The Homocysteine Revolution
The medical discovery of the causative role of homocysteine in vascular disease and the hypothesis implicating homocysteine in the pathogenesis of arteriosclerosis were new and startling when first proposed in 1969. 1 In the succeeding years a new pathway of medical research was uncovered that relates homocysteine to arteriosclerosis, cancer, aging, normal growth and development and the degenerative diseases of aging. This pathway was taken by the author, his colleagues and students and by increasing numbers of other independent medical investigators. This "trail of research" was inspired by and expanded upon past discoveries by Vincent DuVigneaud and other pioneers in knowledge about homocysteine and related sulfur amino acids. 2
What began as a chance observation in children with rare inherited diseases of homocysteine metabolism now promises to unlock mysteries that have long puzzled medical scientists. In 1969 it was difficult to predict the extent to which a seemingly unimportant byproduct of protein breakdown, homocysteine, could unite understanding of how vital nutrients, including vitamins A, C, B12, B2, folic acid, E and B6 function within cells and tissues to control the basic processes of life itself. These processes include cellular respiration, cell and tissue growth, cell removal and replacement, maintenance of connective tissues, expression of genetic information, reproduction and embryonic development. Over three decades ot medical research on homocysteine have yielded a true revolution in the understanding of the role of this amino acid in living cells and tissues, in aging, and in the degenerative diseases of cancer and arteriosclerosis.
During the past decade and especially since 1990 a large number of human studies, both epidemiological and clinical, have confirmed the validity of the homocysteine approach to arteriosclerosis. A veritable avalanche of new data has supported its basic tenets, as explained in previous chapters. This work by increasing numbers of international medical investigators has begun to rewrite the current concepts of the cause of arteriosclerosis in human populations. The simplicity of the homocysteine theory has provided a convincing explanation for the decline in coronary heart disease mortality in the U.S. since the mid-1960s and led to important new proposals for the control of the disease in the population. Assuring adequacy of dietary intakes of vitamin B6 and folic acid is now understood as a promising strategy for prevention of vascular disease through control of elevated blood homocysteine levels in the population at risk." 5 Dietary intake of vitamin B12 is adequate for the population, except for a very few strict vegetarians who consume no animal products at all. The decision of the U.S. Food and Drug Administration to add folic acid to enriched foods beginning in 1998 is a milestone in the long journey toward substantial reduction of arteriosclerosis in the U.S. population. A goal for the future is for the F.D.A. also to require addition of vitamin B6 to enriched foods to replace the quantity of the vitamin lost in food processing and preservation. Not only will the addition of these two vital nutrients to the food supply continue the decline in arteriosclerosis, it will improve the general health and promote increased life expectancy in the population.
The important studies of the relation of risk factors for arteriosclerosis to homocysteine levels in susceptible populations, as exemplified by the Hordaland Homocysteine Study, 4 give guidance for prevention of the disease in those at risk, as explained in Chapter 5. These studies explain the supreme importance of lifelong consumption of an optimal diet combined with cessation of smoking, adequate exercise and other simple measures. Any person can assure adequate dietary intake of vitamins, minerals, fiber, antioxidants and other beneficial nutrients to prevent or delay the onset of elevated blood homocysteine levels. This strategy for prevention of arteriosclerosis is revolutionary because it shifts the emphasis toward consumption of beneficial nutrients and places the consumption of sugars and fats in a new context. These highly processed foodstuffs are a rich source of calories, but a poor source of the vitamins and other nutrients which prevent induction of arteriosclerosis by homocysteine. This approach is revolutionary because it emphasizes that depletion of essential nutrients is the key factor in understanding the origin of the disease. Until recently the traditional view has been to incriminate consumption of cholesterol, fats and sugars because of supposed damage to arterial cells and tissues by these foods. Today, we can view arteriosclerosis as a deficiency disease, rather than a disease of excess.
A wide range of discoveries by medical scientists over the past three decades in basic biochemistry, cell physiology and oncology has shown how the homocysteine revolution is illuminating understanding of cancer and the aging process as well as arteriosclerosis. These exciting discoveries are in the early stages of development because their application to human studies is as yet very limited. Nevertheless, a theoretical basis for understanding, based upon experiments with the cells and tissues of animals, must be developed prior to initiating human studies.
This final chapter is written for the reader who has some acquaintance with the concepts and terminology of biochemistry and basic medical science. It gives a general description of how experiments with cells and tissues led to the synthesis of thioretinamide and thioretinaco, substances of great importance to cellular biochemistry and physiology. These new compounds were discovered by organic synthesis from homocysteine in its reactive thiolactone form, from vitamin A in its retinoic acid form, and from vitamin B12 in its cobalamin form. The functions of thioretinamide and thioretinaco are the focal points in understanding how homocysteine participates in controlling cell division, cell function, and the biochemical processes of living cells and tissues. These substances orchestrate the functions of many vitamins and other trace substances, including folic acid, flavins, ascorbic acid, retinoic acid, cobalamin, pyri-doxine and ubiquinol in the vital processes of living cells. Understanding how thioretinamide and thioretinaco may be altered in the aging process and how their functions change in degenerative diseases of aging, such as arteriosclerosis, cancer, arthritis and autoimmune diseases, can lead to revolutionary new approaches to controlling these diseases in human populations.
Biochemistry and Physiology of Homocysteine
A century ago the brilliant medical investigator Sir Archibald E. Garrod began his studies of a rare inherited disease, alcaptonuria, which led to the concept of a genetic "error of metabolism."" In his lectures and monograph entitled Inborn Errors of Metabolism, Garrod developed the idea that a mutation in a single essential gene could cause an inherited disease because of formation of an abnormal enzyme in all cells of the body. 6 An enzyme is a protein which facilitates a specific chemical reaction within the body's cells and tissues. The chemical reactions and transformations of constituents of food within the body are known as metabolic processes or metabolism. Garrod's brilliant and productive concept has led to the elucidation and treatment of thousands of genetic diseases during the past century, as described in detail in the classic text The Metabolic Basis of Inherited Disease. 1
In the case of homocystinuria, Garrod's concept was shown to be correct by the demonstration that cells cultured from the skin of children with this inherited disease contain an abnormal enzyme, cystathionine synthase, that fails to convert homocysteine into cystathionine normally. 8 By studying these abnormal cells from a patient with homocystinuria, perhaps some clue could be observed that would explain why these children develop arteriosclerosis at an early age. The lack of the enzyme cystathionine synthase in these cultured cells might lead to an abnormality of metabolism that could explain features of this inherited disease.
