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Purpose: There is a strong association between severe hyperhomocysteinemia and myopia. Thus we studied the hypothesis that even moderately increased levels of homocysteine (Hcy) might be a potentially treatable risk factor for myopia., Methods: The Gutenberg Health Study (GHS) is a population-based, prospective, observational cohort study in Germany, including 15,010 participants aged between 35 and 74 at recruitment. The baseline examination was conducted from 2007-2012. Refraction was measured using autorefraction (HARK 599, Carl Zeiss AG, Jena, Germany). Hcy was measured by an immunoassay. We included only phakic participants without a history of corneal surgery or corneal laser treatment. We used linear regression models to evaluate the potential association between Hcy and refraction at baseline, and between Hcy and change in refraction between baseline and 5-year-follow-up examination. We used generalized estimating equation models to account for the correlation between fellow eyes., Results: We included 13,749 participants, categorized as having no myopia (spherical equivalent > -0.75 D, 65.2%), low myopia (-0.75 D--2.75 D, 21.5%), moderate myopia (-3.00 D- 5.75 D, 9.8%) and high myopia (≤ -6 D, 3.5%). Median Hcy levels were similar in all groups (μmol/l). We observed no association of Hcy with refraction or 5-year change in refraction in the models adjusted for age, sex and socioeconomic status., Conclusion: We found no evidence for an association of Hcy levels and refractive error., Competing Interests: The authors have read the journal's policy and have the following potential competing interests to declare: the Gutenberg Health Study is funded in part through contracts with Boehringer Ingelheim, and PHILIPS Medical Systems. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Determining blood concentrations of the amino acids homocysteine, tryptophan, tyrosine and phenylalanine in children is of value in the clinical practice. Over the past decades, the use of blood spot samples to examine amino acid concentrations is increasing rapidly. In children, the use of blood spot samples is especially of relevance, as this method is much less invasive than venous blood sampling. Currently, no paediatric reference values for amino acids in blood spots are available. The aim of the current study was to establish reference values for blood spot concentrations of total homocysteine, tryptophan, tyrosine and phenylalanine in school-age children. Dried blood spots were obtained in a community sample of 104 healthy children, aged 6-12 years old (52% males). Blood spot concentrations of total homocysteine, tryptophan, tyrosine and phenylalanine were determined by positive electrospray liquid chromatography-tandem mass spectrometry. Parents of participants completed questions regarding demographic characteristics. Our sample consisted of healthy children from various ethnic backgrounds, with varying levels of socioeconomic status, in line with the composition of the Dutch society. Blood spot concentrations of total homocysteine, tryptophan, tyrosine and phenylalanine were similar in males and females, and independent of age. In conclusion, paediatric reference values for blood spot concentrations of total homocysteine, tryptophan, tyrosine and phenylalanine were established, which could be of use in the clinical practice.

Nutritional and genetic deficiencies of folate and vitamin B 12 lead to elevation of cellular homocysteine (Hcy), which translates in increased plasma Hcy. The sources and role of elevated plasma Hcy in pathology continues to be a subject of intense scientific debate. Whether a cause, mediator or marker, little is known about the molecular mechanisms and interactions of Hcy with cellular processes that lead to disease. The use of folic acid reduces the incidence of neural tube defects, but the effect of Hcy-lowering interventions with folic acid in cardiovascular disease and cognitive impairment remains controversial. The fact that levels of Hcy in plasma do not always reflect cellular status of this amino acid may account for the substantial gaps that exist between epidemiological, intervention and basic research studies. Understanding whether plasma Hcy is a mechanistic player or an epiphenomenon in pathogenesis requires further investigation, and this research is essential to improve the assessment and potential treatment of hyperhomocysteinemias., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Inherited methylation disorders are a group of rarely reported, probably largely underdiagnosed disorders affecting transmethylation processes in the metabolic pathway between methionine and homocysteine. These are methionine adenosyltransferase I/III, glycine N-methyltransferase, S-adenosylhomocysteine hydrolase and adenosine kinase deficiencies. This paper provides the first consensus recommendations for the diagnosis and management of methylation disorders. Following search of the literature and evaluation according to the SIGN-methodology of all reported patients with methylation defects, graded recommendations are provided in a structured way comprising diagnosis (clinical presentation, biochemical abnormalities, differential diagnosis, newborn screening, prenatal diagnosis), therapy and follow-up. Methylation disorders predominantly affect the liver, central nervous system and muscles, but clinical presentation can vary considerably between and within disorders. Although isolated hypermethioninemia is the biochemical hallmark of this group of disorders, it is not always present, especially in early infancy. Plasma S-adenosylmethionine and S-adenosylhomocysteine are key metabolites for the biochemical clarification of isolated hypermethioninemia. Mild hyperhomocysteinemia can be present in all methylation disorders. Methylation disorders do not qualify as primary targets of newborn screening. A low-methionine diet can be beneficial in patients with methionine adenosyltransferase I/III deficiency if plasma methionine concentrations exceed 800 μmol/L. There is some evidence that this diet may also be beneficial in patients with S-adenosylhomocysteine hydrolase and adenosine kinase deficiencies. S-adenosylmethionine supplementation may be useful in patients with methionine adenosyltransferase I/III deficiency. Recommendations given in this article are based on general principles and in practice should be adjusted individually according to patient's age, severity of the disease, clinical and laboratory findings., Competing Interests: Compliance with ethical standards This article does not contain any studies with human or animal subjects performed by the any of the authors. Conflict of interests None.

