Balz Frei

Questions and Answers with Dr. Balz Frei

Balz Frei, Ph.D.
Professor of Biochemistry & Biophysics
Director and Endowed Chair
Linus Pauling Institute

The following is a question and answer session between the editor and Dr. Balz Frei.

Q: How did you become interested in vitamin C?

A: When I was a post-doc in Bruce Ames' lab in Berkeley, I investigated antioxidant defenses in human plasma. I exposed plasma to different types of oxidizing conditions to determine which endogenous antioxidants are able to inhibit lipid peroxidation. It turned out that vitamin C formed the first line of antioxidant defense and was the only antioxidant capable of completely preventing detectable lipid peroxidation. The other antioxidants in plasma, like urate, bilirubin or vitamin E, were consumed after vitamin C, and unlike vitamin C, they could not completely prevent lipid peroxidation. I didn't expect to see this kind of strong antioxidant protection by vitamin C—much stronger than all the other antioxidants. This got me interested in further exploring the antioxidant effects of vitamin C. Based on its chemical properties and one electron reduction potential, vitamin C is one of the strongest biological antioxidants. It can regenerate other antioxidants, like vitamin E or urate, from their oxidized forms. In the cells and tissues of our bodies, these antioxidants all seem to interact with each other in an antioxidant network. One could propose that vitamin C is the hub of this network because it can interact with and regenerate most of the other antioxidants. For example, fat-soluble vitamin E can be regenerated by vitamin C, which is water-soluble, in an interaction that allows for an electron to go back and forth between these compartments and provide antioxidant protection in the aqueous phase as well as membranes and lipoproteins. Similar interactions occur with other antioxidants.

Q: Why have flavonoids garnered so much interest as antioxidants?

A: Flavonoids are very good antioxidants in the test tube. If you compare flavonoids in test tube experiments with vitamin C, many of them turn out to have a greater antioxidant capacity than vitamin C, which means they can provide more electrons per molecule than vitamin C does. However, vitamin C is more reactive than most flavonoids and, therefore, scavenges free radicals and other reactive oxygen species more readily. In addition, flavonoids are less efficiently absorbed from the diet than vitamin C, i.e., bioavailability is very limited for flavonoids. You can absorb perhaps a few percent of the flavonoids from the food you ingest, but you can absorb 100 percent of up to 200 mg of vitamin C. Vitamin C is present at much higher concentrations than any of the flavonoids in human plasma and tissues. Therefore, it is not clear whether flavonoids can make a significant contribution to the overall antioxidant capacity in the body where relatively high concentrations of vitamin C are present. I believe that some of the health benefits ascribed to flavonoids are explained by effects other than a general antioxidant activity.

Q: What kind of chemical and biological activities other than antioxidant functions do flavonoids and vitamin C have?

A: Flavonoids have been suggested to inhibit a family of enzymes called tyrosine kinases, which are important for signal transduction. Signal transduction refers to a process in which chemicals or proteins interact with receptors on the surface of cells, triggering a reaction inside the cell that sends a signal into the nucleus and alters gene activity. Flavonoids may inhibit tyrosine kinases by a mechanism that is not a general antioxidant effect. The same seems to be true for vitamin C, i.e., some of its effects are related to a general antioxidant activity, and some are not. The functions of vitamin C are related to its reducing capacity because it is a very strong redox compound and can donate electrons very easily. This may manifest itself in two different mechanisms. The first one is an antioxidant mechanism by which reactive oxygen species are scavenged by vitamin C and thereby prevented from causing oxidative damage to proteins, lipids, or DNA. The other mechanism is a "reducing" mechanism. For example, by keeping a chemical called tetrahydrobiopterin in the reduced form, vitamin C can enhance the activity of the enzyme, nitric oxide synthase. Similarly, the function of vitamin C in enzyme reactions is to keep metal ions, such as iron or copper, in the enzyme's active center in the reduced and catalytically active form. Again, strictly speaking, that is not a free radical scavenging mechanism—it's a reducing mechanism.

Q: Why do you think that the intake of supplemental vitamin C is still so controversial thirty years after Linus Pauling advocated it for the common cold?

