Immune response and safety:
by Richard A. Passwater, Ph.D.
I keep a very close watch on vitamin safety issues and I occasionally write and lecture on the subject of vitamin safety. Readers may remember my ten-part series on the safety of vitamins published in this column in from October 1985 through December 1986. I have discussed vitamin safety in my books and have often included chapters devoted entirely to this important subject. I have also spoken on vitamin safety at national and regional NNFA meetings. However, I never consider my research complete until I double-check with Dr. Adrianne Bendich. Through the years, I have found her to be my most knowledgeable and up-to-date reference resource on the subject.
Dr. Adrianne Bendich is Clinical Research Scientist in Human Nutrition Research at Hoffmann-La Roche, Inc. She is an Associate editor of the Journal of Nutritional Immunity and was on the editorial board of the Journal of Nutrition. She is the coeditor of three books entitled "Micronutrients and Immune Functions," "Vitamin Intake and Health," and "Micronutrients in Health and in Disease Prevention,." as well as two Annals of the New York Academy of Sciences and chapters in other technical books. Dr. Bendich has chaired or co-chaired several scientific conferences and she has published more than 75 scientific articles on vitamin biochemistry and safety.
One of my most vivid memories of Dr. Bendich stems from the Dykstra hearing held at the National Institutes of Health in 1991. One of the FDA committee members was making a mountain out of a molehill which brought Dr. Bendich to the microphone to tell the FDA that they had more important health questions to address instead of being on a witch hunt about supplements. She suggested that they worry more about health problems such as smoking, excess alcohol consumption and over-the-counter drugs such as aspirin and acetaminophen.
Dr. Bendich has done much to further the health of people of all ages, and she is a protector of the scientific truth. Perhaps, this is why she won the Roche Award in 1992. Her travel schedule is heavy because she attends many scientific conferences, but recently I was able to chat with her at length concerning the latest safety issues and vitamin safety and public health issues..
Passwater: Dr. Bendich, why did you become interested in nutrition, and especially vitamins and immune function? Why did you become interested in vitamin safety, especially vitamin A and beta-carotene?
Bendich: My first experiments to examine the immunological effects of nutrients involved vitamin E. Dr. Lawrence Machlin asked me to collaborate on a study of the overall effects of vitamin E deficiency in a laboratory animal model.  Dr. Myron Brin, the head of Roche's vitamin research at the time, in the early 1980's, was convinced that essential micronutrients, such as vitamin E and vitamin C, were critical for mounting optimal immune responses.
The objective of our first experiments was to see whether recommended intakes were sufficient to give the best immune responses in young, healthy, unifected laboratory animals.
Passwater: Now that is a start with two very prestigious vitamin researchers. Our readers may remember my "beyond deficiencies" chat with Dr. Machlin in the July 1992 issue. What did your experiments show?
Bendich: We found that diets low in vitamin E could protect against certain signs of frank vitamin E deficiency, such as weight loss and testes degeneration, but the low levels of vitamin E were not sufficient to give the best immune responses. We were the first to show that you needed three-to-five times the recommended dietary levels to see the optimal immune responses. Following these initial experiments, I was asked to join the Vitamin Research Department and we continued examining the effects of vitamin E, then vitamin C and vitamin E, and finally, beta-carotene, on immune responses. I was very fortunate to be the first to document the importance of beta-carotene as an immunoenhancer separate from its role as a source of vitamin A.
The most satisfying part of this research was our ability to move the research from laboratory animal studies to humans. Dr. Jeffery Blumberg was crucial in our beginning the studies at the U.S.D.A. Center of Nutrition and Aging at Tufts which showed that high dose vitamin E supplements safely enhanced immune responses in healthy elderly.  Dr. Simin Meydani, who was the primary investigator in these studies, has continued this research and expanded it further. She has recently also shown that beta-carotene supplements enhance certain immune functions in healthy older men.  In addition, to our collaboration with the Tufts group, we encouraged Dr. John Bogden at the University of Medicine and Dentistry of New Jersey to examine the effects of a multivitamin-mineral supplement on immune responses in healthy elderly, and last year, he clearly showed that this simple, inexpensive, and safe supplement, taken daily for one year, also enhanced immunity. 
