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Oregon State University

The

Linus Pauling
Institute

R E S E A R C H

N E W S L E T T E R

From theDirector

Vitamin D Gets
An “A”

Balz Frei, Ph.D.
LPI Director and Endowed Chair
Distinguished Professor of
Biochemistry and Biophysics

An Interview with Adrian Gombart, Ph.D.
Associate Professor of Biochemistry
and Biophysics
LPI Principal InvestigatorAs director of LPI, I am often asked what supplements
I take—after all, thinking about and researching
micronutrients every day, I should know what dietary
supplements are most important. While I think eating ahealthy diet, exercising regularly, maintaining a healthy
body weight, and avoiding tobacco are of utmost importanceto maintain good health, I also think that some dietary
supplements are critical for optimum health.
First, I take a daily multivitamin/mineral with 100% of
the Daily Value (DV) or Recommended Dietary Allowance(RDA) of most vitamins and essential minerals. I take thismultivitamin primarily as “health insurance”; that is, tomake sure I get at least the recommended amounts of mostvitamins and minerals in case I don’t get each and every
one of them from my daily diet. This multivitamin is not
an excuse not to eat a healthy diet; instead, I consider
taking a multivitamin part of a healthy lifestyle. Plus, it’sdirt cheap—you can buy a year’s worth of multivitamins
for less than a nickel a day!
Next, I take 500 mg of vitamin C every day, in keepingwith LPI’s recommendation to get at least 400 mg daily,
which should be enough to saturate my vitamin C bodypool. I also get 60 mg of vitamin C from my multivitaminand another 100-200 mg from my diet, including a glassof fresh grapefruit juice every morning and additional fruitand vegetables as part of my regular diet. I take the freeacid supplement of vitamin C—ascorbic acid—as I don’t
have any stomach or gastrointestinal problems with its
acidity; but if you do, try a mineral salt of vitamin C like
calcium or magnesium ascorbate.

Q. When did you decide on a career in science?
A. I was interested in science when I started college, andI was thinking about becoming a veterinarian. After
gaining some practical experience in vet clinics and notgetting 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 aposition open in the Linus Pauling Institute. I knew that
LPI had moved to Oregon State and that it was wellknown 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.

continued on page 2

continued on page 3— DESIGNATED BY THE NATIONAL INSTITUTES OF HEALTH AS A —Center of Excellence for Research on Complementary and Alternative Medicine

Continued from cover — From the Director
I also take a daily supplement of 2,000 IU of vitamin D,
which is in addition to the 400 IU I get from my multivitamin.
Vitamin D deficiency is a major health problem in the U.S.
The majority of Americans have serum 25-hydroxyvitaminD levels less than 32 ng/mL, putting them at increased risk
for osteoporosis and bone fractures and possibly hypertension, several cancers, and multiple sclerosis. In addition,
the work by LPI’s Fritz Gombart has shown that adequate
vitamin D intake is essential for healthy immune function
and protection against infectious diseases like tuberculosis.
I had my serum vitamin D level checked a little over a year
ago, and to my dismay it was barely sufficient at only 30
ng/mL. The doctor put me on 50,000 IU of vitamin D per
week for 8 weeks to load up my body stores for the winter,and now I am on a maintenance dose of 2,000 IU. Thisdose also is consistent with the Institute’s recommendation,
which is higher than the government’s recommendation of
400 IU (rumored to be increased soon to 1,000 IU).

lipoic acid is well known to stimulate the insulin receptor
and improve glucose metabolism, and is used in Europe totreat diabetic complications.
My lipoic acid-ALCAR supplement also provides 300
mcg (100% of the DV) of biotin and 150 mg (15% of theDV) of calcium, which complements my multivitaminthat contains only 30 mcg of biotin (the current RDA)
and 200 mg of calcium. Multivitamins do not contain thedaily recommended calcium dose of 1,000 mg because it
would make the tablets too bulky. Therefore, it is important
to make up the difference with an extra calcium supplement
or from your diet. I close the gap from the 200 mg in mymultivitamin with 150 mg from the lipoic acid-ALCAR
supplement and my daily 16-oz cappuccino—providing
about 250 mg of calcium—and an 8-ounce low-fat
probiotic yogurt with another 400 mg.
My daily supplements in combination with my dietand regular exercise closely reflect LPI’s Rx for Health(see http://lpi.oregonstate.edu/lpirx2.html), except that I
don’t take a supplement of 200 IU natural source alphatocopherol (d-alpha-tocopherol). I get 30 IU of vitamin E
from my multivitamin and additional vitamin E from myfish oil supplement. Since I have experienced an increased
tendency to bleed if I take both fish oils and extra vitamin
E, I do not take the latter on a regular basis. My health isexcellent, and I hope to remain healthy for a long time.
Our Rx for Health is designed to help you live longer—
with the best health, too. LPI

Honors for LPI Faculty
Furthermore, I take a daily 1,200-mg fish oil supplement,
which contains 360 mg of eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA). These omega-3 fatty acids
are important for cardiovascular health and have beenshown to lower the risk for heart attacks and sudden cardiac
death. In addition, they have strong anti-inflammatory
properties. Chronic inflammation increases with age andis a critical contributing factor to many chronic diseases,
including heart disease and certain types of cancer andneurodegenerative diseases. Omega-3 fatty acids also areimportant for brain health, and accumulating evidence
suggests that DHA may slow cognitive decline in older
adults and lower the risk for Alzheimer's disease and othertypes of dementia. And some very recent evidence suggeststhat fish oils may help maintain the ends of DNA called
telomeres. Telomere shortening has been suggested to be
an indicator of biological aging and may be associatedwith an increased risk of heart disease.
Finally, I take 400 mg of lipoic acid and 1,000 mg ofacetyl-L-carnitine (ALCAR) daily. This is based on the
research by LPI’s Tory Hagen on the role of these “ageessential” micronutrients in improving mitochondrial
function and energy metabolism with age, and the researchin my own laboratory indicating that lipoic acid has
anti-inflammatory properties and lowers body weight andserum triglycerides in experimental animals. In addition,

2

Spring/Summer 2010

O

n September 23, 2009,
Dr. David Williams, an LPI
Principal Investigator and Professor
of Environmental and Molecular
Toxicology, was honored with
the OSU Alumni Association
Distinguished Professor Award.
The award is given for“outstanding professional
achievement through teaching and scholarship,
service to the university and the community, andprofessional leadership, nationally and internationally.”
Dr. Balz Frei, LPI’s Director andProfessor of Biochemistry andBiophysics, was named an OSUDistinguished Professor on
January 26, 2010, for achieving“national and international
stature as a result of [his]contributions to scholarship/
creative activity, research,education, and service, and whose work has been
notably influential in [his] fields of specialization.”
Congratulations to our Distinguished Professors!

Continued from cover — Interview with Dr. Adrian Gombart

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 andprotein 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 withvery different interests. I think that happens more here
than in medical centers. The university is a little lesspolitical—people are more interested in achieving theirresearch goals rather than protecting their territorial
ambitions. That being said, I have some greatcollaborators in Los Angeles, and our projects are stillongoing and productive.

Q. Are you always thinking about science or doyou 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 playorganized sports, and we spend the weekends runningaround 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 donemuch 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 acritically important issue, though, because you have tocontinue to get funding to be successful.

