Volume 43, Issue 10

Early concepts of the origin of arteriosclerosis were introduced in the 19th century by Rokitansky and Virchow, who described mural thrombosis, inflammatory damage to arterial intima, increased intimal permeability to plasma, mucoid degeneration of arterial wall, deposition of plasma lipids in plaques, and fibrosis and calcification of plaques. Experimental production of arteriosclerosis by feeding animal foods to rabbits was attributed to protein intoxication by Ignatowsky in 1908 and to dietary cholesterol by Anitschkow in 1913. Newburgh confirmed the protein hypothesis in 1915–1925 but failed to identify which amino acid produced plaques because methionine (1922) and homocysteine (1932) had not yet been discovered. Cases of homocystinuria from inherited deficiency of cystathionine synthase were found to be associated with thrombosis and vascular disease 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. The theory explains experimental arteriosclerosis by deficiency of vitamin B 6 in monkeys, choline deficiency in rats, thyroid deficiency in rats, and methionine deficiency in monkeys. The thrombogenic and atherogenic effects of homocysteine were demonstrated in rabbits, baboons and other species, 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 for arteriosclerosis in the general population, supporting the validity of the theory.

High plasma homocysteine levels are associated with a moderately increased risk of cardiovascular disease, particularly of atherosclerotic events. We review the association of plasma homocysteine with heart failure, with a specific focus on a series of previously published investigations from the community-based Framingham Heart Study that evaluated the relations of plasma homocysteine levels with overt heart failure, and with its key antecedents, echocardiographic left ventricular (LV) mass and hypertension. In the Framingham sample, higher plasma homocysteine levels were associated with increased risk of new-onset heart failure in both men and women, with a more continuous and graded relation being observed in women. A positive relation between homocysteine and LV mass was observed in women, but not in men; this may underlie the stronger relations of homocysteine to heart failure risk in women. Plasma homocysteine was not associated with hypertension incidence prospectively in either sex. The relations of increased homocysteine to heart failure (in both sexes) and to greater LV mass (in women) noted in the Framingham sample should be confirmed in other community-based samples. Secondary analyses of heart failure outcomes in ongoing randomized clinical trials may provide insights into whether lowering of plasma homocysteine levels is associated with a reduction in LV mass and/or a reduction of heart failure risk.

Cardiovascular disease is a major problem in diabetes, and risk factors presumably unrelated to diabetes, such as hyperhomocysteinaemia, may be related to the development of cardiovascular complications in diabetic individuals. Plasma homocysteine levels are usually normal in diabetes, although both lower and higher levels have been reported. Homocysteine levels in diabetes are modulated by hyperfiltration and renal dysfunction, as well as low folate status. Insulin resistance does not appear to be a major determinant of plasma homocysteine level. Hyperhomocysteinaemia has been associated with microalbuminuria and retinopathy in type 1 and type 2 diabetes. In patients with type 2 diabetes, plasma homocysteine concentration is a significant predictor of cardiovascular events and death. This relation seems to be stronger in subjects with diabetes than without. The underlying pathophysiological mechanism of this increased vascular risk remains unexplained, but may be related to worsening of endothelial dysfunction and/or structural vessel properties induced by oxidative stress. Because homocysteine and diabetes have apparent synergistic detrimental vascular effects, patients with diabetes are candidates for screening and treatment with folic acid until the results of ongoing clinical trials are available.

In this review we discuss the hypothesis, and current evidence, that a decreased concentration of the endogenous purine-nucleoside adenosine contributes to the increased cardiovascular risk of patients with hyperhomocysteinemia. In hyperhomocysteinemia, the reaction equilibrium of the reaction catalysed by S-adenosylhomocysteine hydrolase will shift towards synthesis of S-adenosylhomocysteine, at the expense of free adenosine. Adenosine receptor stimulation induces several cardiovascular protective effects, such as vasodilation, inhibition of thrombocyte aggregation, of inflammation and of vascular smooth muscle cell proliferation. A decreased adenosine concentration could, therefore, well contribute to the cardiovascular complications of hyperhomocysteinemia. Previous animal studies have shown that administration of homocysteine decreases extracellular adenosine, associated with increased synthesis of S-adenosylhomocysteine. Recently, we showed that in patients with classical homocystinuria, cellular adenosine uptake is enhanced, thus limiting adenosine-induced vasodilation. These observations provide us with additional pharmacological targets, such as adenosine uptake inhibition, to reduce cardiovascular risk in patients with hyperhomocysteinemia.

Homocysteine may have deleterious effects on the cardiovascular system. It has been hypothesized that these effects may be brought about by a decrease in the adenosine concentration via the S-adenosylhomocysteine hydrolase reaction. A requirement for this causal relationship is proof of a reduction in vascular adenosine concentration during conditions of elevated homocysteine concentrations. In the present communication we summarize published data obtained during systematic variation of the arterial homocysteine concentration. Most of the results reported show that an increase in homocysteine concentration to 100μM is associated with a 20–50% decrease in vascular adenosine concentration and an increase in tissue S-adenosylhomocysteine level. Homocysteine effects on the adenosine concentration seem to be more pronounced under conditions of impaired oxygenation. Further experiments, in particular on organs and tissue that release high amounts of homocysteine, i.e., the liver, are warranted to study the potential effects of homocysteine on vascular and tissue adenosine concentrations and consequent effects on organ function. The evidence obtained may be relevant for future assessment of risk indicators in conjunction with homocysteine pathogenicity, which might potentially be extended to measurements of adenosine or S-adenosylhomocysteine levels.

