Elevated Plasma Total Homocysteine Research Articles

Traffic-related Particles Are Associated with Elevated Homocysteine

Recent epidemiologic studies have shown that homocysteine, a sulfur-containing amino acid formed during the metabolism of methionine, is a risk factor for atherosclerosis, myocardial infarction, stroke, and thrombosis. Particulate air pollution has been related to cardiovascular death and hospital admission, but the underlying mechanisms are not fully elucidated. We examined the associations between ambient particulate air pollution and plasma concentrations of homocysteine among 960 community-residing older men (mean age, 73.6 +/- 6.9 yr). Total homocysteine in plasma, measured using high-performance liquid chromatography with fluorescence detection, was regressed on each ambient particulate pollutant (black carbon, organic carbon, sulfate or PM(2.5)), and effect modification by plasma and dietary B vitamins (folate, B6, and B12) was examined. The median concentration of total homocysteine was 10.6 micromol/L. Statistically significant positive associations of total homocysteine were observed with traffic-related particles (black carbon and organic carbon). No association was observed with sulfate, an indicator of coal combustion particles, or PM(2.5) (particulate matter < or = 2.5 microm in aerodynamic diameter). The effects of black carbon and organic carbon were more pronounced in persons with low concentrations of plasma folate and vitamin B12. Exposures to ambient particles, particularly from traffic, are associated with elevated plasma total homocysteine. Homocysteine may be a component or biological marker of the oxidation pathways underlying the effect of ambient particles on the cardiovascular system.

Association of Plasma Total Homocysteine Levels With Subclinical Brain Injury

Elevated plasma total homocysteine (tHcy) levels have been associated with increased risk of dementia and stroke, but it is uncertain whether the mediating mechanisms are predominantly cellular, vascular, or both. To evaluate the relationship between tHcy levels and findings at brain magnetic resonance imaging (MRI) in a community-based sample. Our sample comprised 1965 participants in the Framingham Offspring Study (1050 women; mean [SD] age, 62 [9] years) who were free of clinical stroke, dementia, or other neurologic disease affecting brain MRI and for whom at least 1 measurement of plasma tHcy level (1991-2001) and a brain MRI (1999-2002) were available. We used multivariate regression analysis to relate initial (1991-1995) and concurrent (1998-2001) plasma tHcy levels to total cerebral brain volume and lobar volumes as measures of neuronal loss and atrophy and to the presence or absence of silent brain infarcts and extensive white matter hyperintensity (log-white matter intensity > or =1 SD above the age-adjusted mean) as separate measures of vascular injury. Mean total cerebral brain volume was 78%. At MRI, 218 participants had silent brain infarcts and 250 demonstrated extensive white matter hyperintensity. Participants with a plasma tHcy level in the highest age- (-0.37%, P = .01) or sex-specific (-0.48%, P < .001) quartile had smaller total cerebral brain volumes compared with participants with lower tHcy levels. Initial tHcy levels were associated with a higher prevalence of silent brain infarct (relative risk, 1.5; 95% confidence interval, 1.1-2.1; P = .02) and concurrent tHcy levels, with smaller frontal (-0.14%, P = .001) and temporal lobar (-0.10%, P = .04) volumes. Prevalence of extensive white matter hyperintensity did not differ according to initial or concurrent plasma tHcy levels (relative risk, both 1.0; 95% confidence interval, 0.7-1.4 and 0.8-1.4, respectively). Higher plasma tHcy levels are associated with smaller brain volume and the presence of silent brain infarcts at MRI, even in healthy, middle-aged adults. Thus, both cellular and vascular mechanisms may underlie the association of plasma tHcy level with brain aging, as reflected by the effects on both subclinical and overt disease.