I am very fortunate to be at the center of much of the research involving essential vitamins and carotenoids and immune function, and I am really excited that the early studies in laboratory animals have resulted in improving the health of the elderly. We are expanding the area of research and continue to support WHO-sponsored studies of vitamin A and reduction in childhood disease morbidity and mortality, and new research areas involving the role of micronutrients in HIV progression. Another new area which has our support is the importance of essential fatty acids in reducing autoimmune disease progression.
Passwater: I understand your satisfaction in helping both children and the elderly. But, how did you get from immune function to safety?
Bendich: My involvement in vitamin safety issues seems to be a natural progression which followed our research in humans. The first vitamin E/immunity studies at Tufts University used 800 IU of vitamin E daily. It was important to document the safety of this level of vitamin E for obtaining Institutional Review Board approval of the study protocol. Similar information was needed for beta-carotene, vitamin A, vitamin C, and vitamin B-6. The review papers that I have written on the safety of each of these micronutrients have involved very careful analysis of the published literature, going back 50 or more years. [5-10] The conclusions about the safety of each micronutrient are based upon a prioritization of the source of the data. The highest priority is given to information published in peer-reviewed journals from placebo-controlled, double-blind studies. Next in priority are studies which did not include a placebo, then individual case studies, and finally anecdotal reports. Following such an in-depth analysis, it is remarkable to find that almost all of the "safety" issues often mentioned are not based on solid data.
Passwater: You have co-chaired conferences bringing together scientists from around the world studying vitamins and health. Do you see growing awareness of vitamins in health beyond deficiency diseases? Is the information getting to the medical profession?
Bendich: I have been privileged to be involved in the organization of two New York Academy of Sciences Conferences. The first on micronutrients and immune functions, co-chaired by Dr. Ranjit Chandra, was a very important meeting because it crystallized the importance of micronutrients in human immunity. All of the major researchers from around the world attended and the take-home message was that well before signs of vitamin deficiency are obvious, immune system reactions have been significantly decreased. Thus, the immune system appears to have higher micronutrient requirements than other organs or tissues of the body. For instance, before vitamin A levels are so low that vitamin A-related blindness occurs in children, their ability to fight infections has already been severely compromised. Thus, marginal deficiencies can really decrease infectious disease resistance, especially in children.
The second conference on "Maternal Nutrition and Pregnancy Outcome," I co-chaired with Dr. C. Keen and Dr. C. Whillhite. This conference also was critical in changing human health practices. We were able to assemble virtually all of the researchers involved in finding that folic acid supplementation significantly reduced the risk of neural tube birth defects. In addition, Dr. Czeizel reported that not only were neural tube birth defects reduced in the women who took a folic acid-containing prenatal multivitamin during the preconception period, but the total number of all types of birth defects were halved. Following this meeting, the FDA finally allowed the health claim for folic acid and neural tube birth defect prevention.
Both of these conferences highlighted the growing importance of micronutrients beyond preventing nutrient deficiency diseases. I think the medical profession is beginning to hear this message with regard to some issues, but there is still a lot of education that must be undertaken in medical schools and through continuing education.
Passwater: Every now and then a preliminary paper or even a letter-to-the-editor will be published that calls attention to a safety concern or possible problem with taking supplements. That original question gets plenty of publicity, but when researchers look into the concern and find that there was no problem in the first place, the media makes no mention of this. It is non-news and doesn't sell papers or make a sound-bite to hold viewers to the evening TV news.
Let's discuss some of these concerns and what further research has found. Does beta-carotene interfere with vitamin absorption or transport?
Bendich: Beta-carotene has many functions including its provitamin A activity, antioxidant and singlet oxygen quenching capacities. In the 1980's, the National Cancer Institute initiated several intervention studies with beta-carotene to determine whether the supplementation could lower cancer risk.
