Beyond Cholesterol
The failure of proponents of the cholesterol/fat approach to formulate a comprehensive coherent theory of the origin of arteriosclerosis is the principal reason that new discoveries and new thinking are desperately needed to understand the causes of the leading killer and disease in America. In addition, the most glaring inadequacies of the cholesterol/fat approach, failure to correlate disease with cholesterol or lipoprotein abnormalities in the majority of cases; failure to predict or explain dramatic increases and decreases in the incidence of arteriosclerosis; failure to relate dietary cholesterol to cholesterol levels in blood; failure to lower risk substantially by lowering cholesterol levels through diet, drugs and lifestyle changes all indicate an urgent need for a new, comprehensive and effective approach for prevention and treatment of arteriosclerosis.
The very beneficial reduction in the incidence of myocardial infarction since the 1960s in America has contributed to modest gains in life expectancy. Yet despite these favorable trends, arteriosclerosis remains the leading cause of death by coronary heart disease, stroke and kidney failure. Several years ago, a conference at the National Institutes of Health was unable to explain the decline in morbidity from coronary heart disease by changes in dietary fat and cholesterol, changes in blood cholesterol levels, changes in medical therapy or changes in smoking, exercise or other aspects of lifestyle. 1
The favorable trend in the incidence of arteriosclerotic heart disease in the U.S. is not observed in all countries. In the countries of Eastern Europe in the post-communist era, there have been alarming increases in incidence of coronary heart disease. In Russia the life expectancy has fallen considerably, and coronary heart disease incidence has increased. There are similar alarming trends among the Japanese, who are experiencing increases in coronary heart disease, and among primitive peoples from various parts of the world. Asian populations, such as the Japanese, Chinese and Indonesians, and primitive peoples, such as the Eskimos and Bantus, have in the past been protected against arteriosclerosis because of their dietary traditions. Increases in heart disease among these formerly protected populations in recent years is very likely to have been caused by the introduction of the dietary practices of industrialized, developed countries. Increased consumption of meat, eggs and dairy products, together with increased reliance on highly processed and packaged foods, are widely believed to be at least partially responsible for this trend.
The traditional cholesterol/fat approach to the prevention and treatment of arteriosclerosis is based on the assumption that cholesterol and fats are toxic, producing damage via lipoproteins to artery walls. The prescription for preventing heart disease, according to this approach, is twofold. First, reduce dietary fat and cholesterol, and second, lower the blood levels of cholesterol and LDL. Advocates of this approach have suggested limiting total dietary fats to 30 percent of calories and consuming fewer than 300 mg per day of cholesterol. 2 If reduction of fat is beneficial, according to this approach, then even more drastic diets containing severely restricted fat and cholesterol should be even better. Such drastic diets have been shown to benefit persons with coronary heart disease and arteriosclerosis, causing apparent relief of symptoms 3 and modest evidence of reversal or regression of arteriosclerotic plaques. 4
I suggest that a completely new approach to understanding the cause of arteriosclerosis is inherent in the medical discovery of the damaging effect of homocysteine on arterial walls and the production of arteriosclerosis in children with homocystinuria, as described in Chapter 1.
In the homocysteine approach, the underlying cause of the disease is interpreted as an imbalance between the methionine of dietary protein and the dietary intake of vitamins B6, B12 and folic acid that are necessary to prevent homocysteine accumulation in the cells and tissues of the body. The particular challenge in developing a new theory of arteriosclerosis, based on the medical discovery of the connection between homocysteine and arteriosclerosis, is to integrate the new findings with the vast body of knowledge about fats and cholesterol acquired in the past 80 years. The challenge is not to discard the cholesterol/fat approach, but to integrate past knowledge with new interpretations.
The Homocysteine Theory of Arteriosclerosis
The development of the homocysteine theory of arteriosclerosis is based on the results of animal experimentation, the study of homocysteine in cells and tissues, and evidence from the study of human subjects and populations at risk of arteriosclerosis. 5 In essence the homocysteine theory relates the underlying cause of arteriosclerosis to a buildup of homocysteine in the blood caused by dietary, genetic, toxic, hormonal and aging factors in susceptible populations.
The importance of the homocysteine theory is that it explains many observations of the disease that cannot be explained by the cholesterol/fat hypothesis. Furthermore, the theory explains why the diet of developed, industrialized countries accelerates the age of onset and the pace of progression of the disease. Obviously, factors other than diet will vary among populations, but these factors cannot explain dramatic increases or decreases of disease incidence occurring within a single population. For example, genetic, hormonal and aging factors are relatively constant in populations that may experience two- or threefold changes in the incidence of the disease. These dramatic changes are attributable principally to changes in dietary factors.
The dietary factors that determine whether blood homocysteine levels are elevated are the total methionine content of dietary protein and the content of vitamins B6, B12 and folic acid in the diet. In the diagram of Figure 1, the conversion of homocysteine to methionine is controlled by vitamins B12 and folic acid, which remethylate homocysteine to methionine. This process is reversible, converting homocysteine back to methionine. The only source of homocysteine in the body is from the methiodietary protein
folic acid B12
formation of protein in tissues
METHIONINE
HOMOCYSTEINE
> homocysteine thiolactone
ARTERIOSCLEROSIS ) B6
cystathionine ^
formation of protein in tissues
excretion of derivatives in urine Figure 1. Homocysteine in Cells and Tissues
cysteine
nine of dietary proteins. The conversion of homocysteine to cystathionine is controlled by vitamin B6. This process is irreversible, and the only way to dispose of excess homocysteine is by converting cystathionine to cysteine for excretion in the urine in the form of sulfate and other compounds containing sulfur.
Folic acid and vitamin B12 protect arteries against the damaging effect of homocysteine by conversion to methionine, which does not cause damage unless it is reconverted back to homocysteine. Vitamin B6 protects arteries by converting homocysteine to cysteine and other compounds that are excreted in the urine.
The amino acid methionine is present in all proteins. Methionine is known as an essential amino acid because all animals, including man, require a supply of methionine from dietary protein for proper growth and maintenance of all cells and tissues of the body. The only source of homocysteine is from the methionine of dietary proteins. Dietary proteins vary in the amount of methionine that they supply.
Proteins from animal sources, such as meat, eggs or milk, are abundant in methionine. Proteins from plant sources, such as grains, legumes or vegetables, are much more limited in methionine, containing only one third to one half of the quantity found in proteins of animal sources. Moreover, most fruits and vegetables, with some exceptions, contain much less protein than foods of animal origin.
The homocysteine theory explains why vegetarians and populations consuming a predominantly vegetarian diet are relatively protected against arteriosclerosis compared with populations that consume abundant meat and dairy products. The low quantities of methionine in the protein of plant based foods put less strain on the body's resources for conversion of homocysteine to methionine or for excretion of homocysteine derivatives in the urine. In contrast, the high quantity of methionine in the protein of animal-based foods requires increased amounts of vitamins B6, B12 and folic acid to keep blood levels of homocysteine down to a safe range.
The homocysteine theory explains why the diets of industrialized, developed countries are so likely to hasten the onset and progression of arteriosclerosis. Vitamins B6, B12 and folic acid are each exquisitely sensitive to destruction by the harsh physical or chemical treatments involved in food processing, refining and preservation. In milling wheat into white flour, for example, 50 to 90 percent of vitamin B6 is destroyed. 6 Losses of vitamin B6 amount to 40 to 50 percent in canning meats and fish and 60 to 75 percent in canning vegetables. Losses of vitamin B6 from the freezing of vegetables average 15 percent. Losses of folic acid from cereals, dairy products, meats and vegetables range from 25 to 75 percent when fresh raw foods are compared with refined, processed and preserved foods. Vitamin B12 is obtained only from foods of animal origin, and the small amount of the vitamin required per day (3 micrograms) is easily supplied in most diets. Thus, strict vegetarian diets can on rare occasions lead to serious vitamin B12 deficiency. More commonly, elderly people or those with inflammation of the stomach fail to absorb sufficient vitamin B12 to prevent a borderline deficiency.
Losses of vitamin B6 and folic acid during the refining, processing and preservation of foods are likely to cause widespread marginal or frankly deficient dietary intakes in populations consuming a high proportion of calories from these foods. The homocysteine theory of arteriosclerosis attributes the origin of the disease to the inadequate dietary intake of vitamin B6 and folic acid and the consequent failure to prevent the damage to arteries caused by elevated blood levels of homocysteine. In primitive peoples or in other populations, such as in some Mediterranean countries where diets are rich in fresh or minimally processed foods, the likelihood of vitamin B6 and folic acid deficiency is negligible, the consumption of foods with abundant methionine is low, and the susceptibility to arteriosclerosis is also low.
Figure 2 illustrates the processes by which homocysteine causes arteriosclerosis. In the liver, methionine, obtained from the breakdown of proteins, is continually converted to homocysteine and back to methionine. This conversion process (remethylation) is dependent upon vitamins B12 and folic acid, and deficiencies of these vitamins lead to a buildup of homocysteine. A second process (transsulfuration), which converts homocysteine to cystathionine, cysteine and other compounds for excretion in the urine requires vitamin B6. Deficiency of vitamin B6 leads to a buildup of homocysteine because the body has no other way to eliminate excess homocysteine by excredietary fats
deposition of fats «<-and cholesterol
/ Liver
dietary protein
LDL
homocysteine thiolactone
LDL - HcyT aggregates
vascular macrophage
arteriosclerosis
y damage to arterial lining cells deposition of mucoid matrix increased muscle cell growth destruction of elastic fibers fibrosis, calcification blood clotting
Figure 2. Homocysteine Theory of Arteriosclerosis tion in the urine. Vitamins B6, B12 and folic acid must be obtained from food because, like other vitamins, the body cannot make these essential substances from other nutrients.
