Bruce Ames, phd, and Rhonda Patrick, phd: Discussing the Triage Concept and the Vitamin D-Serotonin Connection

Bruce Ames, phd, is a senior scientist at Children’s Hospital Oakland Research Institute (CHORI), director of its Nutrition & Metabolism Center, and a professor emeritus of biochemistry and molecular biology at the University of California, Berkeley. He is a member of the National Academy of Sciences and he was on its Commission on Life Sciences. He was a member of the board of directors of the National Cancer Institute, the National Cancer Advisory Board, from 1976 to 1982. He was the recipient of the General Motors Cancer Research Foundation Prize (1983), the Tyler Environmental Prize (1985), the Gold Medal Award of the American Institute of Chemists (1991), the Glenn Foundation Award of the Gerontological Society of America (1992), the Lovelace Institutes Award for Excellence in Environmental Health Research (1995), the Honda Prize of the Honda Foundation, Japan (1996), the Japan Prize, (1997), the Kehoe Award, American College of Occupational and Environmental Medicine (1997), the Medal of the City of Paris (1998), the US National Medal of Science (1998), the Linus Pauling Institute Prize for Health Research (2001), and the American Society for Microbiology Lifetime Achievement Award (2001). He has more than 550 publications, which have resulted in his being among the few hundred most-cited scientists (in all fields).

Rhonda Perciavalle Patrick, phd, is a postdoctoral fellow at Children’s Hospital Oakland Research Institute with Dr Bruce Ames. She earned her doctoral degree in biomedical science from the University of Tennessee Health Science Center in Memphis while conducting research at St Jude Children’s Research Hospital in Memphis. She also has a bachelor of science in biochemistry/chemistry from the University of California, San Diego. She has done extensive research on aging, cancer, and nutrition. In Memphis, she investigated the link between mitochondrial metabolism, apoptosis, and cancer. Her groundbreaking work discovered that a protein that is critical for cell survival has 2 distinct mitochondrial localizations with disparate functions, linking its antiapoptotic role to a previously unrecognized role in mitochondrial respiration and maintenance of mitochondrial structure. Her dissertation findings were published in the 2012 issue of Nature Cell Biology.1 Currently, she conducts clinical trials looking at the effects of micronutrients (vitamins and minerals) on metabolism, inflammation, DNA damage, and aging. In addition, she is investigating the role of vitamin D in brain function and other physiological functions. In February of 2014, she published a paper in the FASEB Journal2 on how vitamin D regulates serotonin synthesis and how this relates to autism.

Integrative Medicine: A Clinician’s Journal (IMCJ): Dr Ames, would you start by providing an overview of your triage concept?

Dr Ames: A genetic toxicologist named Jim MacGregor came to my lab on sabbatical. He had been previously looking at chromosome breaks in mice. He would irradiate the mice to get more double-strand breaks and the chromosome would fall apart. He was studying various aspects of this.

One day, all of his control mice were full of chromosome breaks. He tracked it down; the company that made his vitamin mix had mistakenly left out all the folic acid vitamin. He then did a dose response on folic acid. As folic acid gets more deficient, chromosomes break. He showed that this was also true in humans.

MacGregor looked up what level of the population was at, below, the level of folic acid where he saw changes in people. It was 10% of the United States population and half of the poor, who mostly eat a terrible diet. When MacGregor told me all of this it rang a bell in my head and I decided that because I am interested in disease prevention, I should get into nutrition. That seemed to be where there would be the low hanging fruit for disease prevention. I have been working in nutrition for the last 10 or 15 years. I am convinced that is where we will learn how to lower medical costs and lengthen lives.

We started taking human cells in culture and making them deficient in 1 vitamin or mineral or another. There are about 30 vitamins and minerals necessary to run your metabolism. Many that we tried or found in the literature—when the amount was restricted, the chromosomes break. That is the most dangerous part of radiation: when you get broken chromosomes.

As I started working in nutrition, I kept on wondering why nature was doing this. One day the answer hit me: That is what nature wants. Through all of evolution, there have been scarcities of vitamins and minerals. Minerals are not spread evenly through the earth. The red soils have a lot of iron and some soils are deficient in iron. Too much selenium will poison you and too little selenium poisons you; patches of both are common.

I came to the conclusion that there must be a rationing built into our metabolism when you get a little low. What nature is selecting for is for you to survive and reproduce, not so much whether you live to 90.

The plausible way to deal with shortage is to favor proteins necessary for short-term survival by starving those proteins preventing insidious damage that only caused disease years later. I wrote a paper postulating that humans did this sort of rationing. I called this idea triage.

