Part II: Pro-oxidants and Singlet Oxygen: An Interview With Dr. Lester Packer
by Richard A. Passwater, Ph.D.
Professor Lester Packer, Ph.D. is the Director of the Membrane Bioenergetics Group and Professor of Molecular Biology at the University of California at Berkeley. His distinguished career in teaching and research has included appointments at Dartmouth Medical School and the University of Texas Southwestern Medical School. Dr. Packer is also a Faculty Senior Scientist in the Energy and Environment Division of the Lawrence Berkeley Laboratory.
Dr. Packer is the author of about 40 books and about 400 scientific research articles. He is the Executive Editor of "Archives of Biochemistry and Biophysics," and serves on the editorial boards of "Free Radical Biology and Medicine," "Journal of Applied Nutrition" and "Journal of Optimal Nutrition."
Dr. Packer's research interests include the role of vitamin E at the membrane and cell level, nutritional and physiological studies in animal and human exercise, and studies of vitamin E in the skin.
To the Reader: The first part of this interview appeared in the February 1993 issue to provide the foundation needed to understand how free radicals and other reactive oxygen species damage low density lipoprotein to start a major process leading to heart disease. Dr. Packer also discussed how antioxidants prevent this damage, which set the stage for the interviews with Drs. Janero, Kritchevsky, Esterbauer and Steinberg. In this installment, Dr. Packer and I discuss the other side of the coin, the potential for involvement of pro-oxidants.
When this interview was conducted in September 1992, the scientific community was scurrying to develop data that would answer the questions raised in a week earlier by a study of Finnish men suggesting a possible involvement of high body-stores of iron.  The researchers, led by one of my favorite Finnish researchers, Dr. Jukka Salonen of the University of Kuopio, studied 1,931 Finnish middle-aged men for three years and found a correlation suggesting those men who had ferritin (an iron-storage protein) levels above 200 micrograms per deciliter of blood were associated with a 2.2-fold greater risk of acute myocardial infarct (heart attack). Several laboratories began examining their stored samples of blood from other studies to see if they could find a similar correlation. As of November 1993, I am not aware of any confirming studies reported at meetings of published in the scientific literature.
At this writing, it is still too early to determine the relevance of the Salonen report. Dr. John L. Beard of Pennsylvania State University has pointed out in Nutrition Reviews that several issues "mitigate the suggestion that high-normal iron stores lead to heart disease."  The mitigating factors include what is often called "the Finnish Factor" which acknowledges the fact that the data from Finnish men are often askew of those from other Countries possibly due to the consequence of either a selenium deficiency or genetic effect. 
Other mitigating factors are that the mean blood ferritin level in this population was high and the prevalence of hemochromatosis was unknown. The blood ferritin levels of the Finnish men who did not have heart attacks were similar to those of the U. S. population. The Finnish men who did have heart attacks were in a small group who had high blood iron and ferritin, which is an indication of possible hereditary hemochromatosis. Hemochromatosis is caused by an autosomal recessive gene defect which is manifested as excessive iron storage, even when dietary iron is low.
Hemochromatosis is really not a "rare" disease. According to Dr. Margit Krikker, professor of medicine at Albany Medical College in New York, and president of the Hemochromatosis Research Foundation, "Hemochromatosis has a gene frequency that's more common than for any other known genetic disorder, including cystic fibrosis, muscular dystrophy, sickle cell anemia, and even familial hypercholesterolemia. The disease's prevalence was once thought to be one in twenty thousand, but it is actually between three and six in one thousand (0.3 - 0.6%). This means that between 600,000 and 1.6 million people in the United States are affected." 
Regardless of future findings, the Salonen report calls attention to several facts. The first is that we must prevent oxidative stress by keeping the antioxidant-to pro-oxidant ratio in favor of the antioxidants. We do need some of the pro-oxidants such as the transition elements iron and copper to produce antioxidant enzymes such as catalase and superoxide dismutase. Keep in mind that no published data has linked dietary iron with heart disease, and that stored iron is independent of dietary iron in "normal" persons not having the defective gene.
