Recently three papers appeared in the medical literature (1,2,3) by Bruce Ames, a respected and very well known professor of biochemistry from the University of California at Berkeley and the Children's Hospital Oakland Research Institute, suggesting that low levels of micronutrients (vitamins and minerals in particular) at the cellular level may actively promote DNA and protein damage and cause disease to a larger extent than is generally realized. Thus certain micronutrients may play both a prophylactic and therapeutic role in a number of disorders related to damaged cellular components. The general term used to describe this phenomenon is genome instability. This view has also been actively promoted by Michael Fenech of the Australian CSIRO (4) and is backed, not only by extensive literature, but also by a recent paper (5) of unusual length (46 pages) in the American Journal of Clinical Nutrition where Ames and coworkers document the role of vitamins and minerals critical to enzyme reactions which at therapeutic doses could correct for reduced enzyme activity. Over 40 genetically related diseased states are discussed.
The multiplicity of critical functions of vitamins and minerals at the cellular level, and especially their role as companion molecules (cofactors) in enzyme reactions that protect genes from mutations and repair gene damage, is probably unrecognized or unappreciated by all but specialists in this field. The Metabolic Tune- Up suggested by Professor Ames makes a compelling case for vitamin/mineral supplements to avoid problems associated with micronutrient deficiency. Ames also promotes taking several antioxidants that do not appear to be in common use. The Metabolic Tune-Up relates to many health issues, including the potential for cancer prevention and the slowing of aging and related degenerative diseases.
Biochemists and nutritional scientists who study the role of vitamins in human biochemistry can list innumerable cellular processes that are vitamin dependent, where a vitamin acts as an essential factor. In fact, it can be argued that the true importance of vitamins in human biochemistry is far from fully elaborated, simply due to the almost incomprehensible complexity of cellular processes. After all, there are approximately 30,000 human genes and over 3800 enzymes currently catalogued, at least 22% of which require a cofactor, in many cases a vitamin, to function.
What is also perhaps not fully appreciated by the general public, and perhaps some physicians, is the essential role that various minerals play in human biochemistry. Critical enzymes require such metals as copper, zinc, manganese, selenium, etc. as an integral part of their molecular structure or mechanism of action, i.e. no metal, no enzyme activity! Deficiencies are thought to produce a broad spectrum of health problems. Minerals of course must come from food and water, air pollution or out of a bottle. It is also true that overloads of some minerals can be disastrous, leading to excessive and potentially harmful oxidative stress, i.e. the metal acts as a center for oxidation by being reduced as it oxidizes an adjacent molecule, in some cases an important cellular constituent, and then, if re-oxidized, is ready to repeat the process. Iron overload is a good example.
The almost complete absence in North America of patients who present with recognized deficiency diseases such as pellagra, rickets, scurvy, acute night-blindness, or beriberi has probably led to a false sense of security and the belief that almost everyone gets enough vitamins from food. Vitamins and minerals have seemingly fallen off the screen for many health care professionals and supplementation viewed as a fad. Interest now seems obsessively focused on toxicity. However, a significant fraction of the North American population appears to not get even the Recommended Daily Allowance (RDA) of some critical nutrients from their diet or supplements (3). In the view of Ames and others, levels of deficiencies that fall between the RDA and the levels that produce recognized deficiency diseases can have serious consequences in connection with what has come to be called our genome integrity.
Ames and others argue that vitamin and mineral deficiencies are common, present a serious health risk and can be corrected by supplementation. Human studies are exceedingly difficult when the goal is to establish evidence based arguments for or against taking supplements, establish a hierarchy of supplements, i.e. which are the most important, and determine what are the optimum amounts of each micronutrient. This is especially true if the goal is to investigate their impact on all aspects of health, from in utero to old age. There are too many variables, too much human variability, too many important endpoints, too many confounding factors, and too few cellular level biomarkers. Financing for intervention studies can be difficult to obtain since pharmaceutical companies, which typically assist in financing large clinical trials, have little or nothing to gain - vitamins and minerals cannot be patented. Also, because of inherent difficulties in achieving good design, execution and statistical power, studies that attempt to connect micronutrients with disease or health have proved to be very easy to criticize or dismiss as inconclusive.
In this review it will be argued that understanding the key role of certain vitamins, minerals and antioxidants in the context of protecting nuclear and mitochondrial DNA, cellular proteins and enzymes from damage may assist in the process of establishing a hierarchy of supplements. The focus of this review will only be on certain micronutrients.
DNA, CHROMOSOMES, GENES, ENZYMES AND COFACTORS - A VERY BRIEF OVERVIEW
The human genome consists of large amounts of deoxyribonucleic acid (DNA), the macromolecule with the famous double helix structure. DNA contains within its structure the genetic information a living organism needs to develop and function, from conception to death. The human genome consists of about 30,000 genes which are the units of genetic information. Genes are encoded in the DNA that makes up a number of rod-shaped cellular constituents called chromosomes that are collected in the nucleus or mitochondria of each cell. The encoding involves the sequence of four organic molecules (bases) that are linked together to form the two long chains that make up the double helix. Only recently has this sequence problem been solved. It is a monumental achievement that will profoundly influence medical genetics for the foreseeable future.
Genes control cellular functions responsible for maintaining the multitude of biochemical processes that characterize a living organism. They serve as blueprints for the cellular production of proteins which include cell receptors, enzymes, hormones, and cytokines (hormone-like proteins which regulate immune responses and are involved in cell-to-cell communication), etc. The functions carried out by these proteins include cell growth, differentiation, metabolism and cell death. Genes are selectively activated or suppressed when molecules such as neurotransmitters, hormones or growth factors bind to and activate cell surface receptors, initiating a cascade of biochemical reactions within the cell in which enzymes play a central role.
