Post-methionine Load Research Articles

Abstract WP251: Dietary Methionine and Homocysteine Level Interaction and Recurrent Stroke

Background: The folate one-carbon metabolism pathway (FOCM) provides vital one-carbon units for epigenetic regulation. Genetic variants in FOCM genes may affect methionine metabolism. Elevated circulating homocysteine (Hcy), a FOCM byproduct, is linked to increased stroke risk. However, the protective effects of dietary regulation of methionine and Hcy levels against recurrent stroke are uncertain. Objective: To investigate dietary methionine (METH) and its interaction with post-methionine load Hcy levels (POST) with recurrent stroke risk; to assess if genetically determined POST and its interaction with METH predict recurrent stroke risk. Methods: We utilized the Vitamin Intervention for Stroke Prevention (VISP) trial to estimate hazards ratios of METH and its interaction with POST on recurrent stroke with a Cox proportional hazards model. We also generated a POST polygenic risk score (PRS) with ridge regression using previously identified polymorphisms, located within ALDH1L1, CPS1, GNMT, and PSPH, as indicators of FOCM function. Results: The participants (n = 2,100; 64% men) had a mean (SD) age of 67 (11) years. Ancestry consisted of 82% European, 12% African, and 5.7% Other. A small portion of participants had missing POST values (7.3%). Clinical POST (HR 1.02; 95%CI 0.98, 1.06) and its interaction with METH (HR 0.99; 95% CI 0.97, 1.01) were not associated with recurrent stroke risk. Similarly, there was no significant relationship observed between PRS POST (HR 0.96; 95%CI 0.80, 1.15) and its interaction with METH (HR 1.02; 95% CI 0.94, 1.10). Conclusion: Dietary methionine and post-methionine load Hcy levels neither interact nor predict recurrent stroke risk. Our PRS approach showed similar negative findings. The relationship of the FOCM and recurrent stroke risk may lie in epigenetics.

Genome-Wide Meta-Analysis of Homocysteine and Methionine Metabolism Identifies Five One Carbon Metabolism Loci and a Novel Association of ALDH1L1 with Ischemic Stroke

Circulating homocysteine levels (tHcy), a product of the folate one carbon metabolism pathway (FOCM) through the demethylation of methionine, are heritable and are associated with an increased risk of common diseases such as stroke, cardiovascular disease (CVD), cancer and dementia. The FOCM is the sole source of de novo methyl group synthesis, impacting many biological and epigenetic pathways. However, the genetic determinants of elevated tHcy (hyperhomocysteinemia), dysregulation of methionine metabolism and the underlying biological processes remain unclear. We conducted independent genome-wide association studies and a meta-analysis of methionine metabolism, characterized by post-methionine load test tHcy, in 2,710 participants from the Framingham Heart Study (FHS) and 2,100 participants from the Vitamin Intervention for Stroke Prevention (VISP) clinical trial, and then examined the association of the identified loci with incident stroke in FHS. Five genes in the FOCM pathway (GNMT [p = 1.60×10−63], CBS [p = 3.15×10−26], CPS1 [p = 9.10×10−13], ALDH1L1 [p = 7.3×10−13] and PSPH [p = 1.17×10−16]) were strongly associated with the difference between pre- and post-methionine load test tHcy levels (ΔPOST). Of these, one variant in the ALDH1L1 locus, rs2364368, was associated with incident ischemic stroke. Promoter analyses reveal genetic and epigenetic differences that may explain a direct effect on GNMT transcription and a downstream affect on methionine metabolism. Additionally, a genetic-score consisting of the five significant loci explains 13% of the variance of ΔPOST in FHS and 6% of the variance in VISP. Association between variants in FOCM genes with ΔPOST suggest novel mechanisms that lead to differences in methionine metabolism, and possibly the epigenome, impacting disease risk. These data emphasize the importance of a concerted effort to understand regulators of one carbon metabolism as potential therapeutic targets.

