Folate and methionine intake, gene-nutrition interaction and homocysteine as cvd risk factor

During the last years, elevated plasma total homocysteine (tHcy) has been one of the most studied risk factors for heart diseases.

Homocysteine (Hcy) is a sulphur-containing amino acid, which is formed from the essential amino acid methionine. Defects in intracellular Hcy metabolism lead to the elevation of plasma tHcy. These metabolic defects can have a genetic or a nutritional background, i.e. an inadequate intake of folate or vitamin B6 or B12 that serve as cofactors or substrates to the enzymes involved in the Hcy metabolism. Approximately two thirds of the cases with elevated tHcy levels have been estimated to be due to low or moderate concentrations of these vitamins, of which folate is considered the most important. Few previous epidemiological studies have addressed the link between folate and the risk of cardiovascular diseases (CVD). In some studies, subjects with lower circulating folate concentrations or lower dietary intake of folic acid have had higher risk of coronary events compared with others, although not all studies have found this association.

Although it has been supposed that elevated plasma tHcy concentration is a risk factor for CVD, the risk-increasing mechanisms are still poorly understood. It has been proposed that high plasma tHcy concentration alters the anticoagulant properties of endothelial cells to a procoagulant phenotype, causes dysfunction of the vascular endothelium or enhances lipid peroxidation.

Gene-nutrient interactions, folate and paraoxonase (PON)

The human serum paraoxonase/arylesterase (PON) is an antioxidative enzyme in HDL, which eliminates radicals in the circulation and protects against coronary diseases. PON has been suggested to account for an important part of the antioxidative property of HDL, and it has been shown that PON protects LDL against oxidation. Its activity is modulated by two common amino acid polymorphisms at positions 192 (Gln Q > Arg R) and 55 (Met M > Leu L) in the paraoxonase gene PON1. A lowered PON activity has been reported also in patients with atherosclerotic heart disease. Low PON activity or polymorphisms in PON1 gene that are associated with paraoxonase levels in serum are also associated with CHD in some prospective studies.

In human plasma Hcy exists in various forms: less than 1% is in the reduced (sulfhydryl) form, remaining part is oxidized and exists as various disulphides, such as Hcy thiolactone. Hcy thiolactone is formed in all cell types in a human and because inadvertent reactions of thiolactone with proteins are potentially harmful, the ability to detoxify Hcy thiolactone is essential for biological integrity. 
Dr. Jakubowski reported that the enzyme Hcy thiolactonase, which hydrolyzes Hcy thiolactone to Hcy, could be in fact paraoxonase. If it is so, paraoxonase can hydrolyze Hcy thiolactone back to Hcy and Hcy may be then converted either back to methionine (by reaction which needs folate and vitamin B12 as co-factors), or condensed with serine to form cystathionine in a reaction that is dependent on vitamin B6 (transsulphuration pathway). In light of Dr Jakubowski's study, it is possible that folic acid supplementation or high folate intake decreases plasma tHcy (and plasma Hcy-thiolactone levels) and affects serum PON activity by this mechanism.

Gene-nutrient interactions, folate, homocysteine and COMT

Catechol-O-methyltransferase (COMT) is an enzyme that has a crucial role in dopamine inactivation. A common functional polymorphism (Val108Met) in the COMT gene is associated with a three- to four-fold variation in enzyme activity. The low activity genotype has been associated with alcoholism and some other psychiatric disorders, such as bipolar disorder and schizophrenia. COMT also catalyzes the O-methylation of various compounds, like catechol estrogens and dietary polyphenols, and is closely involved to homocysteine metabolism. Therefore we wanted to test the hypothesis that the functional polymorphism in COMT gene could modify the coronary event risk by increasing effect of serum tHcy. Our manuscript "Interaction of serum total homocysteine and folate concentration and the catechol-O-methyltransferase (COMT) gene with risk of acute coronary events: the Kuopio Ischaemic Heart Disease Risk Factor Study" is now submitted.