B-vitamin trials failed to demonstrate beneficial effects on cardiovascular outcomes, but hyperhomocysteinemia still stands out as an independent cardiovascular risk factor, particularly in elderly individuals. B-vitamins may influence early vascular dysfunction, such as endothelial dysfunction, or may have adverse effects, for example on inflammation. We investigated the effect of B-vitamins on endothelial function and inflammation within an interventional study. This study was conducted within the framework of the B-PROOF trial, which included 2919 hyperhomocysteinemic elderly individuals, who received daily vitamin B12 (500 μg) and folic acid (400 μg) or placebo for 2 years. Using an electrochemiluminescence platform, we measured intercellular adhesion molecule 1 (ICAM-1), vascular adhesion molecule 1 (VCAM-1), serum amyloid A (SAA), vascular endothelial growth factor (VEGF) and C-reactive protein (CRP) at baseline and follow-up in a subsample of 522 participants (271 intervention group; 251 placebo). Treatment effects were analyzed with ANCOVA. The participants had a mean age of 72 years, and 55% of them were male. At the 2-year follow-up, B-vitamins did not change the ICAM-1 (+36% change in the intervention group versus +32% change in the placebo group; p = 0.72), VCAM-1 (+27% vs +25%; p = 0.39), VEGF (-1% vs +4%; p = 0.40), SAA (+34% vs +38%; p = 0.85) or CRP levels (+26% vs +36%; p = 0.70) as compared to placebo. In conclusion, in elderly patients with hyperhomocysteinemia, vitamin B12 and folic acid are unlikely to influence either endothelial function or low-grade systemic inflammation. ClinicalTrials.gov Identifier: NCT00696514., (© The Author(s) 2016.)

Background: Results from a number of long-term follow-up studies have suggested that hypertensive disorders in pregnancy are associated with increased risk of cardiovascular disease later in life. More recently, this putative relationship has been substantiated with findings of elevated cardiovascular risk factors, such as lipid profiles and glucose, in women with a history of hypertensive pregnancy disorders. Homocysteine is a sensitive indicator of increased risk but data on homocysteine levels in women with a history of hypertensive pregnancy disorders are inconsistent., Design: This cohort study included 279 women with a history of hypertensive pregnancy disorders at term and 85 women with a history of uncomplicated pregnancies who participated in the Hypitat Risk Assessment Study (HyRAS)., Methods: Blood samples for total homocysteine determination were taken 2.5 years postpartum. Homocysteine levels were determined in plasma using an immunoassay., Results: Women with a history of hypertensive pregnancy disorders had significant higher median homocysteine levels (10.66 μmol/L) 2.5 years postpartum compared with women with a history uncomplicated pregnancies (9.82 μmol/L; p=0.002). Women with a history of hypertensive pregnancy disorders had a higher risk of having a homocysteine level in the highest quartile (odds ratio 3.4, 95% confidence interval 1.5-7.6)., Conclusion: At 2.5 years postpartum, women with a history hypertensive pregnancy disorders had higher homocysteine levels than women who had uncomplicated pregnancies. Although higher homocysteine levels might be a potential link between a history of hypertensive pregnancy disorders and increased cardiovascular disease risk later in life, the clinical implications remain an area for future research.

Betaine is the substrate of the liver- and kidney-specific betaine-homocysteine (Hcy) methyltransferase (BHMT), an alternate pathway for Hcy remethylation. We hypothesized that BHMT is a major pathway for homocysteine removal in cases of hyperhomocysteinaemia (HHcy). Therefore, we measured betaine in plasma and tissues from patients and animal models of HHcy of genetic and acquired cause. Plasma was collected from patients presenting HHcy without any Hcy interfering treatment. Plasma and tissues were collected from rat models of HHcy induced by diet and from a mouse model of cystathionine β-synthase (CBS) deficiency. S-adenosyl-methionine (AdoMet), S-adenosyl-homocysteine (AdoHcy), methionine, betaine and dimethylglycine (DMG) were quantified by ESI-LC-MS/MS. mRNA expression was quantified using quantitative real-time (QRT)-PCR. For all patients with diverse causes of HHcy, plasma betaine concentrations were below the normal values of our laboratory. In the diet-induced HHcy rat model, betaine was decreased in all tissues analysed (liver, brain, heart). In the mouse CBS deficiency model, betaine was decreased in plasma, liver, heart and brain, but was conserved in kidney. Surprisingly, BHMT expression and activity was decreased in liver. However, in kidney, BHMT and SLC6A12 expression was increased in CBS-deficient mice. Chronic HHcy, irrespective of its cause, induces betaine depletion in plasma and tissues (liver, brain and heart), indicating a global decrease in the body betaine pool. In kidney, betaine concentrations were not affected, possibly due to overexpression of the betaine transporter SLC6A12 where betaine may be conserved because of its crucial role as an osmolyte., (© 2015 Author(s).)