Current thinking about how homocysteine causes plaques in the arteries theorizes that a buildup of homocysteine in the body leads to overproduction of a highly reactive form of homocysteine that causes LDL to become aggregated. 7 This reactive form, homocysteine thiolactone, is made from methionine in the liver by an enzyme that participates in protein formation and by other less well-understood processes. The LDL-homocysteine thiolactone aggregates are released into the blood from the liver. Then these aggregates are taken up by macrophages of the artery wall, many of which are derived from wandering monocytes of blood, to form foam cells of early arteriosclerotic plaques. These foam cells degrade the LDL-homocysteine thiolactone aggregates and release fat and cholesterol into developing plaques. The foam cells also release homocysteine thiolactone into surrounding cells of the artery wall, affecting the way cells handle oxygen. As a result, highly reactive oxygen radicals accumulate within cells, damaging the lining cells of arteries, promoting blood clot formation and stimulating growth of arterial muscle cells which form fibrous tissue, mucoid matrix and degenerative elastic tissue.
The homocysteine theory explains why populations that consume foods of animal origin with abundant methionine and foods that are highly processed, refined and preserved with depletion of B vitamins are susceptible to arteriosclerosis. Other factors, known as risk factors, are of major importance in influencing the onset and progression of the disease. As previously noted, these major risk factors include family history, advanced age, male gender, postmenopausal status, cigarette smoke and other toxins, certain drugs and hormones, diabetes and kidney failure, thyroid deficiency, hypertension, lack of exercise and elevated blood cholesterol. How do these factors control homocysteine and its damaging effect on arteries? Is there evidence that these major risk factors influence the way homocysteine is processed in the body?
Homocysteine and Genetic Risk Factors
A family history of early-onset heart disease is related to predisposition to the disease because of genetic inheritance from mother and/or father. As explained in Chapter 2, familial diseases of cholesterol and lipoproteins predispose to arteriosclerosis by causing extreme elevations of blood cholesterol or production of small, dense LDL particles in plasma. In recent years familial factors affecting homocysteine have been discovered among individuals with early-onset heart disease, stroke or peripheral vascular disease. In general, these familial or genetic factors cause greater susceptibility by increasing the quantities of dietary folic acid, vitamin B6 or B12 needed to prevent a buildup of homocysteine in the blood.
As explained in Chapter 1, deficiencies of three different enzymes are known to cause hbmocystinuria, elevation of blood homocysteine and generalized arteriosclerosis. When defective genetic copies of the DNA codes for one of these three enzymes are inherited from both parents, the pure form of the disease, known as the homozygous state, is produced in the affected child. When only a single defective genetic copy of the DNA which codes for one of these enzymes is inherited from one parent (a condition known as the heterozygous state), a mild or hidden form of the disease is produced in the affected child. While the fullblown disease, homocystinuria, is rare in the homozygous form (about 1 in 50,000 to 1 in 150,000), the mild or hidden form in the heterozygous state is much more common, about 1 or 2 per 100.
In the most common form of homocystinuria caused by homozygous deficiency of cystathionine synthase, about one-half of the children respond to moderately high doses of vitamin B6 by eliminating homocystine through the urine and by the reduction of blood homocysteine to normal levels. In the heterozygous or hidden state of the disease, blood levels of homocysteine may be normal. However, after receiving an oral dose of methionine, the precursor of homocysteine, the blood levels of homocysteine become more elevated after two to six hours than what is observed in normal individuals without this hidden genetic defect.
The heterozygous state for cystathionine synthase deficiency can also be detected by direct analysis of the abnormal enzyme function in cultured cells or in liver biopsy tissue. One early study failed to detect increased risk of heart disease in heterozygotes for cystathionine synthase deficiency. Recent studies have also failed to detect the heterozygous state of this defective enzyme in a small sample of familial, early-onset cases of arteriosclerosis, using molecular genetic analysis. 9 Studies are currently under way to detect the true incidence of defective genes for cystathionine synthase in the population, using direct analysis of the gene on chromosome 21 by methods of molecular biology.
The next most common cause of homocystinuria, as Chapter 1 explains, is the defective enzyme methylenetet-rahydrofolate reductase. In the homozygous state, in which defective copies of this gene are inherited from both parents, the affected child has homocystinuria that responds to increased doses of folic acid. In addition, this genetic defect produces an unstable form of the enzyme that loses its normal activity when heated in a test tube experiment. In the heterozygous state, in which a single copy of the defective gene is inherited from one parent, a subtle hidden change causes a slight elevation of blood homocysteine and increased risk of coronary heart disease. Recent studies, using techniques of molecular biology, have estimated that the frequency of this heterozygous condition is as high as 38 percent among French Canadians. 10 The importance of these findings is that a significant proportion of the population requires an increased amount of dietary folic acid to prevent mild elevation of blood homocysteine.
The third type of genetic defect in homocystinuria, deficiency of methyl transferase, is quite rare, and there are no current estimates of the frequency of this defective gene in populations. Taken together, the hidden genetic factors, heterozygous cystathionine synthase deficiency and heterozygous methylenetetrahydrofolate reductase deficiency, have been estimated to be factors in one-third or more of cases of early-onset arteriosclerosis in which no abnormality of cholesterol or lipoproteins is found. 11, 12
Homocysteine and Aging
One of the strongest risk factors for development of arteriosclerotic heart disease, stroke and peripheral vascular disease is aging. The overall risk and incidence of arteriosclerosis closely parallel the aging process, leading to the dictum, "you are as old as your arteries." The incidence and risk of all forms of arteriosclerotic disease are highly correlated with age. It is in the seventh, eighth and ninth decades of life that arteriosclerotic disease affects large segments of susceptible populations, strongly affecting life expectancy. Accordingly, declines in the rate of coronary heart disease and stroke since the 1960s in America have been correlated with modest increases in life expectancy.
The aging process affects the ability of the body to dispose of excess homocysteine. A number of studies have shown a gradual increase in blood homocysteine levels, starting in the seventh decade and increasing in the eighth and ninth decades, closely paralleling the aging process. Typically, the levels of blood cholesterol also rise gradually throughout adult life, reaching a peak in the seventh and eighth decades and levelling off or declining thereafter.
The factors that control blood homocysteine levels in aging are only partially understood. It is clear that food consumption declines with aging, and the dietary intake of vitamins B6, B12 and folic acid parallels this decline. As one ages, the ability to consume food and burn calories gradually declines. The decline in blood levels of vitamin B6 with aging is quite striking, leading to levels only one-fourth to one-third the levels found in babies and young children. 13 This major decline in vitamin B6 is only partially counteracted by vitamin B6 supplementation, suggesting that absorption and retention of the vitamin within the body are affected by the aging process. Folic acid and vitamin B12 levels also decline somewhat with aging, and dietary intake, absorption and retention of these vitamins are also affected by the aging process.
A recent survey of elderly subjects enrolled in the Fra-mingham Heart Study showed that consumption and blood levels of vitamins B6, B12 and folic acid determine the elevation of blood homocysteine in an aging population. 14 The effect was particularly striking in the case of folic acid, where both low plasma levels and low intake of the vitamin were correlated with significant elevation of the level of blood homocysteine. The level of blood homocysteine was significantly correlated with age in the 67-to 96-year-old age groups.
Homocysteine and Elevated Blood Cholesterol
For many years elevation of blood cholesterol has been known as a major risk factor for arteriosclerosis. As explained in Chapter 2, the underlying causes for elevation of blood cholesterol in arteriosclerosis are insufficiently understood. Epidemiological and nutritional surveys have shown that dietary fat and cholesterol are related to blood cholesterol in a general way. Many other factors influence blood cholesterol, however, and these factors are also only partially understood. Dietary cholesterol causes the liver and other tissues to decrease their synthesis of blood cholesterol. When dietary cholesterol is decreased, the liver begins to make more cholesterol to keep the blood cholesterol at levels sufficient for the body's needs.
Drastic diets limiting fat content to less than 10 percent of calories and cholesterol to less than 200 milligrams per day have only partial success in lowering blood cholesterol and preventing or reversing arteriosclerotic heart disease, as explained in Chapter 2. Careful examination of these diets reveals a major reliance on foods of vegetable and fruit origin with emphasis on fresh, raw or minimally processed foods, and the quantities of dietary fats and sugars are strictly limited. These diets contain greater quantities of vitamins B6, B12 and folic acid than diets that rely on foods of animal origin, fats and sugars. Moreover, the switch from protein of animal origin to protein from plant sources limits the quantity of methionine that must be prevented from forming homocysteine by these three vitamins, as illustrated in Figure 1. Thus diets with drastically reduced calories from fats are those predicted to be beneficial by the homocysteine theory of arteriosclerosis.