As described in Chapter 3, the growth of cells in culture from patients with homocystinuria revealed that the cells produce an aggregated matrix substance called proteoglycan that has reduced solubility compared to the finely fibrillar proteoglycan produced by normal cells in culture. 9 Labelling experiments with radioactive sulfate ions showed that this aggregated matrix proteoglycan is more heavily sulfated than the proteoglycans of normal cells. 10 The molecular basis for this aggregated proteoglycan matrix was suggested to be conversion of the normal helical structure of protein to an unfolded random coil that binds more sulfate because of displacement of potassium ions by homocysteine thiolactone cation from the surface of the protein. 11 Because of the chemical properties of homocysteine thiolactone cation, this conversion of fibrillar helical proteins to aggregated unfolded proteins of decreased solubility may have wide implications in the solubility of different types of proteins. Future studies may show that not only proteoglycans but also lipoproteins, nucleopro-teins and membrane proteins may be converted to a less soluble aggregated form because homocysteine thiolactone cation and increased numbers of sulfate ions bind to the surface of these proteins. Because of the decreased solubility of aggregated proteoglycan matrix produced by cultured cells lacking cystathionine synthase activity, the accumulation of sulfated proteoglycans in the arteriosclerotic plaques of children with homocystinuria is attributed to the change in proteoglycan conformation induced by homocysteine. 1011
A second important lesson taught by study of cultured cells from children with homocystinuria is that a previously unknown pathway for activation of sulfate was discovered by using homocysteine thiolactone labelled with radioactive sulfur in the culture medium of these cells. 10 The classic pathway for formation of bound sulfate was discovered in the 1950s by Philip Robbins and the famous biochemist Fritz Lipmann. 12 In this pathway, free sulfate ions, derived by metabolism from cysteine, react with ATP (adenosine triphosphate) to form PAPS (phosphoadenos-ine phosphosulfate), which transfers bound sulfate groups to proteoglycan matrix. In the experiments with cultured cells that are deficient in cystathionine synthase, this classic pathway could not explain the rapid and complete conversion of homocysteine to sulfate because their enzyme abnormality prevents homocysteine from conversion to cystathionine, cysteine and sulfate.
Subsequent experiments suggested that the new pathway for PAPS formation in cystathionine-synthase-deficient cells involves the participation of thioretinamide, a compound synthesized from homocysteine thiolactone and retinoic acid (vitamin A acid) by organic synthesis. 13 In this scheme thioretinamide reacts with superoxide, a reactive oxygen radical, to form sulfite and retinoic acid, and sulfite is converted to sulfate by the enzyme sulfite oxidase for activation by ATP to form PAPS. 14 This scheme explains the previously discovered participation of vitamin A (retinoic acid) in the formation of PAPS and bound sulfate."
The third important lesson taught by study of cultured cystathionine-synthase-deficient cells is that the pattern of growth in the culture dish is abnormal, resembling the growth of malignant cells in culture. 9 10 When malignant cells are cultured they grow abnormally, piling up in multiple layers, whereas normal cells form a beautiful layer, one cell thick, covering the surface of the culture dish. The cystathionine-synthase-deficient cells grow in multiple layers, like cancer cells, forming a distinctive pattern with sparse areas alternating with piled up areas. When additional vitamin B6 is added to the cell culture medium, growth of the cells is stimulated and the proteoglycan matrix becomes partially fibrillar in addition to the aggregated form. If homocysteine thiolactone is added to the culture medium, the cells degenerate, detaching from the surface of the culture dish. Added vitamin B6 in the culture medium prevents the toxic effect of homocysteine on the cultured cells, because enough pyridoxal phosphate, the coenzyme form of the vitamin, is produced to activate traces of the abnormal cystathionine synthase enzyme, converting excess homocysteine to cystathionine.
These observations on growth disturbances in cystathionine synthase-deficient cells are important for two reasons. First, the smooth muscle cells of developing arteriosclerotic plaques multiply and form piled-up layers within the artery wall because of disturbed control of cellular growth. Second, the observations on abnormal cellular growth led to studies of homocysteine thiolactone in malignant cells and to the discovery of thioretinaco, as described in the next section of this chapter.
Vitamin C, also known as ascorbic acid, was first isolated, crystallized and characterized chemically by the brilliant biochemist Albert Szent-Gyorgyi in the 1930s. The human vitamin deficiency disease caused by lack of vitamin C in the diet is called scurvy, a fatal illness that develops about two to four months after deprivation of fruits and vegetables. A characteristic abnormality of the blood vessels in scurvy is bleeding, especially from the gums and into the skin. Investigation of experimental scurvy in guinea pigs caused by feeding a vitamin C-defi-cient diet shows that the sulfated proteoglycans of blood vessel walls become attenuated and thinned. As a result the blood vessels begin to leak because of lack of cohesive-ness of the artery walls and because of abnormalities of the platelets, the specialized blood-cell particles that are needed for blood clotting. In addition, the animals abruptly stop growing because the matrix of the cartilage in the growth centers of the bones fails to bind sulfate when deprived of vitamin C.
Homocysteine reacts more rapidly with ascorbic acid than its chemical relative, cysteine, in biochemical experiments. Furthermore, ascorbic acid levels within cells are known to change characteristically during cell division. In some ways the abnormalities of the blood vessels and platelets in scurvy are opposed to the arteriosclerosis and increased reactivity of platelets found in children with ho-mocystinuria. In addition, the cell cultures from children with homocystinuria reveal a new pathway for conversion of the sulfur atom of homocysteine thiolactone to sulfate. By studying the biochemistry of homocysteine in scurvy, therefore, the function of ascorbic acid in the reaction of homocysteine with oxygen could be investigated.
Experiments with normal guinea pigs and those deprived of vitamin C in their diet showed that experimental scurvy impairs the reaction of homocysteine with oxygen. 16 After radioactively labelled homocysteine thiolactone was injected into the guinea pigs, the livers of the scorbutic guinea pigs were found to accumulate the unoxi-dized form of homocysteine, but in the livers of normal guinea pigs, the homocysteine thiolactone reacted with oxygen to form homocysteic acid and sulfate. By synthesis of labelled homocysteic acid by reaction of homocysteine with hydrogen peroxide, it was further found that normal liver converts the sulfur atom of homocysteine to PAPS, phosphoadenosine phosphosulfate, as shown by isolation of the labelled sulfating coenzyme.