Q. Early in your career at the University ofWashington, you investigated the genetics ofviruses, 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 ofthat has to do with nutrition. If you are malnourished,
you don’t fight infections very well. The vaccine formeasles, while quite effective, is not effective enough toeliminate the disease. Measles is one disease like poliothat 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 thatis used by the measles virus to increase the number ofproteins that its RNA genome can code for by making
changes to the messenger RNA sequence.

Various iterations of RNA editing are used by higherorganisms, as well. At the time, it was a very intriguingmechanism.

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 theprotein 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 theirfunctional 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 likethe 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 thecell cycle.

Q. Is cancer always associated with altered activityof 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 theretinoblastoma tumor suppressor protein functions isvery common in most cancers.

Q. Are there ways to influence the activity oftumor 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 greatinterest in using vitamin D and vitamin A compounds
to induce the tumor cells to change their properties
and stop growing. Vitamin D induces the expressionof p21.

continued on page 5

The Linus Pauling Institute3

Preventing Cancer with Clayflatoxins are naturally occurring toxins formed bymolds. Corn and peanuts are often affected, especiallywhen grown in drought conditions or stored at hightemperature or humidity. These toxins can produce severe
liver damage or cancer in humans. Even with the best
agricultural practices, some contamination by aflatoxins,
such as aflatoxin B1 (AFB1), is unavoidable. In the UnitedStates, strict regulations and testing by industry and government
agencies have been largely effective at minimizing aflatoxin
levels in the nation’s food supply, though at a cost of
approximately 1.4 billion dollars per year. However, thescreening process is not foolproof. Despite the fact that raw
materials are screened, every few years an outbreak of fatal
aflatoxicosis occurs in dogs, a species far more sensitive thanhumans to the acute toxic effects of aflatoxins. The same
company implicated in the recent peanut butter Salmonella
outbreak had previously been cited by the FDA for distributing
peanut products containing unacceptable levels of aflatoxins
on multiple occasions. In many developing countries, theprocess of screening and discarding contaminated grains isjust not economically feasible. Worldwide, over 4.5 billion
people are unavoidably exposed to chronic, high levels ofaflatoxins, and this exposure is believed to be a major factor
in the high rates of liver cancer in some countries. There isan urgent need to develop safe and effective methods toreduce the impact of accidental or unavoidable aflatoxin
exposures on human health.
One strategy to minimize dietary aflatoxin exposure is
to include substances in the diet that bind to these agentsand slow their uptake. LPI investigator Dr. George Bailey
and colleagues have demonstrated that the successful cancer
chemopreventive agent chlorophyllin (CHL) acts primarily
through this mechanism. Another agent that appears toblock aflatoxin uptake through a similar mechanism is a
type of clay known as NovaSil (NS).
Geophagy, the deliberate consumption of dirt or clay, hasbeen a well-documented phenomenon for centuries. One
hypothesis for the consumption of clay by certain tropicalbirds is that the clay reduces absorption of plant toxins andallows the consumption of plants that would otherwise be
toxic. Native American recipes for acorn bread frequently
included mixing the meal with clay and water, a practice that
reduced the levels of bitter-tasting tannins. In some cultures,geophagy is common among pregnant women who reportthat the practice reduces the nausea and vomiting that iscommon in the first trimester. This may be due to the abilityof the clay to adsorb toxins, buffer the gastrointestinal tract,
or provide supplemental calcium. Other clays might provide
protection towards toxins like aflatoxins.
NovaSil is an anti-caking additive commonly added toanimal foods. In the late 1980s our collaborator, Dr. Timothy
Phillips from Texas A&M University, first reported that thisparticular clay bound to aflatoxin with high affinity and highspecificity. His research team subsequently reported that NS
could prevent the acute toxicity induced by aflatoxin in a

4

Spring/Summer 2010

wide variety of animals, including rats, mice, chickens,turkeys, cattle, lambs, and pigs. In poultry, NS was so
effective that it was able to provide almost complete
protection against poisoning from aflatoxins even when the
AFB1 levels were hundreds of times above the toxic levels.
Recently, Dr. Phillips’ laboratory found that supplementation
with NS reduced levels of aflatoxin biomarkers in the bloodand urine of humans from Ghana who are exposed to highlevels of aflatoxin in the diet.
Althoughthesestudies
1 tumor per animal
clearly showed
2-4 tumors
6
5 or more tumorsthat dietary
clay could
5
protect against
the acute4
toxicity ofaflatoxins,3
there was nodirect evidence2that NS could
prevent cancer
1in any animalmodel.
0
In 2006, I
Initiated
2,500 ppm 4,000 ppm
controls
NovaSil
NovaSil
received an
LPI pilot
Inhibition of AFB1- initiated tumor incidence by NS.
project grant
to determine if NS could prevent aflatoxin-induced DNA
damage and cancer in rainbow trout. Trout were selected
for this project for several reasons. Trout are exquisitely
sensitive to aflatoxin-induced liver cancer; indeed, the firstevidence that aflatoxin caused cancer was from studies
conducted in the 1960s by OSU researchers using rainbow
trout. Studies involving hundreds of animals can be
conducted in this model at a modest cost. There is a long
history of using rainbow trout to study chemopreventive
agents, including the seminal work by Dr. George Bailey’slaboratory that established CHL as a highly effective
chemoprotective agent against aflatoxin-induced liver cancer.
7

Tumor Incidence (%)

A

Gayle Orner, Ph.D.
Assistant Professor (Senior Research)

Two separate tumor studies were conducted to determine
if NS inhibited AFB1-induced DNA damage and liver cancer.
In the first study, trout were given diets containing AFB1 atthree times the level allowed in U.S. food supplies, plus oneof two levels of NS or CHL. In each study, NS significantly
reduced the amount of AFB1 bound to liver DNA. The
figure above shows the effect of either 2,500 or 4,000
ppm NS at inhibiting the liver tumor incidence induced
by 60 parts per billion (ppb) AFB1. NovaSil and CHL had
comparable effectiveness in inhibiting tumor incidence
induced by 160 ppb AFB1 (not shown).
Our pilot studies confirmed that dietary NS is effectiveat reducing both DNA damage and liver cancer in AFB1treated trout. NovaSil, like CHL, has the potential toreduce the global impact of aflatoxin exposure on humanhealth. Future studies will examine the effectiveness ofthese two agents when given in combination. LPI

Continued from page 3 — Interview with Dr. Adrian Gombart

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 betumor suppressors. Kinases are enzymes that modify
protein activity by phosphorylation, and cyclin-dependent
kinases regulate the cell cycle. If you can inhibit thosekinases, 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, weanalyzed 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 cellbecause 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 theearly 1980s, there was a lot of interest in using vitamin D
because it can cause cell differentiation or maturation andinhibit 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 theirbeneficial 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 fromthe 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 yourdiet—your body doesn’t synthesize it. Vitamin D wascalled a vitamin because of the discovery that vitamin
D-deficient animals could be fed irradiated food to curerickets. 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 ofproteins called steroid hormone receptors. These transcription factors bind to certain kinds of hormones andother 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’shydroxylated and becomes 25-hydroxyvitamin D, whichis 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 inour 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.