Metabolic conversion of homocysteine (Hcy) to a chemically reactive metabolite, Hcy-thiolactone, catalyzed by methionyl-tRNA synthetase is the first step in a pathway that is suggested to contribute to Hcy toxicity in humans. The accumulation of Hcy-thiolactone is detrimental because of its intrinsic ability to modify proteins by forming N -Hcy-protein adducts, in which a carboxyl group of Hcy is N -linked to the ε -amino group of a protein lysine residue. N -linked Hcy occurs in each protein examined and constitutes a significant pool of Hcy in the blood. N -Hcy proteins are likely to be recognized as neo-self antigens and induce an autoimmune response. Indeed, we found that autoantibodies specific for an Nε -Hcy-Lys epitope on N -Hcy-proteins occur in humans. Serum levels of anti- N -Hcy-protein autoantibodies are directly correlated with plasma tHcy, but not with plasma cysteine or methionine levels. In a group of male patients with stroke, levels of anti- N -Hcy-protein autoantibodies and tHcy were significantly higher than in a group of healthy subjects. In a group of male patients with angiographically documented coronary artery disease, seropositivity for anti- N -Hcy-protein autoantibodies occurred five-fold more frequently than in controls and was an independent predictor of coronary artery disease. These findings show that the formation of N -Hcy-proteins has important physiological consequences and support a hypothesis that N -Hcy-protein is a neo-self antigen that contributes to immune activation, an important modulator of atherogenesis.

The aim of this work was to study the association between plasma thiol levels and percentage carotid narrowing in a group of 68 patients who underwent a carotid endarterectomy, pertained as a risk factor for vascular and cardiovascular disease. Total plasma thiols were measured by capillary electrophoresis laser-induced fluorescence. The mean values of the hematological parameters studied were within normal limits and 25% of the patients were hyperhomocysteinemic (homocysteine >15μmol/L). Pearson's correlation between carotid narrowing degree and the most common risk factors for atherosclerosis showed a positive relationship only between carotid narrowing degree and cysteine levels (r=0.252; p<0.05). Stepwise multiple linear regression with carotid narrowing degree as the dependent variable, and cysteine, homocysteine, age, triglyceride and low-density lipoprotein-cholesterol as independent variables confirmed that cysteine was significantly associated with these variables. By regrouping the population according to cysteine and homocysteine concentration percentiles, we found positive correlation between these parameters and median values of carotid narrowing degree. Our study provides experimental evidence to confirm that plasma homocysteine and cysteine are involved in carotid narrowing after carotid endarterectomy intervention, suggesting that cysteine may be involved in the deleterious molecular mechanisms active in carotid stenosis.

Mild hyperhomocysteinemia is established as an independent risk factor for atherothrombotic disease, including ocular pathologies such as retinal vascular occlusion and non-arteritic ischemic optic neuropathy (NAION). Low intake or low status of B-vitamins explains elevated total homocysteine (tHcy) concentrations only in part. The underlying cause for disturbed homocysteine metabolism requires further insight. We investigated whether the combined determinations of plasma tHcy, methylmalonic acid (MMA) and cystathionine provide more information on the causes of impaired homocysteine metabolism as compared with vitamin B 12 , vitamin B 6 and folate in patients with ocular ischemic vascular disease. A total of 51 hyperhomocysteinemic (>12μmol/L) patients with retinal vascular occlusion (n=42) and NAION (n=9) were included. Mild renal dysfunction was an important determinant of tHcy, indicated by the positive correlation between creatinine and tHcy (r=0.47, p=0.001). The assessment of MMA in addition to tHcy identified at least 12 out of 51 patients (23%) who were most likely to have a functional vitamin B 12 deficiency. An additional 14 patients (27%) with elevated MMA and cystathionine levels also had slightly elevated concentrations of creatinine, pointing to the need for discrimination between renal dysfunction and vitamin B 12 deficiency in this group. In contrast, measurement of cystathionine is very sensitive for renal dysfunction and this marker was strongly related to serum creatinine (r=0.56, p<0.001) and to tHcy (r=0.50, p<0.001). Measurement of the vitamins folate, vitamin B 12 and vitamin B 6 in plasma did not provide sufficient information on intracellular disturbances in homocysteine metabolism. In conclusion, the metabolites homocysteine, cystathionine and MMA are sensitive indicators and valuable for discrimination of the underlying cause of mild to moderate hyperhomocysteinemia, with implications for therapeutic targeting.

