Effect of B-group vitamins and antioxidant vitamins on hyperhomocysteinemia:a double-blind, randomized, factorial-design, controlled trial

Mild hyperhomocysteinemia is accepted as a risk factor for premature cardiovascular disease. In a population with a high prevalence of cardiovascular disease, we screened a group of clinically healthy working men aged 30-49 y (n = 509) for plasma homocysteine and 5,10-methylene tetrahydrofolate reductase (MTHFR) genotype status. Those with mildly elevated homocysteine concentrations (> or = 8.34 micromol/L) were selected for intervention. In a randomized, factorial-design, controlled trial we assessed the effects of B-group vitamins and antioxidant vitamin supplementation on homocysteine concentrations. The 132 men were randomly assigned to one of four groups: supplementation with B-group vitamins alone (1 mg folic acid, 7.2 mg pyridoxine, and 0.02 mg cyanocobalamin), antioxidant vitamins alone (150 mg ascorbic acid, 67 mg RRR-alpha-tocopherol, and 9 mg beta-carotene), B-group vitamins with antioxidant vitamins, or placebo. Intervention was double-blind. A total of 101 men completed the 8-wk intervention. When homocysteine concentrations were analyzed by group, significant (P <0.001) decreases (32.0% and 30.0%, respectively) were observed in both groups receiving B-group vitamins either with or without antioxidants. The effect of B-group vitamins alone over 8 wk was a reduction in homocysteine concentrations of 27.9% (95% CI: 22.0%, 33.3%; P <0.001) whereas antioxidants alone produced a nonsignificant increase of 5.1% (95% CI: -2.8%, 13.6%; P = 0.21). There was no evidence of any interaction between the two groups of vitamins. The effect of B-group vitamin supplementation seemed to depend on MTHFR genotype. Supplementation with the B-group vitamins with or without antioxidants reduced homocysteine in the men with mildly elevated concentrations, and hence may be effective in reducing cardiovascular risk.

The common 'thermolabile' variant of methylene tetrahydrofolate reductase is a major determinant of mild hyperhomocysteinaemia

Mild hyperhomocysteinaemia is a major risk factor for vascular disease and neural tube defects (NTDs), conferring an approximately three-fold relative risk for each condition. It has several possible causes: heterozygosity for rare loss of function mutations in the genes for 5,10-methylene tetrahydrofolate reductase (MTHFR) or cystathionine-beta-synthase (CBS); dietary insufficiency of vitamin co-factors B6, B12 or folates; or homozygosity for a common 'thermolabile' mutation in the MTHFR gene which has also been associated with vascular disease and NTDs. We quantified the contribution of the thermolabile mutation to the hyperhomocysteinaemic phenotype in a working male population (625 individuals). Serum folate and vitamin B12 concentrations were also measured and their relationship with homocysteine status and MTHFR genotype assessed. The homozygous thermolabile genotype occurred in 48.4, 35.5, and 23.4% of the top 5, 10, and 20% of individuals (respectively) ranked by plasma homocysteine levels, compared with a frequency of 11.5% in the study population as a whole, establishing that the mutation is a major determinant of homocysteine levels at the upper end of the range. Serum folate concentrations also varied with genotype, being lowest in thermolabile homozygotes. The MTHFR thermolabile genotype should be considered when population studies are designed to determine the effective homocysteine-lowering dose of dietary folate supplements, and when prophylactic doses of folate are recommended for individuals.

In utero exposure to cigarette chemicals induces sex-specific disruption of one-carbon metabolism and DNA methylation in the human fetal liver

Background: Maternal smoking is one of the most important modifiable risk factors for low birthweight, which is strongly associated with increased cardiometabolic disease risk in adulthood. Maternal smoking reduces the levels of the methyl donor vitamin B12 and is associated with altered DNA methylation at birth. Altered DNA methylation may be an important mechanism underlying increased disease susceptibility; however, the extent to which this can be induced in the developing fetus is unknown.

Methods: In this retrospective study, we measured concentrations of cobalt, vitamin B12, and mRNA transcripts encoding key enzymes in the 1-carbon cycle in 55 fetal human livers obtained from 11 to 21 weeks of gestation elective terminations and matched for gestation and maternal smoking. DNA methylation was measured at critical regions known to be susceptible to the in utero environment. Homocysteine concentrations were analyzed in plasma from 60 fetuses.

