treatment: a review

Homocysteine (Hcy), a sulfur amino acid, is the only direct precursor for L-methionine synthesis through a reaction that requires vitamin B₁₂, representing a connection with "one-carbon" units metabolism. Hcy catabolism requires vitamin B₆ and as a consequence, alteration in folic acid and B vitamins status impairs Hcy biotransformation. Numerous studies have indicated that Hcy is an independent risk factor for cardio- and cerebrovascular diseases. In the last decade, several clinical trials have investigated the possible correlation between the use of folic acid and vitamins B₆ and B₁₂ for lowering Hcy plasma concentration and the reduced risk of stroke or its recurrence. This review is aimed to present some aspects of Hcy biochemistry, as well as the mechanisms through which it exerts the toxic effects on the vascular endothelium. We also discuss the results of some of the clinical trials developed to investigate the beneficial effects of vitamin therapy in the prevention and management of stroke.


INTRODUCTION
Stroke is a major cause of morbidity and mortality worldwide.Many studies indicate a plethora of conventional risk factors for stroke (i.e., hypertension, dyslipidemia, smoking, diabetes mellitus, obesity, and family history).Nevertheless, cerebrovascular events can occur sometimes in individuals without any of the previously mentioned risk factors.As a consequence, it is very likely that other risk factors exist.Hyperhomocysteinemia, defined as an elevated plasma total homocysteine (tHcy) concentration (>10 µM), is one such factor.There is epidemiological evidence suggesting a relationship between hyperhomocysteinemia and increased risk of a broad range of neurodegenerative conditions, including Alzheimer and Parkinson disease, and stroke (Mattson & Shea, 2003;Zou & Banerjee, 2005).

MeTaBOlIC paTHWays Of HOMOCysTeINe
In mammalian cells, Hcy metabolism involves one pathway for biosynthesis and three Hcy consuming path-ways (Fig. 1).There is also a strong connection between some of these metabolic pathways and the metabolism of "one carbon" donors like S-adenosyl methionine (SAM) and N 5 -methyl tetrahydrofolate (N 5 -methyl THF).
Demethylation of the essential, diet-derived amino acid methionine (l-Met) represents the only reaction through which Hcy is synthesized in humans.This conversion involves two enzymes, SAM synthetase/l-Met adenosyltransferase (EC 2.5.1.6)and S-adenosyl homocysteine (SAH) hydrolase (EC 3.3.1.1).SAM synthetase activates l-Met in a reaction with ATP leading to SAM synthesis.SAM is used as a methyl donor in a variety of cellular biosyntheses (i.e., creatine, epinephrine, carnitine, phospholipids, proteins, nucleic acids and polyamines).Demethylation of SAM during the synthesis of the aforementioned compounds generates SAH, which undergoes hydrolysis mediated by SAH hydrolase.Despite its reversibility, the latter reaction is responsible for SAH hydrolysis in vivo, due to the rapid removal of its two products.One consequence of an increased intracellular Hcy concentration is the accumulation of SAH through the reverse reaction catalyzed by SAH hydrolase.When present in excess, SAH acts as an inhibitor for SAM-dependent methylation reactions (Cantoni, 1985).
Resynthesis of l-Met from Hcy occurs via two reactions catalyzed by methionine synthase (EC 2.1.1.13)and betaine-Hcy methyltransferase (EC 2.1.1.5).Methionine synthase is one of the two enzymes that use vitamin B 12 as coenzyme.The second substrate required by methionine synthase is N 5 -methyl THF.The reaction catalyzed by this enzyme sets a link between Hcy and "one carbon" units' metabolism (Castro et al., 2006).N 5 -Methyl THF is synthesized from N 5,10 -methylene THF through a reaction catalyzed by N 5,10 -methylene THF reductase (MTHFR) (EC 1.1.1.68).This reaction requires NADH, being regulated by SAM and SAH as a negative and positive regulator, respectively.