Association between the 2756A&gt; G variant in the gene encoding methionine synthase and myocardial infarction in Tunisian patients

Elevated plasma total homocysteine (tHcy), a risk factor for coronary artery disease (CAD), is due to defects in genes encoding for enzymes involved in tHcy metabolism or from inadequate status of vitamins involved in tHcy disposal. Methionine synthase (MS), a vitamin B(12)-dependent enzyme, catalyses the remethylation of homocysteine to methionine using a methyl group donated by 5-methyltetra-hydrofolate, which is the major circulating form of folate in the body. Functional genetic variants of the MS may alter tHcy as well as folate levels which are independent risk factors for CAD. The influence of a common genetic polymorphism 2756A>G of the MS gene (MTR) on plasma tHcy, folate and vitamin B(12) levels and its relation to the risk of myocardial infarction (MI) in a Tunisian case-control study was investigated. A total of 321 Tunisian patients with MI and 343 healthy controls were included in the study. The 2756A>G variant of the MTR was determined by polymerase chain reaction-restriction fragment length polymorphism analysis. Plasma tHcy was assessed with a fluorescent polarising immunoassay method. Plasma vitamin B(12) and folate were determined by microparticular enzyme immunoassay and ion-capture, respectively. A significant difference in genotype distribution and allele frequency was observed between patients and controls. Patients with MI had a frequency of 1.9% for the GG genotype, 26.2% for the AG genotype and 72% for the AA genotype. Controls had a frequency of only 0.9% for the GG genotype, 18.7% for the AG genotype and 80.5% for the AA genotype (chi(2)=6.97, p=0.03). The MI patient group showed a significant higher frequency of the G allele compared to controls (0.149 vs. 0.101; OR 1.55; 95% CI 1.10-2.18; p=0.008). The association between the 2756A>G variant in the gene encoding MS and MI was no longer significant after adjustment for other well-established risk factors. When clinical and laboratory values were compared amongst genotypes in the study groups, no significant differences were noted. The present study showed a significant but not independent association between the 2756A>G polymorphism of the MTR (presence of G allele) and MI in the Tunisian population.

The reduced folate carrier (RFC1) 80G>A and folate hydrolase 1 (FOLH1) 1561C>T polymorphisms and the risk of colorectal cancer: A nested case‐referent study

Objective. Polymorphisms in genes involved in folate uptake and metabolism may affect folate status and, thereby, the risk of cancer. In this nested case‐referent study, we related two such polymorphisms, reduced folate carrier (RFC1) 80G>A and folate hydrolase 1 (FOLH1) 1561C>T, to the risk of colorectal cancer, taking into account pre‐diagnostic plasma folate and total homocysteine concentrations and the MTHFR 677C>T polymorphism, which were analysed in a previous study. Material and methods. Subjects were 220 cases and 414 matched referents from the population‐based Northern Sweden Health and Disease Study. Results. The RFC1 80A‐allele was associated with reduced plasma folate and elevated plasma total homocysteine concentrations, but the result was statistically significant only for folate. In contrast, the FOLH1 1561T‐allele was associated with higher plasma folate and reduced plasma total homocysteine concentrations, and the result was statistically significant only for homocysteine. Neither polymorphism was related to the risk of colorectal cancer, either in univariate analysis or after adjusting for body mass index, current smoking, recreational and occupational physical activity and alcohol intake. Further adjustment for folate or homocysteine status or the MTHFR 677C>T polymorphism did not affect risk estimates. Subjects with the RFC1 80AA genotype in combination with low plasma folate concentrations or the MTHFR 677TT genotype had a reduced risk of colorectal cancer of borderline statistical significance. Conclusions. These findings suggest that although the RFC1 80G>A and FOLH1 1561C>T polymorphisms may influence folate status, they are not likely to have a major independent role in the development of colorectal cancer.