How does limiting the dietary intake of the methionine found in animal protein and increasing dietary sources of vitamins B6 and folic acid help to prevent elevation of blood cholesterol? Experiments with animals have shown that homocysteine, in its reactive thiolactone form, is capable of increasing the formation of fats in the form of triglycerides and cholesterol in the form of low-density lipoprotein in the liver. Thus diets that emphasize less intake of methionine by consuming proteins of primarily plant origin and greater intake of vitamins B6 and folic acid from minimally processed or raw foods will decrease formation of homocysteine and its secondary effect on triglyceride and lipoprotein formation in the liver.
Dietary fats and dietary sugars of all kinds are examples of calorically rich, highly processed foodstuffs that contain no vitamins, minerals or proteins. For this reason fats and sugars are sometimes referred to as "empty calories." The greater the consumption of dietary fats and sugars, therefore, the greater is the reliance on the remainder of the diet to supply sources of vitamins B6 and folic acid. A diet that contains 40 percent of calories from sugars and 40 percent of calories from fats leads to a nutritional imbalance in which chronic deficiency of folic acid and vitamin B6 fails to prevent overproduction of homocysteine from the methionine of proteins. Such a diet also may contain excessive amounts of animal protein, leading to further buildup of homocysteine because of limited body stores of the essential B vitamins.
The homocysteine theory of arteriosclerosis explains why populations that consume a high proportion of dietary calories as fats, sugars and other highly processed, refined and preserved foods have increased risk of the disease. These diets also exacerbate the tendency of blood cholesterol and lipoprotein levels to increase with age. In contrast, the diets of primitive peoples living their indigenous lifestyle contain few processed, refined or preserved foods with excessive calories from fats and sugars. The natural intake of vitamin B6 and folic acid from native diets is high, protecting the arteries from the damaging effect of excessive homocysteine and preventing excessive rise in blood levels of cholesterol and lipoproteins.
As primitive peoples begin to eat more processed foods, they begin to experience increased cholesterol and lipoprotein levels, increased homocysteine levels and increased risk of heart disease, 15 though recent studies have suggested that genetic factors may protect South African blacks from elevation of blood homocysteine compared with whites consuming the same diet.
The Male Gender, Postmenopausal Women and Hormones
In susceptible populations men have a much higher risk of coronary heart disease than women of the same age. The onset of the disease in men typically occurs in the fifth or sixth decade of life, whereas in women the onset of coronary heart disease is typically delayed until the sixth decade, after menopause. The disease rapidly increases in postmenopausal women until, in the seventh and later decades, the severity and incidence become similar to that of men. The protection against arteriosclerotic heart disease in women is related to estrogen production by the ovaries. The gradual loss of estrogen secretion by the ovaries after menopause or the sudden loss of estrogen secretion following surgical removal of the ovaries causes the arteriosclerotic process to increase rapidly in women of susceptible populations. Recent studies have suggested a protective effect of estrogen against coronary heart disease in postmenopausal women taking hormone replacement therapy.
Studies over the years have consistently shown that women have slightly lower levels of homocysteine in the blood (about 6 to 10 micromoles per liter) than men (about 8 to 12 micromoles per liter). After menopause, however, the blood levels of homocysteine increase in women to values similar to those found in men of the same age. Blood homocysteine levels continue to rise gradually with age, reaching higher values of 10 to 14 micromoles per liter in both women and men in the eighth and ninth decades of life.
It is clear that the blood levels of homocysteine in women and men correlate with relative susceptibility to arteriosclerotic heart disease. Thus in women before menopause the protection against arteriosclerosis is explained by the effect of estrogens and other ovarian hormones on blood levels of homocysteine. The precise reasons for this effect are incompletely understood at a biochemical level, but differences in enzyme activities in the liver and the lower mass of muscle in women compared with men have been suggested to be factors. After menopause or surgical removal of the ovaries, decreased secretion of estrogens and other ovarian hormones contributes to increased blood levels of homocysteine and increased susceptibility to coronary heart disease.
In contrast to the protective effect of natural ovarian hormones on susceptibility to arteriosclerosis, the administration of synthetic estrogens and progesterones in contraceptive hormones has been found to increase the risk of developing blood clots and arteriosclerotic plaques in young women. The use of low-dose contraceptive hormones has reduced the risk of these complications in recent years. These synthetic contraceptive hormones have been found to cause mild episodic increases in blood homocysteine levels, explaining the slightly increased risk of blood clots and arteriosclerotic plaques in the women who take them. Young women who smoke cigarettes and take contraceptive hormones are at greater risk of arteriosclerosis than nonsmoking women who take these hormones.
The reason synthetic contraceptive hormones lead to increased levels of blood homocysteine is that they antagonize the functions of vitamin B6 in the body. This effect is shown by the greater quantities of dietary or supplemental vitamin B6 needed to restore chemical imbalances in the processing of several amino acids in the liver. In experiments with human volunteers and with animals, diets that are deficient in vitamin B6 have been found to cause elevation of blood levels of homocysteine and excretion of trace amounts of homocystine and small quantities of cystathionine in the urine. Women who smoke while taking contraceptive hormones are at greater risk of blood clots and arteriosclerosis because cigarette smoke also antagonizes vitamin B6 in its vital functions in the body, increasing further the quantity of dietary or supplemental vitamin B6 needed to prevent elevation of blood homocysteine levels.
Recently the antiestrogenic, chemotherapeutic drug tamoxifen has been found to decrease the risk of fatal heart attacks by 50 percent in women under treatment for breast cancer. Tamoxifen causes a moderate reduction in blood levels of cholesterol and lipoproteins, and studies of blood homocysteine levels show a decrease of 30 percent in women under treatment for 9 to 18 months. 1 Tamoxifen may act in the body by affecting estrogen function, by increasing the effectiveness of folic acid or by facilitating the processing of oxygen by an antioxidant effect within arterial wall cells, causing a reduction in blood homocysteine and its damaging effect on arteries. Although tamoxifen may benefit the arteries of women under treatment for cancer, the drug is not advised for women without cancer because of its sometimes toxic side effects.
Homocysteine, Drugs and Toxins
In the 1950s and 1960s a study of English workers in the rayon manufacturing industry showed that among production workers exposed to the industrial solvent carbon disulfide the risk of developing coronary heart disease doubled compared with workers and employees not exposed to the solvent. Carbon disulfide is known to antagonize vitamin B6 by chemically combining with an active form of the vitamin in the liver. 18 This effect explains why carbon disulfide increases the risk of coronary heart disease since it leads to increases in blood homocysteine levels by decreasing its conversion to cystathionine.
Cigarette smoke contains small quantities of carbon disulfide among the 600 or more toxic chemicals it contains. Cigarette smoke also contains large quantities of carbon monoxide. Cigarette smoke, like carbon disulfide, antagonizes vitamin B6, probably because of the reaction of carbon monoxide with a form of the vitamin in the liver, causing inactivation. The result of the toxic actions of both carbon disulfide and cigarette smoke is that deficiency of active forms of vitamin B6 decreases the ability of the liver to dispose of homocysteine by conversion to cystathionine, leading to elevated blood homocysteine levels and damage to artery wall cells.
In the 1970s dermatologists began using the chemother-apeutic drug azaribine to treat cases of psoriasis that were resistant to treatment by other methods, especially coal tar and ultraviolet light. A number of patients receiving azaribine developed blood clots in peripheral arteries, heart attacks and strokes soon after starting the drug. When the blood homocysteine was analyzed, the levels were found to have been considerably increased by the drug. Azaribine was found to antagonize vitamin B6, causing excretion of homocysteine and other amino acids in the urine and producing elevation of blood homocysteine. Because of these findings the Food and Drug Administration withdrew its approval of the use of azaribine in the treatment of psoriasis in 1976, the first such recall in history.
Methotrexate, a chemotherapeutic drug widely used for the treatment of leukemia and cancer, has also been found to increase the blood levels of homocysteine. Methotrexate exerts its pharmacological action in the body by antagonizing folic acid, resulting in a buildup of homocysteine because of decreased conversion to methionine.
The widely used anesthetic gas nitrous oxide ("laughing gas") also causes elevation of blood homocysteine levels. Nitrous oxide acts in the body by antagonizing vitamin B12, preventing the conversion of homocysteine to methionine. Other important drugs, including anticonvulsants and antidiuretics, have been found to increase the blood levels of homocysteine, but the effect of this elevation on risk of blood clots and vascular disease has not been determined. 20 Some anticonvulsants like phenytoin act by antagonizing folic acid, reducing the conversion of homocysteine to methionine. The reasons for the action of other drugs on the elevation of blood homocysteine and the relative risk of arteriosclerosis remain to be determined in many cases.
Diabetes and Kidney Failure
Diabetes mellitus (sugar in the urine) is a very common disease that strongly predisposes affected persons to rapidly advancing arteriosclerosis. Sufferers from diabetes frequently are affected by heart attack, stroke, kidney failure, blindness and gangrene of the toes and feet, all caused by severe arteriosclerosis. In fact, arteriosclerosis is the leading cause of death among diabetics.
Diabetes is a complex disease related to insufficient production of insulin by the pancreatic islets or the inability of insulin to transport blood sugar (glucose) into cells for production of energy. As a result, all cells of the body become starved for sugar and switch into a starvation mode of cellular activity. The excess blood sugar in diabetes reacts chemically with the hemoglobin of red blood cells and with the membranes around small blood vessels and capillaries, narrowing the lumen and interfering with the passage of red blood cells. In the kidney, the clogging of small arteries gradually leads to failure of kidney function, a frequent complication of diabetes.