Joyce McCann, phd, in our lab tested this idea. She analyzed vitamin K, which is required by 16 proteins, and the mineral selenium, which is necessary for 25 proteins. Most of the vitamins and minerals are required by many different proteins. What I postulated is that when you get a little short, you ration; those proteins that get the vitamin or mineral—first are the ones essential for survival and reproduction. The price that you pay is disabling of long-term functions. For vitamin K, one of the proteins prevents calcification of the arteries, so that protein gets disabled so you can survive but it means you increase your risk of heart disease.

Then Dr McCann wrote 2 reviews showing that it is true for the mineral selenium and true for vitamin K. With these 2 nutrients the body does do rationing. I think it is going to be true for all of the vitamins and minerals. You pay a price for moderate deficiency in that you disable your long-term protection mechanism.

For example, all the DNA repair enzymes use magnesium as a cofactor. Half the country is short of magnesium. Are you starving DNA repair enzymes so you can survive? We do not know that yet, but Dr Patrick is trying to figure that out right now. You are certainly making trade-offs between long-term survival and short-term survival.

Dr Patrick: I would like to add on to Dr Ames comments regarding vitamin K. It is a perfect example of triage because vitamin K is also involved in clotting of blood cells. Some of those proteins Dr McCann discussed in her review article are involved in clotting of blood cells and need vitamin K as a cofactor to function. There are other vitamin K-dependent proteins that are involved in preventing calcification of the arteries.

You can imagine that for short-term survival you do not want to hemorrhage and bleed out. That is something that will kill you immediately. You want to make sure that any vitamin K you have goes toward that clotting mechanism, because that is going to keep you alive right now, whereas having calcification of your arteries, which can lead to heart disease or even vascular brain disease, is something that rears its head later in life. The vitamin K you get in your diet is going to go first towards that short-term clotting survival mechanism.

IMCJ: So you are saying the body accomplishes this differentiation through the intensity of the bonds?

Dr Ames: You can the do it that way or you could do it in other ways. In fact, the vitamin K system and the selenium system use completely different mechanisms for accomplishing this, but they both accomplish it.

One way is binding constants. You just make them better; that is good for vitamins but doesn’t work so well for minerals because minerals look a lot like each other. Magnesium and calcium are one above the other on the periodic table and the body really cares about the ratio. The sodium to potassium ratio is important also.

It is hard to do just using binding constants. In fact, for selenium, the body uses a different mechanism that we discuss and you can read about in Dr McCann’s reviews, but the important thing is that there is a mechanism.

Vitamin K is used in photosynthesis in plants. Animals have hijacked it for a different purpose. It is used to put an acid group on a glutamic acid in proteins that are already made. You have 2 acid groups sticking out that bind calcium, so all of vitamin K-dependent proteins are calcium-binding proteins. The body sends this phylloquinone from plants directly to the liver and you do all the coagulation proteins, which are really important for short-term health, in the liver and then you convert the vitamin K to another compound, which goes to all the tissues equally, and there is a system worked out for doing this. Selenium uses a very different system, but the important thing is that there is rationing.

IMCJ: How are the recommendations for nutrients currently set?

Dr Ames: There is a level of intake the experts call the EAR, the estimated average requirement. If your intake is below that, the committee says you have inadequate levels. The RDA is set at 2 standard deviations above that, so this is an even lower level for determining inadequacy.

Even with supplements and fortifications, the American diet is abysmal. Here are the actual numbers of people below the EAR: Vitamin D is 70%; vitamin E, 60%; magnesium, 45%; calcium, 38%; vitamin K, 35%; vitamin A, 34%; vitamin C, 25%; zinc, 8%; vitamin B⁶, 8%; folic acid, 8%; and then I just put “et cetera.”

IMCJ: People talk about the premise that the RDAs are probably set much lower than they need to be—that they are sufficient to quell acute deficiency responses such as beriberi, rickets, and scurvy, but way too low to hit optimum levels.

Dr Ames: The nutrition community does not yet understand triage. We are slowly convincing people, but it takes years for people to change their minds. If you consider the most vulnerable protein, which affects your long-term health, then basically the diseases of aging are accelerated through some kind of insidious damage, such as DNA damage that leads to cancer decades later. If you assure that this damage is occurring because you do not get enough magnesium, the RDA does not take that into account.

In the future, you will put a finger in a machine to assay the most vulnerable protein that is turned off first when you do not have enough magnesium—maybe some DNA-repair enzyme. That is the one you should be measuring. Then we may find that we need some of these nutrients more than they are telling us.

IMCJ: How would this affect the interpretation of the recent multivitamin trials that failed to demonstrate measurable beneficial outcomes from supplementation?