Association between Helicobacter pylori-infection, C-reactive protein and status of B vitamins

Some investigations, but not all, found that a chronic infection with Helicobacter pylori (Hp) is associated with deficiencies in B vitamins, elevated plasma total homocysteine concentrations (tHcy) and increased plasma levels of proinflammatory acute-phase proteins. It has been suggested that these factors promote atherogenesis and therefore could mechanistically explain why people infected with Hp might have an increased risk for cardiovascular diseases (CVD). Therefore we evaluated the association between Hp-infection, concentration of C-reactive protein (CRP), and status of various B vitamins in apparently healthy subjects. In 69 subjects with proved Hp-infection and 21 healthy control subjects identified in a cross sectional study, blood samples were collected to determine serum folate, serum vitamin B12, serum methylmalonic acid (MMA), serum CRP and plasma vitamin B6 and plasma total homocysteine (tHcy). The mean concentration of CRP was significantly higher in the Hp-positive collective than in controls. Although mean concentrations of vitamin B12, B6, and MMA differed between the groups, statistical significance was missed. However, the mean concentrations of homocysteine and folic acid were nearly the same in both groups. In univariate analysis a significant impact of Hp-status was shown on cobalamin (p=0.028; eta square: 0.055), and in multivariate analysis of variance the Hp-status had an impact on vitamin B12-values (p=0.028; eta square 0.057). In this study Hp-infection shows no significant impact on status of B vitamins, but has a significant influence on CRP concentration. However, this study does not support the hypothesis that Hp-infection is related to CVD via elevated levels of tHcy.

S-Adenosylhomocysteine—a better indicator of vascular disease than homocysteine?

It is widely accepted that elevated plasma total homocysteine is an independent risk factor for vascular disease. The relation is believed to be causal, but there is no generally accepted mechanism for the pathophysiology involved. The metabolic precursor of homocysteine in all tissues is S-adenosylhomocysteine (AdoHcy). AdoHcy is present in normal human plasma at concentrations approximately 1-500th of those of homocysteine, a fact that presents difficulties in measurement. The requirement for specialized equipment, complicated time-consuming methodology, or both is a reason that measurement of plasma AdoHcy has not generally been carried out in large studies. A recently published rapid immunoassay for AdoHcy in human plasma should make measurement of this important metabolite available for general use. Advantages of the measurement of plasma AdoHcy include 1) a smaller overlap of values between control subjects and patients, and thus the possibility of observing significant differences in fewer samples, 2) an accepted mechanism of metabolic activity as an inhibitor of all S-adenosylmethionine-mediated methyltransferases, and 3) evidence (from recent studies) that a higher plasma concentration of AdoHcy is a more sensitive indicator of vascular disease than is a higher plasma concentration of homocysteine.

S-Adenosylhomocysteine—a better indicator of vascular disease than homocysteine?

It is widely accepted that elevated plasma total homocysteine is an independent risk factor for vascular disease. The relation is believed to be causal, but there is no generally accepted mechanism for the pathophysiology involved. The metabolic precursor of homocysteine in all tissues is S-adenosylhomocysteine (AdoHcy). AdoHcy is present in normal human plasma at concentrations approximately 1-500th of those of homocysteine, a fact that presents difficulties in measurement. The requirement for specialized equipment, complicated time-consuming methodology, or both is a reason that measurement of plasma AdoHcy has not generally been carried out in large studies. A recently published rapid immunoassay for AdoHcy in human plasma should make measurement of this important metabolite available for general use. Advantages of the measurement of plasma AdoHcy include 1) a smaller overlap of values between control subjects and patients, and thus the possibility of observing significant differences in fewer samples, 2) an accepted mechanism of metabolic activity as an inhibitor of all S-adenosylmethionine–mediated methyltransferases, and 3) evidence (from recent studies) that a higher plasma concentration of AdoHcy is a more sensitive indicator of vascular disease than is a higher plasma concentration of homocysteine.