A very striking effect of kidney failure, whether from diabetes or from other causes, is a remarkable buildup of homocysteine in the blood. 21 The levels of homocysteine may become extremely high, reaching two to three times the normal value, and the degree of elevation parallels the severity of kidney failure. These high levels of blood homocysteine subject all arteries of the body to damage and rapidly progressive arteriosclerosis. In the case of kidney failure from diabetes, the remarkable buildup of homocysteine leads to the vascular complications that result in disability and death from the disease. Recent studies of persons with early diabetes without kidney failure have not revealed an abnormality of homocysteine blood levels.
The effect of various treatments on blood levels of homocysteine has been studied in persons with kidney failure. Dialysis with an artificial kidney machine causes a temporary fall in the blood homocysteine level, but after one to two days, the blood homocysteine returns to its previously elevated level. Using vitamin therapy, the most effective treatment is with large doses (5 milligrams per day) of folic acid, which partially decreases the homocysteine level. Supplementation with vitamin B12 or vitamin B6 does not decrease the homocysteine level further. The exact cause of elevation of blood homocysteine in kidney failure, whether from diabetes or from another disease, is insufficiently understood.
Homocysteine and Thyroid Hormone
For many years deficiency of thyroid hormone secretion has been known to predispose to arteriosclerotic heart disease. In persons with a serious deficiency of thyroid hormone, the ability of the cells of the body to use oxygen is impaired. The basal metabolic rate is slowed in hypothyroidism, and the liver begins to make increased quantities of cholesterol and triglycerides. As a result the cholesterol and lipoprotein levels become elevated, and the risk of coronary heart disease increases. Administration of potent thyroid hormone preparations such as thyroxine to persons with severe hypothyroidism and elevated blood cholesterol increases the risk of heart attack. Subtle or marginal deficiencies of thyroid hormone, detected by measuring basal metabolic rate, are found to be widespread in populations with a high risk of arteriosclerotic heart disease.
In West African studies of thyroid deficiency and goiter resulting from insufficient dietary iodine, analysis of the amino acids of plasma revealed elevation of blood homocysteine levels. 22 These findings have been confirmed by several subsequent studies of patients with thyroid abnormalities. The patients at increased risk for arteriosclerotic heart disease with hypothyroidism have elevated blood homocysteine levels, and patients with overactive thyroid glands and hyperthyroidism have decreased levels of plasma homocysteine compared with normal values. In experiments with rats, following surgical removal of the pituitary gland, the growth response of the animals to ho-mocyste acid, the fully oxidized form of homocysteine, was found to require thyroid hormone.
The Scientific Evidence for the Homocysteine Theory
Animal Studies
Chapter 1 explained that feeding homocystine or its precursor methionine to monkeys or rats causes weight loss and lower levels of blood cholesterol because of the toxicity of these amino acids. Before designing my first research project, I considered these questions: How could the elevation of blood levels of homocysteine be produced in animals? Would the elevation of blood homocysteine cause arteriosclerosis and blood clots in animals as it did in children with homocystinuria? How could homocysteine be given to animals that could not eat a toxic diet? Which chemical form of homocysteine should be given to animals to reproduce or mimic the disease homocystinuria in children?
Since no medical investigator had looked for arteriosclerosis after homocysteine was given to animals, I decided in 1969 that the first experiment should be done by direct injection into rabbits. Although the method was somewhat artificial, homocysteine was injected subcutaneously in a dilute solution of glucose and water. In this way, a known amount of homocysteine would be absorbed gradually over a period of hours, simulating the situation in children with homocystinuria. The thiolactone form of homocysteine was injected because it is stable in solution and readily converted to homocysteine by normal enzymes of plasma and tissues. An experiment with radioactively labelled homocysteine thiolactone showed that the amino acid is rapidly converted into a series of labelled compounds in the serum.
Our first results showed that, just as was expected, early arteriosclerotic plaques were found in the coronary arteries of yearling rabbits after only three weeks of twice-daily injections of homocysteine thiolactone! 24 When young weanling rabbits were injected once daily for five weeks, early arteriosclerotic plaques were found in the coronary arteries, aorta and arteries of the other organs. If the animals were fed cholesterol and also injected with homocysteine, the arteriosclerotic plaques were found to contain fat deposits. If the animals were given a diet that was deficient in vitamin B6 and also injected with homocysteine, the plaques became more prominent and more widespread. We had for the first time produced arteriosclerosis by injection of an amino acid, reproducing many of the features of arteriosclerotic plaques found in children with homocystinuria, both in rabbits fed cholesterol and in rabbits given a vitamin B6-deficient diet. When these results were presented at a national meeting, nobody made a comment; the audience maintained a "stony silence," in the words of Dr. Moses Suzman, following one of his lectures on homocysteine and arteriosclerosis.
This response to what I thought was an extraordinary experiment confirming my conclusions about the arteriosclerotic effect of an amino acid was very disappointing. Investigators interested in the traditional approach of feeding cholesterol and fat to animals totally ignored our results and went back to studying cholesterol and lipoproteins some more. One group of investigators in the homo-cystinuria field offered to collaborate by repeating my experiments in their laboratory, sending me the slides from the arteries of their rabbits to examine independently. In my opinion, their results also showed early arteriosclerotic plaques in young rabbits, just as we had found two years earlier. However, these investigators published a contradiction of our earlier findings in their paper in 1974, claiming that the plaques were "spontaneous and of no significance." They illustrated their report not with the slides they had sent me, but with a photograph of a normal artery! 25
I felt totally betrayed by this episode. If only I had photographed their slides before returning them, I could have published an illustrated rebuttal. Subsequently, the investigators refused further collaboration and returned to their studies of sulfur amino acids in children with homo-cystinuria and in newborn children. The only way I could counteract the effect of this contradictory report was to repeat my experiments with rabbits, giving larger doses of homocysteine for longer periods of time.
The original experiments with rabbits 24 were designed to study the effect of moderate doses of homocysteine thi-olactone, comparable with the dose that a human adult might receive when eating a diet consisting predominantly of animal protein. Our results had shown early arteriosclerotic plaques, as I had predicted. If the dose of homocysteine thiolactone was increased fivefold to overload the capacity of the rabbits' tissues to eliminate the amino acid, would more dramatic effects be observed? I remember very well receiving an emergency call to see several rabbits that had died after one month of injections with high doses of homocysteine thiolactone. When the animals were examined, I found that blood clots had formed in the veins of the legs and abdomen and travelled to the lungs, causing bleeding and dead areas of the lungs. In animals that were given injections of vitamin B6 as well as homocysteine, no blood clots formed and the animals survived until the experiment was finished after two months. 26
These results were tremendously exciting because high doses of homocysteine thiolactone had produced in animals the dramatic complication of blood clots in the lungs that had killed some of the children with homocystinuria. However, the experiment was quite artificial because the amino acid had been injected rather than fed in the diet. I needed to design an experiment that would force the rabbits to eat large quantities of homocystine or methionine which had been found to be toxic when fed in an experimental diet by other investigators.
In order to make the experiments resemble the human situation more closely, a synthetic diet was made with agar containing special chemical forms of these amino acids and fed to rabbits. Different groups of rabbits received homocysteine thiolactone in the perchlorate form, methionine converted to methyl homocysteine thiolactone by acid and homocystine treated with hydrogen peroxide. To our great surprise, these forms of homocysteine and methionine stimulated the growth of the rabbits, producing giant rabbits! My associates in the pathology department made several visits to the animal farm to observe them.
At the conclusion of the experiment, when the arteries of the rabbits were examined, arteriosclerotic plaques were found that closely resembled the arteriosclerosis found in children with homocystinuria. We had not only reproduced the vascular disease associated with the genetic disease homocystinuria, we had also produced the complication of blood clot formation and embolism to the lungs. Finally, we had also suppressed the formation of blood clots by vitamin B6, although the vitamin did not prevent the formation of arteriosclerosis that was produced by injecting the very large doses of homocysteine thiolactone or methionine. 26
After publication of our findings, scientists in Japan repeated our experiments and made very similar observations on the formation of blood clots and arteriosclerosis, completely confirming our findings. 27 These scientists also repeated the experiments of Rinehart with vitamin B6 deficiency in monkeys, observing arteriosclerosis after prolonged periods of partial deficiency of the vitamin. They went on to show that vitamin B6 therapy caused reversal and regression of the arteriosclerosis that had been induced by vitamin B6 deficiency. Because their report was published in Japanese in a Japanese journal, however, I was not aware of their important confirmation of our results until I received a review in English from the author some years later.