Secondly, since hemochromatosis is fairly common and it can cause great suffering and death, can be easily treated by donating blood and/or chelators, physicians should routinely screen for hemochromatosis. If one's blood iron level is high, then serum ferritin level is a concern. As discussed later, ferritin is a transporter of iron. The ferritin of interest is the haloferritin, not the apoferritin.
For screening purposes, transferrin saturation is a good initial screen, followed by a ferritin test if transferrin saturation is high.  Persons having hemochromatosis should be under the care of a physician.
In the hemochromatic patient, the iron is believed not to be present in a form to interact with vitamin C. If stored iron does interact with small amounts of vitamin C in the body as in the Fenton reaction to produce free radicals, greater amounts of vitamin C and other antioxidant nutrients would serve to neutralize these free radicals.
Some have shown concern that vitamin C itself could become a pro-oxidant in the body like it can in the test tube. Harvard researchers have shown that vitamin C is always an antioxidant in the body and never a pro-oxidant. 
There is also some confusion about vitamin C and iron absorption. Vitamin C improves absorption of non-heme iron in individuals with low iron stores, but there is no good evidence that it does so when body stores are high. Ascorbate also helps chelating agents remove iron via urinary excretion.
With this brief update completed, let's get back to the interview with Dr. Packer. When we left off, Dr. Packer had just described the supplements that he took.
Passwater: It seems that most of the researchers that have been involved in antioxidant nutrient research for some time take supplements themselves. Most of our readers are familiar with vitamins C and E, and the carotenoids -- I take those antioxidants plus the bioflavonoid, Pycnogenol -- but many of our readers have never heard of alpha-lipoic acid. In fact, I didn't pay much attention to it myself until your research pointed out that it was an important member of the antioxidant team. And, since it is a sulfur-containing antioxidant, I became very interested.
You have even called lipoic acid an important partner in recycling other antioxidant nutrients.
Packer: Lipoic acid (also called thioctic acid) is a disulfhydryl coenzyme that is a co-factor in at least two major energy producing reactions in the body. My studies have focused on its role in the antioxidant cycle. Lipoic acid and Coenzyme Q-10 are partners in vitamin C recycling, which in turn recycles vitamin E. Together, lipoic acid, coenzyme Q-10, vitamin C and vitamin E help conserve the carotenoids in tissues.
Passwater: Dr. Packer, in Part I, we discussed the basic oxygen-radical/antioxidant relationship and its role in health. You mentioned that iron can donate an electron to oxygen to produce a superoxide radical. Many people are concerned about the possible implications of a recent Finnish report suggesting that heart disease may be linked to the amount of iron found in the blood in the form of ferritin. (1) Many people are concerned that dietary iron can increase blood ferritin levels, and thus, increase their risk for heart disease. Some physicians have singled out iron-containing supplements as being dangerous. In fact, the newspaper reports quoted several physicians who saw a chance to gang up on all vitamin and mineral supplements.
What do we know about the dangers of the dietary pro-oxidants such as iron?
Packer: Free iron, that is, iron that has been momentarily liberated from iron-containing proteins can convert mildly reactive oxygen radicals into highly reactive oxygen radicals, such as hydroxyl radicals. The research led by Dr. Jukka Salonen of the University of Kuopio recently reported was in Circulation . The study is an epidemiological study. Such studies do not prove anything -- they just verify associations rather causal or not. And, one such study is in no way definitive.
What intrigues me about the study is that although the association was reported to be with the amount of ferritin in the blood, there was also mention of a correlation with dietary iron. However, this data is not presented in the tables or discussed in further detail.
Passwater: OK, lets hold off the discussion of how dietary iron may possibly be related to heart disease, and review the background or iron transport for our readers. As I discuss in my book "Trace elements, hair analysis and nutrition," iron, in contrast with most other minerals, is regulated in the body primarily by absorption rather than by excretion.  A number of studies have confirmed the critical importance of the gastrointestinal tract in controlling the total body iron stores. [8-10] Urine contains very small amounts of iron, and the only iron found in feces is that unabsorbed from the diet.
