Homocysteine, Folic Acid and Coronary Artery Disease - medIND

Review Article

Homocysteine, Folic Acid and Coronary Artery Disease: Possible Impact on Prognosis and Therapy D. Djuric1, V. Jakovljevic2, A. Rasic-Markovic1, A. Djuric3 and O. Stanojlovic1 Institute of Medical Physiology, School of Medicine, University of Belgrade1, Serbia; Department of Physiology, Faculty of Medicine, University of Kragujevac1, Serbia; and Health Centre Vracar3, Belgrade, Serbia

ABSTRACT Within the past four decades, the efforts of investigators worldwide have established the amino acid homocysteine (Hcy) as an important factor in arteriosclerosis and ageing. The amino acid homocysteine is a unique candidate for the study of different age-related pathological conditions, namely vascular diseases, dementia disorders and late-life depression, due to its multiple roles in different pathways leading to atherosclerosis and neurotoxicity. Especially, the role of homocysteine in predicting risk for atherothrombotic vascular disease has been evaluated in several observational studies in a large number of patients. These studies show that the overall risk for vascular disease is small, with prospective, longitudinal studies reporting a weaker association between homocysteine and atherothrombotic vascular disease compared to retrospective casecontrol and cross-sectional studies. Furthermore, randomised controlled trials of homocysteine-lowering therapy have failed to prove a causal relationship. On the basis of these results, there is currently insufficient evidence to recommend routine screening and treatment of elevated homocysteine concentrations with folic acid and other vitamins to prevent atherothrombotic vascular disease. [Indian J Chest Dis Allied Sci 2008; 50: 39-48] Key words: Coronary artery disease, Arteriosclerosis, Depression, Stroke, Dementia disorders, Homocysteine, Vitamin

INTRODUCTION Current understanding of cardiovascular disease risk is derived largely from studies of Caucasians of European origin. However, people of certain ethnic groups experience a disproportionately greater burden of cardiovascular diseases including coronary heart disease and stroke. Adoption of a Westernised lifestyle has different effects on metabolic and vascular dysfunction across populations, e.g. South Asians have a higher prevalence of coronary heart disease and cardiovascular mortality compared with Europeans. African-Americans demonstrate higher rates of coronary heart disease and stroke while African/Caribbeans in the UK have lower coronary heart disease rates but higher stroke rates than British Europeans. Other non-European groups such as the Chinese and Japanese exhibit consistently high rates of stroke but not coronary heart disease, while MexicanAmericans have a higher prevalence of both stroke and coronary heart disease, and North American native Indians also have high rates of coronary heart disease.1

HOMOCYSTEINE THEORY OF ARTERIOSCLEROSIS: HISTORICAL PERSPECTIVES Within the past four decades, the efforts of investigators worldwide have established the amino acid homocysteine

(Hcy) as an important factor in arteriosclerosis and ageing. Newburgh confirmed the protein hypothesis in 1915-1925 but failed to identify which amino acids produced plaques because methionine (1922) and homocysteine (1932) had not yet been discovered. After its discovery in 1932, homocysteine was demonstrated to be an important intermediate in the metabolism of amino acids. Cases of homocystinuria from inherited deficiency of cystathionine synthase and with mental retardation, accelerated growth, dislocated ocular lenses, and frequent vascular thrombosis were found to be associated with thrombosis and vascular diseases in 1964. The index case of methionine synthase deficiency (cobalamin C disease) was found by McCully in 1969 to be associated with arteriosclerosis, leading to the homocysteine theory of arteriosclerosis.2 The theory explains thrombogenic and atherogenic effects of homocysteine by deficiency of vitamin B6 in monkeys, choline deficiency in rats, thyroid deficiency in rats, and methionine deficiency in monkeys, reproducing the pathological findings found in homocystinuria. Clinical and epidemiological studies in the past two decades have demonstrated that elevated plasma homocysteine is a potent independent risk factor (or risk indicator) for arteriosclerosis in the general population, supporting the validity of the theory.3 As conventional risk factors for arteriosclerosis fail to account for part of the cases, homocysteine, a “new and emerging” risk factor, is being viewed with great

Correspondence and reprint requests: Dr Dragan M. Djuric, Professor and President, Serbian Physiological Society, and Director, Institute of Medical Physiology, University School of Medicine, str. Visegradska 26/II, P.O. Box 783, 11000 Belgrade, Serbia; Phone: 381 11 36 11 754; Fax: 381 11 36 11 261; E-mail: [email protected].

