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Research Newsletter-Fall/Winter 2010

A New Twist on Vitamin C and Beer!

An interview with Fred Stevens, Ph.D.
Associate Professor of Medicinal Chemistry
LPI Principal Investigator

Q. You earned your Ph.D. in The Netherlands and spent some years as a post-doctoral research associate in Europe. You've been at OSU for about eight years and with LPI for the last five years. Is the academic research environment in Europe much different from what you find here?

A. Of course, I've only seen it here at OSU as a faculty member. Here, we're much more focused on grant writing and getting money for research, while in Europe more time is spent on the research itself and writing papers.

Q. How is academic research funded in Europe? Are there government agencies to which you apply for funding?

A. In The Netherlands, the Ministry of Education allocates research funds to the universities, and they distribute that to the departments. Every department has state-funded professors, lecturers, technicians, and graduate students. At Oregon State University, only professors and instructors have state-funded positions. In The Netherlands there are opportunities to apply for national or European grants that are generally smaller in size.

Q. What do you like to do when you're not working in the lab?

A. I like to swim every day.

Q. Whats your position at LPI?

A. I'm a Principal Investigator, and my academic home is the Department of Pharmaceutical Sciences in OSU's College of Pharmacy.

Q. You also serve as co-director of the Biomolecular Mass Spectrometry Core Lab. What does that lab do?

A. That lab provides mass spectrometry services for OSU. I provide expertise and instrumentation for analysis of small organic molecules, which includes metabolites. We analyze metabolites made from lipid peroxidation products and xenobiotics. We do a lot of work for people in the Colleges of Pharmacy and Veterinary Medicine, analyzing drugs and case studies.

Q. You worked for many years on a phytochemical called xanthohumol found in beer. What is xanthohumol and what does it do in cells?

A. Xanthohumol is a flavonoid found in hops. We've known about it for a long time, but it never received any attention from beer brewers because it has no taste. I got interested in xanthohumol back in 1995 when I became a post-doc at Oregon State University with Professor Max Deinzer. We were investigating the chemistry of hops and focused on xanthohumol, which we were able to isolate. Professor Don Buehler, Val Miranda, and I investigated the activity of xanthohumol in cancer cells and found that it induces phase 2 enzymes—enzymes that detoxify carcinogens. That is good for cancer chemoprotection. We also found that xanthohumol inhibits phase 1 enzymes that activate pro-carcinogens. So the effect is two-fold: xanthohumol prevents activation of pro-carcinogens into carcinogens by phase 1 enzymes and helps detoxify carcinogens by the phase 2 process. We can clearly say that xanthohumol is a cancer chemoprotective compound in cell culture.

Q. One problem with the cell culture model is that it doesn't necessarily predict what will happen in the body because compounds need to be absorbed into the blood stream to have many physiological effects. Have you studied the bioavailability of xanthohumol—how much actually gets into the blood stream?

A. We have measured pharmacokinetics in a handful of people and found that xanthohumol is absorbed and may be distributed to the tissues, which seem to hold onto it for a long time. It has a long half-life in the body. Although the plasma levels are relatively low, tissue levels may be much higher. Low plasma levels don't necessarily imply a lack of biological activity.

Q. Does xanthohumol act as an antioxidant in the body?

A. It has antioxidant activity in vitro. We don't think that it functions as an antioxidant in the body.

Q. Is xanthohumol found in other dietary plants or in lagers and wines?

A. It is found in any beer that is produced from hops but not in dietary plants. It's not found in beers that are made from hop extracts.

Q. One of the hop flavonoids you studied, 8-prenylnaringenin, is a potent phytoestrogen. How may that affect health?

A. Prenylnaringenin was identified as a metabolite of isoxanthohumol, which is the isomer made from xanthohumol when you brew the beer. However, the level of prenylnaringenin in beer is very low, and it's very doubtful that the low amount in beer would be estrogenic in people. In the last couple of years, studies have shown that some people have certain micro-organisms in the gut that are able to convert isoxanthohumol into prenylnaringenin. Xanthohumol supplements in those people may be problematic, but the amount in beer is not a concern.

Q. Is there any way to determine which people have microorganisms that convert isoxanthohumol to prenylnaringenin?

A. There is no easy test to identify these people.

Q. How might xanthohumol help prevent prostate cancer?

A. We published a paper with Emily Ho, another LPI Principal Investigator, showing that xanthohumol interferes with NFκB signaling. NFκB is a protein and master switch for the regulation of about 400 genes that are involved in inflammatory processes. Xanthohumol binds to a certain chemical group in a structural subunit of NFκB and prevents its signaling, thereby blunting or reducing the inflammatory response. NFκB-driven inflammation is an important process in the development of prostate cancer.

Q. You studied another kind of flavonoid, proanthocyanidins. What foods contain these?

A. Virtually all plants make proanthocyanidins, also called condensed tannins. They are found in hops, grapes and their seeds, apples, cocoa, tea, and many fruits. Anything that has an astringent taste probably contains a lot of proanthocyanidins.

