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Research Newsletter-Spring/Summer 2010

VITAMIN D GETS AN "A"

An interview with Adrian Gombart, Ph.D.
Associate Professor of Biochemistry and Biophysics
LPI Principal Investigator

Q. When did you decide on a career in science?

A. I was interested in science when I started college, and I was thinking about becoming a veterinarian. After gaining some practical experience in vet clinics and not getting into vet school the first time I applied, I grew impatient and applied for a master's program in genetics at Oregon State University, where I was an undergraduate. I completed the program and published two papers on baculovirus research with George Rohrmann. By that time I was pretty convinced that I didn't want to go to vet school anymore; I wanted to pursue a career in research. I earned my Ph.D. in microbiology at the University of Washington in Seattle and went to Los Angeles for postdoctoral work.

Q. What brought you to the Linus Pauling Institute?

A. I had been in Los Angeles for 15 years working at Cedars-Sinai Medical Center. My family and I enjoyed Los Angeles, but it wasn't really a place where we wanted to stay permanently. Near the end of my tenure in LA, my research had started to focus on vitamin D and its effect on the innate immune system. We made a very exciting discovery that resulted in an NIH grant award, which gave me the opportunity to start looking for positions elsewhere. It turned out that there was a position open in the Linus Pauling Institute. I knew that LPI had moved to Oregon State and that it was well known for excellent research. My own research dovetailed nicely with LPI's research programs, and I felt that it would be a really good match to bring our vitamin D research on immunity to LPI's Healthy Aging Program.

Q. Did you know much about Linus Pauling?

A. I knew that Linus Pauling was an Oregonian and had attended Oregon State University. His name came up frequently because of his scientific stature, especially his Nobel Prize-winning work on the chemical bond and protein structure. I was aware of his Nobel Peace Prize, too.

Q. What do you like about the university environment?

A. The university is a very collegial environment that promotes good interactions among researchers with very different interests. I think that happens more here than in medical centers. The university is a little less political—people are more interested in achieving their research goals rather than protecting their territorial ambitions. That being said, I have some great collaborators in Los Angeles, and our projects are still ongoing and productive.

Q. Are you always thinking about science or do you have other interests as well?

A. I think about science a lot! It's what I really enjoy, but I do have other interests, like travelling. I enjoy activities with my family—my wife and three boys—like walking, biking, basketball, and other sports. My kids play organized sports, and we spend the weekends running around to practices or games, too. Also, I enjoy reading, music, and movies.

Q. What obstacles do scientists often confront?

A. Time management! Balancing family and social life with your career is very challenging. I haven't done much classroom teaching, but I think I will do more now that I'm at a university instead of a medical center. I feel that my time will be spread even thinner with that additional activity, but I find it enjoyable.

Q. What about competition for grants?

A. Competition for grants is difficult. The funding continues to be flat or even decreased because of inflation. It's a critically important issue, though, because you have to continue to get funding to be successful.

Q. Early in your career at the University of Washington, you investigated the genetics of viruses, especially measles. What did you learn from that work?

A. Despite the measles vaccine, measles is still a major killer, mainly of children in developing countries, and a lot of that has to do with nutrition. If you are malnourished, you don't fight infections very well. The vaccine for measles, while quite effective, is not effective enough to eliminate the disease. Measles is one disease like polio that could potentially be eliminated because humans are the only reservoir. There is still interest in improving the vaccine and understanding the biology of the virus. I learned some interesting basic biology from studying the virus, especially something called RNA editing that is used by the measles virus to increase the number of proteins that its RNA genome can code for by making changes to the messenger RNA sequence.

Various iterations of RNA editing are used by higher organisms, as well. At the time, it was a very intriguing mechanism.

fruit and juice

Q. Does that make it more virulent?

A. Yes, the process produces a protein that is probably really important for gene transcription—creating RNA copies from the genome. Subsequent research findings suggested that the protein made by RNA editing contributes to disease severity. The virus lacking the protein would be considered less virulent.

Q. Somewhat later when you worked at UCLA, you got involved in cancer research and studied mutations to tumor suppressor genes and their functional effects. How do tumor suppressor genes work?

A. In cancer cells there are oncogenes and tumor suppressor genes. The oncogenes are considered the accelerator—they promote the growth of the tumor when they become altered. So overexpression of oncogenes pushes cell proliferation. The tumor suppressor genes are like the brakes in the cell—they prevent cells from growing too rapidly or even growing at all.