Results: In addition to identifying baseline sex differences, we found that maternal smoking was associated with sex-specific alterations of fetal liver vitamin B12, plasma homocysteine and expression of enzymes in the 1-carbon cycle in fetal liver. In the majority of the measured parameters which showed a sex difference, maternal smoking reduced the magnitude of that difference. Maternal smoking also altered DNA methylation at the imprinted gene IGF2 and the glucocorticoid receptor (GR/NR3C1).

Conclusions: Our unique data strengthen studies linking in utero exposures to altered DNA methylation by showing, for the first time, that such changes are present in fetal life and in a key metabolic target tissue, human fetal liver. Furthermore, these data propose a novel mechanism by which such changes are induced, namely through alterations in methyl donor availability and changes in 1-carbon metabolism.

Plasma total homocysteine and carotid intima-media thickness in type 1 diabetes: A prospective study

Objective: Plasma total homocysteine (tHcy) has been positively associated with carotid intima-media thickness (IMT) in non-diabetic populations and in a few cross-sectional studies of diabetic patients. We investigated cross-sectional and prospective associations of a single measure of tHcy with common and internal carotid IMT over a 6-year period in type 1 diabetes. Research design and methods: tHcy levels were measured once, in plasma obtained in 1997–1999 from patients (n = 599) in the Epidemiology of Diabetes Interventions and Complications (EDIC) study, the observational follow-up of the Diabetes Control and Complications Trial (DCCT). Common and internal carotid IMT were determined twice, in EDIC “Year 6” (1998–2000) and “Year 12” (2004–2006), using B-mode ultra-sonography. Results: After adjustment, plasma tHcy [median (interquartile range): 6.2 (5.1, 7.5) μmol/L] was significantly correlated with age, diastolic blood pressure, renal dysfunction, and smoking (all p < 0.05). In an unadjusted model only, increasing quartiles of tHcy correlated with common and internal carotid IMT, again at both EDIC time-points (p < 0.01). However, multivariate logistic regression revealed no significant associations between increasing quartiles of tHcy and the 6-year change in common and internal carotid IMT (highest vs. lowest quintile) when adjusted for conventional risk factors. Conclusions: In a type 1 diabetes cohort from the EDIC study, plasma tHcy measured in samples drawn in 1997–1999 was associated with measures of common and internal carotid IMT measured both one and seven years later, but not with IMT progression between the two time-points. The data do not support routine measurement of tHcy in people with Type 1 diabetes.

Homocysteine is lower in the Third Trimester of Pregnancy in Women with Enhanced Folate status from continued Folic Acid Supplementation.

Background: In many countries current recommendations are that women take a daily 400ug folic acid supplement, from before conception until the end of the 12th week of gestation, for the prevention of neural tube defects. Low folate status is associated with an elevated concentration of plasma total homocysteine (tHcy), a risk factor that is associated with pregnancy complications such as pre-eclampsia. Methods: In a longitudinal study, tHcy and corresponding folate status were determined in 101 pregnant women at 12, 20 and 35 weeks of gestation, in 35 non-pregnant control subjects sampled conconcurrently, and in a subgroup (n=21 pregnant, 19 non-pregnant women) at 3 days post-partum. Results: Plasma tHcy concentrations were significantly lower throughout pregnancy compared with control subjects, with values lowest in the 2nd trimester before increasing toward non-pregnant values in the 3rd trimester. Importantly, tHcy concentrations were lower in pregnant women taking folic acid supplements compared to those not, an effect which reached significance in the 3rd trimester (5.25 umol/l v 6.89 umol/l, P <0.05). Furthermore, during the 3rd trimester, tHcy concentrations were significantly higher in pregnant women with a history of miscarriage compared to those with no previous history (7.32 umol/l v 5.62 u­mol/l, P <0.01). Conclusion: This is the first longitudinal study to show that homocysteine levels rise in late pregnancy towards non-pregnant levels; a rise which can be limited by enhancing folate status through continued folic acid supplementation. These results indicate a potential role for continued folic acid supplementation in reducing pregnancy complications associated with hyperhomocysteinaemia.

Homocysteine, Methylenetetrahydrofolate Reductase C677T Polymorphism, and Risk of Retnal Vein Occlusion: A Meta-analysis

Objective: To assess the role of plasma total homocysteine (tHcy) concentrations and homozygosity for the thermolabile variant of the methylenetetrahydrofolate reductase (MTHFR) C677T gene as risk factors for retinal vascular occlusive disease.