Dr Ames: Dr Patrick is into Web-based media and she has a whole video where she demolishes all those papers. The problem is that if you give a vitamin or mineral to people, you do not know who is deficient and who is not deficient. If 10% of the population is short of vitamin X and you do a trial without measuring who is deficient and whether the dose of X actually remedies that, you are diluting the 10% that are deficient with 90% who have enough. It is a poorly designed experiment that should not be funded. You cannot take the randomized, double-blind, clinical trial and apply it to nutrition without thinking about it. The people who do these tests and the people who take them seriously are polluting the field and confusing the public.

Scientists who know nutrition do it the right way: They measure the level of vitamin before supplementation of the vitamin and use the people that are really deficient. When you see there is a vitamin D deficiency, bring it up to adequate levels and then ask whether something happened to those people. You leave out all the people with optimal levels in your trial; otherwise, you do not see anything of interest.

Dr Patrick: Randomized, controlled trials are the gold standard for pharmacological studies because most people that start the trial have zero levels of any sort of pharmacological drug in their system. The same is not true for micronutrients.

As Dr Ames mentioned, if we are talking, for example, about vitamin D—where 70% of the population has insufficient levels and you are not measuring the levels of vitamin D at baseline and then you are giving them a supplement, but not an adequate dose. Say you bring them the RDA, which is 600 IUs, and give them 600 IUs a day for a month. Then, at follow-up you still find that their levels are deficient or insufficient; what is it proving? It is proving that giving 600 IUs per day for a month to someone who is severely vitamin D-deficient is not going to raise them to an adequate level. Any outcome of vitamin D supplementation that is being investigated becomes irrelevant because the person still has inadequate levels of vitamin D after the trail. This biochemical analysis of plasma vitamin D concentration is key and yet so often not used in clinical trials.

It is not that getting more micronutrients is going to have added benefits. NHANES data has demonstrated that people are not getting enough micronutrients and they need to get more to prevent diseases of aging, to prevent things like DNA damage and oxidative damage and inflammation. These are all types of insidious damage that occur a little bit at a time every day but ultimately accumulate into long-term effects and diseases of aging such as cancer, neurodegeneration, and cardiovascular disease.

There are a variety of polymorphisms in genes that are involved in micronutrient transport into the cell, absorption, and binding constants. There is a lot of variation in clinical trials because you have people who have different gene polymorphisms. That variability is due to the fact that people are different. The interaction between genetics and nutrition is also very important and needs to be investigated more in the future. Again, this is why measuring the concentration of the particular micronutrient being investigated at baseline and follow-up is critical. You need this biochemical data to be able to make meaningful interpretations.

There is also another problem with some of these nutrition clinical trials and that is context. If you start with a population of people that is already sick—for example, has cancer or has previously had cancer—and you give them a vitamin supplement such as vitamin E, an antioxidant, and look at the effect, then you cannot generalize that effect on someone who has cancer to someone who does not have cancer.

Vitamin E is a perfect example because it is a very potent antioxidant so it binds and sequesters reactive oxygen species, which are well known to initiate DNA damage. It is really good to have adequate levels of vitamin E because you can prevent DNA damage, which is a well-known initiator of cancer. Preventing anything that is an initiator of cancer is a good thing.

However, if you already have cancer and you give someone supplemental vitamin E, as has been shown in mice, it may not be such a good thing. For example, when you have DNA damage the tumor suppressor gene p53 is activated and induces apoptosis of the cell because it senses something was wrong and it kills the cell. However, if you give vitamin E to someone who already has cancer, then you may not activate that p53 pathway because the reactive oxygen species is sequestered by vitamin E and that apoptotic pathway does not get activated.

IMCJ: If the triage concept is in effect, giving somebody vitamins in the short term of a study wouldn’t necessarily be measurable, would it? The effects of the body’s triage system would take decades to measure.

Dr Ames: The diseases of aging are all those diseases with insidious damage like calcification of the arteries that eventually leads to heart disease or DNA damage that eventually leads to cancer. It is hard to show that, so you need to look at biomarkers. When you do that, there are lots of positive results in nutrition.

Dr Patrick: Part of the problem is that in these clinical trials, they are looking at disease endpoints. They are looking at cardiovascular disease; they are looking at cancer incidence. As Dr Ames mentioned, biomarkers are actually a better way to do that because we do not want to wait 3 decades to look at cancer incidence or cardiovascular disease. Why not look at things that are known to play a role in initiating cancer like DNA damage? That is a short-term biomarker that would be representative of something that could lead to these diseases of aging. That needs to be incorporated into some of these clinical trials. They need to start looking at biomarkers—biochemical biomarkers of diseases of aging—such as cancer and neurodegeneration.