Q. Is the vitamin D found in supplements the same
form of vitamin D that’s made in the body orfound in food?
A. Supplements contain either vitamin D2, also called
ergocalciferol, or, usually, vitamin D3, also known ascholecalciferol. Both are converted in the kidneys to theactive 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 chemicallymodified 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, andsardines, 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 isconsidered 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 theparathyroid tells your body it needs morecalcium. 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 notconsuming enough vitamin D or if
levels of vitamin D are low,then you can’t produce
enough 1,25-dihydroxyvitamin D needed tosequester calcium.

continued on page 6

The Linus Pauling Institute5Continued from page 5 — Interview with Dr. Adrian Gombart

Q. And that explains the development of rickets?
A. Yes. Instead of getting calcium from the diet, the bodytakes calcium out of the bones. The parathyroid hormone
is regulated by the active form of vitamin D, 1,25dihydroxyvitamin 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 importantin every stage of life.
A. Yes, vitamin D levels are very important throughout
life—to prevent rickets in childhood and to ensure bonehealth as we age. Also, it’s becoming apparent that it isimportant 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 inmany 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 bywhere 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 skinbecomes 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 deficientand 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 toincreased levels of inflammation. Inflammation is importantfor fighting infection, but chronic inflammation probably
contributes to the development of many of the diseases

6

Spring/Summer 2010

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 vaccinesand provides long-term protective immunity. T cells andB 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 andneutrophils 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 inmacrophages and neutrophils. Those genes make small
peptides called cathelicidin that punch holes in thepathogen’s membrane and disable it.

Q. Is vitamin D absolutely required for thecathelicidin 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 showedthat the antimicrobial response was dependent on vitamin
D in cultured cells. Their work indicated that low vitaminD levels in the blood would not support the production ofthis 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, butcathelicidin is critical. There’s a knock-out mouse modelin which cathelicidin isn’t present, and the mice aresusceptible 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 relationshipbetween 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 andlooking 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 themacrophage-like cells that we were studying. As we were
getting ready for publication, another paper came outthat reported the observation that I made—that there arevitamin 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 enhancescathelicidin production in numerous cell types and in
macrophages, and reported that the cathelicidin genehas been evolutionarily conserved in primates as animportant part of innate immunity. We followed that up
with a paper last year showing that the expression ofcathelicidin gene is controlled by so-called “junk DNA”
and has been conserved in the human lineage for 55 to
60 million years.

Q. Have there been any epidemiological studies thatlooked at blood levels of vitamin D and rates ofinfection 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 weredeveloped, 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 withreactivation of the disease. For example, in TB patientsfrom Southeast Asia who move north, the disease getsreactivated because they get less sunlight and tend to bevegetarian and covered more in clothing. A recent reportanalyzing data from the NHANES epidemiological study
showed a correlation between vitamin D deficiency andincreased 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 becauseof 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 riskof death from infection. How big was the effectand have there been any follow-up studies using
vitamin D supplementation to try to raisecathelicidin levels in the blood?
A. Cathelicidin is secreted into the blood and is present at
pretty high levels. Most antimicrobial peptides arepackaged in neutrophils, which are white blood cells,
but cathelicidin is also actively secreted into the bloodfor some unknown purpose. There isn’t any research that
correlates vitamin D status with cathelicidin levels inblood. Most of our patients had pretty high levels ofvitamin 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. Somestudies have found that hemodialysis patients who takevitamin D have a sharply reduced risk for cardiovascularmortality. In our dialysis study, we measured vitamin D
and cathelicidin in the blood of several hundred patientsin a prospective cohort of over 10,000 patients undergoing hemodialysis. Those with the lowest level ofcathelicidin had a two-fold increased risk of death from
infection within the first year of hemodialysis comparedto those with the highest levels of cathelicidin. We
found a positive association between the active form ofvitamin D—1,25-dihydroxyvitamin D—and cathelicidin
levels. We are following this up with a clinical study atCedars-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 formorbidity, 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 thatvitamin 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 patientsin 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 vasculardisease in dialysis patients. How is that explained?
A. It’s not well understood, but it may involve therelationship between vitamin D and parathyroid
hormone levels or the effects of vitamin D on bloodpressure, 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 healthand 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 isimportant in the calcium regulation of muscle activity.
There are also intriguing correlative studies suggesting thatvitamin D may improve cognitive function in the elderly.

continued on page 8

The Linus Pauling Institute7

Continued from page 7 — Interview with Dr. Adrian Gombart

Q. Why have clinicians been interested in thetreatment 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 beenshown 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. Does taking supplemental vitamin D seemsensible to you?
A. LPI recommends 2,000 IU per day, and I agree withthat. I take about 2,500 IU per day. It is importantto 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! LPI

Q. The expert consensus on the recommended
daily intake of vitamin D seems to bechanging. For example, pediatricians recently
recommended higher intakes for infants andchildren. Do you think that the Food andNutrition Board of the Institute of Medicine
will increase the dietary recommended intakesof vitamin D for adults?
A. They are considering that right now. I think that
there will be an increase because there is a lot ofevidence to support it.

Vitamin E andChemotherapy
Debbie J. Mustacich, Ph.D.
Assistant Professor (Senior Research)
Linus Pauling Institute and OSU
Department of Biomedical SciencesT

he American Cancer Society estimates that more than
1.4 million new cases of cancer were diagnosed andmore than 500,000 people died of cancer-related deaths in
the United States during 2008. Lung cancer accounts for
29% of all cancer deaths and is the leading cause of cancer
deaths in both men and women. Non-small-cell lung cancer(NSCLC) accounts for 75-80% of lung cancers and has afive-year survival rate of ~15%. In women, ovarian cancer
has the highest mortality rate of all the female reproductive
system cancers, accounting for 6% of all cancer-related deaths
in women. The high death rate for both of these cancers isdue, in most cases, to patients having advanced disease at
the time of diagnosis. In contrast, prostate cancer is often
diagnosed in the early stages when the tumors are responsive
to androgen ablation therapy. However, over time prostatecancers nearly always undergo androgen-independent
progression and become refractory to hormonal manipulationsuch that prostate cancer remains the second leading causeof cancer-related deaths in men. These dismal statistics

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Spring/Summer 2010

indicate a need for innovative improvements in treatment
modalities, particularly with respect to improved therapies
for patients with advanced stage cancer.

Why use cisplatin?
Cisplatin, a platinum-containing compound, is currently
one of the most effective anticancer drugs available fortreating a variety of solid tumors, including lung, ovarianand prostate cancer. Cisplatin-based chemotherapy has beenshown to significantly increase overall survival rates whenused as adjuvant treatment in NSCLC patients following
lung resection surgery, particularly in late-stage cancer.
In addition, clinical trials have demonstrated an increased
response and median survival in hormone refractory prostatecancer (HRPC) patients treated with therapies that include
platinum compounds. In 2006, the National Cancer Instituterecommended that the standard treatment for women withlate-stage ovarian cancer should be surgical removal of thebulk of the tumor followed by intraperitoneal (IP) injectionof cisplatin in combination with a taxane, such as paclitaxol,
given either IP or intravenously (IV). Use of IP cisplatin in
ovarian cancer patients increased median survival by 16months compared to IV cisplatin. Compared to IV
administration, IP administration of chemotherapy drugs
allows longer exposure of the peritoneal cavity, the principalsite of disease in ovarian cancer, to higher doses of the drug.
Unfortunately, like many other anticancer agents, platinumcompounds are neurotoxic, and development of peripheral
sensory neuropathy often limits the dose and duration oftreatment. Notably, neurologic side effects occurred at ahigher rate in patients receiving IP cisplatin such that mostpatients receiving IP cisplatin did not complete as manytreatment cycles as patients receiving IV cisplatin. The
mechanism of cisplatin neurotoxicity remains unknown, thus
hampering the development of optimal adjuvant treatments
to prevent platinum-induced neuropathy and allow patientsthe full life-extending benefits of cisplatin treatment without
the debilitating side effects that reduce quality of life.

