Homocysteine (Hcys) is a homologue of the amino acid cysteine, at the same time being a derivate of methionine, from which it differs by the lack of a methylene group at the sulfur atom (fig. 1). Hcys was discovered in the 1930s, together with cysteine and glutathione. These three compounds constitute a group of low-molecular-mass biological thiol compounds. Elevated plasma level of homocysteine is considered to be an unequivocal risk factor of cardiovascular diseases. There are many literature reports available showing elevated Hcys level as an independent factor of premature atherosclerosis development (besides hypercholesterolemia). The role of Hcys in pathogenesis of vascular disorders, as well as its procoagulant effect, results from its influence on blood vessel walls, blood platelets (fig. 2) and the coagulation/fibrinolysis proteins. Detailed descriptions of the effect of Hcys on selected hemostasis elements, including blood platelets and coagulation, have been provided by Karolczak and Olas (1), as well as by Malinowska et al. (1). Elevated homocysteine level (hyperhomocysteinemia) in blood can result from genetic, congenital metabolic anomalies or from environmental factors (acquired, in connection with lifestyle). It is estimated that every one in ten Europeans has an elevated blood Hcys level, which leads to increased cardiovascular risk, including atherosclerosis and myocardial infarction.

Moreover, increased blood Hcys levels are observed in patients with chronic renal failure, hypothyroidism, various cancer types, malignant anemia, liver diseases, and in persons with malnutrition, especially with low folic acid intake. It should also be noted that an increase in Hcys levels correlates with the severity of some other disorders, including Alzheimer's disease and other types of dementia, Parkinson's disease, multiple sclerosis, and chronic fatigue syndrome.

Proteins that are absorbed with food are metabolized in the human body into amino acids. One of the products of protein degradation is the amino acid methionine (Met). Some of the Met is recycled and used again for protein synthesis, while the remaining portion may be metabolized into homocysteine (2). Hcys is a thiol amino acid, which is synthesized in all types of animal (including human) cells. S-adenosylmethionine transferase (SAM synthetase) catalyses the reaction of adenosine transfer (from ATP) to methionine. S-adenosylmethionine (SAM) is the main donor of the methyl group (–CH₃) for various methylation reactions. After the methyl group is transferred by SAM synthetase to various compounds, SAM turns into S-adenosylhomocysteine (SAH). Subsequently SAH is hydrolyzed by specific SAH hydrolase into adenosine and homocysteine (fig. 3) (3), and its plasma level can be measured. Moreover, it is known that Hcys does not have its own codon, so it cannot be incorporated into protein chains in the way that other amino acids can (4). But it can bind with them through the ε-amino group of lysine. Hcys can also bind to proteins and/or cysteine via disulfide bonds. It has been shown that approximately 33% of the total Hcys circulates as oxidized low-mass disulphide compounds, 1% as a free, reduced form, and about 66% is bound to proteins (as N-homocysteinylated – or S-homocysteinylated proteins) (5, 6).

Literature reports describe 2 pathways of Hcys metabolism. The first one is based on re-methylation of Hcys into methionine, with substantial involvement of folic acid, which acts here as a methyl group donor (7, 8). Vitamin B₁₂ (cobalamin) and Vitamin PP (niacin) are necessary co-factors for metabolism of tetrahydrofolate derivates (9). The second pathway leads to cysteine through Hcys transsulfuration, with pyridoxal-5-phosphate as a co-factor (active form of vitamin B₆) (fig. 4) (10, 11).

Another form of Hcys in plasma is its thiolactone, homocysteine thiolactone (HTL) (fig. 5). HTL is much more reactive than Hcys. Its synthesis results from erroneous homocysteine activation, e.g. by methionyl-t-RNA synthetase (MetRS) (12). It is worth noting that homocysteine thiolactone may undergo non-enzymatic hydrolysis, but mostly it is metabolized by plasma HTL hydrolase, also known as homocysteine thiolactonase (HTLase) or paraoxonase (PON 1).

It has been observed that impaired metabolism of homocysteine leads to a rise of its level in the blood and further to various abnormalities. Normal blood Hcys levels range from 5 to 15 μM. A preprandial level of Hcys exceeding 15 μM is termed hyperhomocysteinemia. There are three types of this disorder – mild, moderate and severe – with plasma Hcys levels 16-30 μM, 31-100 μM and above 100 μM, respectively. Although the severe form is rare, 5-7% of the general population suffer from mild hyperhomocysteinemia. Subjects with mild form of hyperhomocysteinemia do not show clinical symptoms until they reach 40 years of age, when signs of premature atherosclerosis start to show. Moderate hyperhomocysteinemia can pose a significant danger for young women, as it increases the risk of complications during pregnancy (13).