Another important experiment was conducted with baboons that were given homocysteine thiolactone by continuous intravenous injection. 28 In this expensive experiment the arteries were found to have been damaged by homocysteine, producing arteriosclerotic changes that were very similar to what we had found in the rabbits that were given subcutaneous injections of homocysteine. The blood platelets were found to have formed very early blood clots at the sites of injury in the arteries, and larger blood clots and arteriosclerotic plaques were found within only two weeks in the arteries of some baboons that were given high doses of homocysteine thiolactone. Scientists in Prague were able to produce similar damage to arteries by force-feeding methionine or homocysteine to rats by stomach tube. 29
Although a few scientists failed to observe arteriosclerotic plaques in animals that were given homocysteine, probably because of different methods of analyzing the tissues, many subsequent studies have confirmed that homocysteine produces arteriosclerosis and blood clots in a variety of experimental animals. For example, recent experiments in France in which large doses of casein (the calcium form of milk protein) were fed to minipigs, showed early changes in the aorta caused by the destruction of elastic tissue activated by the enzyme elastase, a result of the elevation of blood homocysteine. 30 In an earlier experiment with vitamin B6-deficient pigs, arterial damage and arteriosclerosis were also related to elevation of blood homocysteine levels. 31
These experiments with a variety of experimental animal species provide abundant evidence that induction of elevated blood levels of homocysteine by direct injection, dietary feeding or by chronic partial vitamin B6 deficiency reproduces the essential features of arteriosclerosis that are observed in children with hereditary homocystinuria. The inadequate, flawed or misinterpreted experiments of a few investigators who failed to observe these effects in animals delayed acceptance of the homocysteine theory. The positive results of the many scientists who had had success in this field support the validity of the theory.
Studies of Cells and Tissues
Why should elevated blood levels of homocysteine cause damage and arteriosclerotic changes in the arteries? How does an amino acid normally produced in the body affect artery cells and tissues, narrowing and obstructing the normal flow of blood through the lumen of the artery? How does homocysteine affect the biochemical functioning and growth of cells and tissues? How does excess homocysteine lead to the formation of blood clots within arteries and veins? These are some of the questions that have been addressed by medical scientists during the quarter-century that has elapsed since discovery of the homocysteine theory of arteriosclerosis.
One of the earliest attempts to answer these questions involved growing cells in culture from the skin of children with homocystinuria. Since the enzyme deficiency of cystathionine synthase in homocystinuria involves a genetic defect in all cells of the body, the cultured cells were found also to be deficient in this enzyme. In observing the growth of these cultured cells, the matrix substance produced by the abnormal cells was found to be clumped and aggregated, compared with the finely fibrillar matrix substance produced by normal cells. 32 This effect recalled the observation that the aorta of children with homocystinuria contains a matrix substance of reduced solubility. 33 When homocysteine thiolactone was added to normal cell cultures, some of the fibrillar matrix became clumped, showing that homocysteine changed the aggregation of ground substance to reduce its solubility. As explained in Chapter 2, one of the earliest changes in arteriosclerotic plaques is an accumulation of mucoid matrix substance of decreased solubility in areas of damage to arterial tissues.
When homocysteine thiolactone is added to cell cultures from children with homocystinuria, the cells show extreme toxicity, detaching from the culture dish and losing viability. If vitamin B6 is also added to the cell cultures from children who respond to vitamin B6 therapy, the toxic effect of homocysteine is overcome, allowing the cells to multiply rapidly. By using homocysteine thiolac-tone labelled with radioactive sulfur, a new pathway was discovered by which the sulfur is converted to sulfate without forming cystathionine since the enzyme for this conversion is absent from these cell cultures. 34 Proof of this new pathway was demonstrated by a study of how homocysteine is processed by the livers of guinea pigs deprived of vitamin C. In this study, the reaction of oxygen with the sulfur atom of homocysteine was shown to require vitamin C in order to form PAPS (phosphoadenosine phosphosulfate), the coenzyme that attaches sulfate groups to the mucoid matrix of cells. 35
Another very interesting feature observed in cell cultures from children with homocystinuria is the distinctive pattern of growth, which resembles the pattern of growth of cancer cells in culture. Furthermore, the muscle cells of arteries grow in a similar pattern in early arteriosclerotic plaques. As explained in Chapter 2, the 19th-century German pathologist Rudolf Virchow likened the increased numbers of muscle cells in atheromas to tumors of the blood vessels. In some way, abnormal homocysteine production induces cells to lose control of growth processes, causing growth of muscle cells in arteriosclerotic plaques. Recent experiments have shown that homocysteine damages cultured endothelial cells and increases the growth of smooth muscle cells. 36 These effects on the cells of artery walls explain in a general way the early phases of production of arteriosclerotic plaques. 5
The observations of growth stimulation in normal guinea pigs and rabbits 26 and the abnormal growth pattern of cultured cells from children with homocystinuria 34 suggest that homocysteine is involved in stimulating the growth of normal cells and tissues. Many of the children with homocystinuria grow rapidly in childhood, achieving taller stature than their unaffected relatives. As previously noted, these children also have long arms, legs, fingers and toes as a result of the accelerated growth. Experiments with rats from which the pituitary gland is removed surgically show that a homocysteine compound containing extra oxygen (homocysteic acid) stimulates growth. This growth response simulates the response to growth hormone and is correlated with release of an insulin-like growth factor into the plasma, provided that thyroid hormone is also given. 23 Insulin-like growth factor is known to promote the growth of cartilage and bone in growing animals by increasing the sulfate content of matrix substances. This process is under the control of growth hormone and is mediated by the formation of PAPS from homocysteine. 35
The fundamental nature of the participation of homocysteine in normal growth suggests a relationship to the disturbances of growth in cancer cells. Experiments with radioactively labelled homocysteine thiolactone revealed a complete inability of malignant cells to add oxygen to homocysteine and to form sulfate. 37 Normal cells and cells from children with homocystinuria perform this conversion easily, rapidly and completely. As a result of this specific abnormality, cancer cells accumulate excess homocysteine thiolactone which reacts with and alters the structure and function of proteins, mucoid substances and chromatin, the nuclear material containing DNA. This study suggested a new interpretation of the origin of cancer cells and led to the discovery of the new anticancer compounds, thioretinaco and thioretinamide, which are described in Chapter 6.
Because of the relation of homocysteine to the growth process in normal and malignant cells, a study was performed in young and adult rats and guinea pigs, comparing the effectiveness of the liver in processing homocysteine. The results show that the livers of older animals accumulate homocysteine thiolactone and the livers of younger animals prevent oxygen from converting homocysteine to the dimer form, homocystine. 38 These findings show that homocysteine is processed differently in aged animals, explaining the gradual rise of blood homocysteine levels with age 14 and the decreased ability of tissues to form ad-enosyl methionine from methionine and ATP. The implications of these findings for the aging process are described in Chapter 6.
The liver cells of children with homocystinuria caused by each of the three enzyme deficiencies known to produce abnormal homocysteine processing are found to accumulate droplets of fat within the cytoplasm. A very striking abnormality of the cytoplasmic organelles (mitochondria) is also found in homocystinuria. The mitochondria are the structures within the cell cytoplasm for utilization of oxygen in the burning of food for production of chemical energy in the form of ATP. In homocystinuria the mitochondria become enlarged, assume bizarre shapes, and become aggregated one with another. A similar effect has been produced in the mitochondria of both normal and hypertensive rats by administering either methionine or the oxycholesterol, cholestane triol. 39 This fundamental abnormality of energy production by the mitochondria is a key process by which the cells become damaged and increase the formation of fats and cholesterol in arteriosclerosis. 8
Because of the prominence of blood clots in children with homocystinuria and in animals given homocysteine, medical scientists have studied the effects of homocysteine on blood clotting in the body. Early studies showed that blood platelets, the circulating cell fragments in the blood that orchestrate the clotting process, are overreac-tive, showing increased adherence to glass beads in test tube experiments. Later experiments showed that freshly synthesized homocysteine thiolactone in the uncharged, salt-free form is extremely active in causing aggregation of blood platelets. 40 In addition, homocysteine has been found to activate multiple blood-clotting proteins and to increase formation of thromboxane, the hormone-like fatty acid derivative (prostaglandin A), causing an increased tendency to form blood clots. Finally, homocysteine increases the binding of lipoprotein(a), a lipoprotein fraction related to the clotting process, to fibrin, the protein component of blood clots. 41 These studies show that homocysteine hastens blood clotting by affecting platelets, protein-clotting factors, lipoproteins and prostaglandins that cooperate in the complex activation process within blood vessel walls and circulating blood components.
These diverse experiments with cells and tissues support the homocysteine theory of arteriosclerosis by describing the pathogenic processes by which a buildup of homocysteine in plasma, cells and tissues leads to arterial damage and arteriosclerotic plaques. In Figure 2 (page 64), components of lipoprotein-homocysteine thiolactone aggregates are taken up by cells of the artery wall, forming foam cells. These cells degrade and store fats and cholesterol from the LDL component, releasing them gradually to form the cholesterol crystals and fatty deposits of advanced arteriosclerotic plaques. The homocysteine thiolactone component is released from foam cells and affects the oxygen utilization process of adjacent arterial cells, causing increased formation of damaging free radical substances. In turn the disturbance of oxygen processing causes increased growth of muscle cells, formation of mucoid matrix from the sulfur atom of homocysteine, destruction of eiastin fibers by activation of elastase, production of fibrous collagen fibers, calcium deposits and activation of blood clotting. 8 These diverse studies of the effects of homocysteine on cells and tissues explain the principal processes by which a buildup of homocysteine causes formation of arteriosclerotic plaques.
