Discovery of the Homocysteine Theory of Arteriosclerosis
The American biochemist Vincent DuVigneaud discovered a new amino acid in 1932 by treating methionine with sulfuric acid. The structure of this amino acid is similar to cysteine, except for one extra carbon atom, hence the name homocysteine. Subsequent investigation established the role of homocysteine as an intermediate in sulfur amino acid metabolism and transmethylation reactions. Little was known, however, about the biomedical significance of homocysteine until 1962, when children with mental retardation, accelerated growth, osteoporosis, dislocated ocular lenses, and frequent thrombosis of arteries and veins were discovered to excrete homocysteine in the urine.
Most children with homocystinuria are deficient in the enzyme cystathionine synthase, a pyridoxal phosphate-dependent enzyme that catalyzes the synthesis of cystathionine from homocysteine and serine. Because of this enzyme deficiency, homocysteine and methionine accumulate to high levels in plasma, and homocystine, the disulfide dimer of homocysteine, is excreted in the urine. In a 1969 review1 of an archival case of homocystinuria in an eight-year-old boy, originally published in 1933, it was discovered that the cause of death was a massive stroke resulting from carotid arteriosclerosis and thrombosis. In addition, arteriosclerotic plaques were found to be scattered through arteries to major organs of the body, suggesting a possible connection between homocysteine and atherogenesis.
In a second case of homocystinuria in a two-month-old boy, caused by deficiency of a different enzyme, methionine synthase, widespread, advanced arteriosclerotic plaques were discovered, scattered through the arteries. Because the enzyme deficiency caused elevated blood levels of cystathionine and homocysteine, and decreased levels of methionine, it was concluded that homocysteine causes arteriosclerotic plaques by a direct effect on the cells and tissues of the arteries, since homocysteine elevation was the only metabolic abnormality shared by these two cases. Several years later, investigators in Chicago demonstrated similar arteriosclerotic plaques in a child with methylenetetrahydrofolate reductase deficiency, the third major type of homocystinuria, independently corroborating the conclusion that homocysteine is an atherogenic amino acid. In the original publication of these cases of homocystinuria, it was suggested that elevation of blood homocysteine is important in the pathogenesis of arteriosclerosis in the general population without these rare inherited disorders of homocysteine metabolism.1 Thus, individuals with hereditary, dietary, environmental, hormonal, metabolic, or toxic predispositions to arteriosclerosis develop arterial plaques from damage by homocysteine to the lining cells and tissues of arteries.
As formulated during the period of 1969 to 1975, the homocysteine theory of arteriosclerosis implicates the elevation of blood homocysteine levels as the key factor in the production of vascular disease in the general population.2 Insufficient dietary intake of the B vitamins folic acid, vitamin B6, and vitamin B12 leads to the elevation of blood homocysteine levels. The dietary sufficiency of B6 and folate is inadequate in populations consuming processed foods because these sensitive vitamins are destroyed by heat, milling of grains, extraction of sugar or oils, chemical additives, and other traditional methods of food processing. Dietary B12 is usually adequate, and this vitamin is stable in most forms of food processing. However, insufficient absorption of B12 may become a problem in older persons because of loss of gastric acid and intrinsic factor, leading to elevated levels of blood homocysteine.
The only source of homocysteine is from metabolism of the essential amino acid, methionine, in the liver. Proteins of animal foods contain about two to three times as much methionine as proteins from plant foods. Metabolic regulation by pyridoxal phosphate, methyltetrahydrofolate, and methylcobalamin controls the production of homocysteine from methionine. Fresh fruits and vegetables contain abundant pyridoxine and folate, and populations consuming these foods have lower homocysteine levels and lower rates of heart disease than populations consuming processed foods that are deficient in these vitamins. The only dietary sources of vitamin B12 are foods of animal origin, such as meat, fish, and dairy foods. Vegans who consume no meat or dairy foods have higher homocysteine levels compared with those consuming these foods. Deficiency of dietary vitamin B6 causes elevation of blood homocysteine after a meal containing protein, and deficiency of dietary folate or vitamin B12 causes elevation of fasting homocysteine levels.
Dietary protein is also a factor in controlling blood homocysteine levels. Deficiency of dietary protein leads to elevation of blood homocysteine, and increased dietary protein leads to lower homocysteine levels. These effects are mediated by adenosyl methionine, a metabolic regulator of methionine metabolism that is synthesized from dietary methionine in the liver. Dietary choline, a constituent of wheat germ, vegetables, meats, liver, eggs and seafood, is converted to betaine for conversion of homocysteine to methionine in liver by the enzyme betaine homocysteine transmethylase. The daily requirement of dietary choline is about 550mg per day. Betaine, a constituent of wheat germ, spinach, beets, liver, and seafood, lowers plasma homocysteine in doses of 1g or more per day.