Dr Ames: Nutrition is an inherently complicated issue, as we have all these human polymorphisms. We have 30 different vitamins and minerals, and then we have the omega-3s—the essential fatty acids—and choline and essential amino acids. There are a lot of fires and there is all this genetics going on. You have to be quite sophisticated to do things right. It is not good enough to just know statistics and do epidemiology; they get the wrong answer half the time.

IMCJ: How does triage concept play into the movement toward personalized medicine?

Dr Ames: I think it will fit in very nicely. We are very interested in that whole area. How do you tune up people? Genetics is going to be part of it and so will nutrition.

In part, it is also about what you are leaving out of your diet. People worry about a part per million of pesticide, but it does not make too much sense because every plant has 100 toxic chemicals to kill you and kill all the bugs that are eating it.

We wrote a paper pointing out that you are eating 10 000 times more of nature’s pesticides than you are of these tiny residues of man-made pesticides. If you do animal cancer tests, nature’s pesticides versus man-made pesticides, the hit rate is exactly the same. It is not very plausible and there is no very good evidence that pesticides are anything to worry about. The important thing is to eat a varied diet with lots of fruits and vegetables and more fish and less red meat. Nuts are healthy and berries are healthy and all the things your mom told you—but people are not doing that.

IMCJ: What would be the best advice for clinicians seeking to begin using the triage concept to affect the health of their patients?

Dr Ames: That is hard right now because we do not have good assays for all of us. There are a few you can measure. Vitamin D: Dr Patrick can tell you about her adventures in the vitamin D field in the brain, but vitamin D is a steroid hormone that is turning on 900 genes, lots of which are in the brain. You can get your vitamin D measured. You can get your omega-3s measured because omega-3s are turning out to be hugely important for cell membranes.

One that most Americans are short of is magnesium. Magnesium is in the center of the chlorophyll molecule. Eat a big plate of spinach or kale to get your magnesium, and you get a little from nuts.

The big ones are vitamin D, vitamin E, calcium, vitamin K, and vitamin A. I think everybody should take a multivitamin with minerals as insurance. I do, and I have an Italian wife who feeds me a wonderful Mediterranean diet.

IMCJ: Do you distinguish between vitamin K¹ and vitamin K²?

Dr Ames: There is a difference. Vitamin K is what we get from plants and it is very lipophilic. It goes right to the liver. It is made into the coenzyme that converts 16 proteins to calcium binding proteins. Vitamin K² and that family go to all the tissues equally; you do not make the long-term proteins in the liver. You make them in the other tissue, so they are really 2 vitamins in there that do almost the same thing. That is how the vitamin K system makes sure that they protect your clotting enzyme, but at the price of strong bones and preventing calcification in your arteries. So, there is a difference.

The Japanese have a health food called nattō. It is Bacillus subtilis fermented soybeans. It is full of vitamin MK-7, a compound that is like vitamin K². People who eat it get less heart disease and less bone fractures. That is all discussed in Dr McCann’s review.

Dr Patrick: But you can—if you’re getting vitamin K¹ from plants, for example, from dark green leafy vegetables, then your body will be able to turn it into K², essentially.

Dr Ames: But if you do not eat your veggies, then you are in trouble. Thirty million people in the United States take a prescription for warfarin—also called Coumadin— which prevents thrombosis because it lowers your blood clotting ability. Those people all have more bone fractures and more calcification in the arteries. They are coming up with new drugs, but it is a pretty blunderbuss way of attacking it. That is why understanding the underlying metabolic mechanism is important.

IMCJ: Beta-carotene is often used synonymously with vitamin A. Is this a fair generalization, or do carotenoids also have a role beyond this conversion?

Dr Ames: Carotenoids are antioxidants that defend you against singlet oxygen, a type of reactive oxygen species. If you have light and a dye, that energy of the light gets transmitted to oxygen to make a very energetic form of oxygen called singlet oxygen. Plants are out in the light; they need the light. Every plant turns orange in the fall because that is the carotenoids becoming visible as you lose the chlorophyll.

The carotenoids are in plants to prevent this oxidation and it is turning out that humans have a lot of carotenoids in them. There are 600 carotenoids in nature and 20 of them end up in humans. They found 2 carotenoids in the back of the eye—in the macular; that is why it is yellow. These carotenoids absorb blue light and they protect you.

The brain, it turns out, has a number of carotenoids that clearly seem to be doing something else, but you can make singlet oxygen in the dark, too. We are quite interested in that area.

Dr Patrick: Carotenoids do have a function on their own, independent of being converted into vitamin A.