Human Studies
One day in 1973 Dr. Bridget Wilcken, a pediatrician from England, visited my laboratory in Boston. She had read several of my articles describing my findings on homocysteine and arteriosclerosis. She was travelling to Australia to join her husband, internist Dr. David Wilcken, who was interested in the possibility of investigating homocysteine in patients with coronary heart disease. During her visit, we discussed ways in which evidence for the homocysteine theory could be established by studying human subjects. Subsequently, in 1976, the Wilckens published a study that showed that, in a group of 25 patients with coronary heart disease, the blood homocysteine became highly elevated in seven patients following an oral dose of methionine, compared with high elevation of blood homocysteine in only one of 22 normal subjects. 42 This significant human study was the first of many subsequent studies of homocysteine and arteriosclerosis to be published by the Drs. Wilcken and their colleagues during the past 20 years, and it was the first study of its kind in the medical literature.
Beginning in the mid-1980s epidemiological studies of human populations were begun for the purpose of comparing the blood levels of homocysteine in patients with coronary heart disease, stroke, peripheral vascular disease and kidney failure with the blood homocysteine levels of normal subjects. The result of these studies is a consensus among medical investigators that elevation of blood homocysteine level is a strong independent risk factor for the development of arteriosclerotic disease. 43 Levels of blood homocysteine greater than 14 micromoles per liter are associated with increased risk of arteriosclerosis, and the higher the homocysteine level, the higher the risk. The consequences of this risk are demonstrated by the finding of a three-fold increase in risk of heart attack in a five-year prospective study of 14,000 U.S. physicians. 44
Elevated blood homocysteine is estimated to account for at least 10 percent of the risk of coronary heart disease in the U.S. population. Accordingly, reduction of blood homocysteine levels by the addition of folic acid to the food supply is estimated to prevent as many as 50,000 deaths from coronary heart disease annually. 43 In 1996 the U.S. Food and Drug Administration adopted new guidelines requiring the addition of folic acid to enriched foods such as flour, pasta and other grain-based foods. This action promises to help counteract elevated homocysteine levels and the consequent risk of vascular disease, continuing the decline in mortality from heart disease and stroke that was attributed to the addition of synthetic vitamin B6 to the food supply beginning in 1961. 5
An early study of patients with cerebrovascular disease and stroke demonstrated increased levels of blood homocysteine before and after an oral dose of methionine compared with normal subjects. 45 A recent study of over 1,000 subjects from the Framingham Heart Study showed that the higher the level of blood homocysteine, the greater the degree of narrowing of carotid arteries to the brain. 46 A subsequent study found that increased risk of early-onset (before age 55) heart disease, cerebrovascular disease and peripheral vascular disease correlates with blood homocysteine levels greater than 14 micromoles per liter. 47 In comparison with traditional risk factors, elevation of blood homocysteine was found to be a greater risk factor (22-40-fold) than elevated blood cholesterol (1.2-3.1-fold), high blood pressure (8-18-fold) or cigarette smoking (3.5-fold) in the selected group of patients with early-onset arteriosclerosis.
A large cross-sectional study of vascular disease risk in over 16,000 subjects estimated the relation between blood homocysteine level and other established risk factors. 48 The homocysteine level correlated with male gender, age, cigarette smoking, lack of exercise, blood pressure, heart rate, blood cholesterol and triglyceride levels. For example, the homocysteine level in male smokers, aged 65 to 67, is almost 5 micromoles per liter greater than in nonsmoking women, aged 40 to 42. In another study of 199 male coronary heart disease patients, elevated blood homocysteine, high blood pressure, decreased HDL, increased LDL, fibrinogen (blood clotting protein), plasminogen (clot dissolving enzyme) and viscosity of plasma were all found to correlate with coronary heart disease risk. 49 A study of 304 patients with coronary heart disease revealed that risk of disease correlated with homocysteine levels, diabetes, smoking, male gender, age and low levels of vitamin B6. 50
A total of 209 published studies of the epidemiological relation between homocysteine and arteriosclerosis were reviewed recently. 43 The consensus of these studies is that elevated blood homocysteine is a strong independent risk factor for arteriosclerosis. A detailed study of coronary arteries by X-ray angiography in 163 males with angina pectoris concluded that the degree of narrowing of the coronary artery by arteriosclerotic plaques correlates better with blood levels of homocysteine than with blood levels of cholesterol. 15 A study of a susceptible population estimated a 40 percent increase in risk of arteriosclerotic heart disease for each increase of 4 micromoles per liter of blood homocysteine. 51
The risk of blood clots in the leg veins for pulmonary embolism (blood clots in the lungs) has also been correlated with blood homocysteine levels. Recent studies have shown that 10-20 percent of patients with recurrent blood clots in leg veins have elevated levels of blood homocysteine. 52 53 The risk of blood clots in young individuals with elevated blood homocysteine was shown to be inherited as a genetic predisposing factor in 26 of 30 families studied. 52 Familial predisposition to elevated blood homocysteine levels in arteriosclerosis has also been documented in numerous other published studies." 1247S4
The reason that the U.S. Food and Drug Administration decided to require the addition of folic acid to foods is that mothers consuming 400 meg per day of folic acid, either in the diet or from vitamin supplements, have a greatly reduced risk of giving birth to babies with neural tube defects of the brain and spine such as anencephaly or spina bifida. Recent studies have shown that these same women have a high level of blood homocysteine, predisposing their babies to birth defects. 55 The effect of folic acid in preventing these birth defects, therefore, may be related to the lowering of blood homocysteine levels in early pregnancy. 9,55
Prevention of Arteriosclerotic Heart Disease and the Homocysteine Theory
Considering the overwhelming evidence that elevated blood levels of homocysteine are associated with increased risk of arteriosclerosis, what is the evidence that lowering homocysteine levels decreases the risk of vascular disease? Is there proof that therapy with vitamins B6, B12 and folic acid reduces the risk of vascular disease by reducing homocysteine levels? Despite repeated suggestions over a quarter-century based on the homocysteine theory of arteriosclerosis that large-scale trials should be initiated to answer this question, no definitive information is yet available in the published medical literature. Only with the relatively recent results of human epidemiological studies in the 1990s has there begun to be interest by governmental agencies in funding trials of this type.
The first suggestion that control of blood homocysteine levels might reduce risk of arteriosclerosis came from studies of children with homocystinuria. A study of homo-cystinuria caused by cystathionine synthase deficiency revealed that about one-half of 629 patients responded to large doses (500-1000 mg per day) of vitamin B6. 56 The risk of blood-clot formation was significantly decreased by vitamin B6 in the responding patients, compared with the patients who showed no response to vitamin B6.
In 1962 Dr. John Ellis of Texas discovered that many victims of carpal tunnel syndrome, a painful disorder of the wrist and hand, respond symptomatically to moderately large doses of vitamin B6 (100-200 mg per day) after two to three months. Many patients with carpal tunnel syndrome, particularly if it involves both hands, have deficient blood levels of vitamin B6 and they respond favorably to vitamin B6 therapy. Dr. Ellis observed that few of his patients receiving vitamin B6 developed angina or heart attack. In a retrospective study of his patients over a five-year period, the risk of chest pain or heart attack was found to have decreased by 75 percent, compared with patients of other physicians in the county who received no vitamin B6. S7 Furthermore, there was an apparent increase in longevity of 7 to 17 years in patients who had taken vitamin B6, compared with patients who had not taken the vitamin.
Although no prevention trials based on the homocysteine theory of arteriosclerosis have been published in the medical literature, there is increasing interest by investigators worldwide in designing and completing a definitive prospective study of this type. The necessary elements for the success of such a trial were discussed by medical scientists working in the homocysteine field at the First International Conference on Homocysteine Metabolism in Ireland in 1995. At least three proposals have been submitted to the National Institutes of Health for consideration for funding within the past two years. Governmental funding agencies over the past quarter-century have repeatedly ignored proposals for a large-scale trial of the homocysteine theory because of resistance by adherents of the cholesterol/fat approach. As a result, funding has concentrated on control of multiple risk factors and a variety of potentially toxic drugs that have yielded inconclusive or disappointing results.
The recent flood of findings on homocysteine and arteriosclerosis in human populations will trigger a series of prospective trials of the homocysteine theory, probably within the next decade. A complicating factor is the addition of vitamin B6 and folic acid to foods, breakfast cereals and other widely distributed sources, potentially accelerating the well-established decline in vascular disease since the 1960s.
The Homocysteine Revolution Arteriosclerosis and the Homocysteine Theory
The development of the homocysteine theory has enabled medical scientists to appreciate the significance of the seminal discoveries of the early pioneers in arteriosclerosis research, M.A. Ignatovsky, Harry Newburgh and James Rinehart. The origin of arteriosclerosis is now understood to be a toxic effect of a by-product of protein breakdown, the amino acid homocysteine. The importance of fats and sugars in the genesis of the disease is now understood to be related to loss of vitamins B6 and folic acid through processing, refining and preservation of foods, creating an imbalance between the abundant methionine of foods of animal origin and the amount of these essential vitamins necessary to prevent a buildup of homocysteine in the body.
The role of antioxidants in arteriosclerosis is related to the effects on oxidative modification of LDL, the carrier of homocysteine by LDL-homocysteine aggregates. The uptake of these aggregates by the cells of the artery wall causes deposition of fats and cholesterol and results in damage to artery wall cells by interfering with normal oxygen processing and allowing accumulation of damaging free radical substances. 8 The unsaturated oils of fish do in fact decrease blood homocysteine levels in men with elevated blood fat levels. 58 In a study of men with elevated cholesterol levels, the administration of vitamins B6, B12, folic acid, riboflavin, choline and troxerutin, an antioxidant of plant origin, decreased both blood homocysteine and LDL levels. 59 Choline is a constituent of lecithin that helps to convert homocysteine to methionine in the body.