Plasma Homocysteine in Patients with Retinal Vein Occlusion

To evaluate total plasma homocysteine (HCY) during fasting and post methionine load test (MLT), serum folate, serum vitamin B12, and methylenetetrahydrofolate reductase (MTHFR) mutation in patients with retinal vein occlusion (RVO) and to examine the association between these risk factors and 2 subtypes of RVO: central (CRVO) and branch (BRVO). This case-control study included 91 Italian patients presenting a first RVO and 71 healthy subjects, matched by age, without history of thromboembolic diseases, glaucoma, or malignancy. Homocysteine fasting and after MLT, serum folate level, serum vitamin B12 level, and other laboratory tests were assessed. Genetic analysis for the C677T MTHFR mutation was performed. Multivariate logistic regression analysis indicated that hypertension (odds ratio [OR] 2.63; 95% confidence interval [CI] 1.30-5.30; p = 0.007), higher values of fasting HCY (OR 1.16; 95% CI 1.01-1.33; p = 0.03), and low concentrations of vitamin B12 (OR 0.99; 95% CI 0.995-0.999; p = 0.01) were independently correlated with RVO. Moreover, the main determinants for CRVO risk were hypertension (OR 2.46; 95% CI 1.06-5.72; p = 0.04), high values of fasting HCY (OR 1.20; 95% CI 1.02-1.41; p = 0.03), and low concentrations of vitamin B12 (OR 0.99; 95% CI 0.994-0.999; p = 0.008), whereas for BRVO risk only hypertension was significant (OR 2.74; 95% CI 1.24-6.03; p = 0.01). Genotype distribution of the MTHFR C677T mutation did not reveal any significant difference between patients and controls. These results suggest that elevated fasting HCY levels, low vitamin B12 levels, and hypertension are associated with a risk of RVO, especially for CRVO. Moreover, our data suggest that only hypertension is associated with BRVO risk.

The Metabolic Burden of Methyl Donor Deficiency with Focus on the Betaine Homocysteine Methyltransferase Pathway

Methyl groups are important for numerous cellular functions such as DNA methylation, phosphatidylcholine synthesis, and protein synthesis. The methyl group can directly be delivered by dietary methyl donors, including methionine, folate, betaine, and choline. The liver and the muscles appear to be the major organs for methyl group metabolism. Choline can be synthesized from phosphatidylcholine via the cytidine-diphosphate (CDP) pathway. Low dietary choline loweres methionine formation and causes a marked increase in S-adenosylmethionine utilization in the liver. The link between choline, betaine, and energy metabolism in humans indicates novel functions for these nutrients. This function appears to goes beyond the role of the nutrients in gene methylation and epigenetic control. Studies that simulated methyl-deficient diets reported disturbances in energy metabolism and protein synthesis in the liver, fatty liver, or muscle disorders. Changes in plasma concentrations of total homocysteine (tHcy) reflect one aspect of the metabolic consequences of methyl group deficiency or nutrient supplementations. Folic acid supplementation spares betaine as a methyl donor. Betaine is a significant determinant of plasma tHcy, particularly in case of folate deficiency, methionine load, or alcohol consumption. Betaine supplementation has a lowering effect on post-methionine load tHcy. Hypomethylation and tHcy elevation can be attenuated when choline or betaine is available.

Abstract 3012: Significant Genome Wide Association Identified Between the Glycine N-Methyltransferase Gene (GNMT) and Post-Methionine Load Test Homocysteine Levels in the Vitamin Intervention for Stroke Prevention (VISP) Cohort