The results of these biochemical experiments showed that vitamin C is necessary for the reaction of homocysteine with oxygen to form the active form of sulfate, phosphoadenosine phosphosulfate. Subsequent work with thioretinamide showed that a form of vitamin A, retinoic acid, is needed for the biochemical reaction of homocysteine with oxygen. 14 15 The importance of retinoic acid in the formation of adenosine triphosphate (ATP) was shown in cultured cells. 17 These experiments, therefore, united homocysteine with formation of ATP and PAPS, combined with participation of vitamins A and C. ATP is an important form of chemical energy within cells formed by reaction of oxygen with hydrogen from the carbon atoms of foods, a process known as oxidative phosphorylation. These experiments showed that homocysteine is intimately involved with the basic processes of living cells by which food is burned with oxygen to produce chemical energy in the form of ATP.
In the growth arid development of bone and cartilage in young animals, the process of adding sulfate to cartilage matrix is under the control of growth hormone secreted by the pituitary gland. In young animals growth hormone causes release of a protein factor known as insulin-like growth factor, which increases the binding of sulfate to the matrix of cartilage. This explains why some children with homocystinuria have accelerated growth, causing them to develop increased stature, long arms, legs, fingers and toes. Homocysteine acts in cartilage to increase binding of sulfate, mimicking the action of insulin-like growth factor. 18
In experiments with rats from which the pituitary gland had been removed surgically, homocysteic acid, the form of homocysteine containing three extra oxygen atoms, stimulates the growth of the animals, as measured by tail growth or by cartilage growth. 19 In order to achieve growth, the animals were also treated with thyroxine, the thyroid hormone that is required for normal growth and development. In the plasma of the growing rats, insulinlike growth factor was demonstrated by increased binding of sulfate when incubated with fragments of pig cartilage. The homocysteic acid and thyroxine treatments were promoting the growth of these animals in the absence of pituitary growth hormone by stimulating the release of insulinlike growth Factor from the liver.
Earlier experiments had shown that feeding or injecting homocysteic acid into normal rabbits or guinea pigs increased the growth rate of these animals, producing giant rabbits in one experiment. 20 To this day pathologists who were present in the 1970s when the experiments were done remember the extraordinary appearance of these apparently normal but giant rabbits. In experiments with cultured cartilage cells from chick embryos, homocysteic acid was found to increase binding of sulfate to cartilage, an action that mimics the effect of insulin-like growth factor on cartilage. 21 In several patients with untreated homo-cystinuria with accelerated growth, increased secretion of growth hormone was demonstrated. 22 Taken together, the physiological experiments and other observations on the action of growth hormone and insulin-like growth factor show that homocysteine plays a vital role in the control of normal growth.
The experiments with cell cultures from children with homocystinuria showed that the abnormal aggregated proteoglycan matrix produced by these cells binds more sulfate than the proteoglycan of normal cells, an effect that is reminiscent of the effect of insulin-like growth factor on cartilage matrix. 9 10 The experiments concerning reaction of homocysteine thiolactone with low-density lipoprotein showed that the thiolated lipoprotein had become aggregated, smaller, more dense and moved more rapidly in an electric field. 23 Additional observations on the properties of malignant cells showed that overproduction of homocysteine thiolactone by cultured cancer cells causes binding of homocysteine to proteins, membranes, nucleo-proteins and proteoglycans, producing aggregation, condensation, stickiness and other abnormalities. 24 These effects of homocysteine thiolactone on proteoglycans of cystathio-nine-deficient cells, aggregated lipoproteins, membrane proteins, nucleoproteins and other constituents of malignant cells are explained by its effect on secondary structure and by binding of sulfate to the charged form of homocysteine thiolactone adhering to the surface of these macromolecules.
Homocysteine and Cancer
Since the discovery of the double helix of DNA in 1953 by Francis Crick and James Watson, 25 the general opinion of most medical investigators has been that alteration of the genes of DNA by the action of carcinogenic chemicals, radiation, oncogenic viruses or other carcinogenic factors is responsible for induction of the malignant state of cellular growth in cancer. In the first half of the 20th century, in contrast, the favorite opinion regarding induction of cancer among medical scientists was the altered respiration theory championed by Otto Warburg. 26 According to this theory, carcinogenic factors act by alteration of the process by which cells combine oxygen with electrons from the carbon atoms of food to produce carbon dioxide, water and chemical energy in the form of ATP.
Over the years there has been no apparent way to reconcile these two major theories of the cause of cancer. In the 1950s and early 1960s Albert Szent-Gyorgi extracted undefined substances from normal tissues that retard the growth of malignant cells in animals by altering the way electrons are processed within living cells. 27 He believed that a substance he termed "retine" is lost from normal cells when they become malignant. If he had been able to establish the chemical identity of this substance, his findings may have been able to unite the genetic and respiration theories of the origin of cancer.
Closely related to the genetic theory of carcinogenesis is the participation of growth-regulating and growth-controlling factors called oncogenes. Over 100 genetic factors have been found to control protein and polypeptide gene products that become activated or deactivated in malignant cells. An example is the P53 oncogene which has been found to become altered during carcinogenesis so that its gene product is unable to restrain the growth of normal cells. This oncogene has been found to be involved frequently in cases of colon, lung and ovarian cancer, among others, allowing growth of these malignancies. Another example is the oncogene involved in breast cancer, BRCA-1, which is altered in familial cases of breast cancer.
When these many oncogenes become altered by genetic, dietary, chemical or environmental factors, there is interference with the normal control of the process of cell division, allowing unrestrained growth of a particular type of cell within the body. Oncogenes also become aliered or activated in other non-malignant types of cellular growth, for example, in regeneration of liver cells in animals. The oncogene theory does not explain at a detailed molecular level how relaxation of the control of cellular growth allows the unrestrained nature of malignancy to develop and to become autonomous, allowing spread and growth of cancer cells throughout the body.