Q. Do you find tumor suppressor genes in all cells?

A. Yes. Quite a few have been discovered, including p53, retinoblastoma, and inhibitors of cyclin-dependent kinases. Cyclin-dependent kinases are important for progression through the cell cycle. If this cycle stops, then the cell can't grow. The cyclin-dependent kinase inhibitors put brakes on progression through the cell cycle.

Q. Is cancer always associated with altered activity of tumor suppressor genes?

A. Yes, all cancers have some tumor suppressor dysfunctions, and p53 is most commonly affected. Also, deregulation of the pathway in which the retinoblastoma tumor suppressor protein functions is very common in most cancers.

Q. Are there ways to influence the activity of tumor suppressor genes in cancer cells?

A. There are a number of ways to turn them on. Chemotherapeutic agents damage cancer cells, leading to an increase in the expression of p53 that inhibits tumor cell growth.

Q. Are there nutritional strategies to help prevent cancer by influencing the activity of tumor suppressor genes?

A. Yes, histone deacetylase inhibitors induce a cyclindependent kinase inhibitor called p21. This stops cells from growing. Rod Dashwood's and Emily Ho's groups in LPI are working on organoselenium and sulforaphane compounds found in garlic and broccoli that act as histone deacetylase inhibitors. Also, there is great interest in using vitamin D and vitamin A compounds to induce the tumor cells to change their properties and stop growing. Vitamin D induces the expression of p21.

Q. What are kinase inhibitors and what do they do?

A. We were interested in cyclin-dependent kinase inhibitors because it became clear that they would very likely be tumor suppressors. Kinases are enzymes that modify protein activity by phosphorylation, and cyclin-dependent kinases regulate the cell cycle. If you can inhibit those kinases, you can interfere with the cell cycle. Interrupting the cell cycle is strategically important in cancer therapy. When it was discovered that cyclin-dependent kinase inhibitors could block the progression of the cell cycle, they became candidates for potential tumor suppressor genes. To find out if they were tumor suppressors, we analyzed mutations in cancers from different patients. The normal cell had an unchanged copy of the gene, but the tumor suppressor gene was altered in the cancer cell because of various mutations.

Q. When did you become interested in vitamin D?

A. When I went to Cedars-Sinai Medical Center, I worked with Phillip Koeffler, a hematologist/oncologist who has worked on both leukemias and cancers involving solid tissues. His focus was on the basic biology of cancer, but he was also looking for ways to treat the disease. In the early 1980s, there was a lot of interest in using vitamin D because it can cause cell differentiation or maturation and inhibit the growth of cells. Some people were interested in analogs of vitamin D, which are compounds that have had the structure slightly changed to enhance their beneficial effects and reduce side effects. Dr. Koeffler was working on the use of vitamin D to cause leukemic cells to differentiate, which would have therapeutic value. So I got involved in that research.

Q. What does vitamin D do in the body?

A. It seems like vitamin D does everything! It's primarily known for its importance in bone growth—making strong bones and strong teeth by maintaining proper calcium levels in our blood through absorption from the gut. But it's becoming quite clear that deficiencies in vitamin D lead to a number of diseases. Vitamin D is involved in a lot of important processes in the body, including the immune system, and recent studies show the importance of vitamin D in cardiovascular health.

Q. If vitamin D is synthesized in the skin on exposure to sunlight, why is it called a vitamin?

A. A vitamin is a substance that you need to get from your diet—your body doesn't synthesize it. Vitamin D was called a vitamin because of the discovery that vitamin D-deficient animals could be fed irradiated food to cure rickets. Vitamin D is now considered a hormone because our bodies can synthesize it. Vitamin D's effects are mediated through a protein that belongs to a class of proteins called steroid hormone receptors. These transcription factors bind to certain kinds of hormones and other compounds to turn on gene expression.