Our earlier results and research interests

We have shown in our Kuopio Ischaemic Heart Disease Risk Factor (KIHD) Study that high serum concentration and dietary intake of folate is associated with significantly lower risk of acute coronary events, but in a nested case-control setting of this same cohort elevated plasma tHcy concentration is not associated with elevated risk of coronary events. In KIHD Study low folate intake is also significantly related to increased CVD and overall mortality (in a Cox' proportional hazards' model adjusted for age, examination years, serum LDL and HDL cholesterol, diabetes, systolic blood pressure and BMI, men in the highest fifth of folate intake had a relative risk of CVD death of 0.50 (95% CI 0.27 to 0.92), the risk of CHD death of 0.54 (95% CI 0.26 to 1.13), and death due to any cause of 0.63 (95% CI 0.47 to 0.86), when compared with men in the lowest fifth of folate intake).In a cross sectional analysis of Antioxidant Supplementation in Atherosclerosis Prevention (ASAP) Study we have shown that high plasma tHcy levels are associated with enhanced in vivo lipid peroxidation in men, as measured by plasma F2-isoprostane concentrations. We have also shown in supplementation study of 40 men that dietary folic acid enhances serum PON activity.Sari Voutilainen's present research interest in homocysteine-folate metabolism is to study association between folate and homocysteine with PON enzyme activity, PON genes and CVD risk. Genes available for these analyses three mutations in PON1 genes and one in PON2 gene. Material available for these analyses is KIHD Study 4-year visit cohort, n=1038 men.

Homocysteine and other CVD risk factors

High circulating Hcy concentrations may increase the risk of CVD when present with other CVD risk factors. For example, there is some evidence that in hypercholesterolemic patients the risk of an atherosclerotic event was about three times higher in patients with high plasma tHcy concentrations compared to those with low tHcy concentrations. Another study done in subjects with elevated LDL cholesterol concentrations suggests that even mildly increased plasma tHcy levels are of crucial importance for deterioration of endothelial function. Increased risk of mortality in the subjects with both elevated cholesterol and tHcy concentrations may at least partly be explained by deterioration of endothelial function.Similar findings to those found in hypercholesterolemic patients have also been reported in case of increased plasma fibrinogen levels or in smokers. The increased risk with increased fibrinogen and tHcy concentrations could be explained by their complementary roles in the platelet activation-aggregation cascade. Fibrinogen represents a major step in platelet aggregation while homocysteine impairs nitric oxide production and also contributes to the generation of oxidized species. In some studies smoking has been shown to increase plasma tHcy concentrations, but in the KIHD study population this is not seen. Smokers have an increased risk of vascular disease in general and according to some recent research the risk is greatly increased in the presence of a raised plasma tHcy concentration when compared with non-smokers.

Homocysteine and methionine

Currently Jyrki Virtanen is studying the effects of high plasma tHcy concentration on CVD risk in men of the KIHD study, who also have other CVD risk factors. These include smoking, high serum total and LDL cholesterol and apo-B apolipoprotein concentrations and high plasma fibrinogen concentration. Preliminary results would seem to indicate that although tHcy alone is not a risk factor for CVD in this study population, it may increase the risk when present with above mentioned risk factors.

Since Hcy is formed from an essential dietary amino acid methionine, high intakes of methionine increase the plasma tHcy concentrations. This happens because the capacity of the transsulfuration pathway is exceeded and Hcy is excreted from cells. Elevation of plasma tHcy occurs, for example, in the oral methionine loading test, in which a large dose of methionine (0.1 g/kg body weight of L-methionine) is ingested to diagnose hyperhomocysteinemia. It could be speculated that a long-term high methionine intake from diet could lead to modest but chronic plasma tHcy concentrations, which in turn could increase the risk of CVD. 