Experiments with cultured malignant cells, inspired by the resemblance of the growth pattern of cells from children with homocystinuria to the growth pattern of cancer cells in culture, resulted in the discovery that the sulfur atom of homocysteine thiolactone fails to react with oxygen to form sulfate in these cells. 24 This finding suggested that normal cells in culture are able to convert homocysteine thiolactone to sulfate because they contain a hypothetical homocysteine thiolactonyl derivative which allows this chemical reaction to proceed. In cancer cells, on the other hand, homocysteine thiolactone accumulates within the cells and reacts with proteins and other cellular constituents, including proteoglycans, nucleoproteins and membrane proteins, to cause aggregation of and alteration in solubility and function of a wide range of cell components. In later experiments, free homocysteine thiolactone was demonstrated in human tumors 28 and in cultured cancer cells. 29
The result of accumulation of excess homocysteine thiolactone within cancer cells causes widespread abnormalities of cellular function according to present concepts. The reaction of homocysteine thiolactone with membrane proteins causes diffuse abnormalities of membrane structure, termed simplification of cellular membranes. The reaction of homocysteine thiolactone with nucleoproteins causes altered formation of specialized proteins of different cell types, for example liver, muscle or nerve cells, leading to decreased or abnormal expression of these characteristic proteins. Aggregation and decreased solubility of nucleoproteins from reaction with homocysteine thiolactone causes abnormalities in chromosome structure, chromosome numbers and appearance of nuclear chromatin, producing the abnormalities that pathologists use to recognize cancer cells under the microscope. Reaction of homocysteine thiolactone with nucleoproteins of oncogenes leads to increased or decreased expression of growth factors and growth inhibitors, causing increased cellular growth. Reaction of homocysteine thiolactone with proteoglycans and proteins of cell membranes alters the appearance of these marker substances to the immune system of the body, leading to recognition of cancer cells as significantly altered immunologically.
The chemical nature of the hypothetical homocysteine thiolactone derivative that is lost from normal cells during their transformation to cancer cells was investigated by chemical synthesis of model homocysteine compounds and by testing the ability of these new compounds to inhibit growth of cancer cells in animals. 14 In this way some insight was gained into the chemical characteristics of this hypothetical regulator of homocysteine thiolactone formation within normal cells. If the chemical model compound was able to modify this process in cancer cells by slowing their growth, then the chemical nature of the hypothetical substance within normal cells could be deduced, and the substance could be made in the laboratory by organic chemical synthesis.
The first approach to the problem was to mimic the reaction of homocysteine thiolactone with oxygen since this reaction was found to be blocked in cancer cells. 24 When homocysteine thiolactone hydrochloride was mixed with perchloric acid, a strong oxidizing substance, a new compound was found to crystallize from the solvent solution, and the chemical structure was demonstrated to be homocysteine thiolactone perchlorate by X-ray analysis of the crystals. 30 When injected into animals with transplanted malignant tumors, the substance caused the tumors to swell and degenerate, stimulating their growth.
When homocysteine thiolactone was mixed with the fatty acids oleic acid or arachidonic acid, new compounds were formed that control the growth of malignant tumors, either increasing or decreasing their growth rate. 31 This finding extended the significance of the effect of homocysteine thiolactone perchlorate since all of these substances require for their anticancer activity the property of solubility in organic solvents or fats. The interpretation is that these anticancer compounds are very likely to be bound to cellular membranes at the site of their action since most fats within cells are present in cellular membranes.
Another interesting compound formed from homocysteine thiolactone and vitamin B6, homocysteine thiolactone pyridoxal enamine, was found to decrease tumor growth if the substance was injected for two weeks prior to transplantation of the malignant tumor. This result was interpreted to indicate that the eramine compound decreased the availability of homocysteine by formation of cystathionine, slowing tumor growth. This interpretation agrees with the results of previous studies by other scientists showing that methionine, the precursor of homocysteine, is needed to prevent induction of malignant tumors.
The more detailed chemical nature of the hypothetical regulator of homocysteine formation in cells was studied by formation of new compounds with maleic anhydride and maleimide, substances that are known to mimic the effects of radiation on normal cells. Although the maleic acid and maleimide derivatives of homocysteine thiolactone inhibit the growth of malignant tumors in animals, they are moderately toxic and require administration with liposomes, a form of artificial cell membrane, for optimal anticancer activity. 32
In yet another model compound, homocysteine thiolactone was modified by reaction with a reactive oxygen-containing reagent, oxalyl chloride. The resulting oily substance, oxalyl homocysteine thiolactone, is soluble in organic solvents and fats. However, this new compound had no effect on growth of malignant tumors in animals unless it was first combined with rhodium trichloride. 33 Rhodium is a metal from the transition group which includes cobait, an essential constituent of vitamin B12. This complex of rhodium trichloride and oxalyl homocysteine thiolac-tone dissolves in fats and has a strong inhibitory effect on growth of malignant tumors in animals. However, the complex is moderately toxic and even caused the development of a new malignant tumor in one of the animals.
Taken together, the results of all of these experiments with model compounds showed that the hypothetical regulator of homocysteine formation in cells is likely to be soluble in or bound to the fats of cell membranes and to contain oxygen atoms and unsaturated carbon atoms that modify the reactivity of homocysteine thiolactone and a transition metal atom, quite possibly the cobalt atom of vitamin B12.
Vitamin A is known to modify or prevent the growth of some malignant tumors in man and animals. Vitamin A is also known to be involved in formation of PAPS and bound sulfate, as described previously in this chapter. 15 Furthermore, vitamin A in the form of retinoic acid had been found to be involved in ATP synthesis in cultured cells, potentially linking homocysteine to the process of cellular respiration by which oxygen reacts with electrons from carbon atoms of food to produce chemical energy for the cells. How could vitamin A be involved as the regulator of both homocysteine thiolactone formation and cellular respiration in normal cells?
In experiments with the free-base form of homocysteine thiolactone, a way was found to make a chemical compound with retinoic acid, the acid form of vitamin A. 13 This new compound, called thioretinamide, was found to slow the growth of malignant tumors in animals and to prevent tumor formation in animals that had been given a chemical carcinogen, urethane. The compound, thioretinamide, like vitamin A, is soluble in fats and organic solvents and has some degree of toxicity when given in large amounts to animals. The results of these experiments supported the findings on the previously studied model compounds of homocysteine thiolactone, but how could thioretinamide interact with a transition metal, such as the cobalt atom of vitamin B12, to form a regulator of homocysteine formation in normal cells? Could such a substance slow the growth of malignant tumors and also affect the process of cellular respiration in normal cells?
Vitamin B12 is formed in nature only by microorganisms and yet is required in vanishingly small amounts for all living organisms. The recommended dietary allowance for vitamin B12 is 3 meg per day for adult women and men. Vitamin B12 forms a beautiful, clear red solution and is sensitive to light, easily breaking down in the presence of strong light sources or radiant energy. Experiments with thioretinamide, the compound of homocysteine thiolactone and retinoic acid, showed that vitamin B12 makes a complex in which two molecules of thioretinamide are bound to the cobalt atom of vitamin B12. 4 When isolated from chemical synthesis, this complex, termed thioretinaco, is deep red-brown in solution and has a rich, fruity odor reminiscent of aged red wine.