Q. How many forms of vitamin D are there?

A. Vitamin D is produced from ultraviolet light hitting the 7-dehydrocholesterol molecule in the skin. The pre-vitamin D molecule travels in the blood to the liver, where it's hydroxylated and becomes 25-hydroxyvitamin D, which is the form that circulates in the blood at high levels. Physicians measure 25-hydroxyvitamin D to determine if you are deficient or sufficient. 25-Hydroxyvitamin D is then hydroxylated in the kidneys to the active compound, 1,25-dihydroxyvitamin D or calcitriol, which binds to receptors and turns on genes. While the kidneys are the primary source of biologically active vitamin D, we now know that different cell types in our bodies can produce active vitamin D. Cells of the innate immune system that kill microbes that they've engulfed can produce the active form of vitamin D.

pills in a spoon

Q. Is the vitamin D found in supplements the same form of vitamin D that's made in the body or found in food?

A. Supplements contain either vitamin D2, also called ergocalciferol, or, usually, vitamin D3, also known as cholecalciferol. Both are converted in the kidneys to the active form of vitamin D. Vitamin D2 is found in plants. Vitamin D3 is the form found in animals. Vitamin D3 in supplements primarily comes from chemically modified lanolin from sheep wool. The forms put into supplements are chemically indistinguishable from the forms that are found naturally in food.

Q. What foods contain vitamin D?

A. Vitamin D is found in fish like salmon, herring, and sardines, and in cod liver oil. Some foods in the U.S. are fortified with vitamin D, including milk, orange juice, and some cereals, grains, and breads.

Q. Mushrooms that we find in the supermarket are typically grown in the dark. Would they contain much vitamin D?

A. They would not have much at all, but if they are flash exposed to ultraviolet light, some vitamin D is made. The longer the exposure, the more vitamin D is produced. Wild or cultivated mushrooms exposed to sunlight can have pretty high levels.

Q. How is vitamin D deficiency determined?

A. It's done by a blood test that measures 25-hydroxyvitamin D. Sufficient levels are 30-32 nanograms (ng) per milliliter of blood. From 20 to 30 ng/ml is considered insufficient. Below 20 ng/ml is deficient.

Q. How is sufficiency determined?

A. It's based on the regulation of the parathyroid hormone, which is how your body regulates calcium levels. If your blood level of calcium drops, then parathyroid hormone secreted from the parathyroid tells your body it needs more calcium. That leads to the production of 1,25-dihydroxyvitamin D by the kidneys, which increases the absorption of calcium from the intestines. If you are not consuming enough vitamin D or if levels of vitamin D are low, then you can't produce enough 1,25-dihydroxyvitamin D needed to sequester calcium.

Q. And that explains the development of rickets?

A. Yes. Instead of getting calcium from the diet, the body takes calcium out of the bones. The parathyroid hormone is regulated by the active form of vitamin D, 1,25-dihydroxyvitamin D. If that mechanism for suppressing parathyroid hormone levels is absent, the parathyroid hormone levels increase, leading to the leeching of calcium from the skeleton. At around 30-32 ng/ml of 25-hydroxyvitamin D in your blood, the parathyroid hormone levels remain normal.

Q. It seems that good vitamin D status is important in every stage of life.

A. Yes, vitamin D levels are very important throughout life—to prevent rickets in childhood and to ensure bone health as we age. Also, it's becoming apparent that it is important for reducing the incidence of diseases that we associate with aging, including cardiovascular disease, muscle weakness, increased inflammation, and poor immune function.

Q. What is the optimum level of vitamin D in the blood, and how does supplemental vitamin D affect that?

A. It's not really known what the optimal level is, and there is a lot of research on that now. Vitamin D is involved in many physiological processes. Vitamin D status optimized for proper immune function may not be optimal for cardiovascular health or for preventing cancer. Supplements can raise your blood levels of vitamin D—every 100 IU raises blood levels by about 1 ng/ml.

Q. What factors influence the synthesis of vitamin D in the skin?

A. The synthesis of vitamin D in your skin is affected by where you live and the season. The further north you go, especially during the winter, the less vitamin D is made in the skin. Here in Oregon at around the 45th parallel, you're not going to produce much. If you live in Los Angeles, you can synthesize vitamin D year round. Sunscreen blocks vitamin D synthesis. The darker the skin, the longer you need to spend in the sunlight. As you get older, your skin becomes less efficient at synthesizing vitamin D, probably due to a reduction in the cholesterol substrate.

Q. How widespread is vitamin D deficiency?

A. It's estimated that about 10% of Americans are deficient and that 70% are insufficient.

Q. The litany of diseases that vitamin D may help prevent is long and includes cancer, heart disease, diabetes, multiple sclerosis, hypertension, autoimmune diseases like rheumatoid arthritis, and infections. How can vitamin D be effective in preventing so many different diseases?