Hyperhomocysteinaemia almost invariably occurs in patients with end-stage renal disease (ESRD), but there is debate whether, within the group of ESRD patients, higher or lower plasma homocysteine concentrations are related to an increased risk of vascular disease. Homocysteine is thought to be vasculotoxic in high concentrations, but it may also lead to elevated levels of its precursor, S-adenosylhomocysteine (AdoHcy), which is a potent inhibitor of the transmethylation pathway, in which S-adenosylmethionine (AdoMet) donates its methyl group to a variety of acceptors. Impairment of this transmethylation pathway in ESRD patients has been suggested by high AdoHcy levels, decreased AdoMet/AdoHcy ratios, decreased protein repair requiring methyltransferases, and by DNA hypomethylation. Stable isotope techniques using labelled methionine have indeed demonstrated a decreased whole body transmethylation flux in ESRD patients. These studies have also shown that folic acid treatment is capable of restoring transmethylation rates to normal values. The remaining hyperhomocysteinaemia after folic acid treatment in ESRD is probably due to a persistent impairment of homocysteine clearance through transsulphuration. DNA hypomethylation with its concurrent alterations in gene expression is largely improved by folate treatment. The adverse effects of hyperhomocysteinaemia in ESRD may thus be related to impaired transmethylation. Normalisation of plasma homocysteine does not seem to be required to restore transmethylation to normal levels in ESRD patients.

Hyperhomocysteinemia is present in the majority of well-nourished chronic renal failure and uremic patients. Most observations reported in the literature come from studies carried out in end-stage renal disease patients treated with hemodialysis. The underlying mechanisms of the toxic effects of homocysteine in uremia related to cardiovascular disease and other disturbances are still under scrutiny. As a consequence, macromolecules (i.e., proteins and DNA) have been found to be altered to various extents. One of the mechanisms of homocysteine toxicity is related to the action of its metabolic precursor, S-adenosylhomocysteine, a powerful methyltransferase competitive inhibitor. Disruption of DNA methylation has been demonstrated to occur as a result of hyperhomocysteinemia, and/or is associated with vascular damage. DNA hypomethylation has been found in the mononuclear cell fraction of uremic patients with hyperhomocysteinemia. Proteins are also targets of homocysteine-dependent molecular damage. The formation of oxidative products with free cysteinyl residue thiol groups has been demonstrated to occur in blood. The latter also represents a mechanism for the transport of homocysteine in plasma. In addition, homocysteine thiolactone has been shown to react with free amino groups in proteins to form isopeptide bonds, in particular at the lysine residue level. Another type of isopeptide bond in proteins may result from the deamidation and isomerization of asparaginyl residues, yielding abnormal isoaspartyl residues, which have been demonstrated to be increased in uremic patients. Folate treatment exerts a partial, but significant, homocysteine-lowering effect in uremic patients and has been shown to improve the changes in macromolecules induced by high homocysteine levels. In conclusion, both DNA and proteins are structurally modified in uremia as a consequence of high homocysteine levels. The role of these macromolecule changes in inducing the clinical complications of hyperhomocysteinemia in these patients, although still conjectural in some respects, is at present sustained by several pieces of evidence.

The role of hyperhomocysteinemia (HHcy) as a risk marker for cardiovascular diseases in renal patients is a matter of controversy. The remethylation of homocysteine (Hcy) to methionine in the kidneys is of great importance for Hcy clearance. Hcy remethylation is markedly decreased in patients on hemodialysis, but transsulfuration remains mostly unaffected. Markedly increased concentrations of methylmalonic acid (MMA), as a metabolic marker of vitamin B 12 deficiency, have been found in approximately 70% of renal patients. This is in contrast to normal concentrations of vitamin B 12 usually reported in such patients. We demonstrated in cell culture experiments that the uptake of vitamin B 12 by mononuclear cells from renal patients was lower than that taken up by cells from controls. The lowering of MMA and Hcy concentrations in renal patients after B 12 administration may indicate the presence of intracellular pre-treatment deficiency. We administered folic acid (5mg) plus vitamin B 6 (50mg) and B 12 (0.7mg) three times per week intravenously to hyperhomocysteinemic dialysis patients. Hcy decreased after 4weeks by 51%. Hcy was normalized in almost all patients, while serum concentrations of MMA and cystathionine were reduced by 28% and 26%, respectively. Cystathionine, an indicator for the transsulfuration pathway, showed a drastic increase in renal disease and was only slightly lowered by B-vitamin treatment. The increased cystathionine/cysteine ratio in renal patients indicates possible impairment of the catabolism of cystathionine by cystathionase. Moreover, renal failure is associated with severe abnormalities in plasma concentrations of S-adenosyl Hcy (SAH) and S-adenosyl methionine (SAM), as well as the SAM/SAH ratio. This ratio is an indicator of the availability of methyl groups from SAM. Therapeutic doses of B-vitamins in dialysis patients led to a limited improvement in the biomarkers of methylation and probably did not have a significant effect on transmethylation potential in the cells. Furthermore, elevated serum levels of asymmetric dimethylarginine (ADMA) in renal patients, which are associated with a poor outcome for such patients, could be lowered, but this effect was confined to patients who had no anemia. Future studies may consider extending the duration of vitamin treatment, as well as agents that may enhance the hydrolysis of SAH and cystathionine.