Thioretinaco, the complex of thioretinamide and vitamin B12, becomes bound to cellular membranes, inhibits growth of malignant tumors in animals and prevents growth of tumors in animals given the chemical carcinogen urethane. It was exciting to find that thioretinaco is nontoxic when injected into normal tissues but strongly inhibits growth of human cancer cells in athymic, immunologically compromised mice when injected into the growing tumors. 35
An extremely interesting effect of thioretinaco on normal mice is that injections of the compound cause the animals to become hyperactive, to consume excess food, to excrete excess urine and feces and yet fail to gain weight normally. Thioretinaco speeds up the processes of food consumption, waste excretion and activity so that weight gain is prevented in the animals. When the thioretinaco injections are stopped, the animals calm down, eat less food, excrete less and resume normal weight gain.
In considering the chemical structure of thioretinaco, it is apparent that the complex is bound to the membranes of mitochondria and endoplasmic reticulum within cell cytoplasm. Mitochondria are the power plants of cells which make chemical energy in the form of ATP by burning the electrons of the carbon atoms of food with oxygen that is transported to cells by the hemoglobin of red blood cells. Endoplasmic reticulum consists of layers in membranes within the cytoplasm of cells, where chemical formation of a wide variety of cellular constituents is accomplished and where toxic chemicals and carcinogens react with oxygen to become activated and excreted from cells. Thioretinaco is bound to these membranes, oriented in such a fashion that the sulfur atoms of the two thioretinamide groups reach the inner membrane surface and the phosphate group of cobalamin reaches the outer membrane surface. 14 Thioretinaco is anchored to the membrane by binding of its thioretinamide groups to the fats, cholesterol and phospholipids of the core of the membrane. In this way the many proteins of the membrane can interact with the cobalt atom of the vitamin B12 (cobalamin) group and the sulfur atoms of the thioretinamide groups of thioretinaco.
A breakthrough in understanding occurred when it was discovered that ozone, a highly reactive triple form of oxygen, can bridge the two sulfur atoms of thioretinaco, forming a cluster of five oxygen atoms that are bound to the active site. 14 Because of the positions of the oxygen and suifur atoms of this thioretinaco ozonide oxygen complex, the active site is capable of binding ATP by two of its phosphate groups. When ATP is formed by reaction of ADP and phosphate on the FjFq complex of inner mitochondrial membranes, the binding to the thioretinaco ozonide oxygen active site pulls the ATP off of the FiF 0 complex and releases it into the inner compartment of the mitochondrion. This interaction causes electrons to flow from the proteins of the membrane into the ubiquinone (coenzyme Q10) of the membrane. The electrons are attracted to the thioretinaco ozonide oxygen complex through the thioretinamide and corrin (vitamin B12-like) groups, where they are added to the bound oxygen to form oxygen radicals.
Vitamin C stabilizes the oxygen radicals by forming mo-nodehydroascorbate, a stable radical form of the vitamin. These bound oxygen radicals cause hydrogen ions (protons) to flow through the F,F 0 complex, where they form water from the bound oxygen through several intermediate forms. During this process six molecules of ATP are formed for each oxygen molecule that is converted to two water molecules. The net result of this process is that chemical energy in the form of ATP is formed by the coupled reduction of oxygen to water. In summary, this complex process occurring in mitochondria allows the electrons from the carbon atoms of food molecules to combine with oxygen to produce chemical energy in the form of ATP for use in powering the vital functions of living cells.
Working independently, scientists in Russia discovered that a chemically modified form of vitamin B12 could interrupt the process of oxidative phosphorylation, preventing the orderly reaction of electrons and protons with oxygen and formation of ATP in mitochondria. 36 This analogue of vitamin B12 presumably interferes with the thio-retinaco ozonide oxygen active site by competing with and short-circuiting its function in producing ATP. Other supporting evidence comes from the observation that an ozonized form of ADP reacts with phosphate to form ATP in a chemical system. 37 Over 30 years ago scientists in Germany and Wisconsin discovered that oxidized sulfur compounds, including homocysteine thiolactone and methionine, are active in producing ATP from ADP and phosphate in a chemical system. 3839 Finally, scientists in India found that rats consuming a high-fat, high-cholesterol diet had decreased ability to form ATP in their livers, and treatment of the rats with vitamin B12 restored normal oxidative phosphorylation. 40 Very recently scientists in Germany found that homocysteine in rats causes abnormalities of mitochondria similar to those found in the livers of children with homocystinu-ria during the earliest stages of damage to arterial walls in arteriosclerosis. 41 All of these scientific studies support the crucial role of homocysteine in the function of mitochondria and point to the importance of these effects in the early stages of arteriosclerosis.
This biochemical theory of cellular respiration, explaining how cells burn the electrons from food with oxygen to produce chemical energy, has profound implications both for the induction of cancer by carcinogenic agents and for the induction of arteriosclerosis by the effects of increased homocysteine. In the case of cancer, the loss of thioreti-naco from the membranes of cancer cells allows a buildup of reactive oxygen radicals within cancer cells. These reactive oxygen radicals interact with DNA, causing the chromosomal damage and gene mutations that are found in malignant cells. Reactive oxygen radicals also damage proteins, fats and other components of cancer cells. Experiments with transformed cells and cancer cells showed that methionine could partially restore the normal processing of oxygen and carbon within these cells. 42 This effect may be due to partial restoration of residual thioretinaco formed from increased methionine within these malignant cells.
The French embryologist L. Rapkine, working in the early 20th century, was able to deduce the extreme importance of normal oxygen processing in the development of embryonic cells during cell division. He discovered that during mitosis, the distribution of chromosomes to daughter cells during cell division, reactive oxygen radicals are greatly increased within dividing cells until the two new daughter cells are reconstituted. 43 This finding means that the normal process of oxidative phosphorylation is temporarily interrupted during cell division. A new complex of homocysteine thiolactone and cobalamin, named thioco, was found to promote cell division in normal and malignant cells, whereas thioretinaco prevents cell division. 44 Thus during cell division thioretinaco is temporarily converted to thioco, allowing the accumulation of oxygen radicals that Rapkine discovered, because of conversion of thioretinaco to thioretinamide and the cobalamin component of thioco. 45 In this way the normal process of cell division is regulated by alternation between a complex which processes electrons, thioretinaco ozonide, and a form of homocysteine thiolactone and cobalamin (thioco) which allows accumulation of oxygen radicals during cell division.