Not all forms of iron are absorbed equally. Normally, six to ten percent of the iron in food is absorbed, but iron-deficient individuals can absorb more than 15 percent. Persons with iron-deficiency anemia may absorb 50 to 60 percent of the same iron.  The iron status of the individual, the individual's red blood cell production rate, and other factors regulate the amount of iron transported across the intestinal mucosa.
Packer: Yes, and keep in mind that even in people who eat a lot of iron, there is not much free iron in the body. People can overload with iron, but little of the excess iron is found not contained in storage ferritin. Even Africans who consume a lot of iron because they cook in and eat from iron pots, have little "free" iron in their bodies. They have abnormal quantities of iron in their livers and spleens stored in organelles that have developed that even have membranes around them. Their bodies have sequestered the iron in these deposits.
Passwater: Speaking of peoples from other countries, that reminds me that there is a significant genetic component to iron storage -- aside from those having a genetic-defect disease. The control of ferritin and the plasma-membrane receptor for transferrin, an iron-binding beta-globulin that aids the transport of iron to bone marrow and tissue storage areas, is by genetic expression. (Transferrin is also called siderophilin) The balance between these proteins regulates iron availability, since the transferrin receptor is required for the uptake of iron, and ferritin is necessary for storage of any iron temporarily in excess of immediate need.
Packer: It can be expected that a lot of individual variability in the dynamics and regulation of iron storage occurs in people through environmental influences and genetic make-up.
Passwater: The body produces ferritin, a protein that binds iron, primarily to store iron in cells.  Ferritin is found primarily in the liver, spleen and intestinal mucosa. The iron stored in ferritin is in the form of a ferric oxide complex which is surrounded by the apoferritin protein shell. For every microgram of ferritin in the blood, there is thought to be about eight milligrams of ferritin stored in cells.
Normally, in healthy adult women, there are 20 to 120 micrograms of ferritin in each liter of blood, with the "mean" value being 46 micrograms/liter. For healthy adult men, the range normally is from 30 to 300 micrograms per liter of blood, with the "mean" value being 127 micrograms/liter.
It is thought by some researchers that maximum iron loading of ferritin stimulates lipid hydroperoxide decomposition, thus, those who can store their iron in the iron-binding proteins in such a way that their ferritin loading never approaches a maximum are at an advantage.
There is also another compound, called hemosiderin, that can be considered to be an iron-storage compound. Hemosiderin is a granular iron-containing yellow pigment formed during decomposition of hemoglobin. When hemosiderin deposits form in tissues during red blood cell breakdown, the condition is called hemosiderosis.
Iron carried in the bloodstream is mostly carried in a protein called transferrin. Normally, in the typical healthy adult, there are 200 to 400 milligrams of transferrin in each deciliter of blood.
Another protective protein is ceruloplasmin. Ceruloplasmin catalyzes the oxidation of ferrous iron [Fe(II)] to ferric iron [Fe(III)] which serves to prevent the formation of hydroxyl free radicals.
Packer: The iron in the body is also incorporated into enzymes, hemoglobin and myoglobin. But, when these proteins breakdown at the end part of their normal "lifespan," they release iron into the blood.
Free "reduced" iron is dangerous because it is readily available to react, therefore, the body goes to great effort to trap free iron ions. The body binds iron to transferrin and carries it in the blood to the cells where the iron is transferred to ferritin for storage in the cells.
The potential danger always lies in releasing the iron from proteins. Not only from transferrin and ferritin, but from the hemoglobin of red blood cells. The release of iron can occur through tissue injury. As an example, when you are running on a hard payment, you crush red blood cells every time your foot hits the ground, usually some iron leaves the red blood cells. Iron can also be released due to some disease processes.
If you have more "free" iron around because there is more iron-containing proteins available to be damaged, then it is logical to expect more iron-catalyzed free-radical production. Therefore, this report is not illogical, and more studies should be made.