A: It is surprising how little we still know about vitamin C metabolism and pharmacokinetics more than sixty years after Szent-Györgyi received the Nobel Prize for his discoveries of vitamin C's biological oxidation processes. When Linus Pauling advocated vitamin C for the common cold, cancer, and heart disease, it wasn't known exactly how much vitamin C the human body can take up and retain. Since then there have been a number of studies showing that humans can fully absorb up to 200 mg of vitamin C, but part of that gets excreted in the urine. One can reach maximal levels of vitamin C in plasma with an intake of about 400 mg per day and in cells and tissues with a daily intake of about 200 mg. However, these results are from studies with a small number of young, healthy men and women. While these studies have given us good insights into the pharmacokinetics of vitamin C in these individuals, the pharmacokinetics may be quite different in older people or in ill individuals. If you have a cold, or suffer from cancer or an infection, you may need much more vitamin C just to maintain normal body levels.

Q: Do you think that we will ever be able to determine an individual's optimal intake for vitamin C?

A: If you define the optimal intake as the intake necessary to reach tissue saturation, I think we can determine this intake by measuring plasma levels and urinary excretion of vitamin C, as well as the levels of vitamin C in, for example, white blood cells. I believe that tissue saturation is the optimal situation where one gets maximal antioxidant protection from vitamin C. Thus, tissue saturation with vitamin C over an extended period of time would be the goal for any individual. As mentioned before, for young, healthy adults this seems to be achieved by a daily intake of about 200 mg of vitamin C.

Q: What about the saturation of certain tissues, such as the brain and other organs, that can't be conveniently measured?

A: Generally, we can make certain predictions and draw conclusions based on the plasma and white blood cell levels of vitamin C. There are certain transport mechanisms by which cells take up vitamin C from the blood and other extracellular fluids. We know enough about these mechanisms to reasonably believe that cells and tissues get saturated before plasma gets saturated. But without actual measurements of vitamin C levels in the organs of living people, which is obviously impossible for many organs, we can't be absolutely certain about saturation of each and every tissue. We assume that the transport mechanisms identified in cell culture or in animals are also operating in vivo in humans, but there may be additional transport mechanisms or unknown inhibitors that may interfere with the uptake of vitamin C into tissues.

Q: How would you summarize the epidemiological and biochemical evidence about the role of vitamin C in preventing heart disease and cancer?

A: Epidemiology is limited in that it can rarely provide conclusive answers or evidence of causal relationships. Having said that, when we reviewed the literature regarding the possible role of vitamin C in the prevention of cancer or heart disease, we found that, based on the observational data and very limited clinical trial data, 100 mg of vitamin C per day seemed to be associated with a reduced risk of cancer and heart disease, and higher intakes of vitamin C didn't seem to provide additional protection. However, it should be noted that these studies usually did not include people who routinely take very large doses of vitamin C, and therefore the possible benefits of these large doses remain unknown. For prevention of cataract, 200 mg per day seemed to provide optimal protection. Again, this is mainly based on associations in observational studies and not on randomized trials where vitamin C is given as a supplement and disease outcome is assessed several years later. The 200 mg-dose seems to make sense vis-ŗ-vis the pharmacokinetic data where an intake of 200 mg was associated with saturation of white blood cells. Thus, one could argue that if tissues are saturated, optimal protection is achieved against these chronic diseases that have an oxidative stress component.

Q: Do you think that the application of vitamin C to treat heart disease or cancer might involve larger amounts than those found to be associated with prophylaxis?

A: Yes. So far we have talked mainly about prevention and optimal intake over a long period of time to lower the risk of chronic diseases, but once a person has cancer, heart disease, AIDS, or some other disease, the need for vitamin C and the amount required to help treat these diseases may be very different from what is needed to just maintain maximal body levels under healthy conditions. For example, in studies with researchers at Boston University, we investigated the effect of vitamin C on vascular function in patients with heart disease. These patients had impaired relaxation of their arteries, which is a sign of impaired production of a compound called nitric oxide. When we gave vitamin C to these patients either acutely (two grams) or over a month period (500 mg per day) they regained normal vasodilation, similar to that seen in healthy individuals. It appears, therefore, that vitamin C allows nitric oxide to function normally. Actually, this has quite far reaching implications because nitric oxide not only causes vasodilation, but also has numerous other functions in maintaining normal, healthy blood vessels. For example, nitric oxide inhibits aggregation of platelets, which is an important component in the formation of atherosclerotic plaque as well as the acute formation of blood clots that can trigger a heart attack or stroke. Nitric oxide also prevents chest pain or angina pectoris—the nitroglycerine taken therapeutically provides a source of nitric oxide. Additionally, nitric oxide can inhibit the interaction of white blood cells with the arterial wall and the proliferation of arterial smooth muscle cells, both important steps in the formation of atherosclerotic plaque. Therefore, nitric oxide and, thus, vitamin C may not only have beneficial effects with respect to an acute coronary event like a heart attack or a stroke because they prevent platelet aggregation and vasoconstriction, but they may also have a more long-lasting effect on inhibition of atherosclerotic lesion formation itself.