Prevalence rates for folate deficiency and hyperhomocysteinemia have been markedly reduced following the introduction of folic acid fortification in the United States. We report the prevalence of hyperhomocysteinemia in a population of community-dwelling elderly Latinos in the post-folic acid fortification era. We measured homocysteine, total vitamin B 12 , holotranscobalamin, red blood cell folate, and serum creatinine in 1096 subjects aged ≥60years. Hyperhomocysteinemia (>13μmol/L) was observed in 16.5% of the subjects. The population attributable risk percentages for hyperhomocysteinemia were 29.7% for total B 12 <148pmol/L, 36.4% for holotranscobalamin <35pmol/L, and 41.4% for creatinine >115μmol/L. In contrast, the population attributable risk percentage for hyperhomocysteinemia was only 0.3% for red blood cell folate <365nmol/L. We conclude that in the post-folic acid fortification era, low vitamin B 12 status has become the dominant nutritional determinant of hyperhomocysteinemia. Steps to either reduce the prevalence of vitamin B 12 deficiency or to identify and treat individuals with vitamin B 12 deficiency could further reduce the prevalence of hyperhomocysteinemia.

Studies linking hyperhomocysteinemia (HHCY) and B-vitamin deficiency to some health aspects in children have been accumulating. Low B-vitamin status inearly life, even as early as the time of conception, may endanger the potential for new life and may negatively influence the health of the offspring. Early abortion, pregnancy complications and poor pregnancy outcomes have been linked to elevated concentrations of total plasma homocysteine (tHcy) and low folate or vitamin B 12 . Maternal vitamin status predicts that of fetuses and neonates. Lactating women are likely to experience low micronutrient status, which might affect breast-milk composition and hence the nutritional status of their breast-fed infants. Elevated concentrations of methylmalonic acid (MMA) is common in infants (age <6months), which may indicate a transient inadequate vitamin B 12 status. Deficiency of B-vitamins might confer deleterious effects on the physical and mental health of the child, such as impaired growth, gross motor function, poor school performance and other adaptive skills. The importance of maintaining adequate B-vitamin status during periods of progressive growth and development should be emphasized because symptoms related to folate and vitamin B 12 deficiency are difficult to detect. Serum levels of tHcy and MMA should be estimated in several target groups of children, pregnant and lactating women and those planning for pregnancy. Concentrations of tHcy and MMA are useful indicators of B-vitamin status in the pediatric laboratory. Using these functional markers may facilitate detecting sub-optimal B-vitamin status in children.

A commercially available holotranscobalamin (holo-TC) radioimmunoassay (RIA) (Axis-Shield, Dundee, Scotland) was evaluated in four laboratories and compared with a holoTC ELISA run in one laboratory. The performance of the holoTC RIA assay was comparable in three of the four participating laboratories. The results from these three laboratories, involving at least 20 initial runs of “low”, “medium” and “high” serum-based controls (mean holoTC concentrations 34, 60 and 110pmol/L, respectively) yielded an intra-laboratory imprecision of 6–10%. No systematic inter-laboratory deviations were observed on runs involving 72 patient samples (holoTC concentration range 10–160pmol/L). A fourth laboratory demonstrated higher assay imprecision for control samples and systematic deviation of results for the patient samples. Measurement of holoTC by ELISA showed an imprecision of 4–5%, and slightly higher mean values for the controls (mean holoTC concentrations 40, 70 and 114pmol/L, respectively). Comparable results were obtained for the patient samples. The long-term intra-laboratory imprecision was 12% for the holoTC RIA and 6% for the ELISA. In conclusion, it would be prudent to check the calibration and precision prior to starting to use these holoTC assays in research or clinical practice. The results obtained using the holoTC RIA were similar to those obtained using the holoTC ELISA assay.

Analyses of total homocysteine (tHcy) and to some extent methyl malonic acid (MMA) have become increasingly used in Sweden, primarily for investigating folate and cobalamin deficiency. This has led to increasing costs for diagnosis and laboratory testing, since clinicians still order the established tests, cobalamin and folate. The purpose of this study was to compare the profile of test parameters in six Swedish counties, using laboratory statistics from 2003, and to relate these to medical prescriptions for folate and cobalamin in the same counties. We also wanted to evaluate whether or not a laboratory algorithm with tHcy as the primary test-parameter, followed by vitamin analyses only when tHcy is above a certain decision limit, could reduce the expanding cost of diagnosing folate and cobalamin deficiency. For this analysis we used patient results from two counties in Sweden collected during 2003. There is a slight positive correlation between resources spent upon tests and resources spent upon treating these deficiencies, and a laboratory algorithm based upon initial analysis of tHcy, instead of conventional clinical requests for all parameters, could save approximately 30% of laboratory costs. A typical annual saving in one of these counties (ca. 260,000 inhabitants) by implementing this algorithm would be ca €100,000.