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interest. However, it was found that hyperhomocysteinemia is also a risk factor for neurodegenerative diseases, osteoporotic fractures and pregnancy complications. Numerous retrospective and prospective studies have consistently found an independent relationship between mild hyperhomocysteinemia and cardiovascular disease or all-cause mortality. This interpretation suggests that homocysteine is important in the pathogenesis of arteriosclerosis in persons with hereditary, dietary, environmental, hormonal, metabolic, and other factors predisposing them to hyperhomocysteinemia.

EPIDEMIOLOGY The decreased incidence of heart attack in Europe during World Wars I and II is explained by the scarcity of animal food, 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 B 6 and folic acid. 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 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 folic acid and vitamin B6 (pyridoxine) in preventing disease. A significant decline of strokemortality (8% to 16%) has been observed in the USA and Canada after fortification of grain products with folate. According to Canadian investigators, the prevalence of hyperhomocysteinemia in the general population is between 5% and 10% and may be as high as 30% to 40% in the elderly population. If population-based studies are correct, Hcy may be responsible for up to 10% of coronary artery disease events and, thus, may represent an important and potentially modifiable risk factor for cardiovascular disease.

Metabolism and Biochemistry-Hcy and Biological Determinants The term homocyst(e)ine is used to define the combined pool of homocysteine, homocystine, mixed disulfides involving homocysteine, and homocysteine thiolactone and protein-bound homocysteine.4-6 Protein-bound (i.cc. disulfide-linked) homocysteine accounts for 70% to 80% of the total pool. Homocysteine is formed through demethylation of methionine, which donates a methyl group in many biochemical reactions. It is metabolised through two enzymatic pathways: trans-sulfuration and re-methylation, but not in all tissues (Figure 1). Homocysteine can be re-methylated to methionine through a vitamin B12-dependent reaction catalysed by methionine synthase, in which 5-methyltetrahydrofolate donates the methyl group. In re-methylation, homocysteine typically receives a methyl group from 5-

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methyltetrahydrofolate (the main circulating form of folate in plasma) in a vitamin B12-dependent reaction, catalysed by the enzyme methionine synthase. 5methyltetrahydrofolate is formed by the reduction of 5,10-methylenetetrahydrofolate, via the enzyme methylenetetrahydrofolate reductase (MTHFR). The TT genotype of the MTHFR enzyme causes thermolability of the enzyme, reduces enzyme activity, and impairs the formation of 5-methyltetrahydrofolate, which explains why this genotype is associated with increased Hcy levels when folate status is relatively low. Breakdown of homocysteine by the trans-sulfuration pathway requires both cystathionine-β-synthase and vitamin B 6 and results in the conversion of homocysteine into cystathionine and cysteine, which are further broken down into products excreted in the urine.7,8 Vitamin B6 is important in homocysteine trans-sulfuration, whereas folate and vitamin B 12 play significant role in homocysteine re-methylation. Homocysteine remethylation is thought primarily to affect fasting casual homocysteine levels (in contrast to post-methionine loading homocysteine levels). Fasting homocysteine may be elevated in subjects with clear vitamin B 12 deficiency. For folate, in contrast, fasting plasma homocysteine levels are inversely related to folate, even in the range considered as adequate intake or plasma levels. The most important biological determinants of Hcy levels are: (i) folate, vitamin B 12, and vitamin B 6 status; (ii) polymorphism (C677T) in the methylenetetrahydrofolate reductase (MTHFR) gene; (iii) gender and sex steroids; and (iv) renal function.

