Fabulous Folate:
Folic Acid in Health and Disease

This article first appeared in the
April, May, June, 1995
issues of VRP's Newsletter

by A.S. Gissen
In 1931, a cure for "tropical macrocytic anemia" was found.The treatment consisted of the yeast extract "marmite" which contains an active ingredient subsequently isolated from spinach ten years later.This active component is today known as folate, a member of the vitamin B complex. A deficiency of folate results in the development of a megaloblastic anemia which is morphologically indistinguishable from that associated with a deficiency of vitamin B-12. Although folic acid's relationship to megaloblastic anemia has been recognized for more than half a century, research in the last few decades has firmly established folic acid's role in many physiological functions essential for health. These expanded roles for folic acid include the prevention of birth defects, carcinogenesis, and cardiovascular disease.

Folate Chemistry and Functions
Folate is a generic term that refers to different forms of pteroyl(poly)- glutamate(s) conjugates. In 1945 folic acid was synthesized, and this synthetic material was found to have therapeutic activity similar to the natural vitamin.3 Pteroylglutamic acid, today known as folic acid, is a naturally occurring pteridine derivative comprising three basic parts- a pteridine, para-aminobenzoic acid, and glutamic acid. (See figure 1) Folic acid is a monoglutamate, containing one glutamic acid residue. This form doesn't occur in nature where folates are always present as polyglutamates with up to eleven glutamic acid residues. In most mammals, including humans, the transport form of the vitamin is a monoglutamate, and this is the form found in blood plasma and extracellular fluids. The active coenzyme forms of the vitamin, however, are polyglutamates with between four and six glutamic acid residues.4

Folic acid is an essential dietary component and has a role in a number of reactions involved in amino acid and nucleotide metabolism. Different coenzyme forms exist as a result of variations in the reduction state of the pteridine ring, the C1 group attached to pteroic acid, and the number of glutamic acid residues (See figure 1).5

Folate Intake and Absorption
Humans are totally dependent on food as a source of folate. Although there are folate producing bacteria in the human digestive tract, they contribute little to folate status. Despite over fifty years of research, the actual folate requirement for optimum health is undecided. The World Health Organization (WHO) has suggested that adults require 400 micrograms per day, and pregnant women should receive at least 800 micrograms per day.6 This twenty-five year old recommendation coincided with the United States Recommended Daily Allowance (RDA) until 1989, when the RDA was lowered to 200 micrograms per day for adults and 400 micrograms per day for pregnant women.7 Using data from a large population survey of dietary intake, it was found that in the United States folate intake ranges from 8 micrograms to 5 milligrams per day.8 The mean folate intake was 242 micrograms per day, and half of all individuals were consuming less than 200 micrograms of folate daily. Because food folates are present as polyglutamates of varying chain lengths in food, whereas folates that circulate in the plasma exist in the form of monoglutamates, deconjugation to monoglutamates is required before absorption. Thus, in the process of assimilation folates in the diet as polyglutamates must be hydrolyzed to the monoglutamate form. The hydrolysis, or digestion, of folates into their utilizable monoglutamate form occurs predominantly in the small intestine.9 Because digestion of folates to folic acid is required for utilization, the bioavailability of folates in food varies. It is low for some foods that contain nutritionally important amounts of folate, such as legumes, orange juice, and tomatoes. Extracts of these foods significantly inhibit activity of the intestinal enzyme that converts dietary polyglutamyl folates into the monoglutamyl form prior to absorption.10 A review of studies on folate bioavailability from numerous food sources showed that estimates of folate bioavailability varied widely.11 Folic acid in dietary supplements, as opposed to the folates found in food, requires no digestion prior to absorption and demonstrates excellent bioavailability.

