Folic Acid, Vitamin B6, Vitamin B12 and Vascular Disease

By Kilmer S. McCully, M.D. (1999)

Introduction

The importance of hyperhomocysteinemia in the pathogenesis of arteriosclerosis was first recognized by study of vascular disease in children with homocystinuria caused by two different enzymatic abnormalities of homocysteine metabolism (47). During the 31 years since I made this original observation, I have acquired extensive experience in the field of homocysteine and vascular disease. Over this period I have conducted numerous experimental and clinical studies on the association of folic acid, vitamin B6 and vitamin B12 with vascular disease. My Curriculum Vitae is attached to this report.

Homocystinuria caused by deficiency of cystathionine synthase, an enzyme requiring vitamin B6 for activity, is complicated by rapidly progressive vascular disease, including arteriosclerosis and frequent arterial and venous thromboses. Cases of homocystinuria caused by deficiency of two different enzymes, methyltetrahydrofolate homocysteine methyl transferase and methylenetetrahydrofolate reductase, are also complicated by rapidly progressive arteriosclerosis. These enzymes require vitamin B12 and folic acid for activity. Because of the different metabolic patterns involved in these three different enzyme deficiencies leading to hyperhomocysteinemia, it was possible to conclude that homocysteine causes vascular disease by a direct effect of the amino acid on the cells and tissues of the arteries. This atherogenic effect of homocysteine was subsequently demonstrated experimentally by administration of the amino acid to rabbits, baboons, pigs and other species.

The discovery of the atherogenic potential of homocysteine led to the development of the homocysteine theory of arteriosclerosis (37,39,42,50). According to this theory, deficiencies of the three B vitamins, folic acid, vitamin B6 and vitamin B12, lead to increased blood levels of homocysteine and cause arteriosclerotic vascular disease in susceptible populations. Deficiency of folic acid causes hyperhomocysteinemia in the fasting state because decreased amounts of the coenzymes, methylenetetrahydrofolate and methyltetrahydrofolate, are available to activate methylenetetrahydrofolate reductase, the enzyme needed to produce methyltetrahydrofolate for remethylation of homocysteine to methionine. Deficiency of vitamin B12 also causes fasting hyperhomocysteinemia because deficient amounts of cobalamin are available to form methylcobalamin, the coenzyme needed to activate methyltetrahydrofolate homocysteine methyl transferase for remethylation of homocysteine to methionine. Deficiency of vitamin B6 causes postprandial or postmethionine hyperhomocysteinemia because of a deficiency of pyridoxal phosphate needed to activate cystathionine synthase, the enzyme required for conversion of homocysteine to cystathionine. Following oral methionine from dietary protein or from pure methionine in a loading test, increased amounts of adenosyl methionine are formed in the liver, activating cystathionine synthase and facilitating excretion of homocysteine by conversion to cystathionine, cysteine, cysteine sulfinic acid, taurine, and sulfate. When all three vitamins, folic acid, vitamin B12 and vitamin B6, are supplied in sufficient quantities in the diet, the remethylation of homocysteine to methionine and the excretion of homocysteine by conversion to cystathionine act synergistically to keep blood homocysteine in the desirable range, below 8 mcM.

The most important cause of dietary deficiencies of folic acid and vitamin B6 in susceptible populations is consumption of processed and refined foods that are depleted of these chemically unstable vitamins. The most important cause of deficiency of vitamin B12 is decreased gastrointestinal absorption of the vitamin because of bacterial infection or aging. Other factors leading to hyperhomocysteinemia, including genetic predisposition, age, sex, postmenopausal status, toxic substances, renal failure, hormonal changes, and drugs, also affect susceptibility to arteriosclerosis.