The Vitamin Intervention for Stroke Prevention (VISP) trial was a multi-center, controlled, double blinded clinical trial, designed to determine whether the daily intake of high dose folic acid, vitamins B6 and B12 reduced recurrent cerebral infarction. Baseline post-methionine load homocysteine levels were predictive of recurrent stroke risk in VISP. We have conducted a genome-wide association study (GWAS), investigating 2100 VISP stroke cases required to have homocysteine levels greater than the 25th percentile at enrolment. Using imputation with 1000 genomes and principal component analysis, we were able to detect an association of p≤6.70x10-17 for SNPs within, or flanking, the glycine N-methyltransferase (GNMT) gene and post-methionine load test homocysteine level. Change in homocysteine levels pre and post-methionine load test also resulted in significant GWAS scores in the chromosome 6 region (p = 2.54x10-23, rs7760250). GNMT catalyzes the conversion of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine and sarcosine via the folate one-carbon metabolism pathway. In addition to being an essential component in the conversion of methionine to homocysteine, GNMT plays a significant role in the global epigenetic state as homocysteine is an important methyl donor. This region displays significant linkage disequilibrium, warranting close analysis of the entire GNMT locus in search of both rare and common variants. Cases who carry the minor allele haplotype at associated loci had a higher level of post-methionine load homocysteine (37.69µM/L) than those who carry the major allele haplotype (27.33µM/L), inferring functional differences due to GNMT variants. Taken together these data suggest a clear and important role for GNMT in post-methionine load test homocysteine level, epigenetic state and stroke risk.

Variability of plasma and urine betaine in diabetes mellitus and its relationship to methionine load test responses: an observational study

BackgroundSince betaine is an osmolyte and methyl donor, and abnormal betaine loss is common in diabetes mellitus (>20% patients), we investigated the relationship between betaine and the post-methionine load rise in homocysteine, in diabetes and control subjects. The post-methionine load test is reported to be both an independent vascular risk factor and a measure of betaine sufficiency.MethodsPatients with type 2 diabetes (n = 34) and control subjects (n = 17) were recruited. We measured baseline fasting plasma and 4-hour post-methionine load (L-methionine, 0.1 mg/kg body weight) concentrations of homocysteine, betaine, and the betaine metabolite N,N-dimethylglycine. Baseline urine excretions of betaine, dimethylglycine and glucose were measured on morning urine samples as the ratio to urine creatinine. Statistical determinants of the post-methionine load increase in homocysteine were identified in multiple linear regression models.ResultsPlasma betaine concentrations and urinary betaine excretions were significantly (p < 0.001) more variable in the subjects with diabetes compared with the controls. Dimethylglycine excretion (p = 0.00014) and plasma dimethylglycine concentrations (p = 0.039) were also more variable. In diabetes, plasma betaine was a significant negative determinant (p < 0.001) of the post-methionine load increase in homocysteine. However, it was not conclusive that this was different from the relationship in the controls. In the patients with diabetes, a strong relationship was found between urinary betaine excretion and urinary glucose excretion (but not with plasma glucose).ConclusionsBoth high and low plasma betaine concentrations, and high and low urinary betaine excretions, are more prevalent in diabetes. The availability of betaine affects the response in the methionine load test. The benefits of increasing betaine intake should be investigated.

Transcobalamin 2 variant associated with poststroke homocysteine modifies recurrent stroke risk

The Vitamin Intervention for Stroke Prevention trial found an association between baseline poststroke homocysteine (Hcy) and recurrent stroke. We investigated genes for enzymes and cofactors in the Hcy metabolic pathway for association with Hcy and determined whether associated single nucleotide polymorphisms (SNPs) influenced recurrent stroke risk. Eighty-six SNPs in 9 candidate genes (BHMT1, BHMT2, CBS, CTH, MTHFR, MTR, MTRR, TCN1, and TCN2) were genotyped in 2,206 subjects (83% European American). Associations with Hcy measures were assessed using linear regression models assuming an additive genetic model, adjusting for age, sex, and race and additionally for baseline Hcy when postmethionine load change was assessed. Associations with recurrent stroke were evaluated using survival analyses. Five SNPs in the transcobalamin 2 (TCN2) gene were associated with baseline Hcy (false discovery rate [FDR]-adjusted p = 0.049). TCN2 SNP rs731991 was associated with recurrent stroke risk in the low-dose arm of the trial under a recessive model (log-rank test p = 0.009, hazard ratio 0.34). Associations with change in postmethionine load Hcy levels were found with 5 SNPs in the cystathionine β-synthase (CBS) gene (FDR-adjusted p < 0.031). TCN2 variants contribute to poststroke Hcy levels, whereas variants in the CBS gene influence Hcy metabolism. Variation in the TCN2 gene also affects recurrent stroke risk in response to cofactor therapy.