Betaine serves as a methyl donor in a reaction converting homocysteine to methionine, catalysed by the enzyme betaine-homocysteine methyltransferase. It has been used for years to lower the concentration of plasma total homocysteine (tHcy) in patients with homocystinuria, and has recently been shown to reduce fasting and in particular post-methionine load (PML) tHcy in healthy subjects. Betaine exists in plasma at concentrations of about 30μmol/L; it varies 10-fold (from 9 to 90μmol/L) between individuals, but the intra-individual variability is small. Major determinants are choline, dimethylglycine and folate in plasma, folic acid intake and gender. Recent studies have demonstrated that plasma betaine is a stronger determinant of PML tHcy than are vitamin B 6 and folate. The betaine-PML tHcy relationship is attenuated after supplementation with B-vitamins, and is most pronounced in subjects with low folate. Betaine shows a weaker association with fasting tHcy (than with PML tHcy), and also this association is most pronounced in subjects with low folate. In pregnancy, plasma betaine declines until gestational week 20, and thereafter remains constant. From gestational week 20 onwards, fasting tHcy shows a strong inverse association with plasma betaine, and betaine becomes a stronger predictor than folate of fasting tHcy. To conclude, betaine status is a component of an individual's biochemical make-up with ramifications to one-carbon metabolism. Betaine status should be investigated in pathologies related to altered metabolism of homocysteine and folate, including cardiovascular disease, cancer and neural tube defects.

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Although there is a growing body of evidence that homocysteine plays a causal role in atherogenesis, specific mechanisms to explain the underlying pathology have remained elusive. This review focuses on chemistry unique to the homocysteine molecule to explain its inherent cytotoxicity. Thus, the high pKa of the sulfhydryl group (pKa=10.0) of homocysteine underlies its ability to form stable disulfide bonds with protein cysteine residues, and in the process, alters or impairs the function of the protein. Albumin, fibronectin, transthyretin, annexin II, and factor V have now been identified as molecular targets for homocysteine, and in the case of albumin, the mechanism of targeting has been elucidated.

Inflammation, immune activation and oxidative stress play a major role in the pathogenesis of cardiovascular disorders. In addition to markers of inflammation, moderate hyperhomocysteinemia is an independent risk factor for cardiovascular disease, and there is a link between the activation of immunocompetent cells and the enhanced formation of homocysteine in vitro. Likewise, anti-inflammatory drugs and nutrients rich in antioxidant vitamins are able to reduce cardiovascular risk and to slow down the atherogenic process. Resveratrol, a phenolic antioxidant synthesized in grapes and vegetables and present in wine, has also been supposed to be beneficial for the prevention of cardiovascular events. Apart from its strong antioxidant properties, resveratrol has also been demonstrated to act as an anti-inflammatory agent. In this study the influence of resveratrol on the production of homocysteine by stimulated human peripheral blood mononuclear cells (PBMCs) was investigated. Results were compared to earlier described effects of the anti-inflammatory compounds aspirin and salicylic acid and of the lipid-lowering drug atorvastatin. Stimulation of PBMCs with the mitogens concanavalin A and phytohemagglutinin induced significantly higher homocysteine accumulation in supernatants compared with unstimulated cells. Treatment with 10–100μM resveratrol suppressed homocysteine formation in a dose-dependent manner. Resveratrol did not influence the release of homocysteine from resting PBMCs. The data suggest that resveratrol may prevent homocysteine accumulation in the blood by suppressing immune activation cascades and the proliferation of mitogen-driven T-cells. The effect of resveratrol to down-regulate the release of homo-cysteine was comparable to the decline of neopterin concentrations in the same experiments. The suppressive effect of resveratrol was very similar to results obtained earlier with aspirin, salicylic acid and atorvastatin; however, it appeared that doses of compounds needed to reduce homocysteine levels to 50% of stimulated cells were always slightly lower than those necessary to achieve the same effect on neopterin concentrations. The influence of resveratrol and of all the other compounds on homocysteine production appears to be independent of any direct effect on homocysteine biochemistry.

Elevated serum homocysteine has been associated with increased risk of Alzheimer's disease. Furthermore, elevated homocysteine levels are related to cognitive dysfunction in the elderly. The aim of the present study was to explore the disease specificity of the relation between serum total homocysteine levels and cognitive function. For this, we summarize data from several studies on homocysteine levels in both normal and pathological conditions performed in our laboratories and evaluate possible mechanisms of effects of elevated homocysteine levels in the central nervous system. Total homocysteine levels were measured in serum of: 1) healthy aging individuals; 2) patients with Alzheimer's and Parkinson's disease and patients with other cognitive disorders; and 3) patients with multiple sclerosis. Increased serum homocysteine concentration was related to worse cognitive performance over a 6-year period in the normal aging population (r=−0.36 to −0.14, p<0.01 for the Word learning tests; r=0.76, p<0.05 for the Stroop Colored Word test). Homocysteine was only increased in patients with Parkinson's disease on L-Dopa therapy (18.9 vs. 16.5μmol/L in healthy controls), and not in dementia patients. Homocysteine was elevated in patients with progressive multiple sclerosis (15.0μmol/L, n=39, compared to 12.0 μmol/L in 45 controls) and correlated to both cognitive and motor function (r=−0.33 and −0.33, p<0.05, respectively). The relationship between homocysteine and cognitive function in non-pathological and pathological situations indicates that changes in its levels may play a role in cognitive functioning in a broad spectrum of conditions.