Enzymes play a critical role in regulating the rates of most of the multitude of biochemical reactions in living organisms. They act as catalysts and are in general not consumed. The critical nature of the proper functioning of enzymes can be appreciated by the obvious necessity of biochemical reactions in living organism being in balance and occurring at appropriate rates, since otherwise either negligible or huge amounts of a given product could be produced, with either result having serious or perhaps even fatal consequences. That an individual is alive and well is in a large part due to thousands of exquisitely controlled biochemical reactions that occur at the right place and time and to the appropriate extent.
Many enzymes require the presence of other compounds, called cofactors, before their catalytic activity is enabled. Thus there is a protein portion plus the cofactor which can be a vitamin or other organic molecule or a metal ion (i.e. the charged form of iron, copper, zinc, magnesium, etc). In what follows, the essential role that metal ions or vitamins play as cofactors will be shown to directly relate to the role of micronutrients in many aspects of both health and disease. It is also well established that DNA, enzymes and other proteins and fatty acids can be damaged by reactive species such as reactive oxygen and nitrogen compounds and other free radicals, and thus antioxidants, which neutralize these reactive species, also play a critical role in maintaining normal cellular functions and providing protection from damage.
There are a large number of mitochondria in each cell and their proper functioning is critical to health. A popular theory of aging involves mitochondrial damage from free radical attacks such as oxidative damage. It is well known that it is in the mitochondria that cells make ATP, a chemical that is involved in energy generation, and at the same time chemical reactions in the mitochondria generate large amounts of reactive oxygen species and free radicals which can attack and damage nearby molecules and mitochondrial DNA. As might be expected, there are elegant defense mechanisms to minimize the oxidative damage that could result. Otherwise, living organisms would presumably have self-destructed a long time ago. As will be discussed below, micronutrient deficiencies can severely impact these defense mechanisms and thus are thought to lead to accelerated aging and other health problems.
We can't prevent the gene mutations with which we are born. Serious inherited mutations can sometimes be dealt with by significant lifestyle changes (e.g. phenylketonuria can generally be controlled by dietary intervention), while others inevitably lead to early death or lifelong disability. Some are innocuous. Mutations also occur throughout ones lifetime. There are repair mechanisms for gene mutations induced by natural, cosmic or diagnostic radiation, mutagens, toxic substances, etc., and anything that interferes with the proper operation of these repair processes puts the individual at increased risk for diseases related to genetic damage. Enzymes play a central role in these repair processes and thus enzymes with impaired activity due, for example, to low concentrations of cofactors or oxidative damage, may be unable to adequately carry out this essential function.
WHAT CAN GO WRONG THAT IS AMENABLE TO INTERVENTION?
The thesis of Professor Bruce Ames and coworkers, as will as other researchers in this field is that much metabolic damage occurs at micronutrient levels between those generated by the recommended daily allowances (RDAs) and those that cause acute deficiency disease. When one component in the metabolic- micronutrient network is inadequate, repercussions are experienced in a specific biochemical process or even in a large number of processes, and can lead to deficiency related diseases. For example, cancer may result from DNA damage, cognitive dysfunction from neuron decay, and accelerated aging and Alzheimer's disease from mitochondrial functional decay. The focus on cellular micronutrient deficiency is illustrated by the established role in genomic stability of abnormally low levels of the following specific micronutrients (1,2,3,4).
The above discussion illustrates the connection between cellular micronutrient deficiencies and metabolic and genetic problems. The mechanisms involve:  DNA damage in general,  reduced reactivity of damaged enzymes and  oxidative damage to DNA and other cellular components which has a particularly serious impact on the components of the mitochondria and induces what Ames and others call mitochondrial oxidative decay (2). It is highly significant that deficiencies in folic acid, vitamins B12, B6, C and E and the metals iron and zinc appear, according to Ames, to mimic radiation damage to DNA by causing single- and double-strand breaks, oxidative lesions, or both (3). The reduction of enzyme efficiency in DNA repair process is also a critical aspect of the micronutrient deficiency syndrome.
The deficiency levels at which these various deleterious processes become significant are not easily studied except in human cell cultures. As Michael Fenech points out (4), "To date our knowledge on optimal micronutrient levels for genomic stability is scanty and disorganized." Extensive research by Ames and coworkers (5) suggests that the danger point occurs somewhere at or below about one-half the RDA, and that a significant fraction of the populations of the developed world has a deficiency of this magnitude in at least one of the above micronutrients, and multiple deficiencies are far from uncommon (3). The potential for profound deficiencies among the malnourished seems clear. Thus there is not only a significant personal health issue here, but also a public health issue of considerable magnitude that extends throughout the world.
DEFICIENCIES IN VITAMINS AND MINERALS - A CANCER RISK?
This question is the title of a review in 2002 by Ames and Wakimoto (19) which followed a review in 2001 by Ames addressing the connection between DNA damage from micronutrient deficiencies and cancer (20). The essence of the argument is that micronutrient deficiency can mimic radiation or chemical damage to DNA causing both single and double strand breaks and oxidative damage (lesions) or both. The double strand chromosomal aberration is a strong predictive factor for human cancer (21). Deficiencies in the micronutrients listed above all show laboratory (i.e. cell culture) evidence of mimicking radiation damage with the evidence ranging from likely to compelling (20). The percentage of the US population that is deficient, i.e. an intake of <50% of the RDA for each of these micronutrients ranges from 2% to greater than 20%. Similar or more serious deficiencies may be present in a significant portion of many populations (20). However, as mentioned above, the human in vivo cellular threshold level of each micronutrient where the rate of DNA or other damage becomes significant, in the context of cancer risk, remains in most cases unknown, and definitive studies may be impossible given the natural history of the disease as well as ethical concerns. Thus it is reasonable to turn to epidemiologic studies to seek further evidence, i.e. a diet-cancer link. However, the micronutrients in question are rather widely distributed in the foods we eat. Consider the richest sources for the following micronutrients (19):
Furthermore, epidemiologic studies of the link between diet and cancer are difficult to interpret because each food item can contain dozens and perhaps even hundreds of different micronutrients, making it difficult to correlate a given food item with a given micronutrient and correct for confounding. In addition, many studies may have lacked the statistical power necessary to provide meaningful results. It is also possible that the upper end of the range for intake of a given food class, e.g. fruits and vegetables, may not be high enough to influence cancer risk in a significant fraction of the particular population studied (22). It is not surprising that studies tend to be inconsistent. Bingham and Riboli (23) believe that prospective cohort studies of the diet-cancer link should involve at least 500,000 initially healthy subjects (about 5-8 times the cohort size in large studies already in the literature) who should be followed for at least ten years in order to observe sufficient cancer cases at the common sites. Such a study, involving a large number of European countries, which includes some genetic typing and extensive examination of serum markers (the EPIC Study) is now in the data analysis stage (23).
