The rise, fall, and rise of folic acid.

In recent years, folic acid has become the most prominent of the B-complex vitamins. The U.S. government and private health advocacy groups such as the March of Dimes have mounted a major effort to get women to increase their intake of this vitamin. Yet despite its essential biochemical function of transporting single-carbon fragments in amino acid metabolism and nucleic acid synthesis, folic acid was not always hailed. Not long ago, it was considered relatively unimportant—even dangerous. The timeline of folic acid starts with a brief period of enthusiasm accompanying its isolation and synthesis, followed by decades of disrepute. Now, it has emerged as an indispensable nutrient in areas far removed from its original indications.

Discovery and synthesis
Tracing the discovery of folic acid first requires a few words about pernicious anemia (PA). A severe, often fatal, condition, PA is characterized by a progressive decrease in red blood cells; the presence of abnormally large red cells; and neurologic disturbances such as numbness, weakness, ataxia, irritability, and even dementia or psychosis. PA is a form of anemia that is neither caused by iron deficiency nor cured by iron supplementation.

PA was recognized as a disease entity as early as 1914. By 1926, it was established that a curative substance is present in liver. This substance, known as “extrinsic factor”, was isolated in pure crystalline form in 1948 and named vitamin B12.

The discovery of folic acid stemmed from research on conditions resembling, but not identical to, PA. In 1930, Lucy Wills and S. N. Talpede, in Bombay, suggested that a vitamin B complex deficiency might be responsible for PA-like symptoms in pregnant women. Wills then studied other women whose blood showed many large, immature red cells but who were free of the neurological complications associated with PA. She called this condition “tropical macrocytic anemia”. Monkeys fed a diet similar to that of the anemic patients developed the same disease. The purified liver extracts normally used in treating PA were worthless against macrocytic anemia, but yeast extract, which is rich in B complex, could cure both humans and monkeys.

During the 1930s, U.S. investigators reported similar observations; anemias characterized by abnormal red cells were noted in monkeys and chickens. An unidentified B vitamin was postulated and given various names: vitamin M, vitamin Bc, and U factor. While attempting to define the nutritional requirements of the bacterium Lactobacillus casei, Edmond Snell and W. H. Paterson at the University of Wisconsin found that an unknown growth-promoting factor present in yeast extract (L. casei factor) was essential. Snell then transferred to the University of Texas and, working with Roger Williams and Herschell Mitchell, isolated from spinach a concentrate of L. casei factor, which they named folic acid (Latin folium, leaf). This proved to be identical with vitamin Bc.

Lederle Laboratories, at that time a division of the American Cyanamid Co., entered the picture. In 1941, Lederle recruited Robert Stockstad, who had coined the name U factor, to conduct research on liver extracts at its Pearl River, NY, facility. In 1943, Stockstad isolated from 1.5 tons of liver some crystals of L. casei factor, which was found to be identical to vitamin Bc and therefore to folic acid. A team of eight scientists from Lederle and eight from American Cyanamid’s laboratories in Bound Brook, NJ, was assembled to work on structure determination and synthesis of folic acid. The synthesis was accomplished in August 1945, and marketing began in 1946.

Having achieved this scientific breakthrough and holding an effective monopoly, Lederle marketed the new vitamin with high hopes. Folic acid was indicated for nutritional macrocytic anemia, the megaloblastic anemias of pregnancy and infancy, and tropical sprue. Lederle brought folic acid to the market in tablets, capsules, injections, as an addition to a multivitamin syrup, and as Folvite Elixir.

In the 1940s, most prescriptions were compounded, many in liquid form. Elixirs of vitamin B1 (thiamine) were very popular prescription vehicles. Among these were Lilly’s Elixir Betalin S, Wyeth’s Elixir Bewon, and Wm. H. Warner’s Lixa-Beta. Doctors would write prescriptions for two or three active ingredients, specifying that the mixture be “q.s.’ed” (diluted to the desired volume) with one of these elixirs. Hoping to promote the use of Folvite Elixir in this manner, Lederle decided to have its chemical compatibility with common prescription ingredients explored in an academic research setting and published.

At that time, I held a research fellowship funded by Lederle at the Philadelphia College of Pharmacy and Science. I was assigned to perform the compatibility study. Lederle collaborated by running folic acid assays on mixtures that were physically satisfactory. Folvite Elixir proved compatible with most of the prescription ingredients tested, and the study was published in the now-defunct American Journal of Pharmacy.

Reversal of fortune
Shortly thereafter, the fortunes of folic acid began to decline. First, it became evident that folic acid was unstable in the multivitamin syrup, and observations by both Lederle and American Cyanamid indicated that some of the other vitamins were causing it to decompose. At Lederle’s request, I began to investigate the action of individual water-soluble vitamins on folic acid. Ascorbic acid effected a reductive cleavage at the aminomethylene linkage, liberating p-aminobenzoylglutamic acid (PABG), which then reacted further with the ascorbate. Riboflavin exerted a more dramatic effect. It catalyzed an oxidative photolysis, producing free PABG and a pteridine aldehyde. Remarkably, this photolysis did not require UV light; it occurred rapidly in visible wavelengths >500 nm.