IMCJ: Together, you recently published a paper on hormonal influences on autism that may reveal a mechanism that may explain the controversial link of autism occurrence to vitamin D status. Can you tell us what you found?

Dr Ames: Vitamin D gets converted into a steroid hormone; you actually make it from UVB radiation from the sun. That converts something in your skin called 7-dehydrocholesterol into vitamin D³, which then gets released into the bloodstream. This goes to the liver. In the liver, it gets converted into 25-hydroxy vitamin D, which is a major circulating form of vitamin D and is what most people measure to determine vitamin D status.

After it gets converted into 25-hydroxy vitamin D, it then goes to the kidneys and gets converted into the active steroid hormone 1,25-hydroxy vitamin D. The active steroid hormone has been shown to change the expression of almost 1000 different genes in the body. The way it does that is that it recognizes when you have this active vitamin D steroid hormone in your body, it binds to a vitamin D receptor in the cell. This heterodimerizes with a vitamin A receptor called the retinoid receptor.

These then go inside the cell into the DNA and they recognize this little telltale sequence in DNA, which is essentially a repeat of 6 nucleotides separated by 3 spacers.

When they sequenced the genome, they found over 900 genes with this telltale sequence in it.

Dr Patrick: So this telltale sequence can actually determine whether or not vitamin D will turn a gene on so that it is active and doing what it is supposed to do or it will turn the gene off, meaning that even though the gene is there, it is repressed so it is not making as much of the protein do what it is supposed to do.

Vitamin D can do either one of these: It can turn genes on or it can turn genes off. Just this sequence alone can determine whether or not it is going to do on or off. What we found is that there are 2 different genes that we make that are responsible for converting tryptophan into serotonin. This gene is called tryptophan hydroxylase. We have tryptophan hydroxylase 1 and tryptophan hydroxylase 2. These 2 different isoforms of tryptophan hydroxylase are found in different tissues.

For example, tryptophan hydroxylase 2 is mostly a brain- or neuron-specific gene. It is found in the dorsal raphe, the midpart of the brain, and it is also found in keratin neurons in the gut.

In contrast, tryptophan hydroxylase 1 is predominantly found in nonneuronal tissues. It is mostly in the gut epithelial tissue. It is also expressed in T cells as well as placental tissue.

These 2 different genes of tryptophan hydroxylase both have this telltale sequence called a vitamin D response element that can bind to vitamin D bound to the vitamin D receptor. What was very interesting is that the sequence was different in these 2 genes. The TPH2 gene, which is the brain one, has an activating sequence, whereas the gut epithelial one has a repressing sequence suggesting that vitamin D may be regulating the expression of tryptophan hydroxylase and the serotonin production in opposite directions in different tissues, turning it on in the brain and turning it off in tissues like the gut.

Dr Ames: Serotonin is a hugely important hormone and neurotransmitter and it is one of the main hormones dealing with social interaction.

Dr Patrick: Yes. So serotonin during early brain development actually acts as a brain morphogen. It simulates the proliferation in migration and differentiation of specific neurons in the developing brain, such that it is very important to shape the structure of the developing brain. It is literally acting as a growth factor in that sense during early brain development. They have shown in mouse models that if they knock out the ability to produce serotonin in early brain development, there are functional and structural consequences in terms of the brain abnormalities that occur. Also, a lot of mice end up growing up to have autistic-like behaviors.

Serotonin is also a very important neurotransmitter and also it acts like a hormone as well. They have done studies in humans where you can actually deplete a person of 90% of their brain serotonin levels through a method called acute tryptophan depletion.

So, tryptophan is the precursor for serotonin. It gets converted into serotonin through tryptophan hydroxylase, which is the rate-limiting step, and tryptophan has to be transported into the brain across the blood-brain barrier. It actually competes with other amino acids—branch-chain amino acids—which are essentially large neutral amino acids like leucine and isoleucine. It competes with them to get transported into the brain and it actually gets outcompeted.

Leucine and isoleucine—these large, branch-chain amino acids—preferentially get transported into the brain. You can actually give someone a big shake full of branch-chain amino acids and you can then outcompete any tryptophan, such that it does not get into the brain. Over the course of 5 or 6 hours, brain serotonin levels drop dramatically. Different neuroscientists and behavioral psychologists have worked together to study the consequences in terms of the behavioral consequences of depleting the brain of tryptophan and, thus, serotonin.

What they found is that there are effects on social behavior. When lacking serotonin in the brain, they do not interact in social groups; they have difficulty interpreting facial expressions, of fear or of anger. They also become more impulsive, a little more aggressive, and throw tantrums—things like that. There is a big effect on social behavior. Actually, I do not want to get too far into that right now, because I am actually writing a paper on that.