Elderly subjects with mild cognitive impairment have a high risk for conversion to Alzheimer's disease or are already in a preclinical dementia stage. By cross-sectionally comparing subjects in prodromal and early phases of dementia with non-demented controls, we tested the hypothesis whether low serum vitamin B 12 and folate and high plasma total homocysteine concentrations precede or are a consequence of dementia onset. From a large population of 623 consecutive subjects seen at the Memory Clinic (Ospedale Beata Vergine, Mendrisio, Switzerland), 433 subjects could be included in the analyses: 79 elderly controls, 218 Clinical Dementia Rating 0.5 subjects, and 136 demented patients (111 with Alzheimer's disease and 25 with vascular dementia). As in an earlier report on a smaller sample of the same population (n=228), the lowest folate tertile was strongly associated with mild cognitive impairment (adjusted OR=3.1) and Alzheimer's disease (adjusted OR=4.0). Hyperhomocysteinemia showed a significant association not only with Alzheimer's disease (adjusted OR=3.1) but, at variance with the previous report, also with mild cognitive impairment (adjusted OR=2.6). Present reanalysis results suggest that subclinical folate deficiency and hyperhomocysteinemia might predate dementia onset, findings to be confirmed by longitudinal studies.

Type 2 diabetes mellitus and higher total plasma homocysteine concentrations are each associated with an increased incidence of cardiovascular disease and with diminished cognitive performance. Relations between homocysteine concentrations and cardiovascular disease incidence are stronger in the presence of type 2 diabetes mellitus. Therefore, we hypothesized that relations between homocysteine concentrations and cognitive performance would be stronger in the presence of type 2 diabetes. We related homocysteine concentrations and cognitive performance on the Mini-Mental State Examination in 817 dementia- and stroke-free participants of the Maine-Syracuse Study, 90 of whom were classified with type 2 diabetes mellitus. Regardless of statistical adjustment for age, sex, gender, vitamin co-factors (folate, vitamin B 6 , vitamin B 12 ), cardiovascular disease risk factors, and duration and type of treatment for type 2 diabetes mellitus, statistically significant inverse associations between homocysteine concentrations and cognitive performance were observed for diabetic individuals. The weaker inverse associations between homocysteine concentrations and cognitive performance obtained for non-diabetic individuals were not robust to statistical adjustment for some covariates. Interactions between homocysteine concentrations and type 2 diabetes mellitus are observed such that associations between homocysteine and cognitive performance are stronger in the presence of diabetes.

Elevated plasma homocysteine (Hcy) concentrations are associated with Alzheimer's disease and vascular dementia. Several recent reports have indicated that L-dopa treatment is an acquired cause of hyperhomo-cysteinemia. Despite the fact that a large proportion of Parkinson's disease (PD) patients develop cognitive dysfunctions or dementia, particularly in the late stages of the illness and after long-term L-dopa treatment, the relationship between Hcy and dementia in PD has not been fully investigated. The aim of this study was to evaluate plasma Hcy levels in a group of L-dopa-treated PD patients with cognitive impairment and to elucidate a possible role of Hcy in the development of cognitive dysfunctions in PD. We compared Hcy, vitamin B 12 and folate levels in 35 parkinsonian patients treated with L-dopa (14 with cognitive dysfunctions, 21 without cognitive impairment). Analysis of the data revealed that mean Hcy levels were significantly higher in the group with cognitive dysfunctions (21.2±7.4 vs. 15.8±4.4μmol/L; p=0.0001), while there was no difference in age, sex, B 12 and folate levels. In addition, logistic regression analysis showed that the risk of cognitive dysfunction progressively increased according to Hcy levels after correction for age, sex and B-vitamin status (odds ratio, 19.1; 95% CI, 1.5–241.4; p=0.02). Our results raise the possibility of a relationship between Hcy levels and cognitive dysfunctions in this group of L-dopa-treated PD patients. However, prospective studies on large cohorts of patients should be performed to clarify such an association.

Osteoporosis is a widespread problem, which frequently has devastating health consequences through its association with fragility fractures. The total number of fractures, and hence the cost to society, will increase dramatically over the next 50years as a result of demographic changes in the number of elderly people. Thus, prevention of osteoporosis by identifying risk factors or risk indicators, as well as the development of new treatment strategies, are major issues. Recent data suggest that homocysteine (Hcy), folate, vitamin B 6 and vitamin B 12 affect bone metabolism, bone quality and fracture risk in humans. Since circulating Hcy depends on folate, vitamin B 6 and vitamin B 12 , Hcy could be suitable as a risk indicator for micronutrient-deficiency-related osteoporosis. Initial experimental results indicate that Hcy is not only a risk indicator, but also a player in bone metabolism. Moreover, existing data open speculation that folate, vitamin B 6 and vitamin B 12 act not only via Hcy-dependent pathways, but also via Hcy-independent pathways. However, more studies are needed to clarify the mechanistic role of Hcy, folate, vitamin B 6 and vitamin B 12 in bone metabolism.