Hcy is also the source of a highly reactive compound, Hcy-thiolactone.The synthesis of Hcy-thiolactone is associated with the misactivation of Hcy by the methionyl-tRNA synthetase (Jakubowski, 1997;Jakubowski, 2008a).
The main disposal pathway of Hcy is its transsulfuration to l-Cys.This process takes place primarily in the liver, kidney, small intestine and pancreas.The first reaction is a condensation between Hcy and l-Ser leading to cystathionine which is further hydrolyzed to l-Cys and α-ketobutyrate.These two reactions are catalyzed by the B 6 -dependent enzymes cystathionine-β-synthase (EC 4.2.1.22)and cystathionine-γ-lyase (EC 4.4.1.1),respectively.α-Ketobutyrate undergoes oxidative decarboxylation to propionyl~CoA, which is converted to succinyl~CoA entering in the Krebs cycle.The transsulfuration pathway is responsible for both l-Met catabolism and sulfur atom transfer from l-Met to l-Ser, yielding l-Cys.l-Cys is a precursor for the synthesis of proteins, coenzyme A, and glutathione.Further catabolism of l-Cys gives rise to taurine and inorganic sulfate, both excreted through urine.
In conclusion, Hcy is central for some metabolic pathways: (i) resynthesis of SAH through reversal of SAH hydrolysis, (ii) remethylation to l-Met, and (iii) conversion to cystathionine.
Total plasma Hcy (tHcy) is defined as the pool of free homocysteine, homocystine, Hcy-S-S-Cys disulfide, as well as protein N-and S-linked Hcy.Total Hcy is used as a predictive risk factor for cardiovascular events, the post stroke evolution, screening for inborn errors of methionine metabolism, and as a supplementary test for vitamin B 12 deficiency.
Hcy can be assessed in blood collected in either the fasting or nonfasting state.A standard dinner (about 50 g protein content) induces a 1.15-fold increase of plasma Hcy concentration (Guttormsen et al., 2004).Total Hcy concentration can be assessed in both plasma and serum samples, despite the fact that the latter give slightly higher values (Rasmussen & Moller, 2000).Plasma has to be isolated very quickly to avoid Hcy leakage from erythrocytes and leukocytes or else blood samples should be treated with a preservative (i.e., sodium fluoride, 3-deazaadenosine, citric acid) (Salazar et al., 1999;Hansrani & Stansby, 2007), albeit preservatives may influence the assay of total Hcy (Refsum et al., 2004).
Assessment of total Hcy concentration requires pretreatment of samples with a reducing agent (i.e., sodium borohydride, dithioerythritol, tris(2-carboxyethyl)phosphine) to reduce all disulfide bonds involving Hcy.This treatment does not release the N-linked Hcy, i.e.Hcy residues bound to ε-amino groups of protein lysine residues.This can be achieved only by protein hydrolysis in an acidic environment and at high temperature (Jakubowski, 2008b).
The methods used to assess Hcy level in different biological samples can be classified into chromatographic methods, enzymatic assays, and combined assays (enzymatic reaction followed by an immunoassay) (Refsum et al., 2004).Plasma tHcy concentration can also be assessed through capillary electrophoresis with a suitable detection system.Very recently, assays based on addition of transition-metal complexes to carbonyl groups were developed (Chen et al., 2007;Hung et al., 2009).Most of the chromatographic methods require sample deri-

DeTeRMINaNTs Of plasMa tHcy CONCeNTRaTIONs
Plasma tHcy levels are influenced by genetic, physiologic (age, sex) and lifestyle factors, and various pathologic conditions (De Bree et al., 2002).The physiological range of plasma total Hcy is defined as the 2.5th to 97.5th percentile interval for the analyzed group (Lentz & Haynes, 2004).This corresponds to a lower limit of 5 µM, while the upper limit varies considerably between laboratories and different populations.As in the case of other biochemical parameters, for Hcy also its own reference interval should be established taking into account the influence of different non-modifiable and modifiable factors.The relationship between different factors and plasma Hcy concentration is beyond the aim of this review as there are several excellent reviews dealing with this subject (De Bree et al., 2002;Pezzini et al., 2007).