Plasma Hcy Levels, Hyperhomocysteinemia and Cardiovascular Risk Values for fasting total plasma homocysteine may vary somewhat depending on laboratory methods, but levels of 5-15 µmol/L are usually considered normal.9-11 It is reported that men to have higher values than women, and post menopausal women to have higher homocysteine values than pre menopausal women. This difference is probably mediated through sex steroids, but the mechanisms by which sex steroids affect Hcy metabolism are poorly understood. Homocysteine values will normally increase with age, giving a reference range among elderly (> 60 years) of 5-20 µmol/L.12,13 Plasma Hcy levels above 12-15 µmol/L in the fasting state and above 40-50 µmol/L after methionine loading is probably associated with increased risk of atherothrombotic disease. The evidence for a link between atherothrombotic disease and postmethionineload hyperhomocysteinemia is less extensive than that for fasting hyperhomocysteinemia. Consequently, it is controversial whether one should assess both fasting and postmethionine-load Hcy levels. If fasting and/or postmethionine Hcy levels are elevated, deficiencies of folate, vitamin B12, and vitamin B6 (pyridoxine) should

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Figure 1. Homocysteine metabolism. Two major metabolic routes for elimination of homocysteine are absent in the brain: betainemediated conversion and transsulfuration (dashed arrows). ATP = Adenosine triphosphate, MTHFR = Methylenetetrahydrofolate reductase; SHMT = Serine hydroxymethyltransferase; THF = Tetrahydrofolate; 5, 10-CH 2 -THF = 5, 10-methylene tetrahydrofolate; 5-CH 3 -THF = 5-methyl-tetrahydrofolate; DNA = Deoxyribonucleic acid, RNA = Ribonucleic acid, AdoHcy = S-Adenosylmethionine.

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be excluded. The causes of hyperhomocysteinemia are inherited or acquired causes. Inherited causes include: disorders of transsulfuration, cystathionine B-synthase deficiency, disorders of re-methylation, defective vitamin B12 transport, defective vitamin B12 coenzyme synthesis, defective methionine synthase, 5,10methylenetetralydrofolate reductase deficiency (rare) and a thermolabile variant (common in most populations). Acquired causes include: diseases (chronic renal failure, acute lymphoblastic leukemia, psoriasis), vitamin deficiencies (vitamin B12, folate, vitamin B6) and drugs: methotrexate, an inhibitor of dihydrofolate reductase; phenytoin and carbamazepine, antagonists of folate; nitrous oxide, an inactivator of methionine synthase; theophylline, an antagonist of vitamin B6; and 6-azauridine triacetate, an antagonist of vitamin B6. However, the two genetic factors (heterozygosity for cystathionine-β-synthase deficiency and homozygosity for the C677T mutation of methylene tetrahydrofolate reductase) which are known to cause moderate hyperhomocysteinaemia have not been shown to be associated with increased risk of vascular disease.14,15 Laboratory testing for Hcy is currently restricted to research centres, and its costs range from $30 to $50 per person. Laboratory-based strategies for its detection and quantification have evolved to meet the increasing need for accuracy in risk prediction. 16 Although new technologies have been developed over the past two decades that have enhanced the precision of measurement, universal guidelines for circulating homocysteine determination remain lacking. Starting at a plasma homocysteine concentration of approximately 10 µmol/L (some authors underline 12 µmol/L), the risk increase follows a linear dose-response relationship with no specific threshold level. Although severe hyperhomocyst(e)inemia is rare, mild hyperhomocyst (e)inemia occurs in approximately 5% to 7% of the general population.4,7 Elevated plasma homocysteine levels (>12 µmol/L; moderate hyperhomocysteinemia) are considered cytotoxic and are found in 5% to 10% of the general population and in up to 40% of patients with vascular disease. 17,18 In a recent meta-analysis, it has been estimated that 10 % of the risk of coronary artery disease in the general population is attributable to homocysteine.19 However, homocysteine should be only a marker of folate and vitamin B6 status. 5,6 Also, an additional interpretation is that increased value of homocysteine may only represent cardiovascular risk when combined with other classical risk factors.7,17 It is also reported that an increase of 5 µmol/L in the plasma homocysteine concentration raises the risk of coronary artery disease by as much as an increase of 20 mg/dL (0.52 mmol/L) in the cholesterol concentration.