This experimental and theoretical explanation of the function of the homocysteine derivatives thioretinamide, thioretinaco and thioco in cellular growth and in processing of oxygen within cells allows new interpretations of the induction of cancer. What began as an observation of abnormal metabolism of homocysteine thiolactone in cancer cells 24 now constitutes a coherent theory that explains how carcinogenic agents induce cancer by depletion and inacti-vation of these newly discovered homocysteine compounds from the membranes of cells. 14
Some examples of carcinogenic agents which lead to depletion of thioretinaco from membranes of malignant cells, according to this theory, are dietary methionine deficiency and the carcinogenic chemical ethionine, both of which interfere with normal processing of methionine within target cells 14 Cultured malignant cells characteristically have low levels of adenosyl methionine and elevated levels of adenosyl homocysteine within their cytoplasm. The DNA of malignant cells is characteristically depleted of methyl groups in cancer cells, an effect that is attributed to lowered levels of adenosyl methionine. Finally, the demonstration that homocysteine thiolactone is carcinogenic when painted on the skin of mice 46 or injected into animals given a chemical carcinogen 34 supports the theoretical formulation of induction of cancer by loss of thioretinaco and conversion to thioco in cellular membranes.
In summary, the discoveries of the role of homocysteine thiolactone, thioretinamide, thioretinaco and thioco in malignant cells offer a new way to unite the respiration, genetic and oncogene theories of the induction of cancer. The loss of thioretinaco ozonide from cell membranes leads to the abnormal respiration of mitochondria in malignant cells, with secondary accumulation of reactive oxygen radicals. The loss of thioretinaco also allows excessive synthesis of homocysteine thiolactone from methionine, causing aggregation and altered activation of nucleopro-teins, abnormalities of cellular membranes, altered immunological recognition and increased growth potential through increased activation of oncogenes and increased formation of thioco.
Homocysteine and Aging
Except for children and young adults who frequently have illusions of eternal youth and immortality, most adults are very concerned about the nature of the aging process, how aging increases risk of disease, and how aging leads to deterioration of functions of the organs of the body. In many respects, the aging of one's body is as mysterious a process as the processes of birth, development, reproduction and death. Comparison of the bodies of newborns or young children with young adults focuses attention on aspects of development, maturation and reproduction. Comparison of the bodies of young adults with persons of advanced age calls attention to the profound and dramatic changes, with aging, in the appearance and function of the skin, eyes, ears, brain, heart, bones, muscles, kidneys and every other organ of the body.
Can the fundamental nature of the aging process be understood from the point of view of the many biochemical and physiological changes associated with aging? What are the current theories of aging which can explain the striking changes in appearance and function of all organs and tissues of the body? What inborn processes limit life span? Can these processes be accelerated or retarded? What is healthful aging and how can understanding of the functions of homocysteine, methionine, thioretinaco and adenosyl methionine contribute to promotion of healthful aging? Is it possible to slow the aging process and achieve healthful enjoyment of advanced years?
The miracle of living organisms, including human beings, is that the living cells and tissues of the body are surrounded by oxygen, a highly reactive gas which interacts with the nutrients of ingested food to power the growth, development, maintenance and declining function of all organs of the body. By combining oxygen with the electrons from the carbon atoms and other constituents of food, enough ATP is generated to operate all of the enzymes, membranes, chromosomes and other components of living cells in a continual process of renewal and regeneration of all the body's tissues.
In the aging process, the ability of the cells and tissues of living animals and humans to handle the reactive gas oxygen begins to decline. Less ATP becomes available to run the machinery of the body, and oxygen in the form of reactive oxygen radical forms begins to attack and degrade the vital constituents of cells and tissues. Less oxygen can be utilized by the tissues of aging animals and humans for maintaining the functions of the body's organs. For example, less ATP is available to power contractions of heart and muscles. Because less ATP is available, the function of brain and nerves to coordinate mental activity and muscular movements declines in advanced age. Because of the inability of aging cells and tissues to utilize oxygen efficiently, reactive oxygen radicals gradually accumulate and begin to degrade all cellular constituents by reaction with the fats of membranes, DNA of chromosomes, sugars of carbohydrates and amino acids of proteins. The principal method of extension of life span in experimental animals is life-long caloric restriction in the diet. By limiting the total amount of food that is converted to ATP by burning of hydrogen with oxygen, the generation of reactive oxygen radicals may be curtailed, decreasing damage to cells and tissues, allowing prolonged maximal survival in the range of 10 to 20 percent.
An example of a chemical degradation product of aging is the pigment called lipofuscin. This pigment gradually accumulates within the cells of aging people through the degradation of the membranes of mitochondria of cell cytoplasm. Lipofuscin is produced by reaction of membrane components with oxygen radicals, forming highly oxidized, polymerized fats and proteins. Because cells cannot dispose of the lipofuscin pigment, it gradually accumulates within many cells of the body during aging. Experimentally, lipofuscin accumulates within the cells of animals deprived of dietary vitamin E, an antioxidant vitamin that retards oxygen radical reactions.
The radiochemical theory of aging based on the gradual reaction of cellular constituents with oxygen radicals was introduced in the 1950s. 4748 More recently the loss of enzymatic function of proteins because of degradation by oxygen has been discovered in the cells and tissues of aging animals. 49 Many other examples of the reaction of oxygen with cellular constituents during aging attest to the soundness of the radiochemical theory of aging. However, the underlying reason for the loss of ability of senescent tissues to utilize oxygen normally and to prevent accumulation of toxic oxygen radicals is not explained in detail by this theory.
In the previous section, the theory which explains the participation of homocysteine in the process of oxygen utilization and ATP production is explained in detail. Thi-oretinaco, which is composed of two thioretinamide groups bound to vitamin B12, reacts with ozone and oxygen to provide an active site for burning of the electrons of food with oxygen to produce water and chemical energy in the form of ATP. In the aging process the decline in the ability of cells and tissues to consume oxygen and ATP was suggested to be the gradual loss of thioretinaco from the membranes of mitochondria and microsomes of all cells of the body. 45 According to this theory, because the active site for energy production by oxidative phosphorylation is gradually lost in aging, reactive oxygen radicals produced from incomplete reaction of oxygen with electrons and protons accumulate within cells and escape into the tissues of aging animals. 50 This hypothesis explains why reactive oxygen radicals accumulate in aging tissues, damaging the fats of membranes, the proteins of enzymes and the DNA of chromosomes, causing chemical deterioration of all of the chemical constituents of the body. 4748
The normal defenses of cells and tissues against excess oxygen radicals are dependent upon antioxidant nutrients, such as vitamin E and carotenoids like lycopene and carotene of vegetables, glutathione within cell cytoplasm and the enzymes superoxide dismutase and catalase. All of these chemicals and enzymes inactivate and dispose of reactive oxygen radicals by direct chemical or enzymatic effects. In aging this complex protective system is gradually overwhelmed by excess oxygen radicals because of loss of the thioretinaco ozonide active sites from cellular membranes.