The molecular processes by which homocysteine causes vascular disease have been studied intensively for three decades. Early studies with cells cultured from children with homocystinuria revealed excessive sulfation of extracellular matrix, resulting from conversion of the sulfur of homocysteine thiolactone, the reactive anhydride of homocysteine, to the sulfating coenzyme, phosphoadenosine phosphosulfate. The oxidized form of homocysteine, homocysteic acid, is a precursor of phosphoadenosine phosphosulfate, a potent neurotransmitter, and a promoter of growth of hypophysectomized rats given thyroxin. More recent studies have demonstrated that homocysteine damages cultured endothelial cells and stimulates growth of cultured smooth muscle cells by increased formation of cyclin messenger RNA. These observations help to explain the origin of the increased sulfated extracellular matrix and the growth of smooth muscle cells in developing arteriosclerotic plaques. Vascular damage to intimal cells by homocysteine has been related to oxidative stress, inactivation of nitric oxide, and inhibition of glutathione peroxidase activity and synthesis. The increased tendency to thrombosis in hyperhomocysteinemia is related to effects of homocysteine on multiple coagulation factors, including platelets, tissue factor, activated protein C, thrombomodulin, thromboxane, lipoprotein(a) binding to fibrin, and factors V,VII and XII. Modification of low-density lipoprotein by homocysteine thiolactone forms small, dense LDL particles that self-aggregate and are taken up by macrophages to form foam cells, leading to intimal damage, oxidative modification of LDL, deposition of cholesterol and lipids, thrombogenesis, and the connective tissue alterations of developing arteriosclerotic plaques. The contributions of the author to the discovery and investigation of the homocysteine theory of arteriosclerosis are included in the accompanying curriculum vitae.

Recommended Health Claim
"As part of a well-balanced diet, rich in fresh whole fruits and vegetables, daily intake of at least 400 mcg of folic acid, 3 mg of vitamin B6 and 5 mcg of vitamin B12 may reduce the risk of vascular disease."

Scientific Evidence Supporting the Health Claim

Among scientists who study the relation between vascular disease and nutrient intake, there is general agreement that deficiencies of folic acid, vitamin B6 and vitamin B12 cause arteriosclerosis by causing hyperhomocysteinemia. There is general agreement that homocysteine is an atherogenic amino acid in man and animals and that deficiencies of these three B vitamins are the most important factors leading to hyperhomocysteinemia in populations susceptible to arteriosclerotic vascular disease. As summarized by Ueland et al, the many clinical and epidemiological studies published over the past two decades support the validity of the homocysteine theory of arteriosclerosis. Three important exceptions to this conclusion (1,16,17) may be explained by genetic characteristics of the Finnish population (1), selection of participants at high risk of arteriosclerosis in both intervention and control groups (16), and study of a susceptible population over too short an observation period (17). In contrast, the study by Wald et al, 1998, for example, demonstrates that the blood level of homocysteine predicts susceptibility to vascular disease over a wide range of concentrations in 21,000 men studied over a nine year period. With few exceptions the vast majority of human studies support the conclusion that hyperhomocysteinemia is a powerful independent risk factor for vascular disease.

When consumed as part of a balanced, nutritious diet, the B vitamins, folic acid, vitamin B6, and vitamin B12 are not only without evidence of any toxicity whatsoever, they are essential for health. The presence of these vitamins is required in the diet because they cannot be synthesized from other dietary constituents. These vitamins are needed for normal growth and development in childhood. Since arteriosclerotic vascular disease begins in late childhood and adolescence, and since homocysteine levels rise to adult levels during adolescence, an adequate dietary intake of folic acid, vitamin B12, and vitamin B6 is needed by children and young adults to prevent hyperhomocysteinemia and vascular disease later in life. These vitamins are also needed throughout adult life and senescence to prevent hyperhomocysteinemia and vascular disease. In adults with early or established vascular disease, or in adults with a genetic predisposition to vascular disease because of thermolabile methylenetetrahydrofolate reductase or other inherited enzyme abnormalities, increased dietary or supplemental intakes of folic acid, vitamin B12 and B6 are needed to prevent hyperhomocysteinemia and progression of vascular disease.

As conclusively demonstrated by the Nurses’ Health Study, daily dietary consumption of less than 400 mcg of folic acid or less than 3 mg of vitamin B6 is associated with increased risk of death from coronary heart disease among 80,000 women over a follow-up period of 14 years (71). Those participants with the lowest intakes of folic acid or vitamin B6 had the highest risk of death, those with marginal intakes had intermediate risk, and those with the highest intakes had the lowest risk of death. The Nutrition Canada study of 5000 individuals associated increased mortality from coronary heart disease with decreased levels of folic acid in plasma (57).