Homocysteine in Pathogenesis of Atherothrombosis The amino acid homocysteine (Hcy) is a unique

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candidate for the study of different age-related pathological conditions, namely vascular diseases, dementia disorders and late-life depression, due to its multiple roles in different pathways leading to atherosclerosis and neurotoxicity.20 The homocysteine theory offers an explanation for observations on human arteriosclerosis that are difficult to explain by the cholesterol/fat approach. 21,22 The greater risk of arteriosclerosis with advancing age is attributed to the loss of thioretinaco ozonide from the membranes of all cells which is responsible for the gradual increase in blood levels of homocysteine with age.23,24 It is generally held that Hcy is a vasculotoxic substance. Another possibility is that high levels of Hcy per se are not toxic, but that they inhibit transmethylation of methionine and thus impair methyl transfers from Sadenosylmethionine to a wide variety of methyl acceptors, which may impair important cell functions. These mechanisms are not mutually exclusive. High Hcy levels may also be a marker of impaired intracellular transmethylation reactions, which may themselves cause vascular damage. It must be emphasised that these concepts are based mostly on experimental data; evidence from studies in humans is still scarce. Main mechanisms by which Hcy can cause atherothrombotic disease are: 1. Homocysteine may increase oxidative stress;25 2. Homocysteine may increase cellular oxidative stress in which mitochondrial thioredoxin, and peroxiredoxin are decreased and NADH oxidase activity is increased and generating peroxinitrite and nitrotyrosine in contractile proteins which causes vascular dysfunction;26 3. Homocysteine may impair endothelial function and bioavailability of nitric oxide; 27-30 4. Homocysteine may impair vascular smooth muscle cell function;11 5. Homocysteine may change extracellular matrix, collagen structure and function;31,32 6. Homocysteine may induce a prothrombotic state;33 7. Homocysteine may affect vascular and tissue adenosine;34 8. Homocysteine may increase the formation of highly atherogenic oxycholesterols, increase lipid peroxidation, and increase the oxidation of lowdensity lipoprotein in vitro;25, 35 9. Homocysteine may increase homocysteine thiolactone and incorporate it into foam cells within nascent atheromatous plaques;11 10. Homocysteine may induce inflammation and apoptosis;25 and 11. Homocysteine may induce cardiac remodeling in which the elastin/collagen ratio is reduced, causing cardiac stiffness and diastolic heart failure in hyperhomocysteinemia.36, 37