Many of the epidemiological studies of arteriosclerosis have shown that human blood levels of homocysteine gradually increase with age. 4 Early studies with animals showed that homocysteine thiolactone accumulates in the livers of older animals. 51 Furthermore, the dimerized and oxidized homocysteine form, homocystine, of predominates in the livers of older animals. The reactive mono-meric form of homocysteine which has not reacted with oxygen predominates in the livers of young animals. The decreased ability of aging tissues to prevent gradual accumulation of homocysteine thiolactone and to prevent reaction of homocysteine with oxygen is also related to gradual loss of thioretinaco from cellular membranes, according to this theory. 45,50 Therefore, homocysteine thio-lactone gradually accumulates in aging tissues, and blood homocysteine increases because of the increased reaction of homocysteine thiolactone with water and the transport of homocysteine from cells into body fluids.
The master regulator of levels of methionine within cells, adenosyl methionine, gradually declines within the aging brain, liver and other organs as well as in the blood of man and animals. 5254 Recent studies have shown that adenosyl methionine is lower in the blood of patients with coronary artery disease than in controls. 55 Thioretinaco ozonide was suggested as an alternative process by which methionine reacts with ATP to form adenosyl methionine. 45 As thioretinaco is lost from cellular membranes during aging, the cells and tissues gradually lose their ability to maintain normal levels of adenosyl methionine because of decreased ability of residual thioretinaco ozonide to form this compound from methionine. Adenosyl methionine is the substance which controls the flow of methyl groups (one-carbon fragments) to modify the chemical structures of hormones, DNA and other vital constituents of cells and tissues. The efficiency of this methylation process by adenosyl methionine declines with aging. Some examples of this process are the decreased production of melatonin, epinephrine and other neuroendocrine hormones in aging animals 56 and the decreased methylation of DNA within senescent cells in culture. 57
As discussed previously, increased blood levels of homocysteine cause vascular damage and arteriosclerosis, and the depletion of thioretinaco ozonide from the membranes of normal cells by carcinogenic agents leads to abnormal cellular growth potential and abnormal utilization of oxygen by malignant cells. During the aging process the gradual loss of thioretinaco ozonide from cellular membranes leads to increased production of homocysteine thiolactone and its complex with vitamin B12, thioco. Dietary imbalance of methionine, folic acid and pyridoxine increases the conversion of methionine to homocysteine during ath-erogenesis, and the loss of thioretinaco ozonide from cellular membranes during aging increases the reaction of homocysteine thiolactone with low-density lipoprotein, explaining the close correlation of susceptibility to arteriosclerosis with age. Similarly, the loss of thioretinaco ozonide from cellular membranes during the aging process increases susceptibility of cells to malignant transformation by carcinogenic chemicals, radiation or viruses, explaining the close correlation of susceptibility to cancer with age. In a similar way, overproduction of homocysteine during aging may increase susceptibility to arthritis and other autoimmune diseases of aging by interference with the function of immune cells 58 and lead to alteration of antigenic properties of tissues in the autoimmune diseases of aging. 45
Although the presumed loss of thioretinaco ozonide from cellular membranes explains important aspects of changing oxygen utilization, energy production and degeneration of cellular components with aging, many additional features of senescence and senility need more complete elucidation by medical science. The degeneration of elastic tissue, the coarsening of collagen fibers of skin and the degeneration of cartilage and bone produce some of the most striking changes in appearance and function of the body in aging. Homocysteine may have a role in these aging changes by effects on the structure of elastin and collagen, which are as yet incompletely understood. 4550
The very characteristic failure of contractility of heart muscle, leading to congestive heart failure in many aged persons, may be related to loss of oxygen utilization and ATP formation because of loss of thioretinaco ozonide from the membranes of heart cells. This interesting possibility needs careful exploration by medical investigators in the future. A striking feature of some children with homocystinuria is the lightening of hair color that is reversed by vitamin B6 therapy. 59 Homocysteine interferes with normal melanin pigment formation by interaction with copper, 60 and this effect may have relevance to the graying of hair in normal aging. The role of homocysteine in the rare human diseases of accelerated aging, progeria and Werner's syndrome, has not as yet been determined. Recently the defective gene coding for helicase, an enzyme controlling DNA structure, has been described in Werner's syndrome. 61
Some other theories of aging are the "error catastrophe" theory of cumulative damage to genes and proteins, 62 the DNA-genetic control theory of aging 63 and the neuroendocrine theory of aging. 56 The error catastrophe theory predicts an accumulation of proteins of abnormal structure because of gradual accumulation of genetic damage from interaction of reactive oxygen radicals with DNA, causing erroneous amino acid sequences of proteins. Except for recent evidence of oxidative changes in enzyme structure with aging, presumably caused by reaction of oxygen with proteins, 49 no clear evidence has been found to support the error catastrophe theory.
The DNA-genetic control theory of aging emphasizes the important role of evolution and genetic control of the life span of animals. 63 Evolution has programmed senescence and mortality into the genetic control of life span so that through death senescent parents make ecological space available for their offspring. Recent studies with fruit flies and roundworms have suggested genetic control by genes that significantly prolong life span. The mode of action of the products of these genes controlling aging is suggested by the finding of extension of life span in transgenic fruit flies containing extra genes for superoxide dismutase and catalase. 64 This result generally supports the important role of reactive oxygen radicals in aging.
The neuroendocrine theory of aging generally attributes control of the aging process to declines in the secretion of a variety of hormones, including melatonin, dehydroe-piadrosterone, testosterone, estrogen and others. The theory generally presumes that a brain center such as the hypothalamus is responsible for the decreased secretion of multiple hormones during aging. While there is no doubt about the decline in secretion of many hormones during aging, there is little evidence for the primary function of the brain in coordinating the many changes in cells throughout the senescent body through these hormonal changes. Secondary effects of oxygen radicals, DNA with altered expression, or loss of thioretinaco ozonide and increased formation of homocysteine thiolactone, however, may well involve altered brain function and declining production of many hormones under the control of brain centers.