HypeRHOMOCysTeINeMIa
The term "hyperhomocysteinemia" does not define a pathological condition.It is rather used to describe a biochemical abnormality which can be a direct consequence of various pathological conditions.Plasma Hcy concentration could represent a gradual indicator of the risk for cardiovascular disease (Lentz & Haynes, 2004).Thus, every increase of 2.5 µM in plasma Hcy can be associated with an increase of stroke risk of about 20 % (Clarke et al., 2002).Moreover, plasma tHcy levels above 20 µM are associated with a nine-fold increase of the myocardial infarction and stroke risk when compared to concentrations below 9 µM (Nygard et al., 1997).

MeCHaNIsMs Of HOMOCysTeINe TOxICITy
Over the years, several theories concerning the toxicity of Hcy have been elaborated, but despite the efforts, none does to completely explain the toxicity of this compound.
One of the first hypotheses suggested that hydrogen peroxide formed as a byproduct in metal ion-catalyzed redox reactions involving the thiol group of Hcy was responsible for the toxicity of this compound.The major drawback of this hypothesis was the fact that l-Cys is not a risk factor for vascular disease, despite its 20-to 30-fold higher concentration than that of Hcy (Jacobsen, 2000).Moreover, the amount of hydrogen peroxide produced with the participation of Hcy is very low and its formation is possible only in the presence of transition metal ions as catalysts (Zappacosta et al., 2001).
A more recent theory suggests that Hcy toxicity is a consequence of covalent binding of this compound to proteins, thus modifying their functions (Fig. 2).The process is called homocysteinylation and can be either Shomocysteinylation, when Hcy binds through a disulfide bond to a free protein sulfhydryl residue, or N-homocysteinylation, when Hcy binds through an amide bond to an ε amino group of a protein lysine residue.
Hcy binds through a disulfide bond to fibronectin, annexin II, and intracellular metallothionein, among other intracellular proteins (Majors et al., 2002;Hajjar et al., 1998;Barbato et al., 2007).S-Homocysteinylation of the first two proteins results in inhibition of fibronectin-fibrin interaction and annexin II-tissue plasminogen activator interaction, respectively.One of the plasma proteins that is subject to modification through homocysteinylation is transthyretin (Hanyu et al., 2009).This modification could be responsible for the denaturation and deposition of this protein, as seen in familial amyloid polyneuropathy.Homocysteinylation of metallothionein could be responsible for redox imbalance.Generally, Shomocysteinylation alters the function of biomolecules through: (i) inactivation of potentially active free thiol groups, and (ii) alteration of redox potential of a protein molecule.N-Homocysteinylation is a consequence of the high reactivity of Hcy-thiolactone towards free amino functions, especially the ε amino group of lysine residues from proteins (Jakubowski, 1997;1999).In vivo, Hcythiolactone targets and modifies blood albumin, hemoglobin, immunoglobulins, LDL, HDL, transferrin, antitrypsin, and fibrinogen (Jakubowski, 2002a;2008a).Also, Hcy-thiolactone acts as an inhibitor of Na + /K + -ATP-ase from the cortex, hippocampus, and brain stem of rats, affecting the membrane potential with deleterious effects for neurons (Rasic-Markovic et al., 2009).
Elevated plasma levels of both Hcy-thiolactone and proteins modified through N-homocysteinylation are a direct consequence of either genetic defects in Hcy metabolism or a methionine-rich diet (Brosnan et al., 2004;Jakubowski et al., 2009).Spiroski et al. (2008) showed that methylenetetrahydrofolate reductase (MTHFR-677 and MTHFR-1298) genotypes and haplotypes can be correlated with increased tHcy plasma levels in patients with occlusive artery disease and deep venous thrombosis.