The bottom line is that serotonin is very important for behavior in the brain. We hypothesize that because vitamin D is important to activate serotonin in the brain through tryptophan hydroxylase 2 that vitamin D may also be very important to make enough serotonin and, thus, affect things like behavior, prosocial behavior, and things like that.

It is also important during brain development. They have shown that if you have vitamin D deficiency during early brain development, you have some similar effects as those caused by low serotonin. There is an increase in this lateral dorsal region of the brain, which is found in autistics as well. Structural defects and things like that occur when you have lower vitamin D during brain development.

Also, the flip side of this is the other gene that is regulated by vitamin D, and THP1, which is in the gut. We think that may be being repressed or turned off by vitamin D. Serotonin in the gut has been shown to be a very strong proliferative signal to T cells. It is expressed in naive T cells and when you have a lot of serotonin, it actually causes T cells to proliferate. They have shown in different mouse models of colitis and also inflammatory bowel disease that when TPH1 is deleted in the gut, it prevent these mice from having serotonin production in the gut. Deleting TPH1 and serotonin production in the gut actually ameliorate the inflammatory symptoms associated with colitis. The inflammatory process is dampened, likely because you are not having so much activation of this immune cells.

That is very interesting because vitamin D is known to be anti-inflammatory in many different cases but this may be one other mechanistic link by which vitamin D can lower inflammation in the gut by decreasing serotonin production in the gut.

Dr Ames: Dr Patrick’s paper on autism discusses all of this.

Dr Patrick: Yes. GI, or gastrointestinal, inflammation is also a major symptom in many autistic children. They also have high levels of serotonin in their blood cells. Platelets actually take up serotonin that’s produced in the gut through TPH1. They do not produce it themselves, but they take it up. Autistics have been shown to have very high levels in their blood platelet cells but low levels in the brain. It is like there is this paradox or anomaly, so to speak. Why are there high levels in the blood if there are low levels in the brain? We think this is possibly because of their lowered vitamin D, which can make brain serotonin levels low and the blood levels high.

IMCJ: Your paper spends a little time talking about the possible origin of autistic symptomatology drawing back to possibly maternal low vitamin D status. Would you explain that?

Dr Patrick: There are a couple of possible ways that maternal low vitamin D status could affect autism incidence.

The first way is because, as I mentioned, serotonin is a very important brain morphogen for brain development, meaning it is important to shape the structure in wiring the developing brain. The developing fetus actually depends on the maternal levels of vitamin D.

Vitamin D is important to activate this enzyme TPH2 in the developing brain to produce serotonin. Then, one could imagine, if you do not have enough vitamin D, then that enzyme may not be activated sufficiently and that may lead to lower serotonin levels in the developing brain and thus may alter the structures and the wiring of the developing brain. That could lead to some sort of aberrant structure in wiring that may also lead to increased autism risk. That is one possibility.

The other possibility is that if the mother has low levels of vitamin D, she may have high levels of serotonin in her gut, blood, and placental cells. Because tryptophan can be used in the pathway to make serotonin, through tryptophan hydroxylase 1, if that gene is being overexpressed because there is low vitamin D, it may be sucking all the tryptophan into that pathway and producing a lot of serotonin, which then means less tryptophan available to this other pathway and that tryptophan also gets converted into something called kynurenine through IDO.

It has been shown that kynurenine generation is essential to make key T regulatory cells. T regulatory cells are very important for recognition, to have self-tolerance— basically to be able to tell your immune system, “This is my cell, it is not a foreign invader, do not attack this cell.” They play a very important role in autoimmunity.

It has been shown, for example, in mice when you have a pregnant female mouse and you delete that IDO enzyme so that they can’t produce kynurenines—those female mice have a very, very strong autoimmune response to the developing fetus; such a strong one that they end up aborting it because the fetus is obviously foreign. Due to a lack of production of T regulatory cells, the immune system recognizes it as foreign and attacks it.

What is interesting is that they have shown that mothers with autistic children have high levels of autoantibodies against fetal brain protein in their blood cells. There is not really a good explanation as to why, but it suggests that something may be aberrant in this immune system where the mothers with autistic children, for some reason, during pregnancy—their immune system is recognizing the fetus as foreign and so they start actually making antibodies to attack it—specifically antibodies against the protein in the developing brain, which could alter the way the brain develops.

Dr Ames: Dr Patrick’s paper explains so many things that were in the literature that nobody understood and everything fits together if you understand the mechanism, which is what she sorted out.