Q. How do proanthocyanidins affect the risk for colorectal cancer?

A. We found that proanthocyanidins kill cancer cells in vitro by producing hydrogen peroxide. Bioavailability, or absorption into the blood stream, is not an issue because the colorectal cells would be directly exposed to ingested proanthocyanidins.

Q. Why are xanthohumol and proanthocyanidins synthesized by plants?

A. Well, a lot of phytochemicals are defense chemicals against herbivores. Of course, unripe fruits contain a lot of tannins and are not palatable to animals, so they don't eat them until the fruit is ripe. Then the animals consume the fruit and disperse the seeds, which helps ensure survival of the plant species.

Q. For the last half-dozen years, you have worked on reactions between vitamin C and rancid fats, or lipid peroxidation products, also called LPO products. How does vitamin C react with these?

A. Well, this is a difficult problem. When fats get oxidized, they make degradation products known as reactive aldehydes.

Q. Does that happen in the body?

A. Yes, we have lots of evidence that these aldehydes are made in the body. Vitamin C can react in vitro with these compounds to make what we call ascorbylated lipids. This has been shown for a very small lipid peroxidation product called acrolein, which reacts with vitamin C in cultured cells. This is an important finding because it shows that vitamin C can compete with a well-known pathway called glutathione conjugation that helps get rid of toxic products. At first we were not very successful in finding these LPO products. It now appears that the ascorbylated aldehydes get further metabolized. We have identified one metabolite and want to find out if it is the final end product or gets further modified. Once we have clearly identified the end products, we will try to determine if this process happens in the body.

Q. What is the physiological significance of LPO products? How are they detrimental to health?

A. Not all of them are bad. Prostaglandins are enzymatic breakdown products of lipid hydroperoxides. Prostaglandins are ubiquitous and have very important functions, including the regulation of platelet activity and inflammation. On the other hand, nonenzymatic lipid perioxidation generates reactive aldehydes that can react with DNA in a nonspecific manner. This can cause DNA mutations that could lead to cancer. Aldehydes can also react with proteins, including enzymes, in a nonspecific manner, causing damage that can lead to cellular dysfunction. For example, elevated levels of the LPO product, 4-hydroxy-2-nonenal (HNE), have been associated with the development and progression of Alzheimer's disease.

Q. Can LPO products be used as biomarkers to measure oxidative stress in the body?

A. We have done three studies on this. The first was a study where we exposed rats to carbon tetrachloride, which is a strong inducer of lipid peroxidation and oxidative stress. We then looked in the urine for metabolites of HNE and a related product called 4-oxo-2-nonen-1-ol (ONO). We were the first group to find evidence for in vivo production and metabolism of ONO. It certainly has value as a biomarker of oxidative stress in rats. In the second study, we used smokers as a model of chronic oxidative stress in humans. We found that the smokers had higher levels of LPO products, but the differences were not statistically significant because people vary widely in levels of oxidative stress. Then we looked at smokers who stopped smoking and found that smoking cessation results in a substantial decrease in these biomarkers. With LPI's Maret Traber, we wanted to know if vitamin C supplementation would affect these new biomarkers. Using mass spectrometric analysis of the subjects' urine, we found that vitamin C supplementation decreases these HNE and ONO metabolites.

Q. Would these be better biomarkers of oxidative stress than F2-isoprostanes or other standard biomarkers?

A. The biomarkers that we measure are present in much higher concentrations, so that's a benefit analytically. Also, some of them are very stable because they have been fully oxidized—they are final end products easy to work with. Although this work is still in the very early stages, we can say that in the study we just completed vitamin C supplementation leads to a decrease in HNE metabolites, whereas F2-isoprostanes did not change.

Q. You have studied a ubiquitous chemical called acrolein that is formed by cooking food, hydrocarbon combustion, cigarette smoking, and other processes. What does acrolein do in the body?

A. HNE is the prototypical lipid oxidation product, but acrolein can also be formed from lipid peroxidation. Its major source is smoking. A study a couple of years ago claimed that acrolein—not tar—is the carcinogenic compound in cigarette smoke. That, of course, received a lot of interest in the media.

Q. What epidemiological evidence links acrolein to lung cancer?

A. Acrolein is a very small molecule and a volatile substance, so when you cook with vegetable oils, it's probably present in the kitchen air. In Hong Kong, wok cooking in unventilated kitchens, not smoking, is thought to be the cause of the high rate of lung cancer in women.