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Clinical Studies and Evidence-based Medicine It is known that homocysteine may be atherogenic.38-40 However, many questions remain regarding the relationship of folate, vitamin B12, and vitamin B 6 to levels of total homocysteine and the relationship of homocysteine to total cardiovascular risk.41,42 Patients with mild hyperhomocyst(e)inemia have none of the clinical signs of severe hyperhomocysteinemia or homocystinuria, and are typically asymptomatic until the third or the fourth decade of life when premature coronary artery disease (CAD), aortic atherosclerosis and stroke develop as well as recurrent arterial and venous thrombosis. The mild hyperhomocysteinemia is also an independent risk factor for venous thromboembolism. A marked increase has been found in the risk of venous thrombosis at the highest plasma homocysteine concentrations. A plasma homocysteine concentration of more than 22 µmol/L increased the matched odds ratio for deep venous thrombosis to 4.0.4 A meta-analysis of 27 epidemiological studies, including more than 4000 patients, estimated that a 5 µmol/L increase in Hcy was associated with an odds ratio for coronary artery disease of 1.6 for men and 1.8 for women.16 Also, plasma total homocysteine values are a strong predictor of mortality in patients with angiographically confirmed coronary artery disease.43-45 The patients with multiple CAD tend to have a higher value of homocysteine as well as patients with previous infarction. 45 In a recent published statement for healthcare professionals it is said that odds ratio for CAD in subjects with hyperhomocysteinaemia was 1.5 in 15 studies (95% confidence interval, 1.5 to 1.9); for stroke it was 2.5 in nine studies (95% confidence interval, 2.0 to 3.0), and for peripheral vascular disease it was 6.8 in five studies (95% confidence interval, 2.9 to 15.8).18 Although there is lack of some of the vitamins involved in the metabolism of homocysteine, the increased values of Hcy are mainly due to impaired removal of homocysteine from the blood by the kidney. Impaired renal function (glomerular filtration rate less than 60-70 mL/min) is strongly associated with decreased whole-body homocysteine metabolism and increased Hcy levels, so that virtually all patients with end-stage renal disease have hyperhomocysteinemia, but how this occurs is again not understood.46, 47 There is growing evidence of an association between hyperhomocysteinemia and geriatric multisystem problems, including coronary artery disease, stroke, peripheral vascular disease, cognitive impairment, dementia, depression, osteoporotic fractures, and functional decline.48 The relations of increased homocysteine to heart failure (in both sexes) and to greater left ventricular mass (in women) noted in the Framingham sample should be confirmed in other community-based samples.36 No strong evidence was found to support an association of the MTHFR 677 C—>T polymorphism and coronary heart disease in Europe, North America,

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or Australia.49 The Hordaland Homocysteine Study (HHS), a population-based study of more than 18,000 men and women in the county of Hordaland in Western Norway, indicated that a raised Hcy level was associated with multiple clinical conditions, whereas a low Hcy level is associated with better physical and mental health.50 Using literature mining approach, the genes (135 genes in 1137 abstracts) that are modulated directly or indirectly by an elevated level of homocysteine has been identified. These genes were then placed in appropriate pathways in an attempt to understand the molecular basis of homocysteine induced complex disorders and to provide a resource for selection of genes for polymorphism screening and analysis of mutations as well as epigenetic modifications in relation to hyperhomocysteinemia. The comprehensive network collated has lead to the identification of genes that are modulated by homocysteine indicating that homocysteine exerts its effect not only through modulating the substrate levels for various catalytic processes but also through regulation of expression of genes involved in complex diseases.51 It must be anticipated that individuals with extensive subclinical vascular disease would have both elevated levels of inflammatory markers and raised homocysteine concentrations associated with low folic acid, B12 or B6 levels.52 It was found that homocysteine increased both endothelial intracellular and extracellular cholesterol in HUVECs. In cardiovascular diseases interaction between lipid-lowering drugs and homocysteine could be of great interest, i.e. it seems that nicotinic acid, cholestyramine and clofibrate increase the concentrations of homocysteine. 53 In addition, simvastatin has no effect on homocysteine concentration, but might increase methionine, which may partly explain its efficacy in coronary heart disease.54 Therefore, it may be argued that the link between Hcy and atherothrombosis is not only well-established epidemiologically but also biologically plausible. However, there are heterogeneous results of prospective studies concerning the relation between high Hcy levels and atherothrombosis. It has also been pointed out that studies exploring the mechanisms by which Hcy may induce vascular disease have to a large extent been conducted in vitro or in animal models under experimental conditions that may not pertain in humans. The possible explanations for this are as follows: 1. Homocysteine may not be involved in the early stages of atherothrombosis but rather plays a role in its progression. Some observations support the concept that Hcy is more strongly related to atherothrombotic disease in high-risk than in lowrisk subjects, and this would be consistent with the possibility that Hcy is involved mainly in later stages of the atherothrombotic process; and

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2. The association between mild hyperhomocysteinaemia and both atherosclerotic and thrombotic disease in adults may be modulated by other factors, such as ethnicity and other cardiovascular risk factors (i.e., type 2 diabetes, smoking), and this, at least in part, may explain the inconsistent results among various studies. However, these findings have not been constant across the studies.