Nevertheless, more than two hundred studies have examined the question of the connection between diets high or low in fruits and vegetables and the risk of developing cancer. The accumulated evidence supports the conclusion that eating large amounts of fruits and vegetables lowers the risk of developing some but not all cancers. In his recent bookEat, Drink and Be Healthy (24) (review published in IHN issue #136, April 2003), Dr. Walter Willett lists seven cancer sites and the associated fruit and vegetable types where epidemiologic research has found anticancer effects, a list that emphasizes the apparent lack of a "blanket anticancer effect."
A recent study employed a new approach to this question. In a case-control study the relationship between gastric cancer risk and the total dietary antioxidant potential was examined (25). Total antioxidant potential of dietary plant food was found to be significantly inversely associated with the risk of gastric cancer. The relative benefit was enhanced in individuals exposed to high oxidative stress on the gastric mucosa (long-term smokers and subjects exposed to H. pylori). This study used only 12 dietary items. Very recently, Wu et al reported the results of a large study where the total antioxidant capacity (fat- and water-soluble antioxidants) of individual food items was measured. This benchmark study should facilitate studies attempting to correlate total dietary antioxidant power and specific fruits and vegetables (26). The top 15 food sources of water-soluble antioxidants were small red beans, blueberries (wild), red kidney beans, pinto beans, blueberries (cultivated), cranberries, artichoke hearts, blackberries, prunes, strawberries, raspberries, apples (red delicious and Granny Smith). For the fat-soluble antioxidants, the top 15 foods were avocado, navy beans, pinto beans, black eye peas, broccoli, black beans, raspberries, cranberries, russet potatoes, spinach, oat cereal and Brazil nuts. Pecans, walnuts and hazelnuts were also high in total antioxidants.
The strength of the association between cancer risk and the consumption of fruits and vegetables, when not stratified by antioxidant power, seems to be diminishing somewhat as recent prospective cohort studies are reported, and in a review published in 2004, the authors (including Willett) now use the word probably in describing the inverse association (27). The huge range of antioxidant power found by Wu et al (26) suggests that just lumping fruits and vegetables together in one category in studies may underestimate their effectiveness. The study on gastric cancer appears to be the first where total antioxidant power was a parameter.
However, no one appears to be suggesting that fruits and vegetables are unimportant. In fact, the study of Wu et al (26) provides a useful guide to selecting fruits and vegetables to emphasize in the diet for maximum antioxidant protection. An inverse correlation of fruits and vegetables with cancer is of course consistent with the action of folic acid and vitamin C in the context of DNA damage, but clearly as outlined above, other food classes (e.g. meat and whole grains, etc.) contribute micronutrients thought as well to be critical, and these foods by and large have not stood out in epidemiologic correlations with cancer, either as good or bad. The possible connection between cancer and specific micronutrients as well as multivitamin/mineral intake will be discussed below.
FOLIC ACID: A MAJOR PLAYER
In the model of Ames for chromosome breaks and the associated risk of cancer, folic acid (folate) is a major player. The form of folic acid in food differs in molecular structure and bioavailability from the synthetic compound found in supplements and used in the fortification of flour and cereals, with the synthetic version being almost twice as bioavailable. However, there appears to be an upper limit on the metabolism of the synthetic form, with intakes of more than about 400 mcg/d resulting in serum levels of free folic acid which do not appear to be utilized. Concern has been expressed (28,29) regarding this since it appears unknown if there are dangers associated with long-term high levels of free serum folic acid which might build up as a consequence of both supplementation and eating fortified foods. Special concerns have been raised in connection with children who may be given supplements and eat large amounts of prepared cereals (28,29). Some estimates of potential daily intake exceed the upper limit for children by 300-400%. There does not appear to be a problem with metabolism of the natural form found in food, except that some individuals may have genetic defects which reduce the bioavailability and this can result in deficiencies. Government mandated fortification in the US and Canada started in 1998. Spot checks of the level of fortification indicate that it is common for foods to contain considerably more folic acid than indicated on the label. In the US the upper limit for the daily intake of the synthetic form is 1 mg. There is now concern that this may be exceeded in an unknown but significant fraction of the population. Note the big gap between the 400 mcg/d above which unmetabolized folic acid appears in the blood and the 1000 mcg/d considered to be the safe upper limit. Also, therapeutic doses as high as 5 mg/d are used (29). There are obviously unresolved issues.
One of these issues involves folate therapy after coronary stenting with plain metal stents. Folate therapy was found to increase in-stent restenosis (recurrent blockage) and the need for target-vessel revascularization. This adverse result was observed in a cohort that had undergone successful coronary stenting, although it was absent in the subgroups consisting of women, patients with diabetes and those with homocysteine levels over 15 mcmol/L at baseline. This result was inconsistent with earlier folate trials aimed at reducing restenosis (30).