Folate and the Fetus
Folates have a fundamental role in cell replication, mediated through their necessity for nucleotide synthesis and amino acid interconversions. More than thirty years ago it was postulated that a deficiency of folate, already known to result in abnormal red blood cells, could adversely effect the developing fetus and placenta.12 In recent years, investigation into this possibility has centered on the relationship between defective folate metabolism and

the occurrence of neural tube defects (NTD). In 1993, the Centers for Disease Control and Prevention (CDCP) released a recommendation that, "All women of childbearing age in the United States who are capable of becoming pregnant should consume 400 micrograms of folic acid per day for the purpose of reducing their risk of having a pregnancy affected with spina bifida or other neural tube defects."13

Unfortunately, the entire story is more complex than it appears. As the CDCP states, "The evidence that consumption of folic acid, one of the B vitamins, before conception and during early pregnancy (the periconceptual period) can reduce the number of cases of neural tube defects has been accumulating for several years. Published data are available from randomized controlled trials, nonrandomized intervention trials, and observational studies."13 While this is certainly true, the supporting evidence presented by the CDCP includes numerous studies that utilized high doses of folic acid (5 mg/day) or a lower dose (400 mcg) together with a multivitamin. It seems a strange coincidence that these recommendations that were made in 1993 identically match the new 1989 US RDA recommendation of 400 mcg/day for pregnant women. Prior to 1989 the US RDA recommendation was 800 mcg/day, which is more in line with the higher doses of folic acid proven to result in up to an eighty percent reduction in risk for neural tube defects. Interestingly, the recommendation for women who have had a prior pregnancy affected by NTDs, and are planning to start a new pregnancy, calls for the consumption of 4 milligrams daily of folic acid beginning one month before pregnancy. They also recommend this high dose of folic acid be taken under a physician's supervision.

Sadly, the entire debate over how much folate is necessary to prevent NTD's has missed an important point: that, research in humans and other animals has shown other nutrient deficiencies to result in NTD. One of these other nutrient deficiencies involves vitamin B-12. In one interesting investigation, researchers found that in pregnancies with NTD, amniotic fluid B-12 was significantly decreased although the maternal serum B-12 was normal.14 Surprisingly, folate levels were normal in both maternal serum and amniotic fluid of pregnancies with NTD. While these researchers cite other research that found similar results, the possibility of vitamin B-12 deficiency playing a role in NTDs has strangely been overlooked. Because folate and vitamin B-12 affect similar biosynthetic pathways in the body, and supplements of folic acid can overcome some clinical symptoms of vitamin B-12 deficiency, it is entirely possible that some or all of the positive effects of folic acid supplementation on reducing NTD incidence may simply be due to partially overcoming a subclinical vitamin B-12 deficiency. Hopefully, we won't have to wait another 30 years for the potential of vitamin B-12 to be recognized.

Folate and Vascular Disease
It is well documented that the homocysteinemia caused by genetic defects in homocysteine metabolism is associated with premature vascular disease.15 The accumulation of excess quantities of homocysteine accompanies errors of metabolism that decrease the conversion of homocysteine to cystathione or methionine (See figure 2). These pathways require vitamin B-6 or folic acid and vitamin B-12 for the proper metabolism of homocysteine. While the connection between elevated homocysteine concentrations and vascular disease in genetic homocysteinemia was recognized in 1969,16 the ability of folate deficiency to elevate serum homocysteine has only been generally recognized in the last ten years.17

An extraordinarily high serum homocysteine level is a firmly established cause of vascular disease in genetic defects causing homocysteinemia. In recent years, however, the possibility that slightly to moderately elevated homocysteine levels may play a role in vascular disease among the general population has been increasingly substantiated. A recent review of more than 20 case-control and cross-sectional studies of over 2000 people showed that patients with cardiovascular disease and stroke tend to have higher blood levels of homocysteine than subjects without disease.18 This is in spite of the fact that in most of these patients the homocysteine values are within what has been considered the normal range for serum homocysteine concentration. While extremely high blood homocysteine levels due to rare enzymatic (genetic) defects in various points of homocysteine's metabolic pathway do occur, they are rare and usually result in life-threatening vascular disease at a young age. Moderate elevations, on the other hand, appear to be much more common. This can result from one of two things; either the result of less severe genetic defects in homocysteine metabolic enzymes, or inadequate amounts of folic acid, and /or vitamin B-6, and/or vitamin B-12.