Plasma concentration of proteins modified through N-homocysteinylation ranges between 0.35 and 0.51 µM (Jakubowski et al., 2008).However, in pathological conditions the plasma concentration of these proteins ranges from a two-fold elevation (renal failure) up to a 24-30fold elevation (genetic defects of MTHFR and cystathionine β-synthase) (Perna et al., 2006).
Generally, N-homocysteinylation alters the function of proteins through (i) introduction of new free thiol groups, and (ii) inactivation of free amino groups, affecting the overall redox potential of proteins.Moreover, it has been found that proteins modified through N-homocysteinylation can act as neoantigens, triggering activation of the inflammatory response, a key component in atherogenesis, atherotrombosis and stroke etiology (Undas et al., 2004).Also, these neoantigens induce an autoimmune response, the concentration of autoantibodies being higher in some pathological conditions (i.e., cerebrovascular disease, renal failure) than in normal subjects (Undas et al., 2007).This situation could explain, at least in part, the association of hyperhomocysteinemia with certain stroke subtypes such as small vessel disease, large vessel disease, and primary intracerebral hemorrhage (Eikelboom et al., 2000;Li et al., 2003;Perini et al., 2005).It has been hypothesized that N-homocysteinylated proteins ex-posed at the luminal face of endothelial cells are recognized by specific antibodies, the neoantigen-autoantibody interaction leading to activation of circulating macrophages, which become responsible for repeated vascular endothelium damage.Moreover, Hcy-thiolactone impairs the ability of the vascular endothelium to regenerate itself through direct inhibition of lysyl oxidase, which is responsible for the correct cross-linking of collagen and elastin in the arterial wall (Liu et al., 1997;Raposo et al., 2004).
One mechanism of protection from N-homocysteinylation is through the action of the enzyme called paraoxonase 1 (PON1, EC 3.1.8.1).PON1, a calciumdependent enzyme synthesized by the liver, is a major component of plasma HDL particles, responsible for the antioxidant protection of LDL particles.For PON1, three enzymatic activities have been described: paraoxonase, arylesterase and lactonase (Billecke et al., 2000).The paraoxonase activity is the ability of the enzyme to hydrolyze different organophosphate compounds (paraoxon, diazoxon, soman, sarin).The arylesterase activity is evaluated using phenylacetate as a substrate, while lactonase activity is evaluated using different aromatic lactones (i.e., dihydrocoumarin, 2-coumaranone, homogentisic acid lactone).It is believed that under physiological conditions, the lactonase activity prevails, one of the enzyme's in vivo substrate being Hcy-thiolactone.PON1 has the ability to hydrolyze Hcy-thiolactone, this activity being a major determinant of plasma N-homocysteinylated protein concentration (Jakubowski, 2000;Perla-Kajan & Jakubowski, 2010).It was found that both in vitro and in vivo the lactonase activity of PON1 negatively correlates with N-homocysteinylated protein concentration.Impairment of the PON1 Hcy-thiolactonase activity is responsible, at least in part, for the severity of cardiovascular disease in subjects with coronary artery disease (Koubaa et al., 2009).Thus, due to the lactonase/Hcythiolactonase activity of PON1, HDL particles could prevent post-translational modification of LDL apoproteins through N-homocysteinylation.
Another report suggests that Hcy exerts its toxicity through induction of endoplasmic reticulum (ER) stress.Increased intracellular Hcy concentration is associated with both alteration of redox balance and post-translational protein modifications through N-and S-homocysteinylation. ER is very sensitive to intracellular Hcy or Hcy metabolites' accumulation due to a local redox imbalance and Ca 2+ release.As a consequence of the local redox imbalance, proteins misfold, initiating an ER stress response (Chigurupati et al., 2009) that triggers the expression of different chaperone proteins, growth arrest and apoptosis (Outinen et al., 1999;Zhang et al., 2001).Intracytoplasmic accumulation of calcium has deleterious effects leading to excessive ATP consumption as cells try to pump Ca 2+ out, and also activates a series of enzymes such as calpain, phospholipase A 2 , nitric oxide synthase etc.