Certain questions arose from laboratory animal studies with very high doses of beta-carotene. Very high levels of beta-carotene had to be added to these diets because rats are very poor absorbers of beta-carotene. The animal studies showed massive levels of beta carotene lowered vitamin E levels in the rats. It was therefore of interest to determine whether relatively small supplements of beta carotene in humans could lower blood vitamin E or other nutrient levels. The data collected from over a dozen papers since the 1980's clearly show that in humans, beta-carotene supplements of even 50 milligrams each day for five years did not effect serum (the clear fluid of blood) vitamin E levels at all. There are, however, two papers from small uncontrolled studies where researchers found that beta-carotene supplements reduced vitamin E serum levels. But, again, the major well-controlled studies did not find this effect. In fact, in most of the well-controlled studies, the serum vitamin E levels either remain the same or even increase when beta-carotene supplements are taken.
Passwater: Most of our readers are aware that vitamin A is "fat-soluble" and stored in fatty tissues and the liver. Since vitamin A can be readily stored, it can accumulate in the tissues. However, few readers understand just how excess vitamin A can actually cause liver damage. What do we know about the toxic mechanism?
Bendich: Excess retinol (vitamin A) causes changes in biological membranes, an effect believed to be due to retinol's surface-active properties. Retinol, however, does not show surface-active effects when it is bound to retinol-binding protein (RBP). (RBP is a specific transport protein for vitamin A that the liver manufactures and secretes to complex with vitamin A to deliver vitamin A to the tissues.) Therefore, toxicity appears to occur only when the amount of vitamin A (retinol) exceeds the capacity of RBP to bind to it. Vitamin A that is not bound to RBP binds to lipoproteins, and it is in this form it may have toxic effects when it comes in contact with membranes and body cells. In other words, in vitamin A toxicity, plasma RBP levels are normal, but concentrations of vitamin A not bound to the specific RBP are increased.
Vitamin A is stored in the liver. Excess vitamin A may result in increases in liver enzyme levels in the blood, which is used as an indicator of potential liver damage. Usually elevated serum liver enzyme levels are reversed when high doses of vitamin A are stopped.
Passwater: Your review of vitamin A published in 1989 concludes that incidences of chronic vitamin A toxicity are rare and have averaged fewer than ten cases per year from 1976 to 1987. You did not find confirmed cases of vitamin A toxicity below 36,000 IU. In December 1994, physicians at the University of Pennsylvania School of Medicine led by Dr. Thomas Kowalski reported that in the last three years they have seen 21 cases of liver disease resulting from high doses of vitamin A. In the paper, they described a 45-year old woman suffering from multiple medical problems that had liver damage -- reportedly from taking 25,000 IU of vitamin A daily for at least six years. She reportedly died from this liver failure within months of diagnoses. The physicians claim that this is the second well-documented case in which liver damage has resulted from a vitamin A dose as low as 25,000 IU.
Bendich: The case reported by Dr. Kowalski's group describes a woman who had high blood pressure, diabetes, an enlarged heart and hypothyroidism. She was also taking several medications. The cause of death was actually not described in the article. Nevertheless, it is possible that a level of vitamin A which does not appear to cause adverse effects in the healthy population, may have unexpected effects in individuals taking multiple drugs which can cause liver damage, as in this case report.
Passwater: Once it was believed that all water-soluble vitamins were without adverse effects at all levels of intake because being water-soluble, they are not appreciably retained in the body and are "washed away" in the urine. Niacin has been used in high doses -- one may say pharmacologically -- to lower cholesterol. This is not a nutritional use and it has been prescribed for this purpose by physicians. Is there a dosage above which niacin can have adverse effects?
Bendich: Niacin is a generic term that includes niacinamide and nicotinic acid...