A well-validated concern with high levels of folic acid intake is related to the possibility of masking a vitamin B12 deficiency and related megaloblastic anemia. It is well known that folic acid reverses the evidence revealed by blood tests, i.e. it eliminates the anemia, but does not eliminate the B12 deficiency, which can go on to cause neurological damage which can be severe, reach the point of irreversibility and mimic Alzheimer's disease. Since the elderly can have problems with the metabolism of B12 from food because they are unable to separate the vitamin from its natural, protein-bound form, B12 deficiencies in this age group are common and a real reason for concern. Also, a deficiency in what is called intrinsic factor prevents normal absorption of any form of B12, but this is rare (31). Because of the potential for high intakes of folic acid, there have been calls for fortification with significant amounts of B12 to accompany folic acid. The B12 present in supplements or as a food additive is generally metabolized easily and normally leads to increased serum and tissue levels of B12, although larger amounts may be needed than found in some multivitamin pills. Individuals with a severe deficiency generally need therapeutic doses given by injection. There appears to be only one study reported that addresses the question of an increase in untreated or masked B12 deficiency since folic acid fortification (32). The study found no increase in B12 deficiency in the absence of anemia over the period 1990 to 2001 in the age group 60-80. This result would argue against the suggestion that there is a B12 deficiency problem masked by the over consumption of folic acid in the elderly due to fortification.
The fortification of cereal products and flour was motivated by the fact that a folate deficiency in the first few weeks of pregnancy can result in birth defects, in particular neural tube defects. As a preventive measure, the folic acid supplement must be taken prior to conception. In the mid 90s there was a campaign to motivate women of child-bearing age to take folic acid supplements, but with 50% of pregnancies unplanned and problems in general associated with convincing a large population to take supplements, this program was a failure. After mandated fortification in 1998, a few studies now indicate a drop in neural tube defects, and in one particularly careful study done in Nova Scotia (33), which included aborted fetus data, over a 50% decrease was found. Finally, there is little doubt that in the post-fortification era, there has been a dramatic increase in average serum and red blood cell folate levels and a modest drop in homocysteine levels (34). Whether this latter change will translate into decreased adverse cardiovascular events remains to be seen. It is of interest that the growing popularity of low-carbohydrate diets, as judged by the lamentations of the processed food industry, may result in reduced effectiveness of the folate fortification program. However, the major advocates of low-carb diets all strongly recommend supplements as a vital and essential part of the program (see Research Report "The Diet Zoo" published in IHN issues #143 and #144).
In epidemiologic studies that specifically focused on folate and cancer, some have found an inverse relationship between folate intake and the risk of adenomatous polyps and colorectal cancer. In the Nurses' Health Study, a high intake of folate from fruits and vegetables was found to lower the risk of colorectal cancer, and supplementation with a multivitamin containing folate was found to offer even greater risk reduction (35). Such a result can be viewed as evidence that even a high level of fruit and vegetable consumption may not provide optimum cellular levels of folate. There have in fact been a large number of studies regarding the role of folic acid in colorectal cancer (see (36) for references), but only about half produced statistically significant evidence of reduced risk. In view of the problems with such studies, perhaps this should be viewed in a positive light.
An interesting chapter in the folic acid story involves its relationship to breast cancer. In the Nurses' Health Study (37) no risk reduction was observed for folic acid except for a very weak inverse association for postmenopausal women. However, there was a strong protective effect found for women who consumed over 15g/d (about one drink) or more of alcohol. Alcohol is known to interfere with the absorption and metabolism of folic acid. This suggests that the cohort of non-drinkers had folic acid levels above the threshold for observing increased breast cancer risk, and alcohol consumption put those with low or moderate dietary folate intake below the threshold.
Other studies that show an inverse disease risk relationship with folate consumption could be discussed. For example, it is well known that a folate deficiency is associated with elevated serum homocysteine, and there now seems to be general agreement that high homocysteine levels are a risk factor for cardiovascular disease and perhaps Alzheimer's and Parkinson's disease. Incidentally, vitamin B12 is also directly involved in the folate- homocysteine chemistry (38). There is also growing evidence that homocysteine per se is implicated in chromosome damage (39), and lower serum homocysteine has been found to correlate with lower risk of colorectal adenoma recurrence (36).
At this point, it seems sufficient to conclude that the epidemiology is very suggestive and provides support for the Ames model. The interested reader is referred to reviews by Ames (20), Fenech (4) and Ames and Wakimoto (19) for detailed information on the epidemiologic and cell culture evidence associated with the important micronutrients in the context of both cancer (19,20) and other health issues (4). References are also provided in the item-by-item list given above. A full discussion would result in a 30-40 page review!
THE ANTIOXIDANT CONNECTION AND PACKER'S ANTIOXIDANT NETWORK
It has only been in fairly recent times that the action and importance of antioxidants has become common knowledge among layman and health-care professionals, and only recently has the synergistic relationship between certain antioxidants been explored. A leader in this field is Professor Lester Packer of the University of California at Berkeley. His research and that of others has identified five antioxidants that operate on a cellular level in a synergistic fashion, which means that a given member will function to regenerate one or more antioxidants which have become inactivated. Packer's list, which he calls The Antioxidant Network, comprises vitamins C and E, a-lipoic acid, coenzyme Q-10 and glutathione (41). The main focus of Ames and other researchers concerned with antioxidants is on mitochondrial DNA mutations caused by oxidation (oxidative stress) and their relation to aging. This is also called mitochondrial aging or the mitochondrial free radical theory of aging.
Numerous mitochondria present in most cells are in fact the greatest source as well as the greatest targets for free radical attack, especially reactive oxygen species, but of course, all cellular components are potentially vulnerable. Ames estimates that there are 10,000 hits per cell per day from free radical attack. Obviously the existence of living organisms depends on defense and repair mechanisms. Free radical damage to cells is implicated in a whole host of disease conditions, including amylodosis, acute pancreatitis, arthritis, inflammatory bowel disease, senile dementia, retinal degeneration, and senile cataract. It is the job of antioxidants to control free radical damage and prevent the associated disorders. Thus the importance of the Antioxidant Network. A whole review needs to be devoted to the Antioxidant Network, especially since there are many varied and complex issues associated with vitamin E. Below is a very brief summary of the action of each micronutrient in Packer's network (41).