Correlations between homocysteine levels and atherosclerosis in Japanese type 2 diabetic patients

Elevated total plasma homocysteine (tHcy) level and aortic stiffness are associated with high mortality in type 2 diabetic patients. We tested the hypothesis that tHcy correlates with aortic stiffness and insulin resistance in type 2 diabetic patients. The study consisted of 40 Japanese patients with type 2 diabetes mellitus and high tHcy levels (mean age ± SD, 57 ± 7 years) and a control group of 45 age-matched patients with normal tHcy levels (mean age ± SD, 57 ± 6 years). Brachial-ankle pulse wave velocity (BaPWV) was measured by an automatic oscillometric method. Brachial-ankle pulse wave velocity was used as an index of atherosclerosis. Body mass index values ( P < .05), waist circumferences ( P < .05), and the waist-to-hip ratios ( P < .05) were larger in the high-tHcy group than in the normal-tHcy group. The BaPWV was higher in the high-tHcy group than in the normal-tHcy group ( P < .0001). Fasting plasma glucose ( P < .005) and insulin concentrations ( P < .0001), and the homeostasis model assessment (HOMA) index ( P < .0001) were higher in the high-tHcy group than in the normal-tHcy group. Multiple regression analysis showed that tHcy levels were independently predicted by BaPWV and the HOMA index. In conclusion, our results indicate that the elevated level of tHcy in Japanese patients with type 2 diabetes mellitus is characterized by increased aortic stiffness and insulin resistance, and that the BaPWV and the HOMA index are independent predictors of tHcy.

Assessing Plasma Total Homocysteine in Patients with End-Stage Renal Disease

Elevated plasma total homocysteine (tHcy) is a risk factor for cardiovascular disease; however, in light of several recent randomized trials, the issue of causality has been cast into doubt. Patients with end-stage renal disease are particularly interesting as they consistently have elevated tHcy and their leading causes of morbidity and mortality are related to cardiovascular disease. In the present article, we review the early evidence for the homocysteine theory of atherosclerosis, homocysteine metabolism, mechanisms of toxicity, and pertinent available clinical investigations. Where appropriate, the sparse evidence of homocysteine in peritoneal dialysis is reviewed. We conclude by addressing the difficulties associated with lowering plasma tHcy in patients with end-stage renal disease and suggest some novel methods for lowering tHcy in this resistant population. Finally, to address the issue of causality, we recommend that clinicians and scientists await the results of the FAVORIT trial before abandoning homocysteine as a modifiable risk factor for cardiovascular disease, as this study has recruited patients from a population with consistently elevated plasma tHcy who are known to respond to vitamin therapy.

Mesna as a Nonvitamin Intervention to Lower Plasma Total Homocysteine Concentration: Implications for Assessment of the Homocysteine Theory of Atherosclerosis

Elevated plasma total homocysteine is independently associated with atherosclerosis. Recent randomized trials show that vitamins lower total homocysteine but do not prevent cardiovascular events, suggesting the need for nonvitamin therapies to evaluate whether a causative relationship exists. Mesna (sodium 2-mercaptoethanesulfonate) is a thiol-containing drug capable of liberating homocysteine bound by disulfide bonds to proteins, facilitating its excretion. The effect of oral mesna on total homocysteine has not been evaluated and was the objective of this study. Eleven healthy volunteers received vehicle or 10 mg/kg mesna in random order, after which serial blood and urine samples were collected over 4 hours. Plasma total homocysteine decreased by 24.2% (P < .0001) following mesna. Urinary homocysteine excretion was significantly greater with mesna (3.9 +/- 2.4 mumol) compared to vehicle (0.4 +/- 0.1 mumol), P < .01. Oral mesna decreases plasma total homocysteine and is a potential nonvitamin treatment for assessing the homocysteine theory of atherosclerosis.