Design: Retinal vein occlusion (RVO) is an important cause of vision loss. Early meta-analyses showed that tHcy was associated with an increased risk of RVO, but a significant number of new studies have been published. Participants and/or Controls: RVO patients and controls.

Methods: Data sources included MEDLINE, Web of Science, and PubMed searches and searching reference lists of relevant articles and reviews. Reviewers searched the databases, selected the studies, and then extracted data. Results were pooled quantitatively using meta-analytic methods.

Main Outcome Measures: tHcy concentrations and MTHFR genotype.

Results: There were 25 case-control studies for tHcy (1533 cases and 1708 controls) and 18 case-control studies for MTHFR (1082 cases and 4706 controls). The mean tHcy was on average 2.8 mol/L (95% confidence
interval [CI], 1.8 –3.7) greater in the RVO cases compared with controls, but there was evidence of between-study heterogeneity (P0.001, I2 93%). There was funnel plot asymmetry suggesting publication bias. There was no evidence of association between homozygosity for the MTHFR C677T genotype and RVO (odds ratio [OR] 1.20; 95% CI, 0.84–1.71), but again marked heterogeneity (P 0.004, I2 53%) was observed.

Conclusions: There was some evidence that elevated tHcy was associated with RVO, but not homozygosity for the MTHFR C677T genotype. Both analyses should be interpreted cautiously because of marked heterogeneity between the study estimates and possible effect of publication bias on the tHcy findings.

Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Primed, constant infusion with [H-2(3)]serine allows in vivo kinetic measurement of serine turnover, homocysteine remethylation, and transsulfuration processes in human one-carbon metabolism

Background: One-carbon metabolism involves both mitochondrial and cytosolic forms of folate-dependent enzymes in mammalian cells, but few in vivo data exist to characterize the biochemical processes involved.

Objective: We conducted a stable-isotopic investigation to determine the fates of exogenous serine and serine-derived one carbon units in homocysteine remethylation in hepatic and whole-body metabolism.

Design: A healthy man aged 23 y was administered [2,3,3 H-2(3)]serine and [5,5,5-H-2(3)]leucine by intravenous primed, constant infusion. Serial plasma samples were analyzed to determine the isotopic enrichment of free glycine, serine, leucine, methionine, and cystathionine. VLDL apolipoprotein B-100 served as an index of liver free amino acid labeling.

Results: [H-2(1)]Methionine and [H-2(2)]methionine were labeled through homocysteine remethylation. We propose that [H-2(2)]methionine occurs by remethylation with [H-2(2)]methyl groups (as 5-methyltetrahydrofolate) formed only from cytosolic processing of [H-2(3)]serine, whereas [H-2(1)]methionine is formed with labeled one-carbon units from mitochondrial oxidation of C-3 serine to [H-2(1)]formate to yield cytosolic [H-2(1)]methyl groups. The labeling pattern of cystathionine formed from homocysteine and labeled serine suggests that cystathionine is derived mainly from a serine pool different from that used in apolipoprotein B-100 synthesis.

Conclusions: The appearance of both [H-2(1)]- and [H-2(2)]methionine forms indicates that both cytosolic and mitochondrial metabolism of exogenous serine generates carbon units in vivo for methyl group production and homocysteine remethylation. This study also showed the utility of serine infusion and indicated functional roles of cytosolic and mitochondrial compartments in one-carbon metabolism.

Determination of metformin in plasma using a new ion pair solid phase extraction technique and ion pair liquid chromatography

This article describes the development of the first ion pair solid phase extraction technique (IPSPE), which has been applied to the extraction of metformin from plasma samples. In addition an ion pair chromatographic method was developed for the specific HPLC determination of metformin. Several extraction and HPLC methods have been described previously for metformin, however, most of them did not solve the problems associated with the high polarity of this drug. Drug recovery in the developed method was found to be more than 98%. The limit of detection and limit of quantification was 3 and 5 ng/ml, respectively. The intraday and interday precision (measured by coefficient of variation, CV%) was always less than 9%. The accuracy (measured by relative error, R.E.%) was always less than 6.9%. Stability analysis showed that metformin is stable for at least 3 months when stored at -70degreesC. The method has been applied to 150 patient samples as part of a medication adherence study. (C) 2003 Elsevier B.V. All rights reserved.