Homocysteine and Atherogenesis
Experiments with rabbits, baboons, and pigs show that injection or feeding of homocysteine causes arteriosclerotic plaques in aorta and peripheral arteries. High doses cause prominent plaques that closely resemble the fibrous plaques found in human arteriosclerosis and in homocystinuria. Some of the animals develop venous thrombosis and pulmonary embolism, abnormalities that are found in patients with homocystinuria. Feeding fats and cholesterol to animals injected with homocysteine results in fibrolipid plaques with prominent lipid deposition. The molecular basis for production of arteriosclerotic plaques is related to the effect of homocysteine on cellular degeneration, damage to arterial intima, cellular growth, connective tissue formation, deposition of lipoproteins in plaques, and enhanced blood coagulation. In each of these critical processes in atherogenesis, homocysteine plays a key role.2
Experiments with cell cultures taken from the skin of a child with homocystinuria show that an abnormal aggregated extracellular matrix results from binding of excess sulfate to the macromolecules. In these cell cultures, biochemical experiments demonstrated a new pathway for conversion of homocysteine to sulfate, mediated by thioretinamide, an amide formed from homocysteine thiolactone and retinoic acid (vitamin A acid). Deposition of sulfated extracellular matrix is a characteristic feature of early developing arteriosclerotic plaques.
Another feature of early plaques is fragmentation and degeneration of elastic fibers. Homocysteine activates the enzyme elastase within arteries, causing fragmentation of the internal elastic membrane. Homocysteine also causes cultured smooth muscle cells to produce excess collagen, explaining the fibrosis that is characteristic of human and experimental plaques. Also, arterial smooth muscle cells proliferate in plaques because homocysteine activates cyclins, signaling proteins that mediate cell division. Homocysteine is involved in skeletal growth by releasing insulin-like growth factor and increasing the sulfation of epiphyseal cartilage of animals, explaining the accelerated skeletal growth in children with homocystinuria and the growth of smooth muscle cells in developing arteriosclerotic plaques.
The initial phase in formation of arteriosclerotic plaques involves damage to endothelial and intimal cells, causing cell death and inflammatory reaction within the artery wall. Homocysteine thiolactone causes inflammation, cell death, intravascular coagulation, and stromal and epithelial proliferation with dysplasia when applied to the skin of mice. These inflammatory, proliferative and prothrombotic effects may be related to increased oxidant stress within cells affected by homocysteine.
Lipoproteins, including low-density lipoprotein (LDL) and high-density lipoprotein (HDL), contain homocysteine that is bound to apoB protein by peptide bonds. The ratio of homocysteine within LDL to homocysteine within HDL is higher in patients with hypercholesterolemia than in normal controls. Reaction of homocysteine thiolactone with normal human LDL in vitro causes increased density, increased electrophoretic mobility, aggregation, and precipitation of LDL particles. These homocysteinylated LDL particles are taken up by cultured human macrophages to form foam cells, a key process in atherogenesis. Deposition of cholesterol and lipids within fibrolipid arteriosclerotic plaques probably occurs by a similar process in vivo.
Epidemiological and Observational Evidence
The first human study of homocysteine in vascular disease in 1976 showed that oral methionine causes increased levels of homocystine and homocysteine cysteine disulfide in the plasma of patients with coronary heart disease (CHD). Many subsequent studies showed that persons with coronary, cerebral, or peripheral arteriosclerosis have elevated levels of homocysteine in their blood.3 In patients with early onset of arteriosclerosis, elevation of homocysteine is a more potent risk factor than cholesterol elevation and is similar in strength to the effect of smoking. The European Concerted Action Study showed that elevation of blood homocysteine potentiates the effect of hypertension, smoking, and cholesterol elevation on cardiovascular risk. The Hordaland Homocysteine Study showed that homocysteine levels are correlated with multiple known risk factors for CHD, such as smoking, lack of exercise, increasing age, lack of dietary fruits and vegetables, male gender, postmenopausal status, hypertension, cardiac hypertrophy, and excess coffee consumption.
The Framingham Heart Study showed that the homocysteine levels of elderly participants are related to dietary deficiencies of vitamin B6 or folate or to reduced absorption of vitamin B12. About two-thirds of participants were deficient in one of these three B vitamins, leading to elevation of blood homocysteine. The degree of carotid arteriosclerosis was found to correlate with the level of blood homocysteine in these participants. The PhysiciansÔÇÖ Health Study showed that physicians with elevated homocysteine levels have an increased risk of myocardial infarction over a five-year period. Retrospective and most prospective studies show a correlation between homocysteine levels and risk of cardiovascular disease. Metanalyses of these studies suggest that elevation of blood homocysteine is responsible for at least 10% of US mortality from cardiovascular disease, and that lowering the homocysteine level of the population by 3╬╝m per liter would be expected to reduce coronary disease risk by 16%, deep vein thrombosis by 25%, and stroke by 24%.