Two recent studies illustrate the potential role of antioxidants in cancer prevention. In one, the age-related increase in the extent of hydroxyl radical-induced DNA damage was significantly related to the risk of developing prostate cancer. The other study involved the evaluation of prostate tissue samples for hydroxyl radical induced changes in DNA. Such changes enable researchers to discriminate among non-cancerous and cancerous tissue and between cancer and benign prostatic hyperplasia (benign enlarged prostate) with nearly 100% diagnostic accuracy (58)! Results strongly support the hypothesis that an important mechanism by which antioxidants may reduce the risk of prostate cancer is through the reduction of the damage caused by free radicals. Obviously, both studies support the Ames hypothesis. In this connection the large (32,400 subjects) ongoing primary prevention trial of selenium (200 mcg/d) and vitamin E (400 IU/d synthetic) supplementation is of interest. This study was prompted by earlier positive results and highlights the role of antioxidants in this area.
Those who refuse to consider micronutrient or antioxidant supplementation unless their effectiveness and safety have been examined in North American double blind, randomized, placebo controlled clinical trials for any endpoint required by a suggested used will probably wait a long time. These five antioxidants cannot be patented and offer no profit potential to the pharmaceutical industry. It is in fact remarkable that there has been and still is so much research on vitamin E.
MICRONUTRIENT DEFICIENCIES AND AGING
There are a several theories of aging. Along with the free radical theory, it has been suggested that so-called advanced glycation end products (AGEs) play a role in the aging process (59,60,61). Also, a change in the balance between anabolic and catabolic metabolism in favor of the latter has been proposed (62) as a fundamental feature of aging. AGEs are formed by a reaction of glucose with proteins, and since the AGEs are irreversibly produced end products, they can profoundly influence the activity and function of enzymes and other proteins. Their formation is favored by high serum glucose levels, and this is thought to explain in part the well- known connection between diabetes and degenerative diseases. AGEs are also thought to be involved in direct attacks on DNA. None of the micronutrients discussed above appear to be directly involved in potential protective actions in connection with AGEs. The accumulated evidence supports the thesis that high concentrations of AGEs are undesirable and this provides an additional reason why diabetes and the elevated serum glucose levels frequently associated with the metabolic syndrome should be avoided at all costs, mainly by diet, weight control and exercise.
The switch to a metabolism favoring catabolism is thought to be primarily hormone driven with DHEA (dehydroepiandrosterone) the principal actor; although, as the downhill spiral toward degenerative disease and death proceeds, oxidative damage to DNA, proteins including enzymes and cell membranes is considered very important (62).
The free radical theory of aging, especially as it relates to oxidative damage to the mitochondria, appears to occupy a pivotal role in the modern view of the aging process (2,63). A large percentage of cellular free radical production occurs in the mitochondria, since this is where most of the cellular oxygen consumption takes place. The mitochondrial DNA is unique, is much smaller in terms of number of bases than the nuclear DNA, and the repair mechanisms available are more limited than in the case of nuclear DNA. Thus antioxidant deficiency in the mitochondria is a very important factor in preventing DNA damage, mutations, and in the decrease in enzyme function. Since the mitochondria are the "cellular powerhouses," any impairment of proper function can have a profound effect on, for example, muscle function, and there are recognized "mitochondrial diseases" which derive from mutations and other malfunctions (64). The Packer antioxidant network is thought to play a critical role in mitochondrial antioxidant defenses, and in addition, there is evidence that providing the substrate acetyl-L-carnitine along with a-lipoic acid and Q-10 can have a profound effect on restoring mitochondrial function, although the evidence derives from rodent studies (2). Marriage et al (47) call this nutritional cofactor therapy.
Studies of the sort that mainstream medicine, by and large, require for validation of proposed interventions are probably impossible when the question concerns aging in general. It is thought that the prelude to clinical manifestations of many age-associated problems may have their origin many years in the past and require years to develop. Thus intervention studies would require 20-40 years if the endpoint was primary prevention. Such studies pose tremendous difficulties in recruitment, follow-up, dropout rates and even funding. Some of the principal investigators might not live long enough to see the outcome! Ongoing prospective cohort studies probably do not or cannot examine questions such as the benefits of coenzyme Q-10 and a-lipoic acid in the context of age related degenerative diseases, and some will not even have good dose data on vitamins C and E. Rodent and cell culture studies (2,65,66,67) can and indeed have been very informative and avoid the natural time-base imposed by human aging, but there will probably always be great resistance from mainstream medicine to the translation of these results into recommendations for the general public regarding preventive or delaying actions. However, studies requiring a shorter time span are possible when the question involves reversing or delaying the progression of existing degenerative diseases associated with aging. The use of vitamin E in Alzheimer's disease is a good example where, on the basis of very limited positive intervention studies, high doses are actually recommended and being used (see Prevention of Alzheimer's Disease).
Another example is age-related macular degeneration (AMD) where oxidative stress and oxidation of unsaturated fatty acids are thought to play a significant role (68,13). Zinc deficiency is also implicated through its importance in a number of critical enzyme processes. In one study the prevalence of AMD in patients with low antioxidant intake and low lutein intake was almost twice that of patients with high intake (68). In a large intervention study coordinated by The Age-Related Eye Disease Study Research Group (69), a beneficial effect on the progression of AMD from an intermediate to advanced stage was observed for supplementation with antioxidants and zinc and copper [vitamin C (500 mg/d), vitamin E (400 IU/d), beta-carotene (15 mg/d), zinc (80 mg/d) and copper (2 mg/d)], but no benefit was found for early use. While there is also much interest in lutein and zeaxanthin in connection with the prevention or delaying of AMD, and the combination is readily available in health food stores, there do not yet appear to be definitive studies indicating a role of these carotenoids in primary prevention.
There have been a number of studies on the role of antioxidants in Alzheimer's disease, and the results have been somewhat inconsistent. A very interesting study just published relates directly to the subject of this review. In a cross-sectional and prospective study of 4740 subjects 65 years or older, it was found that the use of vitamin E and C supplements in combination was associated with very significant reduced AD prevalence at the start of the study and incidence 3-5 years later. Note, as discussed above, vitamin C regenerates vitamin E. No protective effect was seen for either of these vitamins used alone, or with vitamin B-complex supplements. In view of the significant public health implications, the authors call for prevention trials (70).