Passwater: Ferritin was chosen as the risk marker in the Finnish study because it usually correlates to total iron stores in the body. However, blood ferritin levels can be raised by inflammation, infection, chronic disease, thalassemia, liver disease or Hodgkin's disease. So a study of the association between ferritin and heart disease must determine if heart disease raises blood ferritin, if some confounding factor increases both ferritin and heart disease, or if increased ferritin levels increase the heart disease incidence. An epidemiological study does not determine which does what, but if additional epidemiological studies support the Finnish study, much effort should be devoted to laboratory and clinical studies.
Epidemiological studies by themselves are not unlike associating umbrellas usage with the incidence of rain -- while there is a high correlation between seeing umbrellas and having rain -- it is not the umbrellas that cause the rain.
Packer: What we can look at in the laboratory is the role that oxidative stress can play in releasing iron from its protein carriers. In a book that I edited, Drs. Barry Halliwell and John Gutteridge discuss how superoxide radicals liberate iron from ferritin and thus promote lipid peroxidation.  Also, it has been reported that an in vitro study showed that a combination of physiological concentrations of hydrogen peroxide and hemin induces rapid peroxidation which releases free iron. There are other studies supporting a possible synergism between oxidative stress, free iron and oxidized LDL. Oxidized LDL is now thought to be a major factor in promoting atherosclerosis.
Passwater: There are other preliminary studies and observations that seem to fit this thesis. The consensus is that women are protected from heart disease until after menopause by estrogen. However, as Dr. Jerome Sullivan of V. A. Medical Center in Charleston has pointed out, this protection could arise from the fact that women have low iron ferritin due to their monthly losses.  Neither hypothesis has as yet been well supported and either could be correct or both may be incorrect. Other data link estrogen with blood platelet aggregation which fits observations involving atherosclerosis and thrombosis very nicely.
Support is found in other common observations that could be given different interpretations to exploit the possible iron link. Aspirin is thought to reduce heart attacks because it reduces the tendency for blood to clot, and thus may reduce the probability of clots from blocking the flow in coronary arteries which is what happens during a heart attack. A new interpretation suggested by Sullivan is that aspirin may be effective because it causes gastrointestinal bleeding, and this blood loss reduces body iron levels.
Some have presented a new interpretation of an old line of thinking to extend this new observation -- as yet unconfirmed -- to explain a suggested link between red meat and heart disease -- not by red meat's fat content, but by its iron content.
Other so-called associations have been reported, but they too are still weak at this time. As an example, there have been reports that carcinogens fed to laboratory rats on a high iron diet have increased numbers of tumors.  This is not a test of body stores, nor necessarily of dietary iron. The effect could be merely due to the fact that ionic iron in the diet reacted with the carcinogen to make it more potent.
In other reports, men with high iron stores were more likely to develop cancer during the ten years of the study than men with lower iron stores. [14,15] However, iron stores were not linked to dietary iron intakes, "which suggests cancer-prone individuals either exhibit altered absorption or metabolism of iron."  Perhaps these men had the faulty gene that leads to hemochromatosis, and that two of the unrecognized "risks" of this inherited disease are cancer and heart disease. Undoubtedly, this will rapidly become a fertile area for research. You have already done some research with iron, free-radicals and heart disease. What have you looked at?
Packer: In studies that we have done in vitro, where iron is studied during ischemia reperfusion of the isolated animal heart, iron catalyzes oxygen damage to the heart tissues. However, if we bind the iron to a sequestering agent in a specific manner, iron is not available to catalyze oxygen reactions and free radical damage is minimized and this is accompanied by greater recovery of the hearts in vitro in terms of mechanical activity (contractility).
Passwater: This will be a practical application for heart surgery. It builds on the natural sequestering that our bodies use. Iron-binding and copper-binding sequestering agents can be important components of our antioxidant defenses because they tie-up transition elements whenever they are in their pro-oxidant ionic states. This allows our bodies to keep adequate amounts of these essential elements in use as components of enzymes and other biomolecules, and then when these larger molecules are broken down so as to momentarily expose the ionic elements, they can be harmlessly swept away.