The Nutrition Canada Study showed that a low plasma level of folate is associated with an increased risk of mortality from CHD.The NursesÔÇÖ Health Study showed that decreased dietary folate or vitamin B6 is associated with increased cardiovascular risk of mortality and morbidity. Dietary intake of less than 3mg per day of B6 or less than 350mg per day of folate significantly increased cardiovascular risk.4 The Third National Health and Nutrition Examination Survey study showed that increased plasma homocysteine level is associated with increased risk of myocardial infarction. Survival studies from Oregon and Israel show that elevation of blood homocysteine is directly correlated with risk of mortality. The Bergen study showed that survival of patients with CHD is inversely related to blood homocysteine levels. The Finland study of stored blood samples from the 1970s showed that mortality from cardiovascular disease is directly related to homocysteine levels in different countries. Countries with homocysteine levels below 8╬╝m/l, such as France, Spain and Japan, have significantly lower risk of mortality than countries with levels in the 10-11╬╝m/l range, such as Finland, Germany, and Ireland.
Homocysteine, Vitamins and Prevention of Cardiovascular Disease
As reported by the Centers for Disease Control and Prevention, the mortality from CHD has declined in the US by more than two-fold since the mid 1960s. At a national conference at the National Institutes of Health (NIH) in 1979, the factors that may be responsible for this dramatic decline were examined in detail. Dietary fat and serum cholesterol levels changed very little, and smoking, exercise, coronary care units, hypertension, and other suspected factors could not account for the decline. Starting in the 1960s, synthetic pyridoxine (vitamin B6) was added to the US food supply in the form of supplements and enriched cereals. Several years later, synthetic folic acid was also added to the US food supply. Addition of pyridoxine and folic acid to the food supply explains the decreased cardiovascular mortality in the past four decades by their effect on lowering homocysteine levels in the US population.
In 1998, the US Food and Drug Administration (FDA) mandated the addition of folic acid to enriched grain foods. Recent studies by the Centers for Disease Control and by the Framingham Heart Study showed that serum folate levels increased dramatically and blood homocysteine levels declined after 1998, leading to an accelerated decline in cardiovascular mortality from heart disease and stroke. This decline was estimated to have saved 48,000 lives per year in the US population from stroke and heart disease by lowering of homocysteine levels by added folate in the food supply.
In an autopsy study of 194 consecutive deaths among veterans, two-thirds of the cases with the most severe arteriosclerosis had no evidence of hypertension, renal failure, elevated serum cholesterol or diabetes.5 Many other studies show a correlation of arteriosclerosis with elevated homocysteine levels. Estimates of the fraction of the US population with elevated homocysteine levels vary from 10% to 40%, depending upon the definition of normal levels. In the US today, the mean homocysteine level is 8.9╬╝m/l, as determined by the Third National Health and Nutrition Examination Survey. In most studies, the predictive value of plasma homocysteine is independent of traditional risk factors. Clinical studies indicate that about 80% of patients with heart disease have at least one of the four traditional risk factors for vascular disease - smoking, elevated cholesterol, diabetes, or hypertension. The Framingham Heart Study showed that serum cholesterol is predictive of vascular disease only in men under age 45, as women and older men reveal no correlation between serum cholesterol and vascular disease risk.
Interventional studies from Canada in patients with cerebral arteriosclerosis showed that the carotid plaques stopped progressing and began to regress with moderate doses of B vitamins. Studies from Switzerland showed that B vitamins significantly delayed restenosis and reduced adverse cardiac events following angioplasty or stenting, although a recent study from Germany failed to show benefit in a highly selected group of stented patients.
Currently, more than 20 current prospective interventional trials involving at least 100,000 participants worldwide are examining whether lowering plasma homocysteine levels by supplemental B vitamins will prevent mortality and morbidity from arteriosclerotic vascular disease. In a current trial sponsored by the Department of Veterans Affairs, 2,050 patients with renal failure are being studied to determine whether high dose folic acid, pyridoxine and vitamin B12 will reduce mortality and morbidity from arteriosclerotic disease.6 Positive results from these trials in the future will add to the evidence supporting the validity of the homocysteine theory of arteriosclerosis.
In summary, the evidence that elevated homocysteine levels are implicated in the pathogenesis of cardiovascular disease is substantial. More than 1,000 new studies of homocysteine and disease are currently being published each year from investigators worldwide. Measures to prevent and treat cardiovascular disease, based on the homocysteine theory, have already proven to be successful in numerous clinical and epidemiological studies. Accordingly, application of these findings to treatment and prevention of heart disease in the general population will increase dramatically in future years. Ôûá