P.5.a.006 Plasma homocysteine as a risk factor for Korean patients with vascular and Alzheimer's dementia

We have modified a high-performance liquid chromatographic (HPLC) procedure based on SBD-F (ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate) pre-column derivatization to obtain an assay that is useful for routine clinical total plasma homocysteine (tHcy) analysis. The introduction of easily handled sodium borohydride instead of the traditional tri-n-butylphosphine in dimethylformamide as a reductant and a 14-min run-time using basic isocratic HPLC equipment are the more notable advantages. The addition of mercaptopropionylglycine as an internal standard contributed to improvements in the reproducibility of the assay, yielding within- and between-run precisions of 1.9 and 4% (C.V.), respectively. Reference values for fasting tHcy were 7.65±2.3 and 8.9±2.4 μmol/l, while post-methionine load gave tHcy levels of 19.9±5.5 and 26.8±5.5 μmol/l, for women and men, respectively (n=40).

Genotype-independent in vivo oxidative stress following a methionine loading test: Maximal platelet activation in subjects with early-onset thrombosis

Backgroundmethionine ingestion (100mg/kg) identifies subjects in whom fasting total homocysteine (tHcy) may be normal but the post-methionine load (PML) tHcy is abnormally high. MethodsIn 96 subjects [54 M/42 F, 40.4±12.3yrs old; 28 with the 68bp844 ins of the Cystathionine-β-synthase gene (CBSins+); 20 homozygotes for the C677T mutation of the methylene-tetrahydrofolate reductase gene (MTHFR++); 13 with the combination of the two, and 35 without any of them], we have evaluated in vivo oxidative stress and platelet activation, as reflected by urinary excretions of 8-iso-PGF2α and of 11-dehydro-TXB2 respectively, before and after a methionine load test (PML). A history of early-onset thrombosis (18 arterial, 32 venous, 2 both) was present in 52/96 of them. ResultsBaseline; tHcy was highest in MTHFR++ carriers (p<0,05); 8-iso-PGF2α and 11-dehydro-TXB2 levels were independent of sex, MTHFR++ and/or CBSins+(p>0.05). PML; The ~3-fold increase (p<0.01 vs baseline) in tHcy reached a plateau within 6–8hrs. Mean PML tHcy was maximal in MTHFR++ carriers (p=0.000). 8-iso-PGF2α and 11-dehydro-TXB2 increase reached a maximum within 4hrs. 11-dehydro-TXB2 increase was highest (p=0.023 vs baseline) in subjects with a history of thrombosis. Baseline 11-dehydro-TXB2 and a history of thrombosis independently predicted PML 11-dehydro-TXB2 (β=0.287, p=0.000 and β=0.308, p=0.026, respectively).The PML increase in 8-iso-PGF2α or in 11-dehydro-TXB2 were comparable in the different genotypes (p>0.05). Conclusionregardless genotypes associated with moderate hyperhomocysteinemia, following a methionine loading test, in vivo oxidative stress and platelet activation occur, being the latter maximal in subjects with a history of early-onset thrombosis.