Calorie restriction is another good example of an anti-aging tactic where decreased metabolic activity may reduce mitochondrial free radical generation and oxidative stress, and if the level of nutrition is still adequate, this should provide beneficial results (71). Calorie restriction also impacts hyperglycemia, the formation of AGEs, and hyperinsulinemia. High insulin levels are in fact thought to be mutagenic. It is well known that animal studies show dramatic life extension with calorie restriction (72). However, there do not appear to be any controlled human studies covering a long period of time. Short term studies show improvements in blood lipid profiles and blood pressure (72,73), and the studies by Willcox et al (74) of the people of Okinawa suggest that calorie restriction contributes to longevity, but there are a number of potential confounding factors and the controls were not totally satisfactory. The impact of wartime calorie restriction on mortality is only tangentially relevant due to the short time interval.
Theories of the mechanism of calorie restriction are reasonably well-developed (75), but again there is no confirmation from long-term human studies. The author is unaware of planned or ongoing studies where a large middle-age adult cohort is on or going to be on a calorie reduced diet for 30 or more years to see if they live longer than average or longer than controls. It is hard to imagine the organization and implementation of such a study. Both obese and non-obese individuals contemplating significant calorie restriction should be aware of the potential need for supplementation, since it is entirely possible that potentially harmful micronutrient deficiencies can accompany a decreased food intake. Micronutrient deficiencies are commonly seen in the elderly, many of who are living an involuntary calorie restricted life due to poor appetite, poverty, depression and perhaps mental decline. Macronutrient deficiencies, especially protein and essential fatty acids, are also possible.
Consider then the question - do antioxidants delay aging? The free radical theory, which is based mainly on animal and cell culture studies, provides a good scientific basis for the hypothesis but how about actual human studies, i.e. clinical trials? Ames in his review "Delaying the Mitochondrial Decay of Aging - A Metabolic Tune- up," is still unable in 2003 to quote supporting clinical studies that would satisfy mainstream medicine and prompt a recommendation to take a variety of antioxidants and other micronutrients, although the three examples quoted above seem to be a good start. Thus while the free radical theory of aging and its related focus on mitochondrial decay appears to be accepted by the scientific community, individuals wishing to take action must realize that they are translating theory into a self-designed intervention program. But if the mix of micronutrients, and antioxidants in particular, is highly likely to be harmless at the doses used, it is hard to argue against this action, given that waiting for the blessing of mainstream medicine may require waiting for a period considerably exceeding ones life expectancy. After all, as will be discussed below, we are still waiting for the go- ahead from high profile segments of mainstream medicine regarding taking multivitamins!
MULTIVITAMIN USE - CLINICAL AND EPIDEMIOLOGIC STUDIES
The role of vitamins and minerals in genomic stability appears well established in the laboratory, but a fair question involves the existence of clinical or epidemiologic evidence that taking multivitamin preparations has detectable health benefits. Ames makes the point that a deficiency in just one of the critical micronutrients can adversely influence genomic stability (3). But most individuals do not know their cellular levels of critical micronutrients or even their total daily intake from food and supplements. Thus the multivitamin/mineral "covers all the bases." Because of synergism, both known and unknown, it seems better to examine the research on multivitamin intake rather than studies involving each micronutrient separately. Also, studies on individual minerals are rare. Generally, subjects in studies who indicate they take a multivitamin may by default also be taking a mineral mix. In some studies of the relationship of multivitamin intake to a particular health issue, the content of the preparations used is either variable or unknown or both. Also, additional supplementation with extra vitamin C and E may go undetected. Nevertheless, the following results are of considerable interest.
These appear to be among the most significant studies that have recently appeared. Studies omitted for lack of space include some with negative results. There have been very few studies, in particular intervention studies that have used high-potency formulations. Both prospective studies and randomized clinical studies may well underestimate the beneficial effects because the studied populations frequently include individuals with good diets who are health conscious, exercise, etc., and in such cases, there might be minimal effects, especially from multivitamins containing just the RDA. Also, follow-up studies that collect data only at enrollment may underestimate beneficial effects when declared non-users of supplements start taking them. At the opposite extreme, individuals with severe deficiencies might need considerably higher doses than found in typical multivitamins. Also, the recommendations for genomic stability involve antioxidants, some of which are either not present in multivitamins or present in low quantities, e.g. vitamins E and C. Nevertheless, the above studies would seem to be highly suggestive and supportive of the recommendations of both Ames and others that multivitamin/mineral supplementation should have a beneficial effect on a number of aspects of health.
Two Harvard medical scientists concur. In a recent communication in the Journal of the American Medical Association (Clinician's Corner) (95), Robert Fletcher and Kathleen Fairfield point out that "Recent evidence has shown that suboptimal levels of vitamins, even well above those causing deficiency syndromes, are risk factors for chronic diseases such as cardiovascular disease, cancer and osteoporosis. A large proportion of the general population is apparently at increased risk for this reason." Furthermore, they go on to recommend that all adults take one multivitamin daily and that the elderly consider a dose of 2 ordinary multivitamins daily, although they suggest it might be safer to take one multivitamin with additional vitamin B12 and vitamin D because of worries that a double dose would provide excessive vitamin A. For women attempting to conceive, they suggest 400 mcg/d of folic acid. They go on to comment that the recommendation of a multivitamin is justified because "a large proportion of the population needs supplements of more than one vitamin." This communication accompanies a detailed review in the same issue of JAMA by these two authors (96) dealing at length with the topic of vitamins for chronic disease prevention. Also, in a paper (40) in the New England Journal of Medicine titled "What Vitamins Should I be Taking, Doctor?," Harvard's Willett and Stampfer present a conservative view on vitamin supplementation, conclude that the likelihood of benefit outweighs that of harm, recommend a multivitamin based on the RDAs and present arguments for why the RDAs for vitamin E (they suggest 400 IU) and folic acid (for cancer prevention) may be too low. Willett in his book Eat, Drink and be Healthy (24) lists five vitamins that "many people don't get enough of from their diets" - folic acid, and vitamins B6, B12, D and E.