Passwater: Excuse me, Dr. Bendich, but perhaps I should interrupt to explain a little about niacin nomenclature to our readers. The Health Food industry has always followed the older system of nomenclature that was used in the United States in the 1940's. The scientific community uses the terminology that you just described. The Health Food Industry uses the term "niacin" specifically to mean "nicotinic acid," while the amide is known as "niacinamide." According to Dr. D. A. Bender of London University, "the name niacin' was coined in the late 1940's when the role of deficiency in the etiology of pellagra was realized, and it was decided that dietary staples should be fortified with the vitamin. It was felt that nicotinic acid' was not a suitable name for a substance that was to be added to foods, both because of its phonetic (and chemical) relationship to nicotine, and because it is an acid." 
The pioneers in our industry followed the same reasoning and thus even today, our labels generally specify "niacin" for "nicotinic acid," and niacinamide for the amide form as you mentioned (but also called nicotinamide). The two forms collectively, but incorrectly, are called "vitamin B-3." For the convenience of our readers, I will show the chemical structures of the vitamers in figure 1 with the scientific nomenclature, and in figure 2 with the nomenclature generally used in the Health Food industry.
It is incorrect to call niacin "vitamin B-3." Nicotinic acid was so named in 1867 when it was isolated as an oxidation product of nicotine. In the 1930's, there was suggestion that pellagra, the classic "4D" deficiency disease having symptoms of dermatitis, diarrhea. Dementia and death, could be a protein deficiency disease or even an unknown water-soluble vitamin deficiency disease. In 1938, Dr. Spies and his colleagues showed that nicotinic acid would cure pellagra, but because its chemistry was already known, it was not assigned a number among the B-vitamins. Although various researchers placed this vitamin between B-2 and B-6 before its identity was known, it is still incorrect to call niacin vitamin B-3, because that was once assigned to pantothenic acid (which is sometimes incorrectly referred to as vitamin B-5)
Pardon my interruption, but I felt that it was important to clarify the various nomenclature preferences for our readers. Please continue.
Bendich: Niacinamide is the molecule commonly used in multivitamin supplements and is safe. Niacinamide does not lower cholesterol levels. Nicotinic acid has been shown to be a very effective agent in lowering cholesterol when given at high doses. Nicotinic acid, as is true for all cholesterol-lowering agents, increases liver enzyme levels and can cause liver toxicity. High doses of nicotinic acid should be taken only under a physician's supervision, which should include the monitoring of liver enzymes. There have been reports that the use of time-release nicotinic acid has resulted in liver damage severe enough to require liver transplant.
Passwater: Yes, there is such a big difference in nicotinic acid and niacinamide, some may wonder how such two different compounds can be the same vitamin. They both prevent pellagra (the "black tongue" disease that produces skin eruptions, gastric disturbances and nervous disorders including insanity), and they both can form nicotinamide nucleotide coenzymes (NAD+, NADP+ and NADH). One -- nicotinic acid -- can cause a temporary skin "flushing" and lower blood cholesterol levels, while the other -- niacinamide -- does neither.
Since the mid-1980's, there has been increasing concern over possible adverse effects of vitamin B-6. Do any of the pyridoxine vitamers cause nerve damage?
Bendich: Vitamin B-6 has been used in pharmacologic doses to treat conditions such as premenstrual syndrome, carpal tunnel syndrome, homocystinuria, galactorrhea and kidney stones. These treatments have generally not been associated with severe adverse effects. However, since 1983, reports have appeared in the literature suggesting that high-dose, long-term administration of pyridoxine produces sensory neuropathy (symptoms such as tingling sensation, loss of feeling, or weakness due to damage or disease of a nerve) in some patients.
Dr. Marvin Cohen and I evaluated the published scientific literature and reported our results in Toxicology Letters in 1986. That study found that the human data on the safety of pyridoxine suggest that oral administration of doses greater than 500 milligrams per day for a prolonged period of time can result in the development of sensory neuropathy. Doses less than 500 milligrams per day appear to be safe on the basis of reports where pyridoxine was administered for periods ranging from six months to six years.
The survey did not reveal any consistent trends for any other adverse effects.
In 1990, we updated our review and examined closely the dose-duration relationship.  We concluded that vitamin B-6 intakes of less than 500 milligrams per day for up to two years were safe. However, daily doses above one gram (1,000 milligrams) or total lifetime intakes above 1,000 grams (1,000,000 milligrams) were consistently associated with neuropathy.