Dr. Packer, let's move away from the transition elements and look at another pro-oxidant, singlet oxygen. Singlet oxygen is not a free radical, but is a reactive oxygen specie. Singlet oxygen is an excited electronic state of oxygen which means that it contains more energy than the "ground" state or normal molecular oxygen. When this excess energy of singlet oxygen is discharged, damage to body molecules can occur. Although this is not free-radical damage, it is another way in which oxygen can be what you like to call "our dangerous friend." Is singlet oxygen a major problem to the body?
Packer: In plants where photosynthesis is occurring, this is a major problem. The photons of ultraviolet energy are being absorbed in the process of converting water and carbon dioxide into oxygen and food. Plants manufacture carotenoids such as beta-carotene to protect against singlet oxygen by converting it back to normal oxygen.
In the human, the action of some enzymes produces singlet oxygen in the dark regions of the body such as myeloperoxidase of macrophages. However, it's not known whether this specie of activated oxygen is damaging in people.
Passwater: You mentioned that beta-carotene can protect against singlet oxygen. How about lycopene and vitamin E. Vitamin E can quench singlet oxygen through an essentially (99%) physical process (Stevens et al, 1974), is this a meaningful action for vitamin E in the human?
Packer: Vitamin E can quench singlet oxygen, although not as efficiently as carotenoids -- but since we don't know how significant singlet oxygen is for human health -- this may be a mute question.
Passwater: Dr. Packer, thank you again for helping to provide a foundation for the understanding of antioxidant nutrients, pro-oxidants and free-radical pathology.
1. High stored iron levels are associated with excess risk of myocardial infarction in Eastern Finnish men. Salonen, Jukka T.; Nyyssonen, Kristiina; Korpela, Heikki; et al. Circulation 86:803-11 (1992)
2. Are we at risk for heart disease because of normal iron status? Beard, J. L. Nutr. Rev. 51(4):112-5 (1993)
3. Increased risk of cardiovascular disease at suboptimal plasma concentrations of essential antioxidants: An epidemiological update with special attention to carotene and vitamin C. Gey, K. Fred; Moser, U. K.; Jordan, P.; at al. Amer. J. Clin. Nutr. 57(Suppl):787S-797S (May 1993)
4. Iron overload diagnosis missed. Anon. Med. World News, p110, (8 June 1987)
5. Screening for hemochromatosis. Edwards, Corwin Q. and Kushner, James P. New Engl. J. Med. 328(22):1616-20 (3 June 1993)
6. Ascorbic acid oxidation product(s) protect human low density lipoprotein against atherogenic modification: Anti rather than prooxidant activity of vitamin C in the presence of transition metal ions. Retsky, Karen L.; Freeman, Mason W. and Balz Frei. J. Biolog. Chem. 268(2):1304-9 (15 Jan 1993)
7. Trace elements, hair analysis and nutrition. Passwater, Richard A. and Cranton, Elmer M. Keats Publishing, New Canaan, CT (1983)
8. Bothwell, T. H. and Charlton, R. W. Annu. Rev. Med. 21:145-6 (1970)
9. Forth, W. and Rummel, W. Physiol. Rev. 53:724-92 (1973)
10. Linder, M. C. and Munro, H. N. J. Proc. Fed. Amer. Soc. Exp. Biol. 36:2017-23 (1977)
11. Role of free radicals and catalytic metal ions in human disease: An overview. Halliwell, B. and Gutteridge, JMC in: Packer, Lester and Glazer, A. N. (eds) Methods in Enzymology, vol. 186, pp 1-85, Academic Press, San Diego, (1990)
12. The iron paradigm of ischemic heart disease. Sullivan, Jerome Amer. Heart J. 17:1177-88 (1989)
13. Iron and the risk of cancer. Stevens, R. G. Med. Oncol. Tumor Pharmacother. 7(2-3):177-81 (1990)
14. Body iron stores and the risk of cancer. Stevens, Richard G.; Jones, D. Yvonne; Micozzi, Marc S. and Taylor, Philip R. New Engl. J. Med. 319:1047-52 (1988)
15. Iron: Health-enhancing or cancer promoting? Somer, Elizabeth Nutr. Rept. 42 (June 1992)
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