Q. How does vitamin C affect acrolein?

A. Acrolein is a reactive aldehyde that can damage bio-molecules. Vitamin C can function chemically as an electron donor. Acrolein is an acceptor, and they react with each other to form an adduct, ascorbylated acrolein. It is a very efficient reaction and is easily done in the test tube. We examined the crystal structure of ascorbylated acrolein with a chemist here at OSU. Since vitamin C adduction to acrolein happens so easily in vitro, we think that this reaction may have biological significance in vivo. Of course, the metabolism of this compound needs to be further studied. When we started, Nicholas Kesinger, my graduate student, challenged cells with acrolein but didn't find anything unusual. That was very disappointing because we had hoped to detect evidence of the chemical reaction we expected. Then we added synthetic ascorbylated acrolein to cells, and it disappeared, although we could easily measure it in a buffer solution. So then we started thinking that ascorbylated acrolein might be metabolized and we discovered that it follows a very unusual metabolic pathway. That was the main finding of Nick Kesinger's Ph.D. project that he completed with me in LPI.

Q. So vitamin C forms an adduct with acrolein that then further degrades through metabolic processes in the body, rendering the acrolein harmless.

A. Right.

Q. Have reactive aldehydes been found to cause diseases in humans?

A. A possible example is Alzheimer's disease. There are a lot of studies showing the involvement of HNE in the pathogenesis of Alzheimer's disease, and, as I mentioned, acrolein is involved in lung cancer in smokers.

Q. How does HNE contribute to Alzheimer's disease?

A. Scientists think that HNE damages proteins that are important for prevention or delaying onset of the disease. For example, HNE can inactivate an enzyme called neprilysin that breaks down the amyloid beta plaques in the brain of Alzheimer's patients.

Q. You've described a reaction between vitamin C and certain phytochemicals that you call ascorbylation, which you've already discussed. How is this relevant to health?

A. This is very controversial because many scientists claim that electrophilic products—chemicals attracted to electron donors—in the body interact with glutathione. The idea that vitamin C plays a role as a nucleophile came from nature. A nucleophile has an excess of electrons—it's an electron donor—and combines with substances that have a deficiency of electrons to make an adduct. About 10 years ago, I isolated a natural product from henna, which contains a dye that's a naphthoquinone. The naphthoquinone reacts readily with vitamin C in the plant material when you crush it to make the dye. That's how I found my first ascorbylated natural product. So I thought, if this reaction happens so readily in plant material, it must have some biological significance. Then we searched more carefully in the plant kingdom for the specific structural moiety allowing the ascorbylation reaction, and to our surprise, we found 33 natural products that had that moiety. In most cases, the authors were not even aware that they had a vitamin C adduct.

Q. Are these adducts formed only in plants, not in the body during digestion or metabolism?

A. Right. These are true natural products. A famous example is ascorbigen, which is an indole-3-carbinol metabolite with vitamin C found in cabbage. There are also tannins that contain the vitamin C moiety. For example, a tea flavonoid—epigallocatechin gallate or EGCG—has been found in an ascorbylated form. There are many, many examples. There must be a really significant reason for this reaction in nature, and that is what we are trying to understand.

Q. Those ascorbylated compounds occur in plants and, of course, may or may not have much relevance to human physiology or health.

A. Right. Plants make vitamin C for their own health; they don't make it just for us! I don't know the relevance of ascorbylation for plants, but if it reduces the reactivity of waste products in the human body, then it could be considered a detoxification pathway.

Scientist working in the lab.

Q. Do ascorbylated compounds contribute to the antioxidant capacity of food?

A. No, because the moiety has been totally modified. Vitamin C has a double bond and two hydroxyl groups. Basically, it functions as an antioxidant because it's a one-electron donor. The ascorbylation reaction is a two-electron reaction, and then the double bond in the vitamin C molecule disappears, and the antioxidant activity is lost.

Q. Where is your research headed in the next few years?

A. We will continue to study the interaction between vitamin C and reactive aldehydes. There are multiple ways in which vitamin C may be helpful in unexpected ways. Last year we compared levels of protein adduction by HNE in human monocytes in vitro with and without vitamin C. We found that vitamin C reduced the level of protein adduction by HNE by 30%. We couldn't attribute all of that to the formation of the ascorbylated products because we didn't know about the metabolism at the time. Nevertheless, there must be other mechanisms by which vitamin C protects proteins from damage by these reactive aldehydes. This could be very important for a number of diseases. We're working on cardiovascular disease, and in the future we will determine the effect of vitamin C in protecting proteins that are important in cardiovascular health.

Q. What kinds of proteins?

A. One enzyme we are studying is aldehyde dehydrogenase. This is an enzyme that metabolizes HNE, but HNE can also damage the enzyme. Of course, if HNE damages the enzyme, then HNE itself cannot be metabolized and more and more of it is accumulated and creates more and more damage. If vitamin C could protect the enzyme, then HNE could be properly metabolized, and there would be less damage.

Q. LPI will move into our new building, the Linus Pauling Science Center, next summer. To which research cluster will you belong?

A. I will be with Balz Frei, Maret Traber, and Don Jump in the cardiovascular and metabolic diseases group.

Q. You must be looking forward to that.

A. I'm very excited!

Last updated November 2010