However, in very sharp contrast, we have the current Establishment view. The American Cancer Society recommends only a well-balanced diet and does not recommend the use of vitamin or mineral supplements to prevent cancer (97). The American Heart Association also recommends that vitamin and mineral supplements not be considered as a substitute for a balanced and nutritious diet designed to emphasize the intake of fruits, vegetables and grains (97). The U.S. Preventive Task Force also takes a similar position, stating that the evidence for or against individual vitamins or multivitamins is insufficient to provide a basis for recommendations (97). Some might argue that the Task Force standards are too high and the position unrealistically conservative! The quantity and quality of evidence they demand may not be available for decades, if ever. It would almost appear that the only deficiency universally recognized and accepted is a prescription drug deficiency! The reader is left to judge just how realistic these Establishment recommendations are in view of the credentials of those favoring supplements, some of whom might well be classed as "Establishment." For example, in his book (24), Willett devotes a whole chapter to the subject of taking a multivitamin for what he calls "insurance." The quotation given at the beginning of this review provides the answer that Professor Ames (2) would probably give - "It should be easier to convince people to take a multivitamin/mineral supplement than to change their diet significantly."
The term "drug-nutrient interaction" generally refers to foods interfering with the action of prescription drugs. The other side of the coin involves prescription drugs interfering with the absorption or action of micronutrients. This can be a very serious problem, especially in the elderly population where multiple prescription drug use is common (10-15 different drugs daily!!). Drugs may influence vitamin status either directly or indirectly (98). The former involves alterations in absorption, metabolism and excretion, whereas indirect effects include altering appetite or taste, gastrointestinal flora and the rate of stomach emptying. Examples of drugs that decrease either serum folate, or B6 or B12, or alter or inhibit enzymes involved with these vitamins (and thus generally increase homocysteine levels) include (98,99):
Prescription drugs can also cause mineral depletion. For example, Seelig and Rosanoff (102) list a large number of drugs that cause magnesium depletion in their book The Magnesium Factor. It is probably safe to assume that detailed studies of vitamin and mineral deficiencies induced by prescription drugs are not routinely done, and thus the overall magnitude of the problem is unknown. This may be just the tip of the iceberg.
MUTIVITAMIN/MINERAL DOSES: HOW MUCH TO TAKE
Given that it appears, at least to some experts, to be a good idea to take a multivitamin/mineral daily, what micronutrient levels are optimum? The answer is that nobody knows, especially if the goal is optimum health rather than simply avoiding deficiency diseases. In the absence of optimum intake information, one is left to improvise. One approach is to consider the supplementation recommendations of two well-known physicians with extensive experience in the use of supplements. The cardiologist Stephen Sinatra uses the following levels of the micronutrients we have been discussing in his daily nutrient formulation, by RDA standards a high-potency formulation. E: 232 IU of natural mixed tocopherols and tocotrienols; C: 400 mg: Folate: 800 mcg; B2: 20 mg; B6: 40 mg; B12: 200 mcg; Magnesium: 500 mg; Zinc: 20 mg. Packer would add 100 mg of a-lipoic acid and 30 mg of Coenzyme Q-10, whereas Sinatra recommends 26 and 30 mg respectively. The late Dr. Robert Atkins, in his book Dr. Atkins' Vita-Nutrient Solution presents a basic schedule that is similar to that of Sinatra except for much more folic acid and vitamin C. A high potency formulation used by the Cooper Institute for clinical studies is also similar to Sinatra's (103,85). Also, they all contain many more vitamins and minerals than are listed above. For comparison, the popular Centrum Silver's formulation provides 150% of the daily RDA for vitamins E (45 IU) and B6 (3 mg), over 400% for B12 (25 mcg), 25% for magnesium (100 mg), while folic acid (400 mcg), vitamin C (60 mg) and B2 (1.7 mg), are just at the RDA. This formulation of course also contains other vitamins and minerals (2003 PC Edition, Physicians Desk Reference). Ames and Fenech both sidestep the question of actual doses expect for folic acid where the recommendation is 400 mcg/d. Thus, how much to take remains controversial.
There are also valid concerns regarding toxicity, although at the dose recommendations discussed above, this does not appear to be an issue. High levels of vitamin E can increase the risk of bleeding and antiplatelet effects. High intake of either vitamin C or Vitamin E is thought to be, under some circumstances, prooxidative rather than antioxidative, i.e. just the opposite of the desired action, but there is little evidence, some of it highly questionable (104). Too much vitamin A, which is fat soluble and can accumulate, may increase the risk of hip fracture (105). The potential problems with high levels of folic acid intake have been addressed above. Zinc is toxic at high levels of intake (13).
Iron appears to represent a special case. While Ames makes a clear case for adequate body stores of iron, excess iron appears to present a significant risk factor for, among other things, type 2 diabetes (106). While it is well known that patients with hemochromatosis, which arises from a genetic defect in iron absorption, are at high risk of developing diabetes (53-82% of patients with hemochromatosis develop diabetes), the very recently reported study in JAMA by Jiang et al found that elevated iron stores were associated with an increased risk of type 2 diabetes in healthy women independent of known diabetes risk factors (106). Iron stores were measured by serum ferritin levels. Normal levels for women range from 12 to 150 mg/ml. In the JAMA study, which was of the prospective case-control type based on the Nurses' Health Study database, it was found that women who developed diabetes had an average ferritin level of 109 vs. 71.5 ng/ml for those who did not. The average age of the cases and controls was about 56, and about 65% were postmenopausal. The authors point out that it has been suggested that the formation of the very active hydroxyl radical catalyzed by iron plays a role in the development of diabetes by attacking cell membrane lipids, proteins and DNA. Trials of iron reduction in type 2 diabetes have shown promise but are nevertheless inconclusive (106). It appears to be generally agreed that men and postmenopausal women should not in general take a multivitamin/mineral containing iron unless there is evidence of anemia. Also, it has been known for decades that iron absorption is closely linked to vitamin C intake in a positive, dose dependent manner.