Dr Patrick: The binding constant of tryptophan, for TPH1, has a much higher binding constant of IDO. In the case where a mom has low vitamin D, they are likely making a lot of TPH1—more than normal—which means it is acting as a tryptophan sink because it binds so tightly to tryptophan and you are making so much of it. All the tryptophan, which is actually a rare amino acid in proteins by the way, is getting sucked into that pathway and making serotonin in the gut cells and that would theoretically not leave a lot of tryptophan to make kynurenine and, thus, T regulatory cells may drop. You may have a case where you would get an autoimmune response in mothers that are vitamin D deficient for example.

Dr Ames: One important thing about vitamin D is that we need ultraviolet B light from the sun to make it in our skin. When you get old like me, your skin does not do that that well. Dark skin is a big advantage for people living in the tropics. It is protecting them from that UVB light that is the burning rays of the sun. So if you put in an Irishman in Australia, they are in deep trouble. The solution there is a hat and sunscreen, but the converse of that is that if you put an African American or an Indian or a Hispanic in Chicago, they are in deep trouble. The solution is a pill that costs a penny or two that has vitamin D.

There is the interaction between genetics and nutrition. In this case, vitamin D, you get relatively little from food. If you eat a lot of fish, you can get some vitamin D but mostly you get it from sunlight. In northern latitudes, you are in trouble. If you have a dark skin, you are in even deeper trouble. I was in Copenhagen at a meeting and there were a few days of beautiful sunshine. It was like California. Every Dane was out soaking up the sun. They would take off most of their clothes and were in the sun.

I asked a few Danes, “How come?” They said, “Well, that is built into us. Whenever there is sun, we try to grab it.” I think it makes them feel good. There is genetics and there is the nutrition and in this case sunlight is also important.

Anyway, 95% of the African Americans in this country are deficient in vitamin D. The solution is cheap. You buy a vitamin D pill. I tell all my dark-skinned friends, Indians and Hispanics and African Americans, that they ought to check their vitamin D. If it is too low, take a pill.

People think that if it is genetic, such as skin, you cannot do anything about it. If you understand the mechanism, then you often can do something about it.

Dr Patrick: Certain polymorphisms have been shown to increase the risk of autism in some of these genes that are actually involved in serotonin synthesis or serotonin release or the response to serotonin, meaning serotonin receptors.

If you already have a polymorphism in a gene that is altering your serotonin production or the way you respond to in it some way, on top of that, being vitamin D deficient may exacerbate that effect. It is really imperative that people who have these polymorphisms and are already not making enough serotonin or having a difficult time responding to it because they do not have enough of the receptor, make sure they are doing what they can through diet to make sure they are getting enough vitamin D to activate that gene to make serotonin.

Dr Ames: In the future, we are going to put our finger in a machine and it will take a finger prick of blood. Already, there is a company in Boulder that can measure 1500 different proteins in a finger prick of blood. Then, there’s another company run by another friend of mine who has put a machine in every hospital in China to measure a couple of dozen proteins of medical importance on a fingerprick of blood. All that is coming quickly. Medicine and most big pharma have abdicated the field of nutrition therapy but they are going to learn. It is going to be part of preventive medicine. It is going to take a decade to work all this out but it is coming fast.

Dr Patrick: The last part of the origins that we discussed in the paper also has to do with estrogen. Estrogen actually activates the same gene, tryptophan hydroxylase 2, the brain isoform of this gene that makes serotonin. It activates it quite robustly, actually.

We think that females may be protected from low serotonin synthesis because they have higher levels of estrogen. When you are talking about a developing fetus or a young neonate, obviously their ovaries are not making estrogen yet.

It has been shown—it is actually difficult to show in the developing fetal brain in humans—that the estrogen levels are different between males and females, but they have sampled amniotic fluid from developing male versus developing female fetuses and they found that there are higher levels of estrogen in the developing female fetus.

Dr Ames: A male fetus gets autism at 5 times the rate of a female fetus.

Dr Patrick: Right, so they have shown that estrogen can actually activate tryptophan hydroxylase 2 and up to 9-fold—really robustly. They have also shown that right after birth, neonatal female brains have higher levels of estrogen in the frontal cortex.

It is possible that at the very least, right after birth, females are making more estrogen in their frontal cortex and therefore can activate TPH2 to produce serotonin.

Dr Ames: The bottom line is vitamin D hormone is a very important steroid hormone. As Dr Patrick said, there are close to 1000 genes involved. Many social interactions use oxytocin a peptide hormone, which bonds the mother to her baby. It goes shooting up in the mother when she has a baby and she bonds with the baby like glue. The social hormones—serotonin, oxytocin, and vasopressin—are all controlled by vitamin D.