Dietary consumption of less than 400 mcg of folic acid or less than 3 mg of vitamin B6 per day leads to elevation of plasma homocysteine levels and narrowing of the carotid arteries, as demonstrated by the Framingham Heart Study (73,74). American adults generally consume less than the desirable amounts of these vitamins, about 250 mcg of folic acid and 1.7 mg of vitamin B6 per day, explaining the frequent occurrence of hyperhomocysteinemia and vascular disease in the population. The amount of vitamin B6 needed to prevent arteriosclerosis in monkeys fed a vitamin B6 deficient diet is the same (3 mg/day) as the amount of vitamin B6 needed to prevent death from heart disease in the human population, when adjusted for body weight (Rinehart et al., 1956). For these reasons, the current Recommended Dietary Intake of 1.3-1.7 mg/day is too low and should be increased to 3 mg/day. The American diet generally contains a sufficient amount of vitamin B12 (5-9 mcg/day) to prevent elevation of plasma homocysteine in most individuals. However, the low intake of vitamin B12 in vegans and in individuals with impaired absorption of vitamin B12 from aging or bacterial infection places them at increased risk of hyperhomocysteinemia and vascular disease. In summary, these studies and numerous other clinical and epidemiological studies have proven conclusively that elevated levels of plasma homocysteine from dietary deficiencies of B vitamins and other factors are a powerful independent risk factor for vascular disease (3,6-12,23,24,5,59,67,72,77,86,87,89,91, Nygard et al, 1995, Wald et al, 1998).

The principal reason for intake of sub-optimal amounts of folic acid and vitamin B6 in the diet is consumption of refined and processed foods that are depleted of these chemically unstable vitamins. In contrast, vitamin B12 is chemically stable and is not depleted from foods by processing methods. Deficiencies of vitamin B12 arise principally from impaired absorption from the gastrointestinal tract. A well balanced, nutritious diet consisting only of fresh whole foods contains sufficient vitamins to provide 400 mcg of folic acid, 3 mg of vitamin B6, and 5 mcg of vitamin B12 per day. Such a diet contains 25-30% of calories as protein, 25-35% of calories as fat, and 35-50% of calories as carbohydrate. In this diet no calories are obtained from refined and processed foods that are depleted of folic acid and vitamin B6 as well as other micronutrients. Numerous epidemiological studies have shown that consumption of diets containing abundant vegetables, fruits and dietary fiber decreases risk of vascular disease, cancer and other degenerative diseases. Individuals who consume such a diet have lower blood homocysteine levels than those who consume a diet of processed and refined foods (Nygard et al, 1995).

Unfortunately, relatively few people have access to or consume an optimal diet that provides desirable amounts of vitamin B6, folic acid, and vitamin B12. Hence, dietary supplements will be needed by many people to provide adequate levels of these essential nutrients in their diets. When consumed as synthetic vitamins in nutritional supplements, folic acid and vitamin B6 reduce the level of homocysteine in the plasma. Small daily doses of these vitamins, 400-1000 mcg of folic acid, and 10-25 mg of vitamin B6, cause a decline of plasma homocysteine to desirable levels, below 8 mcM, within 2-3 weeks in most individuals (85). These studies are highly reproducible, leading to agreement between many investigators in clinics, laboratories and hospitals worldwide. Recent epidemiological studies have demonstrated that in countries with a low death rate from coronary heart disease (Spain, France, Japan) the plasma homocysteine levels are lower ( 7-8 mcM) than in countries (Germany, Scotland, Finland) with a high death rate from coronary heart disease (10-11 mcM) (Alfthan et al, 1997). Moderately large doses of folic acid (2.5-5 mg/day), vitamin B6 (25 mg/day) and vitamin B12 (250 mcg/day) stop progression of carotid arteriosclerotic plaques (Petersen et al, 1998). Multiple supplements, including vitamin B6 (45 mg/day), folic acid (390 mcg/day) and vitamin B12 (90 mcg/day), decrease calcification of coronary arteriosclerotic plaques (Rath et al, 1996). Vitamin B6 therapy (50-200 mg/day) causes an apparent 75% reduction in risk of myocardial infarction and increased longevity of about 8 years in patients with carpal tunnel syndrome and related conditions (15). Those vascular disease patients who take multiple vitamin supplements have an apparent 65% decreased risk of new vascular events (23). As yet, no large-scale prospective studies showing prevention of vascular disease by supplemental vitamin therapy have been published, although at least 10 such studies are currently in progress worldwide.