As a precaution, I would not recommend the use of vitamin B-6 supplements at levels greater than 200 milligrams per day. Higher doses, if needed, should be used under the supervision of a physician who monitors neurological function.
Passwater: You mentioned that folic acid (folate or folacin) reduces the risk of neural tube birth defects. Yet some hesitate to recommend folate supplements because of a fear of masking B-12 deficiency. Would a supplement containing vitamin B-12 in addition to the folic acid, eliminate this worry? Isn't the risk of birth defects greater in young women than the risk of a hidden vitamin B-12 deficiency?
Bendich: The U.S. Public Health Service recommendation is that all women of child bearing potential consume 400 micrograms a day of folic acid for the purpose of preventing neural tube defects. There are no data that indicate that this level of folic acid provided through multivitamin supplementation increases the risk of masking vitamin B-12 deficiency. In fact, a recent report showed that elderly who routinely took a multivitamin, which usually contained both folic acid (400 micrograms) and vitamin B-12 ( at 6 micrograms) had a significantly reduced risk of low vitamin B-12 status. Moreover, vitamin B-12 deficiency is rare in women of child bearing potential and the level of vitamin B-12 in the multivitamin would probably help ensure against vitamin B-12 deficiency in young women as well as other age groups.
I continue to recommend folic acid-containing multivitamin supplements as the most practical, inexpensive, and safe source of folic acid for all women of child bearing potential. Another important source is fully fortified breakfast cereals containing 100 percent of the daily value (DV) of folic acid and other essential vitamins and minerals..
I do not recommend that women choose a single supplement of folic acid alone because of three major reasons: first, the research showed added benefits with the multivitamin supplement, such as reduction in the risk of other birth defects in addition to neural tube defects. Second, many young women have numerous marginal deficiencies, including iron, vitamin E, and vitamin B-6 to name a few. The multivitamin can help to eliminate these deficiencies. Third, if a woman is going to change her daily habits and begin to take a "pill" every day, then wouldn't it be best that the "pill" include all the essential vitamins and minerals?
My answer is Yes!
Passwater: Does vitamin C cause kidney stones?
Bendich: Several studies have found no evidence that vitamin C increases the risk of kidney stone formation. 13-17] Most kidney stones are composed largely of calcium oxalate, and urinary oxalate levels are used as a marker for kidney stone risk. Recently, Dr. Theodore Wandzilak and his team at the University of California at Davis investigated the claim that vitamin C increases urinary oxalate levels. Previous studies suggesting that vitamin C may increase urinary oxalate levels have been flawed because vitamin C interferes with most of the previous methods used to measure urinary oxalate levels. Dr. Wandzilak's group used a new ion chromatography procedure in which vitamin C does not interfere. Their data show that the ingestion of increasingly large quantities of vitamin C did not cause an increase in the urinary excretion of oxalate. They concluded, "Therefore, the safety concerns raised about increased urinary oxalate level and, as a consequence, an increase in kidney stone formation in healthy subjects are not supported by our findings." 
Passwater: That information, plus the new evidence that calcium does not increase kidney stone formation, shows that the conventional wisdom is not always correct, and that tests must be conducted before valid conclusions can be made. Does vitamin C destroy vitamin B-12?
Bendich: In 1980, Dr. M. Marcus showed that an earlier report suggesting this possibility was due to an artifact of the analytical procedure. 
Passwater: Do people have rebound scurvy when they skip or stop taking vitamin C?
Bendich: There is an indication that there is some degree of "conditioning" in white blood cell levels of vitamin C, but this effect is temporary and blood levels of vitamin C do not indicate that vitamin C deficiency develops.
Passwater: Does vitamin C increase iron overload disease incidence?
Bendich: The overall scientific data do not support the premise that vitamin C causes iron overload in normal persons. However, persons with the genetic disorders of hemochromatosis or thalassemia major may find that vitamin C increases the iron toxicity that they are suffering from by mobilizing more of their stored iron. Persons with genetic iron disorders should consult their physicians about the use of vitamin C supplements.