Similarities between cisplatin toxicity andvitamin E deficiency neuropathies
Vitamin E deficiency occurs rarely in humans. When it
does, it is usually the result of a genetic abnormality in thealpha-tocopherol transfer protein or a fat malabsorption
syndrome. A distinct pattern in the progression of neurologicalsymptoms resulting from vitamin E deficiency in humans has
been described, with symptoms characterized by ataxia (lossof muscle coordination), paresthesia (prickling, tingling
sensation), and loss of reflexes. These symptoms result froma loss of the large caliber axons of the sensory neurons.
Pathologic studies indicate that the dorsal root ganglia are
the neural tissue primarily affected by vitamin E deficiency.
Remarkably, the description of neuropathy in cancer patientstreated with cisplatin closely resembles that of human vitamin
E deficiency. In one study, 10 of 11 ovarian cancer patients
receiving cisplatin developed a distal sensory neuropathy,
manifested by decreased vibratory sensation—the firstdetectable symptom in vitamin E deficiency. With continued
cisplatin therapy, four patients developed paresthesia. At
autopsy, platinum levels were highest in the dorsal root
ganglia, as compared to other neural tissue, the same
nervous tissue affected by vitamin E deficiency.

Benefits of vitamin E supplementation
during cisplatin treatment
Many anticancer drugs cause oxidative stress in normal
tissues while killing cancer cells via non-oxidative stressmechanisms. Importantly, cisplatin-based regimens have beenshown to decrease plasma vitamin E (alpha-tocopherol) levels
in patients. Thus, antioxidants, including vitamin E, may
represent an important adjuvant to standard chemotherapy
regimens. However, clinical trials investigating the use ofalpha-tocopherol in combination with chemotherapeutic
drugs have been sparse, in part due to the concern thatantioxidant vitamins, due to their ability to prevent oxidative
stress generated by free radicals, may reduce the efficacy ofsome anticancer drugs. However, numerous cell culture studies
have found that vitamin E, particularly at high pharmacologic
doses, potentiates the apoptotic (programmed cell death)and growth inhibitory effects of anticancer drugs, including
platinum compounds.

depletion, thereby preventing the neurologic side effects ofcisplatin; and 3) alpha-tocopherol supplementation will notdecrease the anticancer efficacy of cisplatin.
We first determined that daily administration ofpharmacologic doses of alpha-tocopherol to rats significantly
increased alpha-tocopherol levels in tissues, including liver(~15-fold), lung (~3-fold), kidney (~2-fold), spinal cord (~2fold), and dorsal root ganglia (~2-fold). Next, we treatedrats with three cycles of five daily injections of either
cisplatin or placebo with and without co-administration ofpharmacologic doses of alpha-tocopherol. As expected, lipid
peroxidation significantly increased and dorsal root gangliaalpha-tocopherol levels significantly decreased in cisplatintreated rats. Importantly, concurrent high-dose alpha-tocopherol administration preserved dorsal root ganglia alphatocopherol levels and prevented increased lipid peroxidationin cisplatin-treated rats. Future studies to further elucidate
the mechanism of cisplatin-induced neuropathy, as well as
determine the efficacy of adjuvant high-dose alpha-tocopherol, will determine 1) the ability of cisplatin to alter theplasma and tissue levels of additional antioxidants; 2) theability of alpha-tocopherol to prevent the morphologicalchanges in dorsal root ganglia associated with cisplatininduced neuropathy; and 3) the anticancer efficacy of
cisplatin in tumor-bearing rats given pharmacologic dosesof alpha-tocopherol.
Understanding the mechanism of cisplatin-induced
neuropathy, as well as determining the ability of alphatocopherol to prevent the biochemical and morphologicchanges associated with cisplatin-induced neuropathy
without decreasing anticancer efficacy, will provide thenecessary evidence-based knowledge to support clinical
trials to determine guidelines for including vitamin E incisplatin-based chemotherapy regimens. Preventing cisplatininduced neuropathies will allow patients to complete, andpossibly increase, their cisplatin treatments; thus allowing
patients to fully benefit from the life-extending potential of
long-term cisplatin treatment without the debilitating side
effects that currently reduce quality of life for both patientand family. LPI

Ongoing studies
Our central hypothesis is that 1) cisplatin depletesalpha-tocopherol in the lumbar region of the spinal cord,
specifically the dorsal root ganglion, by an oxidative stressmechanism whereby platinum acts as a catalyst for lipid
peroxidation resulting in an alpha-tocopherol-deficiency
neuropathy; 2) pharmacologic doses of alpha-tocopherol
will prevent the cisplatin-mediated alpha-tocopherol

The Linus Pauling Institute9Vitamins E and C and the Recovery from
Anterior Cruciate Ligament Surgery
Tyler Barker, LPI Graduate FellowT

he anterior cruciate ligament (ACL) is an integral
component of knee structure and function. Its role is tolimit excessive knee rotation and provide stability to theknee joint. Unfortunately, every year approximately 200,000
Americans injure their ACL. In nearly half the cases, thisinjury to the ligament requires reconstructive surgery.
Immediate and persistent muscle dysfunction commonly
follows the injury and surgical repair of a ruptured ACL.
Despite advances in orthopedic surgery, physical therapy,
and medicine, muscle dysfunction, especially weakness, cancontinue for years. Although impaired neurological feedback
from the surgically repaired knee and reduced physicalactivity contribute to the muscle dysfunction, the reasonsfor muscle atrophy and weakness remain a mystery.
We are interested in ACL injury because oxidative stress
may play an important role in exacerbating muscle
degeneration. Both oxidative stress and pro-inflammatory
molecules called cytokines induce muscle dysfunction during
aging and in chronic heart failure patients. Therefore, ourfindings may be applicable to people other than those with
ACL injury. Because vitamins E and C are potent dietary
antioxidants that modulate diverse inflammatory cytokines,
we conducted a randomized, double-blind, placebo-controlled study to investigate the influence of vitamin E and C
supplementation on the recovery from ACL reconstructive
surgery. We made three major discoveries that we discuss
further below.
1. Vitamin E and C supplementation lowers the increaseof an anti-inflammatory cytokine (interleukin-10 or
IL-10) and stabilizes the depression of a pro-toanti-inflammatory cytokine ratio (i.e., IL-6:IL-10)
immediately following surgery.
2. The recovery of leg strength following ACL reconstruction
is associated with vitamin C levels in blood prior tosurgery and with elevated vitamin C levels post-surgery
following vitamin E and C supplementation.
3. Greater strength gains after ACL surgery are associatedwith lower oxidative stress and a higher pro-to-antiinflammatory cytokine ratio but only in the subjects
supplemented with vitamins E and C.