Q: Do you think that the most opportune time in life to intervene in this very long pathological process of atherosclerosis might be in adolescence, which ironically, may be the time in life when many people would be unlikely to maintain good vitamin C status?

A: Yes. Atherosclerosis is a disease that starts at birth. Babies of mothers with high cholesterol levels already have the first signs of atherosclerosis when they are born—they have foam cells and fatty streaks in their arteries. Therefore, it is very important to eat a healthy diet and maintain optimal vitamin and mineral levels throughout your whole lifetime to decrease the risk of heart disease and cancer, which also develops over many years or even decades. One should always try to have plasma and tissue saturation of vitamin C by eating a healthy diet with lots of fruits and vegetables and possibly by taking supplements.

Q: In recent years a number of different factors have been postulated as playing an important role in contributing to heart disease, including bacterial infections, oxidative stress, and insufficient intake of antioxidants like vitamin C and vitamin E. Can you comment on how these different factors may be related to heart disease?

A: I have no doubt that inflammation is a very important component of heart disease. The accumulation of white blood cells in the arterial wall, followed by a myriad of inflammatory responses, is a pivotal step in the formation of atherosclerotic lesions. There is also accumulating evidence that plasma levels of C-reactive protein, which is a marker of inflammation, are very tightly correlated with heart disease risk. In fact, I believe that inflammation is a very important component of many human chronic diseases, including certain types of cancer and neurodegenerative diseases, such as multiple sclerosis, Parkinson's disease, and Alzheimer's disease. If one can prevent or suppress inflammation, one might be able to lower the risk of many of these diseases. It may be that atherosclerosis and other chronic diseases develop in spurts that correlate with periods of infection and inflammation in our bodies. During an acute inflammatory response, the body is subjected to a lot of oxidative stress due to activation of white blood cells, which generate nasty oxidants like hypochlorous acid, which is the same as household bleach, and peroxynitrite. Therefore, it is important to maintain adequate antioxidant defenses to prevent oxidative damage during those times of inflammation. Vitamin C is one of the foremost antioxidants in preventing oxidative damage by hypochlorous acid and white blood cells.

Q: What projects are now under way in your laboratory?

A: We are primarily interested in understanding and preventing the early stages of atherosclerosis. One of the first steps appears to be oxidative modification of low-density lipoprotein (LDL), the "bad" cholesterol. One of the projects in my lab is to investigate the mechanism of oxidative modification of LDL by activated white blood cells and how this might be prevented by antioxidants. We examined the modification of LDL by hypochlorous acid or activated white blood cells from humans and how vitamin C scavenges hypochlorous acid and prevents, and sometimes even reverses, some specific oxidative modifications to the protein and lipids in LDL.

The second project focuses on endothelial cell function and how it contributes to atherosclerosis. Endothelial cells are the cells that line the artery and thus face both the blood stream and the artery wall. The endothelial cells regulate the transport of LDL and white blood cells from the blood stream into the arterial wall. There are certain molecules expressed on the surface of these endothelial cells, known as adhesion molecules, that interact with the white blood cells and help them get across the endothelial cell barrier. We are interested in how expression of these adhesion molecules is regulated by oxidative stress and antioxidants. One of the antioxidants that we found to be particularly effective in preventing adhesion molecule expression is alpha-lipoic acid, while vitamin C was ineffective. We are further pursuing this line of investigation by examining the effects of lipoic acid in whole animals. One study will look at the effects of lipoic acid on adhesion molecule expression in mice, and the second experiment will find out whether feeding lipoic acid can prevent or inhibit atherosclerosis in transgenic mice prone to the disease.

The third area of interest in my laboratory—also related to endothelial function—goes back to what I mentioned earlier about nitric oxide. We are interested in how the cell's redox environment affects the synthesis and biological activity of nitric oxide.

Q: And what do you mean by redox environment?

A: It refers to the balance between oxidants or reactive oxygen species and antioxidants, which scavenge these reactive oxygen species. The question is whether there is a good balance between oxidants and antioxidants without oxidative damage, or whether that balance is out of equilibrium, which then could lead to oxidative damage and dysfunction of the endothelial cells.