Folic acid supplements have been shown to lower the homocysteine concentrations of both homocysteinemic patients19 and non-folate-deficient subjects.20 Because vitamin B-6 and vitamin B-12 are also involved in homocysteine metabolism, most recent studies have examined the role of all three nutrients on elevated homocysteine levels. Compared with a group of healthy controls, subjects with an elevated plasma homocysteine concentration had significantly lower plasma concentrations of vitamin B-6, vitamin B-12, and folic acid.21 In a placebo controlled, follow-up study using the same patients, a daily supplement of 10 mg. vitamin B-6, 1 mg. folic acid, and .4 mg. vitamin B-12 normalized the elevated plasma homocysteine concentrations within six weeks. These authors concluded that, "Because hyperhomocysteinemia is implicated as a risk factor for premature occlusive vascular disease, appropriate vitamin therapy may be both efficient and cost-effective to control elevated homocysteine concentrations."22

A recently published study examining the role of these three vitamins on homocysteine concentrations demonstrated that inadequate folic acid intake is the main determinant of homocysteine-related vascular disease.23 Other recent studies have shown that homocysteine levels rose inversely with folate status.24 Interestingly, homocysteine concentrations didn't reach a stable low level until folate intake reached approximately 400 micrograms per day or more. Shockingly, the Recommended Dietary Allowance for folic acid was recently lowered from 400 to 200 micrograms per day. Additional data has suggested that as much as forty percent of the population is not consuming an adequate amount of folic acid to keep homocysteine concentrations low.25 Even in persons with elevated homocysteine levels that are unresponsive to high dietary levels of folic acid (400 mcg./day), folic acid supplements in the range of 1-2 mg per day are usually effective in lowering elevated homocysteine concentrations. It appears that certain people, due to their genetic endowment, require different amounts of dietary folic acid to properly metabolize homocysteine.

As fascinating as the evidence for homocysteine's role in vascular disease is, the exact mechanism(s) by which homocysteinemia induces vascular disease has been somewhat elusive. One hypothesis has been that the damage comes from the toxic effect of homocysteine on vascular endothelium by impairing the production of endothelium-derived relaxing factor.26 Another line of thinking has suggested that homocysteine directly stimulates the proliferation of smooth-muscle cells in the vascular endothelium, resulting in atherogenesis.27

Perhaps the most interesting theories of how homocysteinemia promotes vascular disease comes from homocysteine's relationship with copper and iron. In the case of iron, it has been demonstrated that homocysteine induces iron-catalyzed lipid peroxidation of low-density lipoprotein (LDL).28 Because oxidized LDL -cholesterol has been strongly implicated in atherogenesis, this data suggests that homocysteine might promote vascular disease through the oxidative modification of LDL-cholesterol. Interestingly, vitamin E was very effective at slowing the oxidation of LDL caused by homocysteine, providing further evidence for the protective effects of vitamin E.

Another documented result of elevated homocysteine concentrations is a detrimental effect on copper status, including decreased tissue levels of copper, decreased activities of copper-dependent antioxidant enzymes like ceruloplasmin and superoxide dismutase, decreased activity of glutathione peroxidase, and a significant increase in lipid peroxidation in cardiovascular tissue.29 Notably, cardiovascular tissue seems especially susceptible to lipid peroxidation resulting from homocysteinemia-induced copper deficiency, because other tissues such as the liver were not similarly affected.

As the data continues to accumulate, and homocysteinemia gains wider acceptance as a determinant of vascular disease risk, we can only hope that the population becomes as aware of its homocysteine concentration as it is of its cholesterol concentration (both easily measured with a blood test); and most importantly, that vitamins B-6, B -12 and folic acid become routinely recommended by physicians as a safe and inexpensive measure to reduce this easily controlled risk-factor for vascular disease, namely homocysteinemia.

Folate and Carcinogenesis
The possible role of folic acid deficiency in the process of carcinogenesis was first suggested by the evidence of chromosomal breakage and abnormalities present in the bone marrow of folate deficient patients, as well as by the fact that these changes were reversed by folic acid supplementation.16 Increased levels of chromosomal aberrations have been observed in lymphocytes from individuals with lower serum levels of folic acid.17 More than twenty years ago it was demonstrated that oral contraceptive therapy was associated with changes in the cervical epithelium analogous to those found in the bone marrow in folate deficiency.18 It was also observed that folic acid supplementation could reverse the localized cellular deficiency within the cervical epithelium, in spite of the fact that no evidence of a systemic folic acid deficiency was found. This original paper initiated serious interest in folic acid's role in carcinogenesis, an idea based on the important role that folate plays in genetic expression and DNA/RNA synthesis. Studies that have looked for an association between human carcinogenesis and folic acid intake have concentrated on cancers of the cervix, colon, esophagus, brain, and lung. A recently published review of this topic provides substantial evidence that folic acid deficiency plays a role in the development of numerous and varied types of human cancers.19