Vitamin E and C supplementation improvesinflammatory status immediately following
ACL surgery
During ACL surgery, a tourniquet is applied to the upper
thigh of the leg that is being repaired. This procedure provides
a bloodless operating field but also creates an ischemic
condition in which blood and oxygen are deprived from theaffected area. When the tourniquet is removed and blood
begins to circulate again, the tissue is subject to what’s

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Spring/Summer 2010

known as ischemia-reperfusion injury. The restored flowof blood causes oxidative and inflammatory damage.
Ischemia-reperfusion injury is potentiated by oxidative
stress, which stimulates pro-inflammatory cytokines thatfurther exacerbate the inflammation. Although it has been
documented that there is an increase in local markers ofoxidative stress following tourniquet removal, the role ofthe circulating inflammatory cytokine and oxidative stressmarker responses to this ischemia-reperfusion insult has
not been well described.
Therefore, we examined oxidative stress and inflammatory
cytokines in the circulation after ACL surgery in subjects who
received either a placebo or an antioxidant supplement ofvitamins E (400 IU) and C (1,000 mg), taken in two equallydivided daily doses starting approximately two weeks prior
to surgery and continuing for three months after surgery.
As expected, we found that antioxidant supplementation
doubled the vitamin E and C plasma concentrations.
Nonetheless, within 90 minutes following ACL surgery, we
found a significant elevation in oxidative stress in both the
supplemented and placebo groups. There were no significant
differences in markers of lipid peroxidation—F2-isoprostanes
or malondialdehyde—between the groups. These markers
decreased to baseline levels a few days following surgery.
Additionally, there were increases in markers of muscle
damage (creatine kinase) three days after surgery.
Inflammation was also increased at three and seven days
following surgery, as indicated by an elevation in the proinflammatory cytokine IL-6 and C-reactive protein. However,antioxidant supplementation did not affect biomarkers ofmuscle damage and inflammation following surgery.
An immune response typically follows surgery, represented
by elevated blood levels of the anti-inflammatory cytokine
IL-10. At 90 minutes following ACL surgery, IL-10
increased markedly in the placebo but not the antioxidantgroup. This is remarkable because it suggests that the
antioxidant supplementation decreased the body’s need toexpress an anti-inflammatory cytokine. Vitamins E and C
potentially prevented an increase in oxidative stress and/or
pro-inflammatory cytokines and provided protection against
post-surgery complications. However, further research
addressing this question is required.
Another approach to assessing inflammation is tocalculate ratios between pro-inflammatory (IL-6) and antiinflammatory (IL-10) cytokines. Changes in the IL-6:IL-10
ratio, for example, may have prognostic value for poor
clinical outcomes, such as infection or death. We observedthat the IL-6:IL-10 ratio decreased immediately following
ACL surgery. However, this depression was ameliorated in
the subjects who received vitamins E and C. Whether thestabilized pro-to-anti-inflammatory cytokine ratio in our
antioxidant group translates to improved recovery following
surgery is unknown but warrants further investigation.

Greater strength gains after ACL surgery areassociated with lower oxidative stress only insubjects supplemented with vitamins E and C
It is unknown if oxidative stress or inflammatory cytokines
are associated with leg weakness following ACL surgery andwhether supplementation with vitamins E and C would modulate this relationship. We found that the maximum isometricforce produced by the injured leg following ACL surgery was
inversely correlated with plasma F2-isoprostanes—markers ofoxidative stress—within the antioxidant group. That is, the lessoxidative stress, the stronger the leg. Furthermore, maximum
isometric force of the injured leg also correlated with theIL-6:IL-10 cytokine ratio in the antioxidant group. Therefore,
our results indicate that antioxidants decrease oxidative stress,
improve the balance of pro- and anti-inflammatory cytokines,
and improve the recovery from muscle damage.

Better recovery of leg strength following
ACL reconstruction is associated with highplasma vitamin C levels
High levels of vitamins E and C are associated withimproved strength and physical performance in the elderly.
Whether supplemental vitamins E and C can improve limb
strength is unknown. Therefore, we measured plasma vitamin

E and C concentrations before and three months after ACL
surgery in our two experimental groups described above.
Not surprisingly, the injured leg was weaker than the otherleg at baseline, and it remained so three months after
surgery. However, greater gains in leg strength after surgery
were associated with higher plasma vitamin C levels prior
to surgery and with elevated vitamin C levels following
supplementation, suggesting that vitamin C aids muscle
recovery from ACL surgery.
Our data indicate that supplemental vitamins E and C
provided protection against undesirable immunologicalactivity after surgery. The reduction of the anti-inflammatory
cytokine IL-10 by antioxidant supplementation following
surgery may have provided protection against complications,as seen in other conditions. Interestingly, in the subjects
supplemented with vitamins E and C, lower oxidative stressand a higher pro-to-anti-inflammatory cytokine ratio wereassociated with greater leg strength three months after ACL
reconstruction. In conclusion, our results suggest that there
is a potential therapeutic benefit of vitamin E and C
supplementation on both immediate surgically-induced
immunological changes and with the short-term recoveryof leg strength following ACL reconstruction. Antioxidant
supplementation may be of therapeutic benefit to patients
suffering from other conditions involving oxidative stressand inflammation. LPI

Do “Antioxidants Prevent Health-Promoting
Effects of Physical Exercise in Humans”?
Scott W. Leonard, LPI Senior Research AssistantA

paper published in 2009 by Ristow et al. in theProceedings of the National Academy of Sciences,“Antioxidants Prevent Health-Promoting Effects of PhysicalExercise in Humans”, cast doubt on the combined healthbenefits of antioxidant consumption and exercise. Whilethe data do not appear to be flawed and are important,
we believe the authors have overstated their findings.
There is no doubt that achieving and maintaining ahealthful weight is a benefit of exercise, but the molecularmechanisms underlying this effect are unclear. Ristow proposedthat reactive oxygen species (ROS) have an essential role inpromoting insulin sensitivity during exercise, one of the known
benefits of exercise. They then claimed that antioxidant use,
leading to a decrease in ROS, is detrimental to the healthpromoting effects of exercise. There are many good studies,including several from our labs, showing benefits ofantioxidant use during exercise in decreasing markers ofoxidative stress. Antioxidants have been shown to decrease
DNA damage, lipid peroxidation, and protein oxidation
following exercise. The combination of antioxidants andexercise has also been shown to delay brain aging, aphenomenon proposed to be related to oxidative stress.
Ristow reported that antioxidant supplementation blocked
the induction of antioxidant enzymes in the body. However,the supplemental antioxidants may have negated the needfor up-regulation of endogenous antioxidant proteins likesuperoxide dismutase and glutathione-synthesizing enzymes.