Methionine-Induced Homocysteinemia Impairs Endothelial Function in Hypertensives: The Role of Asymmetrical Dimethylarginine and Antioxidant Vitamins

Nitric oxide synthase (NOS) inhibitor asymmetrical dimethylarginine (ADMA) is synthesized by the methylation of arginine as part of the methionine/homocysteine cycle. However, the mechanisms regulating ADMA synthesis in hypertension are unclear. We investigated the role of ADMA and antioxidants in endothelial dysfunction during methionine-induced homocysteinemia in hypertensives. Thirty-nine hypertensives and forty-nine normotensive controls underwent methionine loading (100 mg methionine/kg BW), after being randomized to receive vitamin C (2 g) and E (800 IU) or placebo. Endothelium-dependent dilation (EDD) was evaluated by plethysmography (baseline and 4-h post-methionine loading (4-h PML)). Hypertensives had higher homocysteine at baseline (P < 0.001) and 4-h PML (P < 0.05), whereas methionine increased homocysteine in all groups. EDD was decreased in both vitamins and placebo groups in controls (P < 0.01 for both) and vitamins- and placebo-treated hypertensives (P < 0.05 and P < 0.01, respectively). In controls, ADMA was increased in both vitamin- and placebo groups (P < 0.01 for both) at 4-h PML. Hypertensives had higher ADMA at baseline (P < 0.01 vs. normotensive) and remained unchanged at 4-h PML (P = NS in placebo and vitamins treated). ADMA is elevated in hypertensives but remains unchanged after methionine loading, suggesting that ADMA plays an important role in endothelial dysfunction in hypertensives, but it is not responsible for homocysteine-induced endothelial dysfunction in these patients.

Folic acid, vitamin B12, and homocysteine levels during fasting and after methionine load in patients with Type 1 diabetes mellitus

To assess plasma concentrations of folic acid, vitamin B12, and total plasma homocysteine (tHCY) during fasting and after methionine load in young patients with Type 1 diabetes mellitus (T1DM). We enrolled 41 young patients with T1DM without any sign of microvascular complications and 123 healthy controls in a 1:3 case-control study. Fasting and post-methionine load (PML) tHCY, folic acid, and vitamin B12 levels were measured in both groups. Data regarding chronological age, metabolic control (assessed by mean values of glycated hemoglobin in the last 12 months) and disease duration were also recorded. Fasting and PML tHCY levels were significantly lower in patients than in controls: 7.3+/-2.7 micromol/l vs 8.3+/-2.5 micromol/l (p=0.01), and 16.7+/-5.8 micromol/l vs 17.3+/-4.3 micromol/l (p=0.01), respectively. No correlation was found between fasting and PML tHCY levels and chronological age, disease duration, metabolic control, and insulin requirement. Patients had significantly higher vitamin B12 levels compared to controls: 767+/-318 pg/ml vs 628+/-236 pg/ml (p=0.003), while folic acid turned out to be lower in patients than in controls: 5.3+/-1.9 nmol/l vs 7.5+/-2.6 nmol/l (p<0.0001). Adolescents and young adults with T1DM without microvascular complications showed lower tHCY both during fasting and after methionine load. Lower folate concentrations in these patients might benefit from food fortification.

Fasting and Post-methionine Homocysteine Levels in Alzheimer’s Disease and Vascular Dementia

The aim of the study is to compare the basal homocysteine levels in patients with impairment of cognitive status, and in controls, to evaluate if the methionine loading test is able to identify any differences between patients with Alzheimers disease and patients with vascular dementia. We enrolled 56 subjects, 20 with Alzheimers disease, 18 with vascular dementia, and 18 normal controls. The data shown that plasma homocysteine levels both basal and post-methionine load were significantly higher in the two groups of demented patients than in the control group. No significant differences were found between Alzheimers patients and vascular dementia patients. The homocysteine percent increase after a methionine loading test was significantly higher in the controls with respect to the two groups of demented patients. Only in Alzheimers patients were vitamin B(12) basal levels negatively correlated with basal homocysteine levels (p<0.05), while positively correlated with the homocysteine percent increase after load (p<0.05). The study confirms the possible role of chronically elevated homocysteinemia in neuronal degeneration in demented patients. Even if the methionine loading test revealed an abnormal homocysteine metabolism in demented patients, it didnt show any difference among patients with Alzheimers disease and vascular dementia.