Hyperhomocysteinemia and central retinal vein occlusion in Iranian population

To evaluate total plasma homocysteine level during the acute phase of central retinal vein occlusion (CRVO) in the Iranian population and determine whether hyperhomocysteinemia is also a risk factor for CRVO. Fifty-four patients with recently diagnosed CRVO were studied. Their fasting total plasma homocysteine level was compared with a matched control group of 51 patients evaluated in the same clinic for a non-retinal disease diagnosis. The mean total plasma homocysteine level was 14.76+/-7.67 micromol/l in cases, and 11.42+/-3.74 micromol/l in control subjects. It showed a significant difference (P=0.005) in mean plasma homocysteine level between cases and control group. Odds ratio of CRVO for individuals with hyperhomocysteinemia was 2.88 (95% CI=1.08-7.71 and P=0.03). The overall multivariable-adjusted odds of CRVO in participants with plasma homocysteine level above 15 micromol/l was 4.71 (95% CI=1.46-15.19 and P=0.009) Hyperhomocysteinemia was not statistically different in each age group (<60 years: 27%, 61-70 years: 33.3%, 71-80 years: 31.6%, >81 years: 33.3%, Chi-square test, P=0.98). Elevated total plasma homocysteine level is an independent risk factor for CRVO in Iranian population. In addition to an evaluation of all conventional cardiovascular risk factors, measurement of total homocysteine for evidence of hyperhomocysteinemia may be important in the initial investigation and management of patients with CRVO.

Testosterone regulation of renal cystathionine β-synthase: implications for sex-dependent differences in plasma homocysteine levels

Elevated plasma total homocysteine (tHcy) is an independent risk factor for ischemic heart disease and stroke. Epidemiological studies reveal that men have higher tHcy levels than women, but the mechanism underlying this sex-dependent difference is unknown. One route for intracellular disposal of homocysteine is catalyzed by cystathionine beta-synthase (CBS). Renal function is known to be an important determinant of tHcy, and, in this study, we demonstrate that renal CBS expression and activity in mice diminished approximately twofold after castration, whereas ovariectomization was without effect. The higher renal CBS activity in males (22.7 +/- 3.1 mmol cystathionine.h(-1).kg kidney(-1)) vs. females (8.4 +/- 3.4 mmol cystathionine.h(-1).kg kidney(-1), P < or = 10(-6)) in C57Bl/6J mice was associated with lower plasma tHcy levels in males vs. females, and this difference was exacerbated in Cbs+/- mice (7.7 +/- 1.9 micromol/l in males vs. 13.8 +/- 6.4 micromol/l in females, P = 0.005). Surprisingly, mammals exhibit a diversity of regulatory patterns for kidney CBS, with females exhibiting lower CBS activity in mice, higher in rats and humans, and being indistinguishable from males in rabbit, hamster, and guinea pig. Our data suggest that testosterone-dependent regulation of human CBS in kidney may contribute to sex-dependent differences in homocysteine transsulfuration.

Plasma Homocysteine, Folate, and Vitamin B12 and the Risk of Hip Fracture: The Hordaland Homocysteine Study

Homocysteine and related factors were evaluated as risk factors for subsequent hip fractures among 4766 elderly men and women. High levels of homocysteine and low levels of folate predicted fracture, whereas vitamin B12 and genotypes were not related to fracture risk. High homocysteine may be a modifiable risk factor for hip fracture. Elevated plasma total homocysteine (tHcy) and deficiencies of folate and vitamin B12 are associated with risk of osteoporosis and fracture. We examined whether plasma levels of tHcy, folate, and vitamin B12 and the methylenetetrahydrofolate reductase (MTHFR) 677C-->T and 1298C-->T polymorphisms predicted hip fracture. This was a population-based prospective study of 2639 women and 2127 men who were 65-67 yr at enrollment in 1992-1993. Information on hip fracture was obtained from computerized records of discharge diagnoses from all hospitalizations in the region in the period between enrollment and November 30, 2005. Cox proportional hazard regression was used to estimate fracture risk according to levels of plasma tHcy, folate, and vitamin B12 and for different genotypes. Over a median follow-up period of 12.6 yr, hip fracture was recorded in 184 (7.0%) women and 90 (4.2%) men. The adjusted hazard ratio (95% CI) for fracture in subjects with high (>or=15 microM) compared with low levels (<9.0 microM) of tHcy was 2.42 (1.43-4.09) among women and 1.37 (0.63-2.98) among men. Dose-response analyses indicated a positive association between plasma tHcy and risk of fracture in both sexes and a negative association between plasma folate and risk of fracture among women only. Plasma vitamin B12 level or MTHFR genotype was not significantly related to risk of fracture after adjustments for confounding factors. The association between tHcy and risk of hip fracture was only slightly weakened by adjustments for plasma levels of vitamin B12 and folate. tHcy seems to be a predictor for hip fracture among elderly men and women. Folate was a predictor among women only, whereas vitamin B12 and MTHFR genotype did not predict hip fracture. Our data corroborate the hypothesis that homocysteine may play a role in the pathogenesis of osteoporotic fractures.