The study’s authors focused on changes in insulin sensitivity,
but it is known that contracting skeletal muscle can take up
glucose independently of insulin during exercise, and glucose
uptake is normal during exercise in diabetics, who were
excluded from the study. The authors tried to link theirfindings to the molecular changes that occur in type 2
diabetics, but their choice of healthy men instead of diabetics
made that problematic. It is well accepted that there arehealth benefits from exercise and antioxidant consumption,
whether from the diet or supplementation, alone or in
combination. Indeed, a diet rich in fruits and vegetables
and regular exercise have long been recognized to decrease
the risk for many chronic diseases.
The statement by Ristow that “fruits and vegetables mayexert health-promoting effects despite their antioxidant
content” seems particularly inappropriate. Humans dependon fruits and vegetables and other dietary sources for theimportant physiological antioxidants, vitamins C and E.
In a recent commentary on the oxidative stress paradigm
discussed at the third international symposium on“Nutrition, oxygen biology and medicine-micronutrients,
exercise, energy and aging disorders” in Paris, Dr. Regina
Brigelius-Flohé commented that “it remained obvious thata lot of work is needed to fully understand the conditions
and individual situations under which ROS are beneficial
or detrimental”. LPI

The Linus Pauling Institute11

Nutrition and
Immunity, Part 1
Victoria J. Drake, Ph.D.
LPI Research AssociateT

he immune system functions to protect the body against
infection and disease. It is a complex and integrated
network of cells and organs that defends against pathogenicorganisms and guards against the development of cancer.
The immune system consists of the innate immune systemand the adaptive immune system. This article explores therole of macronutrients and vitamins on immune function.
An article in a forthcoming newsletter will address the roleof minerals and other factors in immunity.

The innate immune system is an immediate, nonspecificresponse to harmful substances. It is composed of physicalbarriers, such as the skin, as well as chemical and microbiological barriers, including the mucous secretions of therespiratory tract and the normal microflora of the gastrointestinal tract. Primary components of the innate immune
system include monocytes, macrophages, and neutrophils—
immune cells that engulf and digest invading microorganisms
in a process called phagocytosis. These cells express surface
receptors that identify pattern-recognition molecules that areconserved among several families of pathogens but uniqueto pathogenic microorganisms. A key component of innate
immunity is the complement system, a biochemical cascadeof at least 20 serum proteins that functions to kill invading
pathogens by direct lysis (cell rupture) or through thepromotion of phagocytosis. Innate immunity also involves
cells that mediate the inflammatory response. Thus, theinnate immune response is a rapid, nonspecific response thatserves as the first line of defense against invading microorganisms. However, if the innate response doesn’t adequately
eliminate microorganisms and resultant infections, theadaptive immune system is summoned to action throughthe signaling of messenger proteins called cytokines.
The adaptive or acquired immune system is a second lineof defense against pathogens. This defense develops over alonger period of time, taking several days to even weeks todevelop. Compared to the innate response, adaptive
immunity is much more complex because it involves antigenspecific responses and immunologic “memory” of pathogenicorganisms. Exposure to a specific antigen on an invading
pathogen stimulates production of immune cells that target
the pathogen for destruction; subsequent responses to thesame pathogen are stronger and more efficient because theantigen is “remembered.” The major mediators of theadaptive immune response are specialized white blood cells,
namely B lymphocytes (B cells) and T lymphocytes (T cells).
B cells produce antibodies and are responsible for humoral
immunity, while T cells are responsible for cell-mediatedimmunity. Although the innate and adaptive immune systems
have unique functions, their components interact and work
together to protect the body from infection and disease.

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Spring/Summer 2010

The ability of the immune system to prevent infection
and disease is strongly influenced by nutritional status of
the host. In fact, malnutrition is the most common cause of
immunodeficiency in the world. Poor overall nutrition canlead to inadequate intake of energy and macronutrients aswell as selected micronutrient deficiencies. These nutrient
deficiencies can cause immunosuppression and dysregulationof immune responses. Specifically, nutritional deficiencies
can impair phagocyte function in innate immunity andcytokine production in adaptive immunity, as well as
adversely affect certain aspects of humoral and cell-mediatedimmunity. Impairment of these responses can compromise
the integrity of the immune system, thereby increasing one’s
susceptibility to infection. Because nutritional status canmodulate the actions of the immune system, the sciences ofnutrition and immunology are tightly linked.
Inadequate intake of macronutrients or selected micronutrients can lead to immune deficiency, impaired hostdefense mechanisms, and therefore, increased susceptibility
to infection and disease. Protein-energy malnutrition(PEM), also called protein-calorie malnutrition, is a common
nutritional problem worldwide. Primary PEM, which is
caused by insufficient intake of protein and/or energy, ismore common in developing nations but is also present incertain subgroups in industrialized nations, such as theelderly and individuals who are hospitalized. Secondary
PEM is more common in developed countries, often
occurring in the context of a chronic disease that interfereswith nutrient metabolism, such as inflammatory bowel
disease, chronic renal failure, or cancer. Regardless of thespecific cause, PEM primarily affects cell-mediated immunity
rather than humoral immunity. In particular, PEM leads toatrophy of the thymus, the organ that produces T cells,
which reduces the number of circulating T cells anddecreases the effectiveness of the memory response to
antigens. Humoral immunity is affected to a lesser extent,
but antibody affinity and response is generally decreased in
PEM. PEM also leads to decreased production of certain
cytokines, reduced levels of several complement proteins,and phagocyte dysfunction. Additionally, PEM compromises
the integrity of mucosal barriers, thereby increasing
susceptibility to infections of the respiratory, gastrointestinal,
and urinary tracts. PEM often occurs in combination with
deficiencies in essential micronutrients, especially vitaminA, zinc, copper, selenium, and magnesium.
Several types of dietary lipids (fatty acids) have beenshown to modulate immune function. Polyunsaturated fatty
acids (PUFAs)—fatty acids with more than one doublebond between carbons—are broadly divided into two mainclasses: omega-3 PUFAs and omega-6 PUFAs. Lipids ofboth classes are essential nutrients because they cannot be
synthesized by the body; therefore, PUFAs must be obtained
from the diet. Dietary sources of omega-3 PUFAs include
oily fish, flaxseeds and their oil, walnuts and their oil, andcanola oil. Food sources of omega-6 PUFAs include vegetableoils (e.g., soybean, safflower, and corn oil), nuts, and seeds.
Eicosanoids (20-carbon PUFA derivatives) are signaling
molecules that play important roles in inflammatory andother immune responses. In general, eicosanoids derived
from omega-3 PUFAs have anti-inflammatory andimmunosuppressive effects, while eicosanoids derived fromomega-6 PUFAs tend to have mostly pro-inflammatory andimmunostimulatory effects, although some omega-6 PUFA
derivatives (i.e., lipoxins) have anti-inflammatory properties.
While high intakes of omega-3 PUFAs may benefit individuals
with inflammatory or autoimmune diseases, increased
omega-3 PUFA intakes, especially at supplemental doses,
could impair host-defense mechanisms and increase
vulnerability to infectious disease. Additionally, conjugatedlinoleic acid (CLA), a class of isomers of linoleic acid (anomega-6 fatty acid), has been shown in one human study toincrease plasma levels of immunoglobulin antibodies IgA and
IgM, decrease pro-inflammatory cytokines, and increase ananti-inflammatory cytokine. CLA is found naturally in meatand milk of ruminants but is also available in supplemental
form. However, more research is needed to confirm theimmune effects of CLA in humans.
In addition to macronutrients, deficiencies in certain
micronutrients (vitamins and nutritionally-essential minerals)
adversely affect both the innate and adaptive immune systems.
Micronutrients play crucial roles in the development andexpression of the immune response, and certain micronutrient
deficiencies lead to immunosuppression and increased
susceptibility to infection and disease. Select micronutrient
deficiencies are common, especially in cigarette smokers,
alcoholics, pregnant and lactating women, and the elderly.
Micronutrient deficiencies often occur in the context of generalmalnutrition; however, common isolated micronutrient
deficiencies include vitamin A, vitamin D, zinc, and iron.
Vitamin A and its metabolites are required for normal
functioning of the immune system. The skin and mucosal cells(cells that line the airways, digestive tract, and urinary tract)function as a barrier and form the body's first line of defense
against infection. Vitamin A maintains the integrity andfunction of these cells; therefore, deficiency in this micronutrient results in loss of integrity of the mucosal barriers andincreased vulnerability to respiratory and diarrheal diseases
and other infections. The vitamin A derivative, retinoic acid,functions as a hormone to regulate more than 500 genes,
several of which are involved in controlling the production,
proliferation, and differentiation of immune cells, including
neutrophils, monocytes, and lymphocytes. Thus, vitamin A
deficiency compromises the function of cell-mediated immunity
and also impairs antibody responses to antigens, adversely
affecting humoral immunity. Additionally, vitamin A deficiency
adversely affects components of innate immunity, such as
phagocyte function and the activity of so-called natural killer
cells, and alters cytokine signals that influence various
immune responses. Vitamin A deficiency is common worldwide, especially in developing nations, and has detrimental
effects on maternal and child health. Severe vitamin A
deficiency in children increases morbidity and mortality
related to measles, malaria, and diarrheal infections; mild
vitamin A deficiency may increase rates of certain infections,as well. Vitamin A supplementation can enhance immunity,
decrease susceptibility to infection, and significantly reduce
infection-related morbidity and mortality in children.
The active form of vitamin D—1,25-dihydroxyvitamin D—
is a potent modulator of the immune system. Within a cell’s
nucleus, 1,25-dihydroxyvitamin D associates with the vitamin D
receptor (VDR), a transcription factor that regulates gene