In recent years numerous data on possible genetic causes of cerebrovascular diseases have been collected. It has been observed that stroke occurs significantly more often in twins, especially monozygotic twins, as well as in subjects whose parents suffered from cerebral stroke (14). A significant clinical role here has been attributed to genetically conditioned alterations of homocysteine metabolism.

An elevated Hcys level may result from genetic defects of enzymes involved in its metabolism, among others cystathionine-β-synthase (CBS), methionine synthase (MS) and methylenetetrahydrofolate reductase (MTHFR). Heterozygous lack of CBS, CBS mutations and methylenetetrahydrofolate reductase gene polymorphism are thought to be the most probable causes of hyperhomocysteinemia. In CBS, Ile278Thr and Gly307Ser are the most common mutations. If they occur homozygously, they may lead to significant elevation of homocysteine level and to an increased risk of atherosclerosis of carotid arteries (15). Methylenetetrahydrofolate reductase gene polymorphism involves substitution of cytosine by thymine in position 677 (C677&ro;T). This nucleotide exchange leads to the synthesis of a thermolabile enzyme form with decreased activity. As a result synthesis of methylenetetrahydrofolate (donor of methyl group in re-methylation of homocysteine) is decreased. Prevalence of this mutation is race dependent. In Asian and Caucasian populations (including Poland) there are 50% C7T heterozygotes and 10-13% T/T homozygotes, while in the Afro-American population cases of this polymorphism are rare (16). T/T homozygotes have on average 2.5 μM higher levels of Hcys than C/C homozygotes. However, this difference has been observed in the case of folic acid and riboflavin deficiency in the diet (17). It has also been observed that A&ro;C transversion in position 1298 of the MTHFR gene leads to substitution of glutamate with alanine in the enzyme, which leads to its decreased activity (18).

Acquired hyperhomocysteinemia may result from various factors. Among others, lifestyle, diet, various diseases, medications, drugs, sex and age influence levels of Hcys. Dietary errors leading to deficiency of vitamins B₂, B₆, B₁₂ and folic acid may in turn cause an increase of Hcys level. Also alimentary tract disorders connected with impaired absorption of these vitamins may play an important role.

An excess of methionine-rich proteins and saturated fat acids in diet also favor elevated Hcys plasma levels (19). Hcys concentration increases with age (in seniors being about twice as high as in children) and depends on sex (in men higher than in women). Moreover, excessive intake of coffee, alcohol and tobacco smoking lead to increased plasma Hcys level. Drinking more than 8 cups of coffee per day leads to an increase of total Hcys level of 28% in women and 19% in men. Alcohol leads to gastric and bowel disorders, which may lead to impaired absorption of vitamins. It also inhibits methionine synthase. Carbon monoxide and carbon disulphide contained in tobacco smoke deactivate vitamin B₆ in the liver, which decreases the rate of homocysteine metabolism. One cigarette daily leads to a 1% Hcys level increase in women and 0.5% in men (20).

Hcys level also depends on kidney function and creatinine level. In patients with chronic kidney failure renal Hcys metabolism and elimination are impaired. Insufficient kidney function and elevation of creatinine level leads to elevation of Hcys levels. Hyperhomocysteinemia is seen in 85-95% of hemodialysis patients (21). Medications with antagonistic effects on absorption and metabolism of folic acid (e.g. trimethoprim, methotrexate, phenytoin, cholestyramine), vitamin B₆ (e.g. niacin, theophyllinum) and cobalamin (e.g. metformin) lead to increase of Hcys level. Other medications, such as L-dopa, fibrates, cyclosporine, aminoglutethimide and some diuretics, have a similar effect. Hyperhomocysteinemia may also occur in the course of other disorders, such as cancer (especially pancreatic, breast and ovarian cancer), hypothyroidism, lymphoblastic leukemia, lupus erythematosus, psoriasis, diabetes, alcoholic hepatopathy and many others. Increased Hcys levels are also observed in patients with Cushing syndrome, in this case associated with increased cortisol levels (22).

