioned that your attention was aroused by Dr. Daniel Steinberg's group's discovery that changed the direction of heart disease. They found that it is not normal LDL that is the problem, but oxidized-LDL, i. e., LDL that has been altered by free radical attack or reaction with oxygen. How does oxidized-LDL differ from normal LDL?

Esterbauer: I would like to have X-ray eyes and be able to actually see an oxidized LDL particle. From the chemical analyses which we made, it seems clear that it must look ugly, the beautiful architecture of normal LDL no longer exists. The antioxidants are destroyed, the polyunsaturated fatty acids and even the cholesterol moiety are heavily oxidized and partly polymerized. A large number of smaller and highly reactive break-down products segregate from the oxidizing lipids and emanate from the particle.

As recently shown by Dr. Edwin Frankel from the University of California, Davis, some of these new products are even volatile and can be detected in the gas phase above solutions of LDL. [7,8] Pathologically, perhaps the most important change in oxidized-LDL is that its protein, the apolipoprotein B, is damaged and altered to such an extent that is now binds to a "scavenger" receptor present on the surface of specialized white blood cells called macrophages.

Passwater: OK, now we are getting to the crux of the issue. Oxidized-LDL is taken up by special white blood cells and make these cells look foamy. What are these "foam cells" and what is their relationship to atherosclerosis?

Esterbauer: Pathological, microscopic and histochemical studies have shown that the fatty streak and plaques which form in the intima region of the major arteries are mainly made up of cells so altered in their appearance by engulfed LDL cholesterol that they are known as foam cells. Most of these foam cells develop from macrophages, which again stem from more general-purpose white blood cells called monocytes. The monocytes immigrate from the circulating blood into the arterial wall.

For a long time it was an absolute mystery how the macrophages engulf so much cholesterol. If macrophages were fed with normal LDL, even in high concentration, they did not become overloaded with cholesterol, nor did they develop to foam cells. A milestone was the discovery published by Dr. Daniel Steinberg's group in 1984 that macrophages fed with oxidized-LDL avidly took up this material and develop to foam cells. The uptake of oxidized LDL occurs in an uncontrolled manner through the macrophage scavenger receptor.

Passwater: Aha, you have just provided the perfect introduction to next month's interview with Dr. Daniel Steinberg. Dr. Steinberg will share with us the "eureka" moments that lead to the discovery of how antioxidant nutrients help protect us against heart disease. It is a very interesting story. But, I still wish to develop a basic overview of this process so that we can better understand what you have elucidated about the interactions of the various antioxidant nutrients in preventing LDL from oxidizing. Tell us more about the consequences of the different activity of oxidized-LDL compared to normal LDL. How do foam cells enter the arterial wall?

Esterbauer: Foam cells develop in an interior layer of the artery called the intima. It is important to realize that the foam cells develop in the arterial intima itself from resident macrophages. Foam cells do not form in the bloodstream as an immigration of foam cells from the circulation into the arterial wall is not possible. On the contrary, there is some evidence that foam cells have the capacity to emigrate from the arterial intima into the bloodstream.

Passwater: You point out that foam cells accumulate in the arteries. The proponents of the old "cholesterol theory," based on solely blood cholesterol levels, could not provide good reasons why cholesterol deposits did not form in veins as well as arteries. After all, the cholesterol concentration is the same in both arteries and veins. They attempted to dance around that issue with various explanations, but like the cholesterol theory itself, the answers did not stand up to scientific investigation. Why do foam cells accumulate in arteries and not in veins?

Esterbauer: The main reason has to do with the difference in pressure of the circulating blood in each. The lower blood pressure in veins causes less LDL infiltration into vein walls, than the higher pressure in arteries cause LDL infiltration into artery walls. Also, monocytes adhere to vein surfaces (endothelium) less than artery surfaces. Therefore, foam cells accumulate in arteries and not veins because arteries have more monocytes adhering on the artery surfaces and because the higher blood pressure causes infiltration of LDL.

The present opinion is that normal LDL which is continuously infiltrated into the intima layer of arteries encounter there an "oxidative stress" This oxidative stress is most likely mediated by activated macrophages which have been recruited at endothelial cells at the sites of injury to the lining of the artery.

Many other studies not directly related to atherosclerosis have shown a remarkable feature of macrophages. If they become activated they respond with an oxidative burst, whereby large amounts of oxygen radicals are formed. These free radicals likely deplete the nearby environment from all water-soluble antioxidants, as for example, vitamin C. LDL entrapped in such oxidizing "fire" would then also be rapidly attacked and oxidized. Once initial deposits of oxidized-LDL are formed in the arterial intima, a self-sustaining and accelerating process can commence, since compounds released from oxidized-LDL stimulate immigration of more and more monocyte-macrophages from the blood to the site, where oxidized LDL is deposed.