As explained in Chapter 5, the lifelong consumption of an optimal diet for prevention of arteriosclerosis decreases risk of vascular disease by counteracting the elevation of blood homocysteine levels. Although medical science currently has no specific therapy or preventive strategy for extension of maximum life span, decreased risk of arteriosclerosis will contribute in a major way to the achievement of healthful aging and increased life expectancy. Lifelong consumption of an optimal diet will also decrease risk of developing cancer, arthritis and other degenerative diseases of aging. In this way the final decades of human life may be enjoyed with relative freedom from disease, even though total life span may not be lengthened significantly.
Medicine for the New Millennium
Medicine in the new millennium will use the medical discovery of the causative role of homocysteine as a starting point in the rational conquest of the diseases of aging. Improved methods will be developed for the complete, rapid, routine measurement of total homocysteine in blood. New methods for the analysis of low-density lipoprotein and homocysteine aggregates will allow closer correlation with the progression of arteriosclerosis. In this way the homocysteine theory of arteriosclerosis will become established in the 21st century as the mainstream path to understanding the cause, prevention and treatment of the most important diseases in the population.
The complex interaction of growth factors, macrophages, endothelial and smooth muscle cells and the signalling substances of immune and inflammatory cells will become understood more clearly in the atherogenic process. For example, the important function of nitric oxide, a reactive radical of one nitrogen and one oxygen atom, in relaxing blood vessels and lowering blood pressure has already been related to reaction with homocysteine to form nitrosohomocysteine. 65 The anesthetic gas nitrous oxide, the less reactive combination of two atoms of nitrogen and one atom of oxygen, interacts with vitamin B12 to decrease formation of methionine, leading to elevated blood levels of homocysteine. The more reactive nitric oxide may be found in future work to interact with thioretinaco ozonide to displace oxygen and decrease production of ATP by oxidative phosphorylation. Thus the reversible relaxing effect of nitric oxide on blood vessels may be related to decreased production of chemical energy in the form of ATP, diminishing the contractility of muscle cells of the arterial wall. Increased understanding of this area of cardiovascular physiology will help medical scientists to understand and to control the effects of hypertension and to develop new approaches to controlling congestive heart failure.
In the future the roles of Chlamydia pneumoniae, 66 cytomegalovirus and herpesvirus will be more clearly defined and related to production of polyamines of viruses from adenosyl methionine, and alteration of homocysteine formation by infected arterial wall cells. These developments will enable medical scientists to understand the importance of controlling a wide range of risk factors in prevention of arteriosclerosis.
Since malignant cells are believed to have enhanced growth potential because of the loss of thioretinaco ozon-ide from cellular membranes, medical scientists may find ways of replacing this substance within malignant cells or preventing its loss from cells that are targeted by carcinogenic chemicals, viruses or radiation. The cytokine interferon is known to prevent the toxic effect of ozone in lung tissue 67 and to decrease the production of reactive oxygen radicals in hepatitis C patients. 68 These discoveries suggest that interferon and other cytokines may be useful in enhancing the anticancer effects of thioretinaco ozonide. The mode of action of interferon may be to affect the membrane binding and orientation of thioretinaco ozonide, preserving and enhancing its function within malignant cells and preventing growth and spread of cancer cells throughout the body.
The closely related substances thioretinaco, thioretinamide and thioco control cell division and cellular growth and function. 45 A better understanding of the role of these substances in organization and function of the membranes of cells will lead to new discoveries in controlling transmission of neural impulses, contractility of heart muscle, blood vessel wall muscle cells and skeletal muscle cells. The deterioration of these functions with aging may be ameliorated by replacement or enhanced function in the membranes of cells of aging animals and man. The homocysteine derivative, homocysteic acid, containing three extra oxygen atoms, is a potent excitor of nerve function. Vitamin B12, vitamin B6 and folic acid may control human psychiatric or neurological abnormalities by affecting the formation of homocysteic acid in the nervous system. 69
The important role of folic acid in prevention of birth defects, particularly neural tube defects such as spina bifida and anencephaly, has been related to effects of homocysteine on mother and developing fetus. 70 This discovery shows that improved understanding of the control of homocysteine formation may lead to important advances in the prevention of common birth defects by introducing folic acid into the food supply of pregnant women as mandated by the U.S. Food and Drug Administration beginning in 1998. In addition, this discovery points to the importance of homocysteine in the control of normal cell division and cellular function in the developing human fetus.
One of the most debilitating aspects of human aging is the degenerative disease of bones and joints known as osteoarthritis. The only currently effective way of ameliorating the effects of this condition known to medical science is surgical removal of the hip or knee joint and implantation of artificial prosthetic joints. By understanding the effects of aging on the cells and tissues of cartilage and bone, theoretically related to loss of thioretinaco ozonide from cellular membranes, it may be possible for the next generation of medical investigators to develop strategies for prevention of osteoarthritis. At present the most promising way to prevent the disease is lifelong consumption of an optimal diet which provides the cells of bone and cartilage with balanced amounts of methionine, folic acid, vitamin B6 and vitamin B12, preventing elevated homocysteine levels. Although osteoarthritis is not caused by complications of arteriosclerosis, the beneficial effects of an optimal diet may slow progression of both degenerative diseases through similar effects on connective tissues of arteries, bones and joints.
Rheumatoid arthritis is an autoimmune disease that is related to but distinct from osteoarthritis. In rheumatoid arthritis antibodies are formed against components of bone, cartilage and synovia of joints, and the immune cells of the body attack the joints of most parts of the body, causing inflammation, fibrosis and joint destruction. Although this disease can affect children in the juvenile form of rheumatoid arthritis, most sufferers from arthritis are middle-aged or elderly. The finding of immune cells that are modified by homocysteine in this disorder' 8 may lead to a new understanding of its cause and prevention. Other autoimmune diseases of aging, including thyroiditis with Grave's disease, pernicious anemia and lupus erythematosus, are diseases that may arise because of alteration of the antigenic properties of the thyroid gland, acid-secreting cells of the stomach and circulating DNA fragments in the blood, respectively. These diseases may be shown by future work to be related to increased homocysteine thiolactone formation secondary to the effects of aging because of loss of thioretinaco from cellular membranes.
The consequences of the homocysteine revolution for medicine in the new millenium will be profound. Through better understanding of the nature of the aging process, the role of diet and other risk factors in the causation of arteriosclerosis, and the importance of thioretinaco and related substances in cancer, better control of the principal degenerative diseases of aging will enable large segments of the population to enjoy healthful aging well into their advanced years. Because of increased longevity and freedom from disease, the elderly population will require less interventional medicine through effective disease prevention. This development will call for major accommodation and changes in the attitudes, actions and philosophy of the medical, pharmaceutical, nutritional and governmental sectors of society.
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