Homocysteine and stroke

Although high plasma tHcy has been suggested to be a risk factor for CVD, its role as a risk factor for stroke is more controversial. Although most case-control studies suggest it to be a risk factor for stroke, the results from prospective studies are conflicting. Two recent meta-analyses, however, have concluded that Hcy might increase the risk of stroke. The mechanisms through which Hcy could cause stroke are its hypercoagulative effects in ischemic stroke and promotion of plaque rupture in hemorrhagic stroke. Jyrki Virtanen's next paper will concern the role of high plasma tHcy concentration in the risk of overall and ischaemic stroke in the KIHD study population. 


Although folate or folic acid intake could lower the risk of CVD through reducing plasma tHcy concentrations, elevated homocysteine may also be only a marker for low folate and/or vitamin B6 status or an indicator of an unhealthy lifestyle rather than a causal risk factor per se. Ongoing intervention trials will indicate whether homocysteine-lowering through vitamin supplementation prevents heart diseases, or are the measured circulating high homocysteine and low folate levels just markers of unhealthy lifestyle.






More information about our studies: sari.voutilainen at, jyrki.virtanen at

Our publications:

Voutilainen Sari et al: Functional COMT Val158Met Polymorphism, Risk of Acute Coronary Events and Serum Homocysteine: The Kuopio Ischaemic Heart Disease Risk Factor Study. PLoS ONE 2007 Jan 31;2:e181.

Virtanen J et al. High dietary methionine intake increases the risk of acute coronary events in middle-aged men. Nutr Metab Cardiovasc Dis 2006;16:113-20.

Virtanen J et al. Serum homocysteine, folate and risk of stroke: Kuopio Ischaemic Heart Disease Risk Factor (KIHD) Study. Eur J Cardiovasc Prev Rehabil 2005;4:369-75.

Voutilainen S, Virtanen JK, Rissanen TH, Alfthan G, Laukkanen J, Nyyssonen K, Mursu J, Valkonen VP, Tuomainen TP, Kaplan GA, Salonen JT. Serum folate and homocysteine and the incidence of acute coronary events: the Kuopio Ischaemic Heart Disease Risk Factor Study. Am J Clin Nutr. 2004;80(2):317-23.

Voutilainen S, Rissanen T, Virtanen J, Lakka TA, Salonen JTS. Low folate intakes are associated with an excess risk of acute coronary events: the Kuopio Ischaemic Heart Disease Risk Factor Study. Circulation 2001;103:2674-2680.

Voutilainen S, Lakka TA, Porkkala-Sarataho E, Kaplan GA, Salonen JT. Low serum folate levels are associated with an excess risk of acute coronary events: the Kuopio Ischaemic Heart Disease Risk Factor Study. Eur J Clin Nutr 2000;54:242-248.  

Voutilainen S, Lakka TA, Hämelahti P, lehtimäki T, Poulsen HE, Salonen JT. Plasma total homocysteine concentration and the risk of acute coronary events: the Kuopio Ischemic Heart Disease Risk factor Study. Journal of Internal Medicine 2000;248:217-222. 

Voutilainen S, Morrow J, Roberts J, Alfthan G, Nyyssönen K, Salonen J. Correlation between Plasma Total Homocysteine Concentration and Plasma F2-Isoprostane in 100 men in Eastern Finland. Arteriosclerosis, Thrombosis and Vascular Biology 1999;19:1263-1266.  

Voutilainen S, Alfthan G, Nyyssönen K, Salonen R, Salonen JT. Association Between Elevated Plasma Homocysteine and Increased Carotid Artery wall Thickness in Men. Annals of Medicine 1998;30:300-306. 

Voutilainen S, Rissanen T, Seppänen K, Porkkala-Sarataho E, Kaikkonen J, Tuomainen T-P, Virtanen J, Lehtimäki T, Malin R, Penttilä I, Kaplan GA, Salonen JT. Folic acid increases serum paraoxonase activity: evidence from a double blind oral supplementation trial in men. Current Topics in Nutraceutical Research 2003;1:175-182.