Homocysteine-lowering Therapy The decision to measure plasma Hcy and to initiate Hcy-lowering treatment should depend on the absolute risk of atherothrombotic events, not on the presence or absence of other risk factors. Established treatments of cardiovascular risk factors (stopping smoking and blood pressure- and cholesterol- lowering treatments), when indicated, should take precedence over Hcy-lowering treatment. The efficacy of treatments that lower homocysteine concentrations in reducing the risk of cardiovascular disease remains controversial. In observational studies, 25% lower homocysteine has been associated with about 10% less coronary heart disease and about 20% less stroke. Treatment of elevated Hcy levels is indicated in high-risk people, such as those with a personal or family history of premature atherosclerosis or a predisposition to develop hyperhomocysteinemia. Despite negative results from secondary prevention trials regarding the cardiovascular risk reduction there is convincing evidence about the effectiveness of B-vitamin supplementation in lowering the risk of stroke (approximately 20%). 39 Presence of elevated Hcy (>12 µmol/L) strongly and independently predicts progression of coronary plaque burden. 40 Based on various calculation models, reduction of elevated plasma homocysteine concentrations may theoretically prevent up to 25% of cardiovascular events. Currently, more than 20 prospective, worldwide, interventional trials involving at least 100,000 participants are examining whether lowering plasma homocysteine levels with supplemental B vitamins will prevent mortality and morbidity from arteriosclerotic vascular disease. Review of the design and statistical power of 12 randomised trials (about 52,000 participants: 32,000 with prior vascular disease in unfortified populations and 14,000 with vascular disease and 6000 with renal disease in fortified populations) was done to assess the effects of lowering homocysteine with B-vitamin supplements on risk of cardiovascular disease. The recent Swiss Heart Study showed that B vitamins slowed restenosis in patients with coronary arteriosclerosis treated with angioplasty.55 A homocysteine-lowering strategy may prevent or slow the development of age-related problems.48 Establishing a prospective meta-analysis of the ongoing trials of homocysteine lowering should ensure that reliable information emerges about the

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effects of such interventions on cardiovascular disease outcomes.56

FOLIC ACID Folic acid, a water soluble B vitamin, has recently gained considerable attention because of its great potential to prevent many disorders through supplementation for the general population.57 Folic acid was repeatedly reported to improve endothelial dysfunction in various clinical conditions, although the mechanisms of this beneficial effect are not fully understood. It seems that folate (main circulating metabolite of folic acid in plasma is 5MeTHF) acts through at least four mechanisms in atherosclerosis: (i) indirectly, to decrease homocysteine level and insure optimal functioning of the methylation cycle, (ii) directly, to produce antioxidant effects, (iii) to interact with enzyme endothelial nitric oxide synthase (eNOS), and (iv) to affect cofactor bioavailability of nitric oxide.57 For folic acid it has been found: (a) to revert dysfunction of eNOS,58 (b) to influence on postprandial endothelial dysfunction,59 (c) to improve endothelial function in coronary artery disease by reduction of intracellular superoxide,60 (d) to inhibit intimal hyperplasia induced by a high-homocysteine diet in a rat carotid endarterectomy model, 61 (e) to reverse endocardial endothelial dysfunction in homocysteinemic hypertensive rats,62 and (f) anti-arrhythmia effects after reperfusion injury in rat heart 63. Further indirect evidence comes from the recent observation that therapy with the antifolate methotrexate may promote atherosclerosis.64 As vascular endothelial dysfunction is a recognised key event in the aetiology of atherosclerosis, an accepted surrogate and “end point” for clinical investigations in cardiovascular disease, and most important matter in atherosclerosis prevention, it is very important to study vascular effects of folic acid. It is also very interesting to study interaction of folic acid or 5MeTHF in production of NO from eNOS which is dependent on optimal concentration of L-arginine and its critical cofactor BH4. 65 In an “oxidising environment”, BH4 is oxidised to its inactive metabolite dihydrobiopterin (BH2), resulting in the electron “uncoupling” of eNOS, further production of damaging superoxide, and endothelial dysfunction. The exact mechanism whereby folic acid or its derivatives can interact with this pathway to increase NO bioavailability is unknown, but chemical stabilisation of BH4, regeneration of BH4 from BH2 and direct interaction of 5MeTHF with eNOS to mimic BH4 have all been postulated. One of the first recognised beneficial effects of folic acid on vascular endothelial function has been attributed to the reduction in plasma tHcy concentrations. Folic acid supplementation was demonstrated to improve endothelial dysfunction in asymptomatic subjects with hyperhomocysteinemia66,67