Plasma Homocysteine Is Elevated in Elderly Patients with Memory Complaints and Vascular Disease

Background: There is an association between elevated plasma total homocysteine (tHcy) and vascular disease in elderly patients suffering from organic and non-organic mental disease. The main objective of the present study was to investigate the relationship between plasma tHcy concentration and vascular disease in elderly patients with memory complaints. Methods: In 108 patients with memory complaints the concentration of plasma tHcy was related to the presence of vascular disease. Furthermore, different determinants of plasma tHcy concentration were measured. Results: Patients with confirmed memory complaints (CMC, n = 86) which fulfilled the criteria of mild cognitive impairment were divided into two categories, with and without vascular disease. The CMC group without vascular disease showed similar biochemical and brain imaging findings to patients with subjective memory complaints (SMC, n = 22). The group of CMC patients with vascular disease had higher age, higher plasma tHcy, lower serum folate and lower renal function than patients without vascular disease. Conclusion: The finding of two subgroups of patients with CMC supports the notion that mild cognitive impairment is a heterogenous clinical entity with multiple aetiological factors. The elevated plasma tHcy in the group of CMC patients with vascular disease is likely to be associated with vascular disease.

Antibodies to N-homocysteinylated albumin in patients with systemic lupus erythematosus

Hyperhomocysteinemia is known to predispose to atherosclerosis and occurs more commonly in patients with systemic lupus erythematosus (SLE) than in the general population. It has been shown that elevated plasma total homocysteine (tHcy) results in protein N-homocysteinylation and production o autoantibodies against N-homocysteinylated (N-Hcy) proteins. The aim of the study was to investigate whether anti-N-Hcy-albumin antibodies occur in patients with SLE and identify factors that determine these antibodies in such population. Patients and methods. In 50 subjects with SLE and 50 age- and sex-matched healthy controls, we determined serum IgG antibodies to N-Hcy-albumin using an in-house enzyme linked immunosorbent assay. Patients had higher plasma tHcy and C-reactive protein (CRP) than controls, while serum folate and witamin B12 were lower in patients. Levels of anti-N-Hcy-albumin were higher in patients with SLE than in controls (medians: 0.31; vs. 0.19; p < 0.0001). In SLE patients, levels of anti- N-Hcy-albumin antibodies correlated with tHcy (r = 0.83; p <0.0001), CRP (r = 0.33; p = 0.02) and the duration of the disease (r = 0.3; p = 0.04). Seropositivity to anti-N-Hcy-albumin antibodies was more frequent in SLE patients than in controls (50% vs. 10%; p < 0.001). In SLE patients tHcy and CRP concentrations, along with the duration of the disease were independent predictors of anti-N-Hcy-albumin antibodies level. There were no associations between a type or levels of antinuclear antibodies patent's, or age with anti-N-Hcy-albumin antibodies. Compared with healthy controls, in SLE patients levels of anti-N-Hcy-albumin antibodies are significantly higher and are largely determined by tHcy, CRP and the disease duration. This novel autoimmune response might contribute to increased risk of vascular events in SLE patients.

High prevalence of hyperhomocysteinemia in Chinese adults is associated with low folate, vitamin B-12, and vitamin B-6 status.