The effect of vitamin C on iron absorption is still an area of active interest. Vitamin C appears to enhance the absorption of non-heme iron consumed in the same meal. However, this effect does not appear to continue to increase with increasing intake of vitamin C. Dr. Marvin Cohen and I reviewed 24 studies which included 1,412 subjects eating meals designed to measure the iron absorption at different levels of vitamin C. We found that vitamin C doses above the RDI level do not increase susceptibility to iron overload in normal individuals.
Passwater: Is vitamin C an in vivo pro-oxidant or mutagenic?
Bendich: Harvard researchers have found evidence that vitamin C acts only as an antioxidant in vivo.  Even in the presence of transition metal ions, the researchers found that vitamin C acted as an antioxidant rather than a pro-oxidant.
Stich and others have shown that old suggestions that vitamin C might be mutagenic was due to a problem with the testing method.  Vitamin C has not been shown to be mutagenic. In fact, many published studies show that vitamin C has anti-mutagenic properties because it is an important antioxidant.
Passwater: Does vitamin C impair copper utilization?
Bendich: This has not been shown in controlled studies. Two groups have investigated this question without finding such a relationship. [22-23]
Passwater: When we were chatting at a scientific meeting a few years ago, you were trying to trace the report that vitamin E could raise blood pressure in some individuals. I remember that the Drs. Shute used to say that this could happen in some diabetics or those who had rheumatic fever. Were you ever able to find evidence to verify these reports? Does vitamin E increase blood pressure in some individuals?
Bendich: No. There are no published reports of increases in blood pressure in any of the placebo-controlled, double-blind studies with dosages up to 2,000 IU per day.
Passwater: Does vitamin E cause tiredness?
Bendich: There are no peer-reviewed, placebo-controlled studies to support this. There have been several studies to examine the safety of vitamin E even at very large doses, and none have reported this as an observation. Usually, the anecdotal reports often suggest increased energy levels.
Passwater: In 1974, I conducted a study of vitamin E usage and health effects . Drs. Linus Pauling and James Enstrom used data from the California subjects in my study and they conducted a follow-up study that showed health benefits in taking supplements.  The data on vitamin E showed benefit at all intakes of vitamin E as shown in Table 1. Although, the data showed greater freedom from disease and longevity at the 300 to 499 IU per day levels than the levels above 1,000 IU per day, there was better longevity at all intakes of vitamin E than among those not taking vitamin E.
Some have incorrectly used these data to imply that the higher level represented a toxic effect. What it was actually reflecting was the fact that some very ill persons began taking a large amount of vitamin E to help fight their diseases. Typically, persons in apparent good health usually decide to take about 400 IU of vitamin E daily if they wish its protection against free radicals. If people have a need for extra action from vitamin E, such as to prevent hot flashes during menopause or to relieve intermittent claudication, they often take about 600-800 IU daily. If they are recovering from a heart attack or have been diagnosed with cancer, they often start with 1,000- 1,200 IU daily. Thus, those who are taking more than 1,000 IU of vitamin E daily are often those already suffering from life-shortening diseases.
Still, the 1,000 IU per day level produced greater health and longevity than found in the control group not taking vitamin E supplements. The proportion that died among those taking more than 1,000 IU daily was 0.37, compared to the higher portion of 0.43 among those taking no vitamin E supplements.
Do doses of 1,000 milligrams per day of vitamin E increase morbidity or mortality?
Bendich: A comprehensive review of the literature concluded that vitamin E is safe at levels of intake of approximately 3,000 IU daily for prolonged periods.  Furthermore, data from the most carefully controlled study in a metabolic ward showed that various measures of body function including hematological, renal, hepatic, and thyroid function were unchanged following vitamin E supplementation at 800 IU daily..
Passwater: Well as you told the FDA during the Dykstra hearing, "there is no public health problem regarding the safety of vitamin supplements."
Thank you Dr. Bendich.