A. One connection between those diseases is the immune system. Vitamin D is important for maintaining a balanced T lymphocyte repertoire. An imbalance could lead to increased levels of inflammation. Inflammation is important for fighting infection, but chronic inflammation probably contributes to the development of many of the diseases you mentioned. A level of vitamin D in blood of 30 ng/ml or higher may be necessary for optimal immune response, function, and control of the inflammatory response.

Q. What is the difference between innate immunity and adaptive immunity?

A. Adaptive immunity is involved in our response to vaccines and provides long-term protective immunity. T cells and B cells are important parts of adaptive immune response. B cells produce antibodies in response to antigens from pathogens like bacteria or viruses. T cells release toxins to kill pathogens. The innate immune system responds quickly to pathogens in a non-specific manner. Cells of the innate immune system like macrophages and neutrophils engulf pathogens and kill them but don't retain a memory of those pathogens.

Q. How does vitamin D help kill pathogens?

A. We discovered that vitamin D turns on genes in macrophages and neutrophils. Those genes make small peptides called cathelicidin that punch holes in the pathogen's membrane and disable it.

Q. Is vitamin D absolutely required for the cathelicidin peptide to be made?

A. Yes. The model that seems to be developing is that activation of toll-like receptors—proteins that recognize microbial molecules—cause macrophages to ramp up production of vitamin D and its receptor, leading to the production of cathelicidin. Much of the early cell culture work in this area was inconsistent, probably because there wasn't much vitamin D present in the culture media. Another group at UCLA led by Robert Modlin showed that the antimicrobial response was dependent on vitamin D in cultured cells. Their work indicated that low vitamin D levels in the blood would not support the production of this important antimicrobial peptide by macrophages.

Q. How important is cathelicidin among these antimicrobial peptides?

A. It seems to be quite important. There are a lot of defenses that work together to battle pathogens, but cathelicidin is critical. There's a knock-out mouse model in which cathelicidin isn't present, and the mice are susceptible to skin, eye, and urinary tract infections, and their gut is colonized by bacteria much more easily.

Q. Is cathelicidin found in most animals?

A. In humans and closely related primates there is only one cathelicidin gene that is regulated by vitamin D. In animals, vitamin D is important for immune response but not as a regulator of cathelicidin.

Q. How was the discovery of the relationship between vitamin D and the cathelidicin antimicrobial peptide made?

A. It was made when I was working with Phil Koeffler at Cedars-Sinai Medical Center. We were working on the differentiation of myeloid cells to become innate immune cells called macrophages and neutrophils. I was studying diseases where there were defects in that process and looking for ways to turn on the antimicrobial peptide genes with compounds that might provide a potential treatment for disease. I discovered that the active form of vitamin D strongly induced cathelicidin in the macrophage-like cells that we were studying. As we were getting ready for publication, another paper came out that reported the observation that I made—that there are vitamin D-binding sites in the cathelicidin promoter. We had a grant submitted and a patent application filed, and our paper was published a few months later. Furthermore, we reported that vitamin D enhances cathelicidin production in numerous cell types and in macrophages, and reported that the cathelicidin gene has been evolutionarily conserved in primates as an important part of innate immunity. We followed that up with a paper last year showing that the expression of cathelicidin gene is controlled by so-called "junk DNA" and has been conserved in the human lineage for 55 to 60 million years.

Vitamin D structure

Q. Have there been any epidemiological studies that looked at blood levels of vitamin D and rates of infection or incidence of disease?

A. There are a lot of anecdotal reports, but most of the published studies deal with mycobacteria or using vitamin D to treat tuberculosis. Before antibiotics were developed, TB patients exposed to sunlight in sanitariums seemed to improve. No one knew what was responsible for that therapeutic effect. There is a lot of evidence showing that deficiency of vitamin D is associated with reactivation of the disease. For example, in TB patients from Southeast Asia who move north, the disease gets reactivated because they get less sunlight and tend to be vegetarian and covered more in clothing. A recent report analyzing data from the NHANES epidemiological study showed a correlation between vitamin D deficiency and increased respiratory tract infections.