Plasma homocysteine level depends among other things on sex, age, smoking, function of the liver and kidneys, diet and physical activity. Proper diet, abstaining from tobacco smoking, and optimal physical activity may influence the levels of this amino acid. There are also various compounds available which lead to reduction of Hcys levels. First, folic acid and vitamins B₆ and B₁₂ should be mentioned. Others include choline, inositol, methionine and zinc compounds. Folic acid and vitamins B₆ and B₁₂ are necessary in metabolism of Hcys; therefore they can be used for both treatment and prophylaxis of hyperhomocysteinemia (23).

Insufficient intake of folic acid leads to limitation of re-methylation of Hcys into Met, which induces hyperhomocysteinemia (24). It has been shown that supplementation with folic acid alone decreases levels of Hcys. A similar effect can be obtained by combination of folic acid with B vitamins. Folic acid has the effect of 60% of all vitamins in that respect, since it acts as a methyl group donor in the process of re-methylation of Hcys into Met. In prevention of hyperhomocysteinemia doses of 400 μg of folic acid, 2 mg of vitamin B₆, and 3 μg of B₁₂ daily are recommended. As the human body is unable to produce folic acid, it has to be supplied from the environment. Folic acid is present in various foods, both animal and plant derived. The highest levels of folic acid can be found in yeast and green vegetables, such as asparagus, spinach, parsley, broccoli, lettuce, cauliflower, Brussels sprouts, soya, beans and peas. Other sources include: fermented dairy products, poultry, eggs, liver, bran, porridge, granary bread, and oranges. It should be noted that folic acid is highly thermolabile, which must be taken into consideration in meal preparation and storage of food products. Vitamin B₁₂ is mostly absorbed from animal-derived foods, among others offal, fish and dairy products. Vitamin B₆ can be supplied via meat, fish, beans, grains, pepper, Brussels sprouts, carrots, spinach, cabbage and bananas (25). Unfortunately, food processing leads to a significant degradation of various nutritional elements; thus additional vitamin supplementation may be necessary. Also, especially vitamin-enriched foods can be used. In the USA, since 1999 fortification of flour with folic acid (to the amount of 400 μg intake per day) and B₁₂ (to the amount of 2.5 μg intake per day) has been obligatory (26). A Mediterranean diet is considered to be the healthiest type of diet. Observations have been made that premature atherosclerosis development is less often seen in Mediterranean countries, with the diet containing large amounts of fruits and vegetables. Often mentioned as a good example is a French diet, which additionally includes pâtés made from the fattened livers of geese, containing large amounts of vitamins B₆ and B₁₂.

As mentioned above, treatment of hyperhomocysteinemia is based on supplementation with higher doses of group B vitamins. Numerous studies show that adequate vitamin B₆ supplementation may lead to up to 25% decrease of Hcys level. The role of vitamin B₁₂ seems to be smaller, since its supplementation leads to 7% decrease of Hcys level (27). Recommended treatment of hyperhomocysteinemia includes daily supplementation with 1 mg of vitamin B₁₂ (p.o. or i.m.), 5 mg of vitamin B₆, 1 mg of folic acid. The dose of 650 μg of folic acid daily leads to 40% reduction of homocysteine level. Concomitant supplementation with 400 μg of vitamin B₁₂ is recommended to avoid resistance against the treatment of this vitamin's deficiency (28). In patients with chronic renal failure the dose of 15 mg daily of folic acid is recommended. Treatment with vitamins leads to normalization of Hcys levels in more than 90% of patients within 6 weeks. However, normal, dietary sources of vitamins necessary for normal metabolic function are preferred over vitamin supplements due to the higher rate of absorption of vitamins. Moreover, normal sources of these vitamins also contain various other desirable compounds which have favorable effects on the cardiovascular system, including antioxidants.

This review article describes present knowledge on prevention of negative consequences of elevated homocysteine levels. This goal can be partially achieved through treatment with folic acid and vitamins B₆ and B₁₂. Of particular interest are observations made in various countries that treatment with these compounds decreases the risk of various cardiovascular diseases. However, in spite of significant progress in this field, many questions pertaining to toxic effects of Hcys remain unanswered. Nevertheless, the use of modern, highly sensitive analytical methods allows us to track the pathways of metabolism of homocysteine and its derivates, which will further improve our understanding of the mechanism of action of these compounds.