Background: Recently, increased plasma homocysteine (Hcy) has been suggested as an independent risk factor for osteoporotic fractures. Therefore, it is tempting to speculate that Hcy adversely affects bone metabolism. This study aimed to analyze the relation between Hcy and biochemical markers of bone metabolism and bone mineral density (BMD). Materials and methods: We investigated 143 peri- and post-menopausal women [median age (25th–75th percentile), 67 (57–75) years]. All subjects underwent a detailed medical examination, measurement of bone mineral density at lumbar spine (BMD-LS) and total hip (BMD-HIP), and fasting venous blood and urine sampling. Osteocalcin (OC), serum calcium (Ca), urinary desoxypyridinoline cross-links (DPD), osteoprotegerin (OPG) and soluble receptor activator of NF-κB ligand (sRANKL) were studied. Results: According to BMD subjects were classified as normal (n=24), osteopenic (n=51) or osteoporotic (n=68). Median Hcy did not differ between normal, osteopenic and osteoporotic subjects (p=0.647). Partial correlation analysis, controlling for the major confounders, age, creatinine, menopause and previous fractures, revealed significant correlations between Hcy and DPD (r=0.193, p=0.022), as well as between Hcy and Ca (r=0.170, p=0.045). After adjustment for the same confounders, subsequent regression analysis confirmed significant associations of Hcy with DPD and Ca. No significant relations could be observed between Hcy and BMD-LS, BMD-HIP, OC, OPG or sRANKL. Conclusion: Our results demonstrate weak, but significant, relations between Hcy and markers of organic and inorganic bone resorption, suggesting a mechanistic role of Hcy in bone metabolism. The relation between Hcy and bone resorption was not dependent on OPG or sRANKL.

The endothelium plays a crucial role in the maintenance of vascular tone and structure by releasing the endothelium-derived vasoactive mediator, nitric oxide (NO). NO is formed in healthy vascular endothelium from the amino acid precursor L-arginine. Endothelial dysfunction is caused by various cardiovascular risk factors, metabolic diseases, and systemic or local inflammation. One mechanism that explains the occurrence of endothelial dysfunction is the presence of elevated blood levels of asymmetric dimethylarginine (ADMA) – an L-arginine analogue that inhibits NO formation and thereby can impair vascular function. Accumulating evidence from prospective clinical studies suggests that elevated plasma or serum levels of ADMA are associated with an increased risk of major adverse cardiovascular events. This article gives an updated overview of the currently available literature on ADMA and cardiovascular disease from prospective clinical trials. Recently, advances have been made in the development of analytical methods that are reliable and fast enough to allow determination of ADMA in clinical routine.

Asymmetric dimethylarginine (ADMA) is a potent inhibitor of nitric oxide synthase and is regarded as a novel risk factor for cardiovascular disease. The metabolic pathways of ADMA and homocysteine are strongly intertwined. First, during synthesis of ADMA, two equivalents of homocysteine are formed. Second, homocysteine has been shown to inhibit the ADMA-degrading enzyme dimethylarginine dimethylaminohydrolase. Finally, homocysteine, either directly or by increasing oxidative stress, may promote release of free ADMA by accelerating protein degradation. Currently used techniques for the quantification of ADMA in plasma are mostly based on liquid chromatography with fluorimetric or mass spectrometric detection. Plasma ADMA has a very narrow concentration distribution, with an inter-individual coefficient of variation of approximately 12%, and even slightly elevated ADMA concentrations are associated with increased cardiovascular disease risk. Therefore, to generate useful results in clinical research, high precision of the assay used for the quantification of ADMA assay is a matter of prime importance. Assays with a high coefficient of variation may lead to low statistical power in clinical trials and to a severe underestimation of the strength of associations in epidemiological studies.

Background: Hyperhomocysteinemia, a proxy measure for the nutritional status of the B vitamins, may be involved in the etiology of preeclampsia via inducing endothelial dysfunction. Asymmetric dimethylarginine (ADMA) is an inhibitor of NO-synthase that may adversely affect the endothelium. Materials and methods: We investigated serum concentrations of folate, vitamin B 12 , B 6 , homocysteine (Hcy) and related metabolites in 139 Syrian preeclamptic women and 93 asymptomatic pregnant women of comparable age, gestational age and socioeconomic status. Plasma concentrations of ADMA were determined in a subset of age- and gestation-age-matched pairs of patients and controls (n=63). Results: Higher concentrations of Hcy, cystathionine and methylmalonic acid (MMA) were closely linked to a lower status of the B vitamins. Higher concentrations of Hcy and cystathionine were observed in the preeclamptic group than in the matched controls (median Hcy 9.3 vs. 6.0μmol/L; median cystathionine 284 vs. 232nmol/L). Serum folate was significantly lower in patients than in controls (16.4 vs. 36.0nmol/L). Folate supplementation was less likely to be used in preeclamptic women. Concentrations of MMA were elevated in patients and controls and did not differ significantly between the two groups. Median plasma concentrations of ADMA were significantly lower in asymptomatic women than in those who developed preeclampsia before the 37th week of gestation (0.61 vs. 0.68μmol/L). Conclusions: Elevated serum concentrations of Hcy, cystathionine and MMA indicate poor status of the B vitamins during pregnancy. The adverse effect of Hcy on endothelial function might be related to ADMA in early-onset preeclampsia. More emphasis should be placed on increasing the intake of B vitamins in pregnant women from developing countries.