Dietary and supplementary betaine: Effects on betaine and homocysteine concentrations in males

Background and aims Betaine is an osmolyte that when catabolised decreases plasma total homocysteine. A betaine-rich meal has acute effects similar to a supplement, but the effects of a longer-term increase in dietary betaine intake need clarification. We compared the effects of two weeks of dietary and supplementary betaine on plasma betaine and homocysteine concentrations both fasting and after a methionine load. Methods and results In a randomized crossover study, 8 healthy males (22–36 y) consumed either a betaine-rich diet (∼800 mg/day) or a betaine supplement (0.5 g twice daily) for 14 days. Fasting blood samples were collected on day −5, −1 (pre-treatment) 0, 2, 6, 9, 13 (treatment), 14 and 18 (post-treatment). Post-methionine load blood samples were collected on day −5, 0, 6 and 13, while 24 h urine samples were collected on day −5, 0, 6, 13 and 14. Plasma betaine, dimethylglycine, homocysteine and urine betaine, dimethylglycine and creatinine concentrations were measured. Plasma betaine concentrations significantly increased for both treatments compared to pre-treatment values ( P < 0.001). Fasting homocysteine levels were minimally affected. Both treatments reduced post-methionine load homocysteine and this effect tended to be greater following a betaine-rich diet ( P = 0.108). Small increases in urinary betaine excretion were observed following both treatments (≈1.5% of supplement; ≈1.3% of dietary betaine). Most was attributable to increased excretion of betaine as dimethylglycine. Conclusions Supplemental or dietary betaine similarly increase circulating betaine concentrations and attenuate the post-methionine load rise in homocysteine concentrations.

Methionine loading does not enhance the predictive value of homocysteine serum testing for all‐cause mortality or major adverse cardiac events

Hyperhomocysteinaemia is independently associated with atherosclerotic disease. Methionine loading could improve the predictive value of hyperhomocysteinaemia by detecting mild disturbances in enzyme activity. The aims of this study were to determine the beneficial effect of methionine loading on the predictive value of homocysteine testing for long-term mortality and major adverse cardiac events (MACE). In an observational study, 1122 patients with suspected or known vascular disease, underwent homocysteine testing, which was measured fasting and again 6 h after methionine loading. Hyperhomocysteinaemia was defined as a fasting level > or =15 micromol/L and post-methionine loading level > or =45 micromol/L or an increase of > or =30 micromol/L above fasting levels. Primary end-points were death and MACE. Multivariate Cox regression analysis was used, adjusting for all cardiac risk factors. During follow up (mean 8.9 +/- 3.4 years), 98 patients died (8.7%), 86 had a MACE (7.7%), 579 patients had normal tests, 134 patients had only fasting hyperhomocysteinaemia, 226 only post-methionine hyperhomocysteinaemia and 183 patients had both. In multivariate analysis, overall survival and MACE-free survival were significantly worse for those with fasting hyperhomocysteinaemia, with hazard ratios of 1.86 (95% confidence interval (CI) 1.20-2.87) and 2.24 (95%CI 1.41-3.53), respectively. The addition of hyperhomocysteinaemia after methionine loading did not significantly increase the risk of death or MACE, with hazard ratios of 0.97 (95%CI 0.52-1.81) and 0.89 (95%CI 0.47-1.69), respectively. The presence of post-methionine hyperhomocysteinaemia did not significantly alter risk of death or MACE in patients with normal or increased fasting homocysteine levels, respectively. In conclusion, methionine loading does not improve the predictive value of homocysteine testing with regard to long-term mortality or MACE.