Elevated plasma total homocysteine (tHcy) concentrations are associated with lower folate, vitamin B-12, and vitamin B-6 status and are considered an independent risk factor for cardiovascular disease in developed countries, but data in developing countries are limited. We conducted a cross-sectional study to explore tHcy status and its association with plasma B vitamin status in 2471 Chinese men and women aged 35 to 64 y, living in the urban and rural areas of the northern and the southern regions of China. Blood samples were also collected in 2 seasons (spring and fall). The geometric mean plasma tHcy concentration was significantly higher in the north (adjusted geometric mean, 95% CI; 13.0 micromol/L, 12.6-13.3) than in the south (9.1, 8.9-9.4) after controlling for gender, area (urban and rural), age, and season (spring and fall). Twenty-eight percent of northerners and 7% of southerners had plasma tHcy concentrations>or=16.0 micromol/L, a level used to define hyperhomocysteinemia. Within each region, men had higher plasma tHcy concentrations than women (16.1 vs. 10.6 micromol/L in the north, and 10.7 vs. 7.9 micromol/L in the south) and 40% of the northern men had hyperhomocysteinemia. Generally, individuals living in the urban areas had 30% (95% CI, 1.0-1.6) greater odds of having high tHcy levels (>or=16 micromol/L) than those living in the rural areas. Low plasma concentrations of folate, vitamins B-12 and B-6, older age, being male, and living in urban areas were all independently associated with elevated tHcy, with low folate as the strongest determinant.

Homocysteine-lowering trials for prevention of vascular disease: protocol for a collaborative meta-analysis

Elevated plasma total homocysteine is a risk factor for cardiovascular disease (CVD), but the randomized trials of dietary supplementation with B-vitamins to lower homocysteine have not yet provided clear evidence of benefit on vascular risk. Cumulative meta-analysis of all randomized trials assessing the effects of lowering homocysteine levels with B-vitamins on risk of CVD. An individual patient data meta-analysis of all randomized trials of the effects on vascular risk of lowering homocysteine with B-vitamins will maximize the power to assess the epidemiologically predicted differences in risk. Among the 12 randomized homocysteine-lowering trials for prevention of CVD, involving more than 1000 participants, data should be available on approximately 52,000 participants (32,000 with prior CVD in unfortified populations; and 14,000 with prior CVD and 6000 with renal disease in fortified populations). To minimize bias, the design and primary analyses to be carried out have been pre-specified. The analyses will include assessment of effects on major vascular events (MVE), stroke, major coronary events (MCE), in addition to venous thrombosis, cancer and fractures. Additional analyses will assess effects on vascular outcomes in sub-groups defined by population, prior disease, per 3 micromol/L difference in homocysteine levels achieved by treatment, pre-treatment vitamin status, duration, age, sex and vascular events excluding revascularizations and, separately, excluding vascular events occurring during the first year of treatment. A cumulative meta-analysis of the homocysteine-lowering trials should ensure that reliable evidence emerges about the effects of lowering homocysteine on risk of vascular and non-vascular outcomes.

Perspective on the efficacy analysis of the Vitamin Intervention for Stroke Prevention trial