It may turn out when much more research is done that just taking a multivitamin/mineral pill containing the RDA of each micronutrient plus a balanced diet rich in fruits and vegetables will be quite sufficient to prevent genome instability. At this point no one really knows. The DNA and protein damage Ames, Fenech and others are concerned about is thought to occur at intakes of 50% or less of the RDA, but studies are far from clear on this point for all critical micronutrients. The multivitamin/mineral has great merit in providing a comprehensive assortment of micronutrients and many would find this approach more convenient and cheaper than taking the individual items. Some may feel that they really want to play it safe and use a more potent supplement, what some call a "designer" multivitamin/mineral. After all, it is a common belief that the RDAs for some micronutrients may be well below that required for optimum health. Also, older individuals may find the designer multivitamin/mineral more attractive, given that they are more prone to dietary deficiency, malabsorption and inadequate tissue and serum levels due to drug interactions. The Life Extension Mix plus their "Booster" is a good example of a state of the art designer multivitamin/mineral formulation, as is the daily nutrient sold on Sinatra's web site. The Life Extension Mix contains 66 micronutrients, including fruit and vegetable extracts.
The mineral content of the multivitamin/mineral supplement should not be ignored since an adequate and balanced mineral intake is far from a given just from diet. Also, multivitamin users need to consider increasing calcium and magnesium, which are generally low even by RDA standards in many multivitamins because even the RDA would make the pills too big or too many would be required daily. Extra vitamin E (where the RDA appears very low), as well as coenzyme Q-10 and a-lipoic acid should also be considered. Strong arguments can be made for using the natural vitamin E (a-d-tocopherol or mixed natural tocopherols and tocotrienols) rather than the synthetic "dl" form. Natural vitamin E succinate is also popular, but it should be mentioned that the anti- cancer activity recently reported in a number of publications is only relevant if this particular derivative is administered intravenously (107). Nevertheless this vitamin E derivative merits close attention as the anti- cancer action is explored more extensively.
There is overwhelming evidence that a number of vitamins and minerals are required as antioxidants or cofactors for enzymes or part of the structure of enzymes involved in DNA synthesis and repair, the maintenance of methylation of DNA and the prevention of oxidative damage. Deficiencies in these micronutrients can cause genome damage and the levels of damage are equal to or greater than that caused by exposure to ionizing radiation or chemical genotoxins. Damage involves DNA, proteins, enzymes, and lipids. It is significant that it may take decades before this damage becomes manifest as symptomatic disease. The importance of this type of damage is illustrated by the fact that eight human enzymes have been identified (glycosylases) that are specifically involved in just the repair of the type of DNA damage caused by deficiencies in either antioxidant micronutrients or folate and vitamin B12 (4). As Challem (108) points out in an interesting article calling for a new "vitamin paradigm," there are a dozen or more nutrients that in this context are essential but there are thousands of conceivable genetic defects (inborn or acquired) that can produce elevated requirements for one or more micronutrients. This in fact complicates the design and interpretation of studies because micronutrient requirements vary greatly among individuals, seem to be higher than generally believed, and also are thought to fluctuate greatly within the individual (108).
The obvious conclusion to be drawn from the accumulated evidence detailed above is that taking a multivitamin supplement merits very serious consideration. The most recent estimate of the use of multivitamins every day found about 34% of usage among Americans. The authors also examined the intake of what they called non- vitamin, non-mineral supplements. It is interesting that neither the super antioxidant a-lipoic acid nor Q-10 made the list, even with a cut-off of 1.4% of the population (109).
Many individuals take multivitamins simply because they think it is a good idea. The rather extensive research supporting the micronutrients discussed above now provides evidence-based justification on a molecular level for such action that goes far beyond the simple notion that vitamin and mineral supplements are "good for one." This molecular point of view also complements the extensive epidemiologic results supporting both the need for a balanced diet including ample dietary intake of fruits and vegetables, as well as the epidemiologic evidence for the merits of taking multivitamin/minerals. It will probably not have escaped notice that the substances featured above are not esoteric products the health food store clerk might have trouble finding, but rather, all are commonly found in food and are available individually or in multivitamin pills. Thus deficiencies could be easily avoided at very minimal cost. The only remaining question has to do with optimum dose levels and determining dietary intake.
The multivitamin/mineral pill can be viewed as a cheap and potent personal or public health intervention, and one that may well be highly effective in prevention of disease, including cancer and degenerative diseases, especially in an aging population. In this review the special deficiency-related problems in developing countries have been ignored, but it is of interest that UNICEF and WHO are planning trials of a multivitamin/mineral to reduce morbidity and mortality among pregnant and lactating women in developing countries (110). Dramatic results might be expected. For example, the supplementation with vitamin A in women of reproductive age living in Nepal yielded a 40% reduction in maternal mortality (111)!
Ultimately, genetic typing will probably become common, and with this information physicians will be able to tailor-make vitamin/mineral combinations with doses adjusted to reflect the genetic profile and the presence of mutations (polymorphisms, which are in fact very common). After all, Ames has already identified over 40 disease-causing mutations that are amenable to vitamin or mineral therapy. This gives a glimpse of one aspect of future medical practice.
Finally, one should not forget that man gave up the hunter-gatherer way of life about 10,000 years ago in favor of eventual urbanization and agricultural sources of food, with an ever evolving toxic environment along with depleted soils, over-nutrition, and in general eating habits which in developed countries are vastly different than those of our forbearers whose genes we carry today, genes that dictate our human biochemistry, our metabolism, and our micronutrient needs.