B-vitamins, homocysteine and gene polymorphism in adults with fasting or post-methionine loading hyperhomocysteinemia

Although fasting and post-methionine loading (PML) homocysteine concentrations are not necessarily related, a high percentage of hyperhomocysteinemia cases would be missed if methionine loading was not performed. The influences of B-vitamins and genetic polymorphism (methylenetetrahydrofolate reductase 677C --> T, MTHFR 677C --> T) on fasting and PML homocysteine concentrations and the relationship between fasting and PML homocysteine were studied. This study was a cross-sectional study. Healthy subjects were divided into either fasting hyper-homocysteinemia (>or=12.2 micromol/l) (fasting hyper-hcy, n = 51), PML hyper-homocysteinemia (fasting homocysteine <12.2 micromol/l but PML homocysteine >or=25.6 micromol/l) (PML hyper-hcy, n = 29), or normo-homocysteinemia (fasting homocysteine <12.2 micromol/l and PML homocysteine <25.6 micromol/l) (normo-hcy, n = 118) group based on elevated fasting and PML homocysteine levels of the 75th percentile of the population. The concentrations of plasma fasting and PML homocysteine, serum folate, vitamin B-12, plasma pyridoxal 5'- phosphate (PLP) were measured. The genetic polymorphisms were determined. Fasting homocysteine, but not PML homocysteine and MTHFR 677C --> T genotype, was significantly and inversely affected by serum folate concentration after adjusting for potential confounders (beta = -0.062, P < 0.01). Fasting and PML homocysteine were highly associated in the fasting hyper-hcy and pooled groups (P < 0.01) but not in the PML hyper-hcy and normo-hcy groups. PML homocysteine did not interact with either serum folate (P = 0.302), vitamin B-12 (P = 0.465), plasma PLP (P = 0.996) or MTHFR 677C --> T genotype (P = 0.136) to affect fasting homocysteine concentration. Approximately one-third (36.3%) of hyperhomocysteinemia cases would be missed if methionine loading were not performed. Even though subjects may have a normal fasting homocysteine concentration, they need further screening for their PML homocysteine.

Drug-induced pertubation of the aminothiol redox-status in patients with epilepsy: Improvement by B-vitamins

Patients with epilepsy have excess morbidity and mortality due to ischemic cardiovascular disease. Many of these patients have elevated concentrations of plasma total homocysteine (Hcy), which is an acknowledged risk factor for cardiovascular disease, venous thromboembolic disease, foetal malformations and dementia. Hyperhomocysteinemia may have negative effects through mechanisms involving oxidative damage. In the present study, we have investigated the aminothiol redox-status in patients on antiepileptic drugs. Thereafter, in a subset of patients with elevated total Hcy, we evaluated the effect of B-vitamin therapy. In the first part of the study, 101 patients on antiepileptic drugs were compared with 101 matched healthy controls. The redox-species of Hcy, cysteine and cysteinylglycine, the major aminothiols in plasma, were analyzed by high-performance liquid chromatography (HPLC). Hyperhomocysteinemia was defined as fasting total Hcy above 12 micromol/L and/or post-methionine load concentrations above 38 micromol/L. In the second part of the study, 33 patients identified with hyperhomocysteinemia were supplemented with three B-vitamins for 30 days; folic acid (B9), pyridoxine (B6) and riboflavin (B2). All redox-species of Hcy were significantly elevated in the patients, except the fasting concentrations of reduced Hcy (p=0.09). The reduced/total ratio of cysteine in fasting plasma was lower in the patients than in the controls: 5.20% vs. 6.19%, respectively (p=0.006). After 30 days of B-vitamin supplementation, the plasma concentrations of reduced, oxidized and protein-bound Hcy species were significantly lowered by 17%, 22% and 28%, respectively. The reduced/total ratio of cysteine rose from 4.9% to 7.9% (p=0.007). Patients on antiepileptic drugs have abnormal aminothiol redox-status associated with hyperhomocysteinemia. This is similar to findings in patients with cardiovascular disease. B-vitamin supplementation partially corrects the abnormal aminothiol redox-status. Possibly, B-vitamin supplementation may be useful in drug-induced hyperhomocysteinemia.

Post-methionine load test: A more sensitive tool to reveal hyperhomocysteinemia in Alzheimer patients?