Unfortunately, as previously discussed, serum levels of folic acid may significantly underestimate the true incidence of subclinical folate deficiency. The equivocal results found in some studies may result from this underestimation, as significant tissue deficiencies may exist in spite of normal serum folic acid levels. In line with this reasoning, a study was conducted on the effect of smoking on folate levels in buccal mucosal cells.20 The folate levels in these cells, taken from the mouth mucosa, were thought to more accurately reflect tissue folate status than serum levels of folic acid. These researchers found that the correlation between plasma folate and oral mucosal folate was not of statistical significance, suggesting that plasma folate levels do not reflect the folate levels in oral mucosal cells. In fact, smokers were twelve times more likely to have a buccal mucosal cell folate deficiency than were nonsmokers, despite the fact that their plasma folate levels were at the low end of the normal range and not considered deficient. As the authors stated, "Much of our knowledge about the relationship between micronutrients and cancer comes from studies in which serum or red-blood-cell nutrient levels have been correlated with cancer incidence. Although blood micronutrient levels are presumed to reflect tissue levels, data on tissue nutrient levels is limited."

Thus, the accumulating epidemiological evidence of folate's role in carcinogenesis may seriously underestimate the role of folate, because almost all of these studies utilize the easily collected serum folate level. It is likely that these studies which suggest a relationship between serum folate deficiency and cancer may be showing us only the tip of the iceberg and that large studies utilizing tissue folate measurement would show a very strong association between localized tissue deficiencies of folate and the development of cancer.

Folate Supplementation
Because of all the evidence that most persons are consuming diets inadequate in folate content for optimum health, many people consume supplements containing folic acid. Although estimates of the incidence of subclinical folate deficiency have varied, some research has suggested that it is widespread. In one well-done study that examined metabolic evidence for folic acid deficiency (elevated homocysteine levels), in addition to the more commonly measured serum folic acid level, it was found that the incidence of metabolic evidence of folic acid deficiency is substantially higher than could be estimated from the serum concentration of folic acid.21 While the incidence of low serum folic acid was found in only five percent of the subjects, metabolic evidence of folic acid deficiency was found in thirty percent. It seems likely that subclinical deficiency is more common than thought, mainly because the commonly tested serum level of folic acid isn't a good measure of true folate status. This is especially relevant to persons with increased folic acid utilization and requirements such as smokers, drinkers, pregnant women, and the elderly.

The safety and toxicity of folic acid has been reviewed, and folic acid is generally regarded as not toxic for normal humans.22 There has been some concern expressed, however, about possible neurological injury when large amounts of folic acid (>1 mg./day) are given to patients with undiagnosed pernicious anemia, a result of vitamin B-12 deficiency. This can result in excess folic acid producing a temporary hematological improvement of the anemia of vitamin B-12 deficiency but not correcting the neurological abnormalities. Although the above review found little documented evidence of this actually occurring (one or two documented cases over a ten year period), persons taking folic acid supplements would be well advised to concurrently consume a vitamin B-12 supplement. Certainly the research supports the idea that folic acid supplementation of 200-1000 micrograms per day is safe for virtually all persons and that this level of supplementation can prevent or correct clinical and subclinical evidence of folate deficiency in the vast majority of persons.

Unfortunately, the optimal intake of folate is undetermined. The supplemental form of folate, folic acid, is more bioavailable than many food folates. Additionally, the necessary dietary intake to ensure both adequate serum and tissue folate levels in different groups of individuals (i.e. smokers) is unknown. Based on the knowledge to date, 500 to 1000 micrograms per day of supplemental folic acid should be adequate and safe for most individuals. Based on bioavailability studies, approximately twice this amount of food folates would be necessary. Few of us do, however, consume this amount on a regular basis. Given its safety and many potential benefits, it is hard to justify not supplementing with folic acid. With all the potential benefits of folic acid, we can only hope for the day when it is required that foods be fortified with folic acid, perhaps along with vitamin B-12. The benefits of this to the population at large, many of which are consuming an inadequate amount of folate in their diet, could be enormous.

Copyright 2000 VRP