The homocysteine theory offers an explanation for observations on human arteriosclerosis that are difficult to explain by the cholesterol/fat approach. The dramatically declining incidence of heart attack and stroke in America, despite relatively constant dietary fat and cholesterol intake and constant blood levels of cholesterol, is explained by the effect of synthetic vitamin B6 in preventing the disease. 5 The low incidence of arteriosclerosis in Eskimos, despite high dietary fat and cholesterol, is explained by the effect of unsaturated fish oils and the abundant vitamin B6 of fish in suppressing blood homocysteine levels. The decreased incidence of heart attack in Europe during World Wars I and II is explained by the scarcity of animal foods, such as meat and eggs, and the reliance of the population on vegetables, decreasing the amount of methionine in the diet and increasing natural sources of vitamin B6 and folic acid.
The effects of drugs, toxins and hormonal status in risk of arteriosclerosis are explained by their effects on blood levels of homocysteine, since effects on LDL and blood cholesterol levels may not be consistently observed. The important effect of kidney failure on risk of arteriosclerosis is explained by large increases in blood homocysteine levels, since cholesterol and LDL levels may be unaffected in many cases. The high risk of arteriosclerosis of the coronary arteries after heart transplantation is explained by the prominent increase in blood homocysteine levels. 6061
As explained more fully in Chapter 6, the greater risk of arteriosclerosis with advancing age is attributed to the loss of a key player in the processing of homocysteine and methionine by all cells of the body, thioretinaco ozonide. The loss of this substance from the membranes of all cells is responsible for the gradual increase in blood levels of homocysteine with age, increasing the risk of arteriosclerosis. The decline in the processing of foods in the liver and other organs by reaction with oxygen leads to the accumulation of damaging oxygen radicals in all aging tissues. 62
While the homocysteine theory of arteriosclerosis needs further development in some respects, the basic elements of the theory that have already been discovered explain the principal processes underlying the genesis of the disease. These principles have already been applied to prevent arteriosclerosis in susceptible individuals and populations. In Chapter 5 the therapeutic measures currently available to individuals at risk for arteriosclerosis are explained in light of the existing knowledge of its cause, the altered processing of homocysteine.
REFERENCES
1. Proceedings of the Conference on the Decline in Coronary Heart Disease Mortality, Richard J. Havlik and Manning Feinleib, Editors. (Bethesda: NIH Publication No. 79-1610, 1979), pp. 1-399.
2. Peter O. Kwiterovich, The Johns Hopkins Complete Guide for Preventing and Reversing Heart Disease (Rocklin, Calif.: Prima Publishing, 1993), pp. 163-176.
3. Nathan Pritikin and Patrick M. McGrady, Jr., The Pritikin Program for Diet and Exercise (New York: Grosset & Dunlap, 1979), pp. 79-105.
4. Dean Ornish, S.E. Brown, L.W. Scherwitz, J.H. Billings, W.T. Armstrong, T.A. Ports, S.M. McLanahan, R.L. Kirkeide, R.J. Brand and K.L. Gould, "Can lifestyle changes reverse coronary heart disease?" Lancet 336:129-133, 1990.
5. Kilmer S. McCully, "Homocysteine theory of arteriosclerosis: Development and current status." In: Antonio M. Gotto, Jr. and Rudolfo Paoletti, editors, Atherosclerosis Reviews, Volume 11 (New York: Raven Press, 1983), pp. 157-246.
Beyond Cholesterol: The Homocysteine Theory of Arteriosclerosis
resulting from processing and preservation of foods." American Journal of Clinical Nutrition 24:562-573, 1971.
7. Marek Naruszewicz, Ewa Mirkiewicz, Andrzej J. Olszewski and Kilmer S. McCully, "Thiolation of low-density lipoprotein by homocysteine thiolactone causes increased aggregation and altered interaction with cultured macrophages." Nutrition, Metabolism and Cardiovascular Diseases 4:70-77, 1994.
8. Kilmer S. McCully, "Homocysteine and vascular disease." Nature Medicine 2:386-389, 1996.
9. Arno G. Motulsky, "Nutritional ecogenetics: Homocysteine-related arteriosclerotic vascular disease, neural tube defects and folic acid." American Journal of Human Gtmetics 58:17-20, 1996.
10. Phyllis Frosst, Henk J. Blom, R. Milos, Philippe Goyette, C.A. Shephard, Rowena G. Matthews, Godfried J.H. Boers, Martin den Heijer, Leo A.J. Kluijtmans, Lambert P.W.J, van-den Heuvel and Rima Rozen, "A candidate risk factor for vascular disease: A common mutation in methylenetetrahy-drofolate reductase." Nature Genetics 10:111-113, 1995.
11. Paul N. Hopkins, Lily L. Wu, Steven C. Hunt, Brent C. Jones, G. Michael Vincent and Roger R. Williams, "Higher plasma homocysteine and increased susceptibility to adverse effects of low folate in early familial coronary heart disease." Arteriosclerosis, Thrombosis and Vascular Biology 15:1314-1320, 1995.
12. Jacques J. Genest, Jr., Judith R. McNamara, Barbara Upson, Deeb N. Salem, Jose M. Ordovas, Ernst J. Schaefer and M. Rene Malinow, "Prevalence of familial hyperhomocysteine-mia in men with premature coronary artery disease." Arteriosclerosis and Thrombosis 11:1129-1136, 1991.
13. A. Hamfelt, "Age variation of vitamin B6 metabolism in man." Clinica Chimica Acta 10:48-54, 1964.
14. Jacob Selhub, Paul F. Jacques, Peter W.F. Wilson, David Rush and Irwin H. Rosenberg, "Vitamin status and intake as primary determinants of homocysteinemia in an elderly population." Journal of the American Medical Association 270:2693-2698, 1993.
15. Johan B. Ubbink, WJ. Hayward Vermaak, J.M. Bennett, Piet J. Becker, D.A. van Staden and S. Bissbort, "The prevalence of homocysteinemia and hypercholesterolemia in an-giographically defined coronary heart disease." Klinische Wochenschrift 69:527-534, 1991.
16. Johan B. Ubbink, J. Hayward Vermaak, Rhena Delport, An-natjie vander Merwe, Piet J. Becker and Hendrik Potgeiter, "Effective homocysteine metabolism may protect South African blacks against coronary heart disease." American Journal of Clinical Nutrition 62:802-808, 1995.
17. G. Anker, P.E. Lonning, Per Magne Ueland, Helga Refsum and E.A. Lien, "Plasma levels of the atherogenic amino acid homocysteine in postmenopausal women with breast cancer treated with tamoxifen." International Journal of Cancer 60:3o5-368, 1995.
18. Edward J. Calabrese, "Environmental validation of the homocysteine theory of arteriosclerosis." Medical Hypotheses 15:361-367, 1984.
19. William Drell and Arnold D. Welch, "Azaribine—homocysti-nemia—thrombosis in historical perspective." Pharmacy and Therapeutics 41:195-206, 1989.
20. Per Magne Ueland and Helga Refsum, "Plasma homocysteine, a risk factor for vascular disease: Plasma levels in health, disease and drug therapy." Journal of Laboratory and Clinical Medicine 114:473-501, 1989.
21. Joy A. Friedman and Johanna T. Dwyer, "Hyperhomocys-teinemia as a risk factor for cardiovascular disease in patients undergoing hemodialysis." Nutrition Reviews 53:197-201, 1995.
22. Yves Ingenbleek, Denis Barclay and Henri Dirren, "Nutritional significance in serum amino acid patterns in goitrous patients." American Journal of Clinical Nutrition 43:310-319, 1986.
23. Pierre Clopath, Virginia C. Smith and Kilmer S. McCully, "Growth promotion by homocysteic acid." Science 192:372-274, 1976.
24. Kilmer S. McCully and Bruce D. Ragsdale, "Production of
Beyond Cholesterol: The Homocysteine Theory of Arteriosclerosis
arteriosclerosis by homocysteinemia." American Journal of Pathology 61:1-11, 1970.
25. Shiela Donahue, John A. Sturman and Gerald Gaull, "Arteriosclerosis due to homocysteinemia. Failure to reproduce the model in weanling rabbits." American Journal of Pathology 77:167-174, 1974.
26. Kilmer S. McCully and Robert B. Wilson, "Homocysteine theory of arteriosclerosis." Atherosclerosis 22:215-227, 1975.
27. Fumio Kuzuya and N. Yoshimine, "Homocysteine theory of arteriosclerosis." Donixakukoka {Journal of Japan Atherosclerosis Society) 6:135-139, 1978.
28. Laurence A. Harker, Russell Ross, Sherrill J. Slichter and C. Ronald Scott, "Homocystine-induced arteriosclerosis. The role of endothelial cell injury and platelet response to its genesis." Journal of Clinical Investigation 58:731-741, 1976.
29. Josef Hladovec, "Experimental homocystinemia, endothelial lesions and thrombosis." Blood Vessels 16:202-205, 1979.
30. Pierre Rolland, Alain Friggi, Andre Barlatier, Philippe Piquet, Valerie Latrille, Marie M. Faye, Joel Guillon, Philippe Char-piot, Heidi Bodard, Odette Ghirnighelli, Raymond Calaf, Roger Luccioni and Danielle Garcon, "Hyperhomocysteinemia-induced vascular damage in the minipig." Circulation 91: 1161-1174, 1995.