The adverse effect of hyperhomocysteinemia on the vascular wall can be partially explained by increasing plasma concentration of asymmetric dimethylarginine (ADMA), a potent inhibitor of nitric oxide synthase. The aim of the study was to compare ADMA and homocysteine levels in three groups of subjects: blood donors with normal homocysteine concentration (group A), patients with hyperhomocysteinemia and normal kidney function (group B) and hemodialysis patients who are known to be hyperhomocysteinemic (group C). Concentrations of homocysteine (enzymatic method), ADMA (enzyme-linked immunoassay) and creatinine (Jaffe method) in EDTA plasma were measured. Plasma ADMA levels were significantly higher in both groups with hyperhomocysteinemia (1.60±0.56μmol/L in group B, 1.81±0.57μmol/L in group C) when compared with those in blood donors (0.82±0.29μmol/L, p<0.001 in both cases). Significant positive correlations were found between concentrations of ADMA and homocysteine (r=0.42, p<0.0001), ADMA and creatinine (r=0.39 p<0.001), homocysteine and creatinine (r=0.69, p<0.0001), age and homocysteine (r=0.47, p<0.001), age and ADMA (r=0.57, p<0.001) and age and creatinine (r=0.37, p<0.001). Increased ADMA concentrations in hyperhomocysteinemic patients were confirmed, but multiple linear regression analysis showed that this significant correlation is only apparent due the dependence of both parameters on age.

Inadequate folate intake and aging are each strongly implicated as important risk factors for certain cancers. Since both folate depletion and aging are strongly associated with hyperhomocysteinemia, genomic DNA hypomethylation, and increased uracil misincorporation into DNA, it appears that each of them enhances carcinogenesis by inducing a derangement of one-carbon metabolism that supplies one-carbons to biological methylation reactions and nucleotide synthesis. Recent studies have demonstrated that inadequate dietary folate and aging may interact and synergistically disturb the normal homeostasis of one-carbon metabolism, thereby provoking subsequent biochemical and molecular aberrations, including alterations in critical gene expression related to carcinogenesis. These studies have further indicated that modest folate supplementation may reverse or partially ameliorate those adverse effects induced by folate depletion and aging.

Since vitamin B 12 serves as a cofactor in the synthesis of methyl precursors for biological methylation and enables methylfolate to be recycled for nucleotide synthesis, B 12 deficiency has been known to induce hyperhomocysteinemia and inadequate DNA synthesis, along with “methylfolate trap”. Even though depletion of B 12 , a common B-vitamin deficiency in the elderly, has not often been invoked as a causative factor in carcinogenesis, a recent animal study demonstrated that a B 12 -deficient diet, which was of insufficient severity to cause anemia or illness, disturbed normal homeostasis of one-carbon metabolism in the colonic mucosa and resulted in diminished genomic DNA methylation and increased uracil misincorporation in DNA, both of which are purported mechanisms for one-carbon metabolism-related colonic carcinogenesis.

Vegetarians have a lower intake of vitamin B 12 than omnivores do. Vitamin B 12 deficiency (holotranscobalamin II <35pmol/L or methylmalonic acid >271nmol/L) was found in 58% of 71 vegetarians studied. Higher homocysteine levels (>12μmol/L) found in 45% indicate disturbed remethylation of homocysteine to methionine. The methylation of DNA is strongly linked to homocysteine metabolism. Since DNA methylation is an important epigenetic factor in the regulation of gene expression, alteration of the methylation pattern has been associated with aging, cancer, atherosclerosis and other diseases. Three observations indicate that DNA methylation could be diminished by a vegetarian lifestyle. The vegetarian diet has a low content of methionine, remethylation of homocysteine is reduced by vitamin B 12 deficiency and elevated homocysteine levels can induce the generation of S-adenosylhomocysteine (SAH), a potent inhibitor of methyltransferases. In our study we observed a significant correlation between SAH and whole-genome methylation (r=−0.36, p<0.01). This observation underlines the role of SAH as a potent inhibitor of methyltransferases. The methylation status was not correlated with homocysteine or S-adenosylemethionine (SAM). These results indicate that the degree of methylation does not depend on the supply of methyl groups and that the reverse generation of SAH has no influence. In addition to whole-genome methylation, the specific promoter methylation of the p66Shc gene was studied. However, the latter did not correlate with SAH, SAM or homocysteine. Obviously, the promoter methylation of the p66Shc gene is controlled in a specific way, without following the general regulating influence of SAH. In conclusion, an inhibitory effect of SAH on whole-genome methylation was found, but from our data no interaction between vegetarian lifestyle and DNA methylation could be determined.