The effect of high plasma Hcy concentration on gene expression is dual.Transcription of the gene coding for glutathione peroxidase-1 (GPx1) is down regulated by Hcy (Lubos et al., 2007).Moreover, Hcy downregulates endothelial heparane sulfate with subsequent decrease of extracellular membrane-bound superoxide dismutase present at the luminal face of the endothelium and as a consequence, the endothelium becomes exposed to deleterious effects of O 2 •- (Yamamoto et al., 2000).Other studies suggest that Hcy induces the expression of superoxide dismutase in endothelial cells leading to con- sumption of NO • and thus impairing endothelial vasorelaxation (Hucks et al., 2004).

HOMOCysTeINe as aN aggRessION faCTOR agaINsT THe eNDOTHelIUM
Hcy is an important endothelial aggression factor promoting endothelial dysfunction through different mechanisms, leading to atherosclerotic plaque formation.Hcy (i) inhibits the growth of endothelial cells, (ii) induces an imbalance between O •-and NO • with deleterious effects on the vascular physiology, (iii) induces the expression of different adhesion molecules, and (iv) promotes the formation of modified LDL particles, which are important players in the etiology of atherosclerotic plaque formation (Fig. 3).
Hcy activates, through different pathways, NAD(P)H oxidase and xanthine oxidase, two important pro-oxidant enzymes that generate O 2 •-in the endothelial cells.
Using human umbilical vein endothelial cells (HU-VEC) it was found that Hcy activates the NADPH oxidase complex through plasma membrane translocation of the cytoplasmic subunit p47 phox (Carluccio et al., 2007).The fact that Hcy-dependent activation of NAD(P)H oxidase plays a central role in endothelial redox imbalance was proved by the ability of the NAD(P)H oxidase inhibitor apocynin which restores both O 2 •-concentration and vascular tonus to control Hcy levels in hyperhomocysteinemic rats (Edirimanne et al., 2007).
Another important source of O 2 •-in the vascular endothelium is xanthine oxidase (Bagi et al., 2002).Administration of the xanthine oxidase inhibitor oxypurinol prevented flow-induced constriction of arterioles from rats with hyperhomocysteinemia.
O 2 •-can interact with NO • , synthesized by the constitutive endothelial nitric oxide synthase (eNOS), producing the peroxynitrite anion (ONOO -).Consumption of NO • through this reaction is responsible for alteration of the vascular tonus.Moreover, ONOO -is responsible for the induction of thromboxane A 2 synthesis in both endothelial cells and platelets, leading to vasoconstriction (Ungvari et al., 2000;Bagi et al., 2002).This is probably achieved through peroxynitrite-dependent inactivation of prostaglandin I 2 synthase, arachidonic acid becoming thus available for thromboxane A 2 synthesis (Zou et al., 1999).
Free radicals induce the conversion of tetrahydrobiopterin (BH 4 ), one of the eNOS cofactors, to trihydrobiopterin (BH 3 • ) leading to eNOS uncoupling (Patel et al., 2002).There is evidence for a direct involvement of Hcy in free radical-induced eNOS uncoupling (Topal et al., 2004).Decreased bioavailability of BH 4 for eNOS changes the activity of this enzyme from NO • to O 2 •- production.Moreover, in animal models for hyperhomocysteinemia, administration of an O 2 •-scavenger (e.g., superoxide dismutase, 4,5-dihydroxybenzene 1,3-disulfonate) reverses endothelial dysfunction, suggesting that at least in part, the Hcy-dependent endothelial dysfunction is mediated by O 2 •- (Weiss et al., 2003).