The pathogenesis of stroke is very different from that of myocardial infarction; most strokes are embolic, and as elevated plasma total homocysteine (tHcy) increases thrombosis, it increases the risk of cardioembolic strokes, particularly in atrial fibrillation, as well as the risk of deep vein thrombosis leading to paradoxical embolism, and elevated tHcy also increases strokes from cortical vein thrombosis. The main study results of the Vitamin Intervention for Stroke Prevention (VISP) trial showed no benefit of vitamin therapy for homocysteine lowering. However, there were a number of problems with the trial including: 1. We did not use a placebo control; we used a multiple vitamin containing low doses of folate and B6, and the recommended daily intake of B12; 2. Patients with low serum B12 levels in both arms of the trial received injections of B12; and 3. Folate fortification of grain products in North America coincided with the initiation of the trial. We therefore conducted a hypothesis-driven analysis of a subgroup of VISP from which patients who could not have responded to the study treatment were excluded, i.e., those with low or very high serum B12, and those with renal failure. In the remaining 2155 patients, there was a significant reduction of stroke, coronary events and death (p=0.049). When the participants were stratified according to the median entry level of B12 (313 pmol/L), it became apparent that serum B12 status was the key determinant of response, those with high serum B12 at entry who received high-dose vitamin had a 33% reduction of events compared to those with low B12 who received low-dose vitamins; the other two groups were intermediate (p=0.02 for survival free of stroke, coronary and death in the Kaplan-Meier analysis). In this efficacy analysis, we found a significant reduction of vascular risk with vitamin therapy. Similarly, the Heart Outcomes Prevention Evaluation 2 trial showed a 25% significant reduction of stroke with vitamin therapy (p=0.03). The authors thought this result was a chance finding, because they did not think that stroke and myocardial infarction would be biologically different; however, stroke and myocardial infarction have very different pathogenesis, so that conclusion is unwarranted. It seems likely that even if vitamin therapy does not significantly reduce coronary events, it will reduce the risk of stroke.

Homocysteine and Stroke Prevention: Have the Trials Settled the Issue?

Recent publicity surrounding disappointing results of clinical trials of homocysteine lowering has led to the claim that 'homocysteine is dead'. However, there is strong evidence that elevated plasma total homocysteine is an important independent risk factor, and the highly plausible biological rationale is the following: total homocysteine increases coagulation, impairs endothelial function, increases oxidative stress and low density lipoprotein oxidation, and treatment with vitamins reverses these effects and halts progression of carotid plaque. Some studies have shown clinical benefit of vitamin therapy in coronary angioplasty and peripheral vascular disease. It has recently become apparent that vitamin B12 absorption is impaired in the elderly, and that metabolic B12 deficiency is much commoner than would be appreciated by statistical definitions of 'normal' serum B12; higher doses of B12 and perhaps other therapies such as betaine and thiols may be needed to achieve adequate reductions of total homocysteine. It remains likely that effective lowering of total homocysteine will reduce stroke and other vascular events.

Differential effects of low and high dose folic acid on endothelial dysfunction in a murine model of mild hyperhomocysteinaemia

The exact mechanism(s) by which hyperhomocysteinaemia promotes vascular disease remains unclear. Moreover, recent evidence suggests that the beneficial effect of folic acid on endothelial function is independent of homocysteine-lowering. In the present study the effect of a low (400 μg/70 kg/day) and high (5 mg/70 kg/day) dose folic acid supplement on endothelium-dependent relaxation in the isolated perfused mesenteric bed of heterozygous cystathionine β-synthase deficient mice was investigated. Elevated total plasma homocysteine and impaired relaxation responses to methacholine were observed in heterozygous mice. In the presence of N G -nitro- l-arginine methyl ester relaxation responses in wild-type tissues were reduced, but in heterozygous tissues were abolished. Clotrimazole and 18α-glycyrrhetinic acid, both inhibitors of non-nitric oxide/non-prostanoid-induced endothelium-dependent relaxation, reduced responses to methacholine in wild-type but not heterozygous tissues. The combination of N G -nitro- l-arginine methyl ester and either clotrimazole or 18α-glycyrrhetinic acid completely inhibited relaxation responses in wild-type tissues. Both low and high dose folic acid increased plasma folate, reduced total plasma homocysteine and reversed endothelial dysfunction in heterozygous mice. A greater increase in plasma folate in the high dose group was accompanied by a more significant effect on endothelial function. In the presence of N G -nitro- l-arginine methyl ester, a significant residual relaxation response was evident in tissues from low and high dose folic acid treated heterozygous mice. These data suggest that the impaired mesenteric relaxation in heterozygous mice is largely due to loss of the non-nitric oxide/non-prostanoid component. While low dose folic acid may restore this response in a homocysteine-dependent manner, the higher dose has an additional effect on nitric oxide-mediated relaxation that would appear to be independent of homocysteine lowering.