Folvite Elixir, though perfectly stable, never became a popular prescription vehicle as Lederle had hoped. By the late 1940s, the prescription trend was beginning to move away from compounding in favor of dispensing proprietary tablet and capsule dosage forms. Furthermore, a clinical issue arose, indicating that folic acid was by no means as benign as vitamin B1.

It had been known from the outset that folic acid cannot cure PA. In 1947, physicians were alerted to a new finding: Folic acid could prevent anemia in PA patients while permitting irreversible neurological changes to proceed unchecked. It could thus mask the disease and hamper early diagnosis. Although it might cause serious problems for PA sufferers, the deficiency states for which it was essential were not widely prevalent in the United States. Reflecting medical opinion, one respected reference source, Extra Pharmacopoeia Martindale, 25th ed. (The Pharmaceutical Press: London), stated in 1967 that it was “potentially dangerous” to include folic acid in multivitamin preparations. For about 30 years, folic acid was relegated to insignificance. Even as late as 1989, the U.S. recommended daily allowance (RDA) was set at only 200 µg.

Back in the limelight
Yet new medical research soon turned the tide. In the United Kingdom, R. W. Smithells and colleagues had noted that a number of tragic birth defects—spina bifida, anencephaly, and encephalocele—occur more frequently in women of lower socioeconomic classes. They hypothesized that these birth defects, categorized as neural tube defects (NTDs), might be caused by the poor nutrition of these women. They performed a placebo-controlled study in women who had already given birth to one baby with NTD. The treatment arm was given a multivitamin plus iron tablet containing 360 µg of folic acid. Their results, published in 1980, showed that among those women who took the vitamin pill regularly, the occurrence of NTD births was only 0.6%, while among the controls and those not compliant with the treatment, there were 5% NTD births. Although the active treatment contained multiple vitamins and a mineral, Smithells focused on folic acid; in 1965, he had reported on a possible link between folate deficiency and NTD in humans.

K. M. Laurence and colleagues in Wales followed up the Smithells report in 1981. They described a double-blind randomized controlled trial of folate treatment prior to conception, again in women who had had a baby with NTD. Among women who complied with treatment, there were no NTDs in 44 births, whereas among controls and noncompliant women, there were 6 NTDs in 67 births. Ten years later, the U.K. Medical Research Council Vitamin Study confirmed that folic acid alone reduced the recurrence of NTD.

Finally, in 1992, A. E. Czeizel showed that even a first occurrence of NTD could be prevented by taking a supplement containing 800 µg of folic acid before and for a few months after conception.

Because of the solid evidence generated in Europe, the U.S. Food and Drug Administration was spurred to action. On March 5, 1996, the FDA issued a rule requiring folic acid to be added to enriched grain products at a level of 140 µg per 100 g of grain. The FDA’s modeling of food consumption data suggested that this level would provide the average woman of reproductive age with about 100 µg of folate a day. Although the Centers for Disease Control and Prevention, the March of Dimes, and the American Academy of Pediatrics recommended fortification with 350 µg per 100 g of flour, the FDA was concerned that too much folic acid might delay the diagnosis of PA. The above requirement went into effect on January 1, 1998.

Surveillance studies in two states that are considered high-risk regions for NTD, South Carolina and Texas, have already documented the effectiveness of the fortification program. American experts advocate a fortification program with a higher level of folate, at least double the current amount, which would be more on a par with that used in some other countries. Others, however, caution that widespread fortification and supplementation may lead to neuropathy in some persons. Nevertheless, it is well established that 400 µg of folic acid per day can prevent the tragedy of spina bifida and other NTDs.

Another health-maintaining effect of folic acid supplementation may be on the horizon. Evidence is growing that high plasma levels of the amino acid homocysteine are a risk factor for atherosclerosis and coronary artery disease. Supplementation with daily doses of 400 µg of folic acid reduces homocysteine to safer levels. It is deemed likely that this reduction in homocysteine concentrations will prevent a significant number of deaths from heart attacks and strokes. Trials to test this hypothesis are under way; these trials may show that folic acid has a protective effect for older people as well as women of childbearing age.

Vitamin discoveries and improved living standards have practically eliminated many deficiency diseases in the United States and other developed nations. Although the timeline of folic acid has not been as straightforward as for other vitamins, unexpected and unrelated health benefits have emerged.

Suggested reading

• Brent, R. L.; Oakley, G. P.; Mattison, D. R. Pediatrics 2000, 106, 825–827.
• Editorial. N. Engl. J. Med. 1947, 237, 713.
• Scheindlin, S.; Lee, A.; Griffith, I. J. Amer. Pharm. Assoc. (Sci. Ed.) 1952, 41, 420–427.
• Smithells, R. W.; et al. Lancet 1980, 1, 339–340.
• Sneader, W. Drug Discovery: The Evolution of Modern Medicines; Wiley & Sons: New York, 1985.