There is a very large margin of safety in consumption of synthetic folic acid, vitamin B6 and vitamin B12 as vitamin supplements. Vitamin B12 has never been reported to have any toxic effects in man or animals, even in massive doses of several grams per day. Vitamin B12, in oral doses of 200-1000 mcg, causes a reduction in plasma homocysteine levels to desirable levels in patients with low vitamin B12 levels in plasma because of inadequate gastrointestinal absorption. A few rare cases of mild sensory neuropathy have been reported following massive doses of vitamin B6 in the range of 1-2 grams per day. A careful review of the literature concluded that doses of vitamin B6 of 500 mg per day or less are not associated with evidence of neuropathy (Bendich, 1991). Treatment of thousands of patients with carpal tunnel syndrome and related conditions over a period of 35 years was not associated with a single case of neuropathy when vitamin B6 was given at a dose of 200 mg/day (15). Children with homocystinuria have failed to exhibit evidence of neuropathy even though vitamin B6 is given in large doses (1-2 g/day) over a period of years.

A concern has been expressed for many years about possible progression of neurological damage in subjects with undiagnosed deficiency of vitamin B12 from folic acid in supplements or fortified refined foods. The likelihood of this outcome is exceedingly rare. From a survey of American hematologists, Butterworth concluded that no more than 2 possible cases of neurological damage by folic acid in undiagnosed vitamin B12 deficiency were identified in a population of over 200 million per decade.

In summary, there is no evidence of toxicity of folic acid, vitamin B6, or vitamin B12 in the usual doses found in foods, multivitamin supplements, or fortified foods that are needed to lower plasma homocysteine and prevent vascular disease. Possible toxicity from massive doses of folic acid or vitamin B6 is negligible because of the extreme rarity of such cases.

The absorption of many nutrients declines with age. Fewer calories are needed to maintain body weight. Poor dentition, depression, and other factors decrease food intake in the elderly. In addition, the absorption of vitamins, especially folic acid, vitamin B6 and vitamin B12, gradually declines with age. The Framingham Heart Study showed that decreased absorption of these B vitamins causes blood homocysteine levels to rise in the elderly (74). In the case of vitamin B12 decreased production of intrinsic factor by the stomach with aging impairs absorption of this vitamin. In the case of folic acid decreased stomach acid because of bacterial infection with Helicobacter pylori or because of impaired acid secretion leads to poor digestion and release of folic acid from foods, decreasing absorption. In the case of vitamin B6, the ability to absorb the vitamin steadily declines from childhood through adulthood to old age. Decreased absorption of these B vitamins with age helps to explain the gradual increase in blood homocysteine levels observed with aging (2,7,23,74,87, Nygard et al, 1995). Decreased dietary intake and decreased absorption of vitamin B12, folic acid and vitamin B6 with resulting elevation of blood homocysteine have been found to be factors in a wide range of diseases of the nervous system, including Alzheimer’s disease, schizophrenia, cognitive impairment of the elderly, and fibromyalgia-chronic fatigue syndrome.

Many drugs antagonize the effects of folic acid, vitamin B6, and vitamin B12 in the body (70, Ueland et al, 1989, Refsum et al, 1998). In some cases large doses of these vitamins are used to counteract side effects of various drugs. Some examples are vitamin B6 to counteract the side effects of isoniazide, folic acid to counteract toxicity of methotrexate, and vitamin B6 and folic acid to counteract side effects of oral contraceptives. Some examples of drugs that cause elevation of blood homocysteine levels because of antagonism of B vitamins are azaribine, which antagonizes vitamin B6, nitrous oxide anesthesia, which antagonizes vitamin B12, and methotrexate, which antagonizes folic acid. Other commonly used drugs that cause elevation of blood homocysteine are antiepileptic drugs, L-dopa, cholestyramine, fenfibrate, and niacin. In general the effect of these drugs is to antagonize the effects of folic acid, vitamin B6 or vitamin B12 within the tissues of the body, rather than impairment of absorption of the vitamins. These B vitamins, even in large doses from supplements, have no effect on absorption of drugs. However, large doses of these vitamins can reverse the effects of drugs in the body, for example, reversal by folic acid of the antiepileptic effects of drugs that antagonize folic acid. The chemically synthesized B vitamins that are used in supplements, including folic acid, pyridoxine, and cobalamin, have never been reported to cause allergic effects. Allergenicity from supplements is usually attributed to reactions to fillers, additives, or botanical constituents in preparations containing these substances. The chemically synthesized folic acid, pyridoxine and cobalamin that are used in supplements and in fortified foods are generally stable, well absorbed, and effective within the body for controlling blood homocysteine levels and other physiological and biochemical effects.