It is critically important to make sure your vitamin D level is adequate. You can get too much for a lot of things, particularly with the minerals, but you can get too much vitamin D, too. Mae West said, “Too much of a good thing is wonderful,” but she was saying that about sex, not vitamins.

IMCJ: In identifying the mechanism of action for vitamin D and autism, what is that leading us toward in terms of preventive action and treatment of symptomatology for autism?

Dr Ames: That is a good question. We think it is certainly relevant in prevention and pregnant women would not have enough and in the prenatal multivitamin. You certainly want to do prevention. Dr Patrick’s hopeful that it might work in treatment, too.

Dr Patrick: It should be standard practice for clinicians to measure 25-hydroxy vitamin D levels in women before they are going to conceive so that they can tailor the right dose of vitamin D for her—to make sure that she is going to get an adequate amount of vitamin D for the developing fetus. I think that is one really important preventative measure.

In addition to that, consider omega-3 fatty acids. I talk about this mechanism more in this paper I am writing now, but it also affects serotonin levels. They affect the release of serotonin and they affect the response to it; actually, they affect the structure of the serotonin receptor. That is because omega-3s are very important for cell membrane fluidity, meaning your cell membrane has a certain fluidity to it and you have receptors and transporters and proteins that are embedded in that cell membrane. If we are talking neurons, neuronal cell membranes, then we are talking about neurotransmitter receptors that are embedded in the membranes.

If the membranes are not getting the proper amounts of these polyunsaturated fatty acids that they require, such as DHA, then that alters their fluidity and consequently the receptors that are embedded in that are affected for the structure. They are not as readily available to bind a neurotransmitter, for example. This has been demonstrated in mice.

Dr Ames: Where do you get your omega-3s from? Fish oil. Most fatty acids are C18, with 18 carbons. There is EPA that you get from fish oil, which is C20, and then there is DHA, a C22, which is the main one in the brain—30% of brain fatty acids are DHA. If you do not eat any fish, you are in trouble. Fish is a really important item in the diet: tuna, salmon. You do not want to eat swordfish every day; you will get mercury poisoning. Again, in moderation. These omega-3 essential fatty acids are going to be more important than the effects of the mercury carried by the fish, though it is a balance. You can buy pills that have them without mercury.

Dr Patrick: Yes, so that might be a very important preventative measure prenatally. Like I said, measuring vitamin D levels is just imperative. It should be a standard part of a prenatal care package. I know they do not do it right now and it really needs to be. They have a standard prenatal vitamin they give to people but it is not one-size-fits- all. You have to figure out what the vitamin D level of this female is before you give her a vitamin D supplement. We have learned that from numerous clinical trials. Giving someone 400 IUs a day is not going to do anything if they are severely deficient. In some cases, they require much more. In some cases, they may already be getting enough. So it is important to measure.

You asked for treatment, as well, and I think that is a little trickier because obviously, you have got already a person whose brain wiring has developed differently and it is difficult to change that. However, I do think severity of symptoms may potentially be modified by making sure a person has adequate serotonin synthesis going on in their brain or adequate oxytocin production. Those 2 things are regulated by vitamin D.

Many autistic children do not go outside a lot. They are not eating a great diet. They tend to have very strict dietary preferences. Many times, they are not eating healthy foods like fish, but rather chicken nuggets or French fries or things that are just not very nutritionally dense food.

Make sure that they do get their source of vitamin D and omega-3. Both of those affect serotonin levels. Vitamin D would also be affecting oxytocin levels, which is also important in social behavior.

At the very least, you can modify the severity of some of these symptoms, making sure that they are producing enough of this neurotransmitter that is affecting behavior.

Tryptophan supplementation is a little trickier because there have been fewer clinical trials investigating what dose to give. There have been some looking at tryptophan supplementation in autistics, as well as other neurodevelopmental and neuropsychiatric orders. I think ultimately you can use things that help alleviate some of the competition between tryptophan and branch-chain amino acids. Exercise is one because branch-chain amino acids get taken up into your muscle cells when you are exercising, so it alleviates competition. You actually end up getting a lot more tryptophan transported into the brain during exercise, which is part of the reason why you feel really good after exercise. You are actually producing more serotonin. That is also another important lifestyle factor.

IMCJ: Any last thoughts before we conclude?

Dr Ames: Like your mom told you, get some exercise; eat your greens; and eat a good, varied diet. We are just not paying attention. We are eating comfort food and refined foods that have low nutritional density.

We are optimistic that it is all going to be sorted out, but nutrition is a pretty muddy field and epidemiology is a very difficult field, so you do not want statisticians just running epidemiology because they do not understand the mechanisms and they get the wrong answer half the time. One needs really smart people. There are plenty of people like that out there, but there is a lot of noise in the system.