expression. The VDR is expressed in most cells of the immune
system; thus, vitamin D status can profoundly impact
immunity. Vitamin D can affect aspects of both innate
and adaptive immunity, including phagocytosis, cytokine
production, lymphocyte differentiation, and antibody
production. Additionally, the active form of vitamin D
stimulates the expression of antimicrobial peptides, which
are synthesized by various immune cells and function as
critical components of the innate immune system. Vitamin D
deficiency has been linked to an increased risk of autoimmune
diseases (e.g., type 1 diabetes, multiple sclerosis, andrheumatoid arthritis) and certain cancers (colorectal, breast,and prostate). For more information about vitamin D and
immunity, see the interview with Dr. Adrian Gombart inthis newsletter.
Other vitamins play critical roles in immunity, such as theantioxidant vitamins C and E. Vitamin C is a highly effective
antioxidant that protects the body’s cells against reactive
oxygen species that are generated by immune cells to kill
pathogens. Immune cells accumulate vitamin C in highconcentrations, which protects them against oxidative damage.
Some studies have shown that vitamin C stimulates the
production and function of neutrophils, lymphocytes, andphagocytes. Cell culture studies have demonstrated a direct
antiviral effect of vitamin C, but clinical studies have had
mixed results. Vitamin C also regenerates vitamin E, a lipidsoluble antioxidant that protects the integrity of cell membranes, from its oxidized form. Vitamin E supplementationin the elderly has been shown to improve age-related declines
in immune function, particularly aspects of cell-mediatedimmunity. Some studies have found that this translates to anincreased resistance against infections. In addition to the
antioxidant vitamins, several B vitamins, such as vitamin B6,
folate, and vitamin B12, are important in immunity. Vitamin
B6 deficiency impairs aspects of both humoral and cellmediated immunity due to its requirement in the biosynthesis
and metabolism of amino acids—the building blocks ofproteins like cytokines and antibodies. Deficiencies in either
folate or vitamin B12 can also adversely affect immune
responses because these B vitamins are needed in thebiochemical reactions that synthesize the nucleic acids
DNA and RNA.
Poor overall nutrition can lead to nutritional deficiencies
that compromise immunity and increase susceptibility toinfection and disease. Even subclinical deficiencies in various
nutrients may have adverse effects on the immune system.
Infection and illness can, in turn, exacerbate states ofmalnutrition in many different ways, for example, byreducing nutrient intake, impairing nutrient absorption,
increasing nutrient losses, or altering the body’s metabolism
such that nutrient requirements are increased. Immune
function also declines with age. For example, older people
often exhibit decreased vitamin D synthesis in the skin andmay have impaired vitamin C uptake. States of malnutrition
and infection can aggravate each other and lead to a vicious
cycle. Eating a healthful diet and taking a daily multivitamin-mineral supplement as nutritional insurance will
help to maintain optimal immune function. Single nutrient
supplements, such as vitamin D, may also be necessary. LPI

The Linus Pauling Institute13

Epigenetics

Rod Dashwood, Ph.D., LPI Principal Investigator
Director, LPI Cancer Chemoprotection Program

E

Drugs that inhibit HDAC have shown promise in the
pigenetics” has finally arrived
clinic as therapeutic agents. They cause aberrantly silenced
front-and-center on the popular
genes to be turned back on, thereby triggering cancer cells
landscape. A recent cover of Timeto commit suicide, also known as apoptosis. An exciting
magazine showed an image of doubleadjunct to this work is that some dietary constituents also
stranded DNA being unzipped next to the words, “Why yourhave the ability to act as HDAC inhibitors. Metabolism of
DNA isn’t your destiny”. The cover stated: “The new sciencefood and its individual constituents generates compoundsof epigenetics reveals how the choices you make can change
like butyrate, sulforaphane, organosulfur and organoselenium
your genes – and those of your kids”. Just how “new” thiscompounds, and indole-3-carbinol derivatives that inhibit
science really is can be debated, since it likely goes back to
HDAC activity and block cancer cell growth. These themes
Darwin and Lamarck and opposing views of nature versus
are being developed in a newly funded $8.45 million program
nurture. Whereas Darwin argued that incremental changes
project grant from the National Cancer Institute (NCI),
underlie the process of natural selection and survival-of-theentitled “Comparative Mechanisms of Cancer Chemofittest, Lamarck postulated that some traits were acquired
prevention”, which has the following central hypothesis:
within a lifetime due to environmental pressures. The Time
article cited the example of Norrbotten county, Sweden.
“Sulforaphane, indole-3-carbinol, and the cruciferous
This cold and desolate area historically has been associatedvegetables from which they derive are effective dietarywith periods of feast-and-famine, and research has shown
chemopreventive agents because they alter the pattern ofthat boys who went from normal eating to gluttony
in a single season produced sons and grandsons
who lived shorter lives. Another well-known
PROJECT 1
Chemoprevention of prostateexample is the agouti mouse, which is fat, has acancer, HDAC inhibition
yellow coat, and is prone to cancer and diabetes.
DNA methylation (E. Ho)
If the diet given to female agouti mice before
HDAC inhibition by
Chemoprotection by
conception is changed by adding chemicals
sulforaphane and broccoli
sulforaphane andsprouts in preclinical models
broccoil sprouts in thecalled methyl donors (e.g., folate), the offspringtransplacental model
ETB Core
have brown coats and are slim, despite having
DNA methylation and(blocking mechanisms)
Epigenetic/Translational
non-histone targets
the same agouti gene sequence. Offspring of the
Biomarkers,
LCMS,
Statistics
mice fed their usual diet look like their parents.
Epigenetic modifications(C. Löhr)
Human biomarker studies(DNA methylation and
How might such rapid changes occur, and in a(colon, prostate)histone status)trans-generational manner? The answer seems
Admin Coreto lie within the realm of epigenetics.
Mechanisms of“