Passwater: Now that's a disastrous chain reaction. Please summarize what you have learned about the relationship between the antioxidant nutrients such as vitamin E and the carotenoids so far.

Esterbauer: I can only refer to our studies on the protection of LDL by antioxidants. One can isolate LDL from the blood and determine its oxidation resistance. One will always observe that LDL is only oxidized when it has lost its antioxidants. The first defense line is alpha-tocopherol and gamma-tocopherol (vitamin E), and the last defense line is the carotenoids, mainly beta-carotene. Much better than I can do it, Brown and Goldstein described the situation with the words "if LDL is depleted from its antioxidants, it is left to the mercy of oxygen" [9]

We could show by an ex vivo study that oral intake of vitamin E at daily doses of 150, 225, 800 and 1200 IU increased the vitamin E content in LDL in a dose dependent manner by about 40 to 110% above baseline values and the oxidation resistance of LDL increased more or less proportionally. [10] We also learned that the antioxidant efficacy of vitamin E varies rather strongly between individuals, the reason for that is still not known. With beta-carotene the situation is even more complex, in some subjects, as for example vegetarians or patients with fat-malabsorption, we found that oral intake of beta-carotene can significantly increase the oxidation resistance of LDL. But in other healthy subjects beta-carotene supplementation for 3 weeks had no effect whatsoever.

For adults the RDA for vitamin E is 15 IU per day, and I think this is too low to provide an adequate protection for LDL. One should also consider that very strong individual variations exist in absorption of vitamin E and its incorporation into LDL. Furthermore, the oxidative stress situation of individuals is variable.

So far, no consensus exists on the optimal dose of vitamin E. Professor Fred Gey from the University of Bern (Switzerland) recommends a plasma vitamin E level of around 30 micro molar. Such a level can perhaps be reached by most persons with an intake of about 100-200 IU per day.

Finally, I want to say that we must not only think of vitamin E but also on all other antioxidant nutrients, such as vitamin C, beta-carotene, selenium and perhaps others, such as flavonoids. In our body these antioxidants usually act in a concerted and synergistic way and only an optimal balance of all of them will ensure, at least in my opinion, an optimal health.

Passwater: Several scientists that I have interviewed in this series have mentioned that they have followed your lead in researching the role of antioxidant nutrients in protection against heart disease. What additional information have they added?

Esterbauer: This is a very kind comment by them. During this interview I have already mentioned several prominent scientists who contributed much more to the LDL oxidation theory of atherosclerosis than I did. Our major contribution, perhaps, was that we introduced quantitative clinical-chemical assays, which enable us and others to measure oxidation resistance of LDL and the protective effect of antioxidants. We have now so many biochemical and epidemiological evidence in support of the oxidation theory, what we need is a support by experimental animal studies, clinical studies and intervention trials. I want to mention in this context the work by Dr. Anthony Verlangieri from the University of Mississippi, who showed that in primates vitamin E is prophylactically and therapeutically effective in atherosclerosis. [11,12]

Dr. Jan Regnstrom from the Karolinska Institute in Stockholm studied the oxidation resistance of LDL in survivors of myocardial infarction and found a significant inverse correlation between severity of coronary atherosclerosis and oxidation resistance of LDL. [13]

Finally, I want to address again the important work by Professor Daniel Steinberg from the University of California, San Diego. He organized and chaired a round table consensus conference on antioxidants in prevention of human atherosclerosis, which was supported by the National Heart, Lung and Blood Institute in September 1991 in Bethesda, MD. The summary of the Meeting was published, and I quote from this paper, "It was the consensus that the evidence available justify a clinical trial of natural antioxidants." [14]

Passwater: What will you be investigating about this relationship next?

 Esterbauer: As I mentioned earlier, the efficacy of vitamin E to protect LDL against oxidation varies strongly between individuals, and we investigate presently the underlying reasons. Perhaps, genetic factors play an important role besides dietary factors. Another project of my group deals with the development of ELISAs (enzyme linked immunosorbent assay) for measuring oxidized LDL and other proteins damaged by oxygen radicals and lipid peroxidation in plasma, tissue and single cells. As you know, many researchers believe that oxidative stress is a major cause of many diseases. If this is so, assays to measure oxidatively damaged proteins should have a prognostic and diagnostic value.

Passwater: Thank you Professor Esterbauer for your lucid explanations in explaining your research to us.