Objective To identify the real number of hyperhomocysteinemic Alzheimer's patients who may benefit from homocysteine-lowering therapy. Methods Basal and post-methionine load homocysteine levels were assessed by rp-HPLC system. Results PML test revealed twice as many hyperhomocysteinemic AD subjects with respect to the fasting analysis. Conclusion PML test resulted useful in detecting higher number of hyperhomocysteinemic AD patients who may have the chance of an early folate treatment.

Dietary and supplementary betaine: acute effects on plasma betaine and homocysteine concentrations under standard and postmethionine load conditions in healthy male subjects

Background: Betaine comes from the diet and from choline, and it is associated with vascular disease in some patient groups. Betaine supplementation lowers plasma total homocysteine.Objective: We compared the acute effects of dietary and supplementary betaine and choline on plasma betaine and homocysteine under standard conditions and after a methionine load.Design: In a randomized crossover study, 8 healthy men (19–40 y) consumed a betaine supplement (≈500 mg), high-betaine meal (≈517 mg), choline supplement (500 mg), high-choline meal (≈564 mg), high-betaine and -choline meal (≈517 mg betaine, ≈622 mg choline), or a low-betaine and -choline control meal under standard conditions or postmethionine load. Plasma betaine, dimethylglycine, and homocysteine concentrations were measured hourly for 8 h and at 24 h after treatment.Results: Dietary and supplementary betaine raised plasma betaine concentrations relative to control (P < 0.001) under standard conditions. This was not associated with raised plasma dimethylglycine concentration, and no significant betaine appeared in the urine. A small increase in dimethylglycine excretion was observed when either betaine or choline was supplied (P = 0.011 and < 0.001). Small decreases in plasma homocysteine 6 h after ingestion under standard conditions (P ≤ 0.05) were detected after a high-betaine meal and after a high-betaine and high-choline meal. Dietary betaine and choline and betaine supplementation attenuated the increase in plasma homocysteine at both 4 and 6 h after a methionine load (P ≤ 0.001).Conclusions: Dietary betaine and supplementary betaine acutely increase plasma betaine, and they and choline attenuate the postmethionine load rise in homocysteine concentrations.

Assessment of pre- and post-methionine load homocysteine for prediction of recurrent stroke and coronary artery disease in the Vitamin Intervention for Stroke Prevention Trial

Assessment of pre- and post-methionine load homocysteine for prediction of recurrent stroke and coronary artery disease in the Vitamin Intervention for Stroke Prevention Trial

Assessment of pre- and post-methionine load homocysteine for prediction of recurrent stroke and coronary artery disease in the Vitamin Intervention for Stroke Prevention Trial

Methionine (Met) loading increases total plasma homocysteine (tHcy) and assesses homocysteine metabolism. We tested the hypothesis that pre- or post-Met tHcy will predict recurrent stroke or coronary artery disease (CAD) in a subgroup analysis of the Vitamin Intervention for Stroke Prevention (VISP) trial. VISP subjects with non-disabling stroke underwent measurement of tHcy at baseline (fasting pre- and post-Met load) and were randomized to high/low-dose B-vitamin therapy for prevention of recurrent stroke or CAD. In the sample cohort of 2124 subjects, mean ± S.D. tHcy levels in μmol/l were pre-Met 13.2 ± 4.3, post-Met 30.4 ± 9.76, and pre/post-Met Δ 17.1 ± 8.3. The hazard ratio (HR) for recurrent stroke was 1.16 ( p = 0.026) for 1 S.D. higher pre-Met tHcy and 1.15 ( p = 0.054) for 1 S.D. higher post-Met tHcy. For CAD, the HR for 1 S.D. higher pre-Met tHcy was 1.27 ( p = 0.001) and was 1.00 ( p = 0.99) for post-Met tHcy. In survival analyses using pre- or post-Met as covariates, the coefficient of pre/post-Met Δ was not significant for stroke and was only marginally significant for CAD ( p < 0.08), but was negative. We conclude that fasting, pre-Met tHcy is as effective as post-Met tHcy or pre/post-Met Δ in predicting the risk for stroke and CAD.