Moreover, Hcy-induced overproduction of O 2 •-is responsible, at least in part, for the activation of the nuclear factor κB (NF-κB), a key regulator of gene expression (Au-Yeung et al., 2004).NF-κB induces the expression of genes coding for pro-inflammatory proteins like monocyte chemoatractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin, and receptor for advanced glycation end products (RAGE) in endothelial cells (Poddar et al., 2001;Hofmann et al., 2001;Carluccio et al., 2007;Hwang et al., 2008).Thus, Hcy enhances monocyte binding to vascular endothelium, a key event in the process of atherosclerotic plaque formation.In vitro studies using endothelial cells, monocytes and macrophages exposed to different concentrations of Hcy for different times, have revealed different degrees of inducfigure 3. some of the biological effects of Hcy at endothelial level.
A metabolic peculiarity of endothelial cells that renders them very susceptible to Hcy toxicity is their inability to metabolize Hcy through the transsulfuration pathway (Jacobsen et al., 1995).Hcy alters gene expression in endothelial cells through different mechanisms: (i) inhibition of the transcription of the cyclin A gene, (ii) inhibition of cyclin A messenger RNA expression, and (iii) transcriptional repression of the gene coding for FGF2 (fibroblast growth factor 2) (Wang et al., 2002;Jamaluddin et al., 2007;Chang et al., 2008).These effects are the result of alteration of the methylation patterns of the promoters of the cyclin A and FGF2 genes.
In vitro studies suggested an additional mechanism for Hcy toxicity through post-translational modifications of LDL particles.Hcy promotes both oxidation of LDL particles by ceruloplasmine's oxidase activity, and nitration (Exner et al., 2002;Griffiths et al., 2006).Modified LDL particles are highly pro-aterogenic promoting the formation of foam cells through uptake by scavenger receptors expressed by monocytes.
Finally, another mechanism that in conjunction with the aggression effect upon endothelium could explain the proatherosclerotic effect of Hcy and Hcy-thiolactone is the ability of the latter compound to covalently modify serum fibrinogen.It was found that clots containing N-homocysteinylated fibrinogen are more resistant to fibrinolysis, leading to increased risk of thrombotic events (Jakubowski et al., 2008;2009).
These processes could provide an important link between hyperhomocysteinemia and atherosclerotic aggression on endothelium.

HOMOCysTeINe as a NeUROTOxIN
Both in vitro and in vivo studies have indicated that Hcy exerts neurotoxic effects inducing neuronal damage and cell loss through excitotoxicity and apoptosis.This could be, at least in part, a direct consequence of the inability of cerebral tissue to metabolize Hcy through the betaine and transsulfuration pathways, favoring Hcy accumulation in the nervous system (Finkelstein, 1998).
Stroke patients are a particular risk group as disruption of the blood-brain barrier exposes nervous tissue to plasma Hcy concentration for prolonged periods (Lindgren et al., 1995).Moreover, it was found that Hcy itself is able to induce blood-brain barrier (BBB) disruption (Kamath et al., 2006).The disruption of BBB is the consequence of at least two different processes.On the one hand, Hcy induces an imbalance between the activity of matrix metalloproteinase 9 (MMP-9) and the level of tissue inhibitor of metalloproteinase-4 (TIMP-4), through increasing MMP-9 activity and decreasing TIMP-4 level (Moshal et al., 2006;Tyagi et al., 2009).As a consequence, MMP-9 acts upon different components of the BBB leading to disruption of this structure.On the other hand, Hcy acts as an excitatory neurotransmitter through γ-aminobutyric acid (GABA) receptors A/B, leading to increased vascular permeability (Tyagi et al., 2005;2007).Hcy acts as an agonist for both group I and group III metabotropic glutamate receptors as well as for Nmethyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5methyl-4-isoxazolepropionate (AMPA)/kainite ionotropic glutamate receptors (Boldyrev and Johnson, 2007;Bleich et al., 2004;Ho et al., 2002).Overstimulation of these receptors triggers cytoplasmic calcium pulses, overproduction of free radicals, and activation of caspases leading to apoptosis.Using spontaneously hypertensive strokeprone rats, it was found that exposure of nervous tissue to Hcy during stroke is followed by secretion of excitatory amino acids, i.e., glutamate and aspartate, which are responsible for further excitotoxic effects on neurons (Ganguly et al., 2008).Activation of neuronal ERK2 (extracellular signal-regulated kinase 2) is a direct consequence of Hcy-induced NMDA receptor stimulation (Poddar & Paul, 2009).Also, after a transient activation of the pro-survival transcription factor CREB (cAMP response-element binding protein) by ERK2, CREB becomes inhibited, favoring neuronal apoptotic death.