31. Lori A. Smolin, T.D. Crenshaw, D. Kurtycz and N.J. Benev-enga, "Homocysteine accumulation in pigs fed diets deficient in vitamin B6: Relationship to atherosclerosis." Journal of Nutrition 113:2122-2133, 1983.
32. Kilmer S. McCully, "Importance of homocysteine-induced abnormalities of proteoglycan structure in arteriosclerosis." American Journal of Pathology 59:181-193, 1970.
33. Nina A.J. Carson, C.E. Dent, C.M.B. Field and Gerald E. Gaull, "Homocystinuria. Clinical and pathological review of ten cases." Journal of Pediatrics 66:565-583, 1965.
34. Kilmer S. McCully, "Macromolecular basis for homocysteine-induced changes in proteoglycan structure in growth and arteriosclerosis." American Journal of Pathology 66:83-95, 1972.
35. Kilmer S. McCully, "Homocysteine metabolism in scurvy, growth and arteriosclerosis." Nature 231:391-392, 1971.
36. Jer-Chia Tsai, Mark A. Perrella, Masao Yoshizumi, Chung-Ming Hsieh, Edgar Haber, Robert Schlegel and Mu-En Lee, "Promotion of vascular smooth-muscle growth by homocysteine: A link to atherogenesis." Proceedings of the National Academy of Sciences USA 91:6369-6373, 1994.
37. Kilmer S. McCully, "Homocysteine thiolactone metabolism in malignant cells." Cancer Research 36:3198-3202, 1976.
38. Kilmer S. McCully, "Growth disorders and homocysteine metabolism." Annals of Clinical and Laboratory Science 5:147-152, 1975.
39. Dietrich Matthias, Curt H. Becker, R. Riezler and Paul H. Kindling, "Homocysteine induced arteriosclerosis-like alterations of the aorta in normotensive and hypertensive rats following application of high doses of methionine." Atherosclerosis 122:201-216, 1996.
40. Kilmer S. McCully and Angelina C.A. Carvalho, "Homocysteine thiolactone, N-homocysteine thiolactonyl retinamide and platelet aggregation." Research Communications in Chemical Pathology and Pharmacology 56:349-360, 1987.
41. Peter C. Harpel, Victor T. Chang and Wolfgang Borth, "Homocysteine and other sulfhydryl compounds enhance the binding of lipoprotein(a) to fibrin: A potential link between thrombosis, atherogenesis and sulfhydryl compound metabolism." Proceedings of the National Academy of Sciences USA 89:10193-10197, 1992.
42. David E. Wilcken and Bridget Wilcken, "The pathogenesis of coronary artery disease. A possible role for methionine metabolism." Journal of Clinical Investigation 57:1079-1082, 1976.
43. Carol J. Boushey, Shirley A.A. Beresford, Gilbert S. Omenn and Arno G. Motulsky, "A quantitative assessment of plasma homocysteine as a risk factor for vascular disease." Journal of the American Medical Association 274:1049-1057, 1995.
44. Meir J. Stampfer, M. Rene Malinow, Walter C. Willett, Laura M. Newcomer, Barbara Upson, Daniel Ullmann, Peter V. Tishler and Charles H. Hennekins, "A prospective study of plasma homocysteine and risk of myocardial infarction in U.S. physicians." Journal of the American Medical Association 268:877-881, 1992.
45. Lars E. Brattstrom, Jan Erik Hardebo and Bjorn L. Hult-berg, "Moderate homocysteinemia—a possible risk factor for arteriosclerotic cerebrovascular disease." Stroke 15:1012-1016, 1984.
46. Jacob Selhub, Paul F. Jacques, Andrew G. Bostom, Ralph B. D'Agostino, Peter W.F. Wilson, Albert J. Belanger, Daniel H. O'Leary, Philip A. Wolf, Ernst J. Schaefer, and Irwin H. Rosenberg, "Association between plasma homocysteine concentrations and extracranial carotid artery stenosis." New England Journal of Medicine 332:286-291, 1995.
47. Robert Clarke, Leslie Daly, Killian Robinson, Eileen Naughton, Seamus Cahalane, Brian Fowler and Ian Graham, "Hyperhomocysteinemia as an independent risk factor for vascular disease." New England Journal of Medicine 324:1149-1155, 1991.
48. Ottar Nygard, Stein Emil Vollset, Helga Refsum, Inger Sten-svold, Aage Tverdal, Jan Erik Nordrehaug, Per Magne Ue-land and Gunnar Kvale, "Total plasma homocysteine and cardiovascular risk profile. The Hordaland homocysteine study." Journal of the American Medical Association 274: 1526-1533, 1995.
49. Arnold von Eckardstein, M. Rene Malinow, Barbara Upson, Jurgen Heinrich, Helmut Schulte, Rainer Schonfeld, Ekkeh-art Kohler and Gerd Assmann, "Effects of age, lipoproteins and hemostatic parameters on the role of homocyst(e)-inemia as a cardiovascular risk factor in men." Arteriosclerosis and Thrombosis 14:460-464, 1994.
50. Killian Robinson, Ellen L. Mayer, Dave P. Miller, Ralph Green, Frederick van Lente, Anjan Gupta, Kandice Kottke-Marchant, Susan R. Savon, Jacob Selhub, Steve E. Nissen, Michael Kutner, Eric J. Topol and Donald W. Jacobsen, "Hyperhomocysteinemia and low pyridoxal phosphate, common and independent reversible risk factors for coronary artery disease." Circulation 92:2825-2830, 1995.
51. E. Arnesen, Helga Refsum, K.H. Bonas, Per Magne Ueland, O.H Forde and J.E. Nordrehaug, "Serum total homocysteine and coronary heart disease." International Journal of Epidemiology 24:704-709, 1995.
52. Isabella Fermo, Silvana Vigano'D'Angelo, Rita Paroni, Guisep-pina Mazzola, Giliola Colori and Armando D'Angelo, "Prevalence of moderate hyperhomocysteinemia in patients with early-onset venous and arterial occlusive disease." Annals of Internal Medicine 123:747-753, 1995.
53. Martin Den Heijer, Ted Koster, Henk J. Blom, Gerard M.J. Bos, Ernst Briet, Pieter H. Reitsma, Jan P. VandenBroncke and Frits R. Rosendaal, "Hyperhomocysteinemia as a risk factor for deep vein thrombosis." New England Journal of Medicine 334:759-762, 1996.
54. Godfried H.J. Boers, Antony G.H. Smals, Frans J.M. Trij-bels, Brian Fowler, Jan A.J.M. Bakkeren, Henny C. Schoon-derwaldt, Win J. Kleijer and Peter W.C. Kloppenborg, "Heterozygositv for homocystinuria in premature peripheral and cerebral occlusive arterial disease." New England Journal of Medicine 313:709-715, 1985.
55. Johan B. Ubbink, "Is an elevated circulating maternal homocysteine concentration a risk factor for neural tube defects?" Nutrition Reviews 53:173-175, 1995.
56. S. Harvey Mudd, Fleming Skovby, Harvey L. Levy, Karen D. Pettigrew, Bridget Wilcken, Reed E. Pyeritz, G. Andria, Godfried H.J. Boers, Irvin L. Bromberg, Roberto Cerone, Brian Fowler, Hans Grobe, Hildgund Schmidt and Leslie Schweitzer, "The natural history of homocystinuria due to cystathionine beta synthase deficiency." American Journal of Human Genetics 37:1-31, 1985.
57. John M. Ellis and Kilmer S. McCully, "Prevention of myocardial infarction by vitamin B6." Research Communications in Molecular Pathology and Pharmacology 89:208-220, 1995.
58. Andrzej J. Olszewski and Kilmer S. McCully, "Fish oil decreases serum homocysteine in hyperlipemic men." Coronary Artery Disease 4:53-60, 1993.
Beyond Cholesterol: The Homocysteine Theory of Arteriosclerosis
59. Andrzej J. Olszewski, Wictor B. Szostak, Magda Bialkowska, Stefan Rudnicki and Kilmer S. McCully, "Reduction of plasma lipid and homocysteine lex els bj pyridoxmc, folate. cobalamin, choline, riboflavin and troxerutin in atherosclerosis." Atherosclerosis 75:1-9, 1989.
60. P. Ambrosi, A. Barlater, G. Habib, Danielle Garcon, B. Kreit-man, Pierre H. Roland, S. Saingra, D. Metras and Roger Luccioni, "Hyperhomocysteinemia in heart transplant recipients." European Heart Journal 15:1191-1195, 1994.
61. Peter B. Berger, James D. Jones, Lyle J. Olson, Brooks S. Edwards, Roger P. Frantz, Richard Rodeheffer, Bruce A. Kottke, Richard C. Daly and Christopher G.A. McGregor, "Increase in total plasma homocysteine concentration after cardiac transplantation." Mayo Clinic Proceedings 70:125-131, 1995.
62. Andrzej J. Olszewski and Kilmer S. McCullv, "Homocysteine metabolism and the oxidative modification of proteins and lipids." Free Radical Biology and Medicine 14:683-693, 1993.