PROJECT 2

indole-3-carbinol

PROJECT 3

Chemoprevention of colon
Transplacental chemoEpigenetics is the science that seeks to explain
and sulforaphanecancer, HDAC inhibition,
prevention of lung tumors
chemoprotection
how changes in gene expression can occur without
histone status (R. Dashwood)and lymphomas (D. Williams)
changes in the underlying DNA sequence. For
Histone modifications,example, a central theme in the cancer research
HDAC inhibition andturnover, DNA methylation
field has been that mutations or large-scalechromosome rearrangements can alter the DNA
histone modifications (acetylation, methylation, phosphorylasequence of key genes that regulate the fate of cells. If a genetion) and HDAC activity in cancer cells, as well as DNA pronormally expresses a protein that acts as a “brake”, and thismoter methylation status, thereby de-repressing epigeneticallygene is mutated, then the corresponding dysfunctional protein
silenced genes that regulate the cell cycle and apoptosis.”no longer blocks unrestrained cell growth, which is a hallmark
of cancer. Such tumor suppressor genes, however, also may
LPI investigators Drs. Emily Ho, David Williams, andhave a normal DNA sequence, and yet the gene is aberrantly
I will lead three integrated projects that focus on dietarysilenced in cancer. One way this can occur is by the addition
chemoprotection strategies for cancers of the prostate,
to DNA of methyl groups. If these methyl groups accumulate
lung, and colon, as well as lymphomas, and include
on the ‘start’ region of the gene, the complex cellular machinerytransgenerational studies in mice. All three projects use
needed to turn on that gene is disrupted. The cellular
facilities and scientific expertise in the centralizedmachinery also can be blocked from even accessing the DNA
Epigenetic/Translational Biomarkers (ETB) Core, headedin the first place. DNA is not naked in a cell but is surrounded
by Dr. Christiane Löhr. Reporting duties to the NCI willby proteins called histones, which dynamically open and close
involve an Administrative Core overseen by me, as Program
to permit access to DNA at the right time and place. In cancers,
Director. An important and exciting aspect of this work isover-expression of an enzyme called histone deacetylase (HDAC)
that it will proceed to clinical trials with human volunteers,
leads to acetyl groups being removed from histone proteins,testing several of the epigenetic hypotheses in the context
tightening their interactions with the DNA, and therebyof real-world food intake patterns. LPI investigators haveswitching off tumor suppressor genes. In essence, the “brake”
been at the leading-edge of research in this area for several
is turned off, and cells continue to replicate without restraint.
years. With this new grant, we hope to go much further

14

Spring/Summer 2010

along the road to understanding the complex interactions
between diet, epigenetics, and cancer prevention. LPIDevelopments
Get your name on LPI’s new homeThe new home for the Linus Pauling Institute isquickly taking shape! After several months ofcareful work on the basement level, the upper floors of
the Linus Pauling Science Center are starting to appear.
When finished in the summer of 2011, this state-ofthe-art, 105,000-square-foot facility will bring theLinus Pauling Institute to a whole new level of
excellence and dramatically enhance the legacy ofLinus Pauling. To see our progress, please visit thelive webcam at http://webcam.oregonstate.edu/lpsc.
There’s still time for you to be a part of this turning
point in the history of the Linus Pauling Institute—and to forever make your mark on its new home.
By making a gift of $10,000 or more to support theinstitute’s world-class research, your name will be
added to the list of donors honored on a wall inside
the building. Naming opportunities for offices,classrooms, and labs start at $25,000 and go up to$1 million, and pledges may be paid over five years.

$25,000 or more for the institute in your will, yourgenerosity may be honored with a named space inside
the building just like a gift that is made in full now.
Please consider including LPI in your estate plans—suggested bequest language can be found at
http://lpi.oregonstate.edu/giving.html.
For more information about naming opportunities inthe Linus Pauling Science Center, contact Kevin Heaney
at 541-737-5495 or Kevin.Heaney@oregonstate.edu.
And, please remember, we appreciate gifts of any size
to support LPI’s work.
We are currently reviewing candidates who haveapplied for the LPI Director of Development position
and will have news in the next newsletter. LPI

LPI is grateful for the bequests we havereceived from the following friends
this past year:
Grace Telenius DavisLouise Marie Sorensen
Joan Dritz

Bequests are another great way you can join thelegacy of Linus Pauling. When you designate a gift ofThank you!
The Linus Pauling Institutegratefully acknowledges grants
from the following companies
and organizations:• USANA Health Sciences, Inc.

(Salt Lake City, UT), to support humanresearch on health benefits of micronutrientsand dietary supplements• Bayer Consumer Care AG(Basel, Switzerland), to support theMicronutrient Information Center• Spirit Mountain Community Fund

(Grand Ronde, OR) and The Juan Young
Trust (Portland, OR), to support the LPI
Fitness and Nutrition Study, a local outreachprogram to improve dietary habits andphysical activity in elementary school
children. This program is also supported bya generous gift from an anonymous donor.

The Linus Pauling Institute15

Linus Pauling Institute

Non-Profit Org.
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PAID

Stephen Lawson, Research Newsletter Editor
Oregon State University
571 Weniger Hall
Corvallis, Oregon 97331-6512

Portland, OR
Permit No. 1006

phone: 541-737-5075
fax: 541-737-5077
email: lpi@oregonstate.edu
Internet Web site: http://lpi.oregonstate.edu

GIVING
to theLinus Pauling
Institute
Gifts in support of research
efforts can be made at any time.
Checks should be payable to
OSU Foundation for
Linus Pauling Institute.
Information on giving isavailable through the
OSU Foundation,
1-800-354-7281, or by
writing to the Institute.

Micronutrient
Research for
Optimum Health
Printed on recyclable paperusing soy inks

The Linus Pauling
Science Center
under construction
To see the progress on
our building, please
visit our real-time Web
cam at http://webcam.
oregonstate.edu/lpsc.
LPSC is scheduled forcompletion in summer
2011.

Look for these informative articles inside!
Page 1 ..... From the Director
Page 1 .....Vitamin D Gets an “A”
Page 2 .... Honors for LPI Faculty

Page 11 ... Do “Antioxidants Prevent
Health-Promoting Effects ofPhysical Exercise in Humans”?

Page 12 ... Nutrition and Immunity, Part 1

Page 4 .... Preventing Cancer with Clay

Page 14 ... Epigenetics

Page 8 .... Vitamin E and Chemotherapy

Page 15 ... Developments

Page 10 .. Vitamins E and C and theRecovery from Anterior Cruciate
Ligament Surgery

Special thanks to Barbara McVicar for editorialassistance and photographs, authors of signed
articles, and Dick Willoughby for the logo
photograph of Linus Pauling.