Expression of NMDA receptor is not confined to neurons.Other cells, including endothelial cells from cerebral tissue, can express this receptor.Free radicals induce up-regulation of the NR1 subunit of the NMDA receptor, increasing the susceptibility of cerebral endothelial cells to excitatory amino acids, favoring BBB disruption (Betzen et al., 2009).
Another consequence of the nervous tissue exposure to Hcy is the activation of poly-ADP-ribose polymerase (PARP) in neurons leading to consumption of ATP and NAD + with deleterious effects upon energetic metabolism, followed by free radical production (Kruman et al., 2000).It was also found that Hcy is able to inhibit neuronal DDHA activity leading to accumulation of ADMA with subsequent inhibition of neuronal NOS (Selley, 2004).
Not only neuronal cells are subject to toxic effects of Hcy, but also microglia, which are brain-resident macrophages.Hcy promotes activation of these cells through induction of NAD(P)H oxidase (Zou et al., 2009).Also, in vivo studies indicated that Hcy exerts toxic effects on endothelial cells from cerebral vasculature through mechanisms previously presented (Dayal et al., 2004).Another consequence of hyperhomocysteinemia is represented by Hcy-induced toxic effect on endothelial progenitor cells (Alam et al., 2009).Exposure of these cells to high levels of Hcy is followed by induction of their apoptosis through caspase-3 and -8 activation and cytochrome c release from mitochondria.

HOMOCysTeINe, sTROKe aND VITaMIN THeRapy
Different clinical trials have recognized Hcy as an independent cardio-and cerebrovascular risk factor.One such trial enrolled 5661 monitored in time (Perry et al., 1995).During the follow-up period 141 cerebrovascular events (stroke) were recorded.Using a study group of 107 from among those that developed stroke and a control group of 118, it was found that the geometric mean of plasma Hcy concentration was significantly increased in the study group (13.7 µM vs. 11.9 µM, P = 0.004).
Due to the dependence of Hcy metabolism on folic acid and vitamins B 6 and B 12 it was suggested that administration of these vitamins could reduce plasma Hcy concentration, lowering the risk of stroke and stroke re-currence.Indeed, many trials have indicated that administration of folic acid and vitamins B 6 and B 12 decreased serum Hcy concentration.However, some of these studies failed to show a direct correlation between vitamin intake and lowered risk for cardio-and cerebrovascular events (Clarke et al., 2002).Some potential causes responsible for the lack of consensus between these trials could be (i) differences between study groups in respect to baseline Hcy concentrations, (ii) inclusion of patients from countries that do not have regulations regarding food enrichment with folic acid, (iii) trial period, (iv) composition of vitamin formulations, (v) gender distribution, and (vi) patients' medication.Also, patients' compliance is an important factor for the success of trials that investigate the impact of some drugs on the course of a pathological condition.