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as well as in hyperhomocysteinemic patients with established coronary heart disease.60,68 Interestingly, this beneficial effect was also observed in subjects without elevated homocysteine concentrations suggesting different effect of folates on the endothelium independent of homocysteine.59,69 It is clear that folic acid will improve endothelial function, and the implication is that homocysteine lowering will result in a significant reduction in cardiovascular risk.66,67,70 Lowdose folic acid supplementation can lower tHcy with no measurable reduction in cardiovascular risk, and highdose folic acid can dilate human coronary circulation without any change in plasma tHcy level. 71 These observations suggest that high-dose folic acid has other “pharmacological” actions independent of its effects on plasma tHcy concentrations. 72 Folic acid therapy has been shown to improve endothelial function in patients with familial hypercholesterolemia via a possible antioxidant mechanism.69 Oral administration of folic acid (5 mg) for four weeks improves endothelial function in patients with hypercholesterolemia treated with statins, with possible beneficial effects on the prognosis of these patients. 73 Several observational studies found a clear association between aging, low folate intake, risk of coronary heart disease and certain cancers.74,75 Since both folate depletion and ageing are strongly associated with hyperhomocysteinemia, genomic DNA hypomethylation, and increased uracil mis-incorporation into DNA, it appears that each of them enhances carcinogenesis by inducing a derangement of one-carbon metabolism that supplies one-carbon to biological methylation reactions and nucleotide synthesis.76-80 Folic acid alone, folic acid combined with vitamin B12 and B6, and vitamins B6 and B12 alone have all been shown to reduce homocysteine concentrations. The minimal effective doses of folic acid and vitamin B 6 have not yet been determined. Normalisation of the plasma homocysteine concentration usually occurs within four to six weeks after the initiation of therapy, but may occur in as little as two weeks. According to Stehouwer 2002 (International Atherosclerosis Society/IAS Commentaries, www.athero.org) fasting hyperhomocysteinemia can be treated with folic acid (1-5 mg/day). However, there are insufficient data on the dose-response relationship within this dosage range. Postmethionine hyperhomocysteinemia can be treated with pyridoxine (50-250 mg/day) plus folic acid (1-5 mg/day). The current recommended dietary allowance (RDA) for folic acid for non-pregnant women is 180 µg per day and the average dietary intake in many countries among adult women is approximately 225 µg per day. Because of evidence that this level of intake may be insufficient to minimise the risk of neural tube defects and possibly coronary heart disease, some have urged that the RDA be reset to the earlier level of 400 µg per day. Each 100 µg per day in American population