Q: What have your investigations on the potentially pro-oxidant effects of vitamin C revealed?

A: It is well known that vitamin C acts as a pro-oxidant in test tube experiments in which free metal ions, such as iron or copper, are present. There has been some concern that people with hemochromatosis, or iron overload, should not take vitamin C supplements because they may cause, rather than prevent, oxidative damage. But one has to realize that the in vitro conditions don't mimic what is going on in cells and tissues or in plasma. Therefore, we have been doing a number of experiments to determine if vitamin C acts as a pro-oxidant under physiological conditions and in the body. So far, we have not been able to find any pro-oxidant activity of vitamin C under those conditions. Quite to the contrary, we have observed that vitamin C very strongly protects against oxidative lipid damage by iron or copper in human plasma, and we also have seen antioxidant protection of vitamin C against lipid peroxidation in animals that were loaded with iron. At this point, there is no compelling evidence that vitamin C has any adverse or pro-oxidant effects under conditions of iron overload.

Q: In recent years there have been a number of negative reports about vitamin C that generated media headlines and scared people about taking supplemental vitamin C. Some of these reports related to putative DNA damage to human lymphocytes, increased carotid artery wall thickness, and the formation of genotoxins by interactions between vitamin C and lipid hydroperoxides. Some of these experiments used modest amounts of vitamin C, such as 500 mg. Do you think that these studies are valid?

A: First of all, the body doesn't distinguish between supplemental vitamin C and vitamin C derived from fruits and vegetables. If vitamin C had adverse effects in the body, they wouldnít be restricted to supplemental vitamin C but would also apply to vitamin C from food. There is overwhelming evidence from epidemiological studies, clinical studies, animal studies, and basic research studies that vitamin C is beneficial and doesn't cause any adverse health effects. If supplemental vitamin C caused harmful effects in humans, one would also have to restrict the consumption of fruits and vegetables, which is, of course, an absurd recommendation. The study on DNA oxidation published in Nature a few years ago turned out to be severely flawed because the methods used to measure DNA damage caused extensive ex vivo artifacts. In other words, the DNA damage products measured in that study were generated during the preparation of the samples and werenít actually present in the samples obtained from the patients. Many other scientists who actually are doing these kinds of DNA oxidation measurements agree with me on this point.

As you indicated, there was an observational study claiming that vitamin C supplementation causes acceleration of atherosclerosis in the carotid artery. That study still hasnít been published—it was a non-peer reviewed abstract at an American Heart Association meeting in San Diego in March 2000. This study disagrees with much larger studies that have been published in peer reviewed journals like Circulation, an American Heart Association journal, showing that the intake of vitamin C is inversely correlated with carotid artery wall thickness. Thus, a single small study, poorly controlled and not peer reviewed, cannot discredit the evidence that vitamin C is beneficial and does not cause arterial wall thickening. If anything, the published data suggest that vitamin C decreases arterial wall thickness.

Regarding the study on lipid hydroperoxides reacting with ascorbate, which was published recently in Science, I have written a comment on this study in the last Newsletter (Fall/Winter 2001). Again, this was only a test tube study that revealed some unusual chemistry of vitamin C, but didnít tell us anything about the relevance of these observations to the situation in vivo. As I mentioned, our investigations did not find any pro-oxidant effects of vitamin C under physiological conditions. Whether any deleterious reaction of vitamin C with lipid hydroperoxides actually occurs in vivo and causes oxidative DNA damage, as the authors of that study suggested, remains to be seen.

Q: Large-scale studies have not revealed any role for vitamin C in kidney stone formation, and your studies do not demonstrate any pro-oxidant effects of vitamin C in vivo. Additionally, the Institute of Medicine's Dietary Reference Intakes report did not note any serious toxicity for vitamin C. Given this evidence, do you feel that there is any reason people should be careful about how much supplemental vitamin C they take?

A: No. I don't think there are any adverse health effects of supplemental vitamin C—certainly not in healthy people. People who have had previous kidney stones and are prone to kidney stone formation may want to avoid very large doses of vitamin C. But the overwhelming majority of the population does not have to be concerned about large doses of vitamin C. The only adverse effects identified by this panel of the National Academy of Sciences were gastrointestinal disturbances and diarrhea. In my view, those aren't serious enough adverse health effects to limit the intake of vitamin C.

Last updated May, 2002

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