Q. Has there been any interest in combining vitamin D supplementation with drug therapy for tuberculosis?

A. There are a number of ongoing trials. One trial was published but wasn't very promising, probably because of the way it was designed. It will be interesting to see the results from the ongoing trials.

Q. Your study in patients undergoing chronic hemodialysis showed that those with the lowest blood level of cathelicidin had an increased risk of death from infection. How big was the effect and have there been any follow-up studies using vitamin D supplementation to try to raise cathelicidin levels in the blood?

A. Cathelicidin is secreted into the blood and is present at pretty high levels. Most antimicrobial peptides are packaged in neutrophils, which are white blood cells, but cathelicidin is also actively secreted into the blood for some unknown purpose. There isn't any research that correlates vitamin D status with cathelicidin levels in blood. Most of our patients had pretty high levels of vitamin D, so we really need to look at a group of people who have marginal or deficient levels of vitamin D. In the study with dialysis patients, vitamin D compounds were used to suppress parathyroid hormone levels. Some studies have found that hemodialysis patients who take vitamin D have a sharply reduced risk for cardiovascularmortality. In our dialysis study, we measured vitamin D and cathelicidin in the blood of several hundred patients in a prospective cohort of over 10,000 patients undergoing hemodialysis. Those with the lowest level of cathelicidin had a two-fold increased risk of death from infection within the first year of hemodialysis compared to those with the highest levels of cathelicidin. We found a positive association between the active form of vitamin D—1,25-dihydroxyvitamin D—and cathelicidin levels. We are following this up with a clinical study at Cedars-Sinai Medical Center with sepsis patients.

Q. Hospital infections are a great cause for concern because of increased morbidity and mortality. One recent study found that the risk for morbidity, organ failure, and length of stay in the ICU among surgical trauma patients was substantially reduced by supplemental vitamins C and E given prior to surgery. Do you think that vitamin D should also be tried in that context?

A. I think it's worth considering. A lot of people going into the hospital have insufficient or deficient levels of vitamin D and are more likely to develop sepsis. The very elderly are even more likely to be deficient in vitamin D. Multivitamin supplements are usually given to patients in the hospital, but I think they could really consider higher doses of vitamin D to boost immunity.

Q. You mentioned the effect of vitamin D on vascular disease in dialysis patients. How is that explained?

A. It's not well understood, but it may involve the relationship between vitamin D and parathyroid hormone levels or the effects of vitamin D on blood pressure, since adequate vitamin D status decreases the risk for high blood pressure. Vitamin D has antiinflammatory properties. Chronic inflammation increases the development of cardiovascular disease and keeping it in check could prove beneficial.

Q. We've talked about vitamin D and bone health and immunity. Are there other roles for vitamin D in older people?

A. It's becoming apparent that vitamin D is important for preventing falls due to its role in maintaining muscle strength. That's especially important in older adults. Vitamin D seems to help prevent muscle atrophy and is important in the calcium regulation of muscle activity. There are also intriguing correlative studies suggesting that vitamin D may improve cognitive function in the elderly.

Q. Why have clinicians been interested in the treatment of breast cancer and prostate cancer with vitamin D?

A. Much preclinical data show that active metabolites of vitamin D prevent the growth of those cancer cells, probably by inducing the tumor suppressor genes and also by causing the cells to differentiate and stop proliferating. The active metabolites have been shown to increase the efficacy of chemotherapeutic agents in preclinical models. Also, high circulating serum levels of vitamin D correlate with lower rates of numerous cancers.

Q. The expert consensus on the recommended daily intake of vitamin D seems to be changing. For example, pediatricians recently recommended higher intakes for infants and children. Do you think that the Food and Nutrition Board of the Institute of Medicine will increase the dietary recommended intakes of vitamin D for adults?

A. They are considering that right now. I think that there will be an increase because there is a lot of evidence to support it.

Q. Does taking supplemental vitamin D seem sensible to you?

A. LPI recommends 2,000 IU per day, and I agree with that. I take about 2,500 IU per day. It is important to keep sufficient vitamin D levels in the blood. It can be taken daily all year, and it's better to keep levels consistently high rather than have them go up and down. And there's no problem with any toxicity at these doses. You'd have to take 100,000 IU or more per day long term to cause toxicity. Our bodies are capable of synthesizing about 20,000 IU/day through sun exposure!

Last updated June 2010