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was associated with a 5.8% (95% CL) lower risk of coronary heart disease (CHD). Somewhere it was suggested that increasing folate consumption by approximately 200 µg per day would reduce total homocysteine concentrations by approximately 4 µmol/L, a reduction that could potentially have a major effect on cardiovascular mortality. Although folic acid supplementation may lower homocysteine concentrations, there is still controversy that it improves the clinical outcome. The dose and combination of vitamins that should be used remain unclear, although doses from 200 µg per day up to 20 mg per day were used in patients with coronary artery disease. Thus, doses of folic acid as low as those found in multivitamin pills might be of use in lowering homocysteine levels in patients with coronary artery disease. Much higher doses of folic acid are needed to lower homocysteine concentrations in patients with renal failure, i.e. doses as high as 15 mg per day or more. Nevertheless, if homocysteine levels are high, and no other risk factors for vascular disease are present, or if episodes of thrombosis are recurrent, vitamin therapy may be a reasonable addition to the usual treatment. In such patients, it is currently used 0.4 mg folic acid daily with re-checking the homocysteine concentration after four to six weeks. Evidence for beneficial effects of folic acid is based on the results of clinical studies and the observations that high-dose folic acid will improve endothelial function, reduce blood pressure, reduce carotid artery plaque size, and reduce coronary artery restenosis rates after angioplasty, given that folic acid is a safe, cheap, and well-tolerated potential treatment.68,81 On the contrary, long-term homocysteine-lowering treatment with folic acid plus pyridoxine is associated with decreased blood pressure but not with improved brachial artery endothelium-dependent vasodilation or carotid artery stiffness found in a two-year, randomised, placebo-controlled trial.82 Diets lower in folic acid and carotenoids are associated with the coronary disease epidemic in Central and Eastern Europe.83 It seems that desirable folate intake depends on a number of genetic, nutritional, environmental and ethnic factors, meaning that a more individual approach may be required. Finally, there are a few very important papers recently published which favour folic acid in treatment, primary and secondary prevention of coronary artery disease. 84-88 Graded associations can be observed between higher intakes of folate and vitamin B6 and the lower risk of CAD.89,90 In this setting one should know fasted Hcy rose on low-folate/high-methionine diet, but high folate ameliorated the effect of high methionine on fasted plasma Hcy.91 A meta-analysis of relevant eight randomised trials was done to assess the efficacy of folic acid supplementation in the prevention of stroke. It was found that folic acid supplementation significantly reduced the risk of stroke by 18% in primary prevention.92 Otherwise, the conclusion drawn from

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previous meta-analyses that folic acid, through lowering homocysteine, has a role in prevention of cardiovascular disease is in some doubt.93

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FOOD FORTIFICATION

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Beginning in January 1998, U.S. products with cereal grain or flour were fortified by the addition of 140 µg of folic acid per 100 g of flour. This measure is intended to reduce the incidence of neural tube defects, but it may also reduce homocysteine concentrations and decrease the risk of atherosclerosis in the general population, although substantially higher levels of folate may be needed to achieve desirable effects in many subgroups. Geographical variability may be due to higher folate intake in North America and Europe or to publication bias. 83 Dietary supplementation with folic acid and vitamin B12 lowers blood homocysteine concentrations by about 25% to 30% in populations without routine folic acid fortification of food and by about 10% to 15% in populations with such fortification. The current fortification programme will increase folate consumption by approximately half of this amount and it remains to be seen whether this dietary supplementation of folate will affect the prevalence of coronary heart disease in the general population. Prospective, randomised clinical trials, however, will be necessary to determine the effect of folic acid and vitamin B supplementation on cardiovascular morbidity and mortality. Fortification, of course, leads to exposure for the total population, which stimulated urgent consideration of potential health risks, especially for elderly and any person with a B12 deficiency.94,95

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CONCLUSIONS Finally, during the last decade, the utility of homocysteine in predicting risk for atherothrombotic vascular disease has been evaluated in several observational studies in a large number of patients. These studies show that the overall risk for vascular disease is small, with prospective, longitudinal studies reporting a weaker association between homocysteine and atherothrombotic vascular disease compared to retrospective case-control and cross-sectional studies. Furthermore, randomised controlled trials of homocysteine-lowering therapy have failed to prove a causal relationship. Although it was proved that folic acid possess clear vasodilator and antioxidant properties in rat and human coronary circulation.71,96 On the basis of these results97, there is currently insufficient evidence to recommend routine screening and treatment of elevated homocysteine concentrations with folic acid and other vitamins to prevent atherothrombotic vascular disease.

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