The VISP (Vitamin Intervention for Stroke Prevention) trial (double-blind, randomized, controlled study) showed no significant reduction in stroke recurrence between the group that received a low-dose vitamin formulation (200 µg B 6 , 6 µg B 12 , 20 µg folic acid) and the group that received a high-dose vitamin formulation (25 mg B 6 , 0.4 B 12 , 2.5 mg folic acid) (P = 0.80).There was also a modest difference in the reduction of Hcy plasma level between the two groups (2 µM) (Toole et al., 2004).One explanation for the failure of that study was the very low baseline level of plasma B 12 vitamin.Excluding the patients that received monthly vitamin B 12 , the patients with high plasma vitamin B 12 concentration (> 637 pmol/L) and those with renal failure generated a new group of 2155 persons.Statistical analysis performed with this new group indicated that stroke incidence in the new high-dose vitamin formulation group was almost 21 % lower than in the other group, suggesting a beneficial role of high doses of vitamin B 12 (P = 0.049) (Spence et al., 2005).At the same time, it was noted that a better capacity of vitamin B 12 absorption associated with the high-dose vitamin B 12 therapy decreased the incidence of cerebrovascular events.HOPE-2 (Heart Outcomes Prevention Evaluation 2) and NORVIT (Norwegian Vitamin) trials indicated a beneficial effect of folic acid and vitamins B 6 and B 12 on plasma Hcy concentration (Bønaa et al., 2006;Saposnik et al., 2009).One major difference between the HOPE-2 and NORVIT trials on the one hand and VISP trial on the other was their duration.The follow-up period for the first two studies was 5 years and 3.5 years, respectively, while the follow-up period for the VISP trial was 2 years.
The HOPE-2 trial indicated that the overall stroke incidence was lower in the vitamin group when compared to the placebo group (P = 0.03, Saposnik et al., 2009).Also the incidence of the two main types of stroke was lower in the vitamin group when compared to the control group (ischemic stroke, 30.62 % vs. 37.98 %; hemorrhagic stroke, 3.1 % vs. 3.87 %).By contrast, full recovery at day 7 or at discharge was more frequent, but not significantly, in the vitamin group (vitamin group vs. placebo group, 15.7 % vs. 9.6 %).In the period 24 h from the moment of stroke onset there was no significant improvement of the neurological deficit between the two groups.The results of HOPE-2 trial indicated that vitamin therapy could be beneficial for some particular categories: persons younger than 70 years (untreated dyslipidemia, no medication for coagulopathies), patients with hyperhomocysteinemia, and people from countries where there is no regulation regarding food enrichment with folic acid.
Another conclusion of the VISP, HOPE-2 and NOR-VIT trials was that correction of vitamin B 12 status in the elderly, in conjunction with folic acid therapy, could decrease stroke risk.
A recent meta-analysis study of several prospective trials from 2007 until 2009 has offered some possible directions for future research in this area (Lee et al., 2010).It was found that Hcy-lowering therapy could be beneficial in patients with vascular disease in early stages.Also, such an approach could be more successful in men than in women.This study suggested that only clinical trials with follow-up periods of 3 years or more report decreased stroke incidence, probably because the biological effect only then becomes apparent.
The supplementation of grain products with folic acid was implemented mandatory by 1998 in the USA and Canada.If high homocysteine concentrations are an independent risk factor for stroke, one might expect a reduction in stroke mortality following folic acid fortification.Indeed, a recent population-based study showed that the decline in stroke mortality accelerated in 1998-2002 in nearly all population in the USA and Canada (Wang et al., 2007).

CONClUsIONs
Despite the beneficial effects of vitamin therapy on plasma Hcy concentration, there are no unequivocal data regarding the impact of lowering Hcy level on the incidence and/or recurrence of cerebrovascular events.This situation could be solved by using larger population trials.Also, the trial period could be an important factor for the success of such studies, enabling the beneficial effects to become apparent.Stroke incidence and recurrence should be investigated in relation with gender, as men have higher incidence of cerebrovascular events than women, along with a plethora of secondary pathologies.Another issue that should be better addressed is the subclinical vitamin B 12 deficiency that affects a large number of elderly people.Correction of vitamin B 12 status, along with administration of the other vitamins required for Hcy metabolism could lead to a decrease in the frequency of cerebrovascular events.

figure 2 .
figure 2. S-and N-homocysteinylation of proteins by homocysteine and homocysteine thiolactone, respectively