Introduction: Classical homocystinuria is a rare genetic disease caused by cystathionine β-synthase deficiency, resulting in homocysteine accumulation. Growing evidence suggests that reduced fat mass in patients with classical homocystinuria may be associated with alterations in choline and homocysteine pathways. This study aimed to evaluate the body composition of patients with classical homocystinuria, identifying changes in body fat percentage and correlating findings with biochemical markers of homocysteine and choline pathways, lipoprotein levels and bone mineral density (BMD) T-scores., Methods: Nine patients with classical homocystinuria were included in the study. Levels of homocysteine, methionine, cysteine, choline, betaine, dimethylglycine and ethanolamine were determined. Body composition was assessed by bioelectrical impedance analysis (BIA) in patients and in 18 controls. Data on the last BMD measurement and lipoprotein profile were obtained from medical records., Results: Of 9 patients, 4 (44%) had a low body fat percentage, but no statistically significant differences were found between patients and controls. Homocysteine and methionine levels were negatively correlated with body mass index (BMI), while cysteine showed a positive correlation with BMI (p<0.05). There was a trend between total choline levels and body fat percentage (r=0.439, p=0.07). HDL cholesterol correlated with choline and ethanolamine levels (r=0.757, p=0.049; r=0.847, p=0.016, respectively), and total cholesterol also correlated with choline levels (r=0.775, p=0.041). There was no association between BMD T-scores and body composition., Conclusions: These results suggest that reduced fat mass is common in patients with classical homocystinuria, and that alterations in homocysteine and choline pathways affect body mass and lipid metabolism., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Accumulation of the homocysteine (Hcy) precursor S-adenosylhomocysteine (AdoHcy) may cause cellular hypomethylation in the setting of hyperhomocysteinemia because of cystathionine β-synthase (CBS) deficiency, an inborn error of metabolism. To test this hypothesis, DNA and protein arginine methylation status were assessed in liver, brain, heart, and kidney obtained from a previously described mouse model of CBS deficiency. Metabolite levels in tissues and serum were determined by high-performance liquid chromatography or liquid chromatography-electrospray ionization-tandem mass spectrometry. Global DNA and protein arginine methylation status were evaluated as the contents of 5-methyldeoxycytidine in DNA and of methylarginines in proteins, respectively. In addition, histone arginine methylation was assessed by Western blotting. CBS-deficient mice exhibited increased (>6-fold) Hcy and AdoHcy levels in all tissues examined compared with control levels. In addition, global DNA methylation status was not affected, but global protein arginine methylation status was decreased (10-35%) in liver and brain. Moreover, asymmetric dimethylation of arginine 3 on histone H4 (H4R3me2a) content was markedly decreased in liver, and no differences were observed for the other histone arginine methylation marks examined. Our results show that CBS-deficient mice present severe accumulation of tissue Hcy and AdoHcy, protein arginine hypomethylation in liver and brain, and decreased H4R3me2a content in liver. Therefore, protein arginine hypomethylation arises as a potential player in the pathophysiology of CBS deficiency., (© FASEB.)

Objectives: To assess the effect of preventive pravastatin treatment on coronary heart disease (CHD) morbidity and mortality in older persons at risk for cardiovascular disease (CVD), stratified according to plasma levels of homocysteine., Design: A post hoc subanalysis in the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER), started in 1997, which is a double-blind, randomized, placebo-controlled trial with a mean follow-up of 3.2 years., Setting: Primary care setting in two of the three PROSPER study sites (Netherlands and Scotland)., Participants: Individuals (n = 3,522, aged 70-82, 1,765 male) with a history of or risk factors for CVD were ranked in three groups depending on baseline homocysteine level, sex, and study site., Intervention: Pravastatin (40 mg) versus placebo., Measurements: Fatal and nonfatal CHD and mortality., Results: In the placebo group, participants with a high homocysteine level (n = 588) had a 1.8 higher risk (95% confidence interval (CI) = 1.2-2.5, P = .001) of fatal and nonfatal CHD than those with a low homocysteine level (n = 597). The absolute risk reduction in fatal and nonfatal CHD with pravastatin treatment was 1.6% (95% CI = -1.6 to 4.7%) in the low homocysteine group and 6.7% (95% CI = 2.7-10.7%) in the high homocysteine group (difference 5.2%, 95% CI = 0.11-10.3, P = .046). Therefore, the number needed to treat (NNT) with pravastatin for 3.2 years for benefit related to fatal and nonfatal CHD events was 14.8 (95% CI = 9.3-36.6) for high homocysteine and 64.5 (95% CI = 21.4-∞) for low homocysteine., Conclusion: In older persons at risk of CVD, those with high homocysteine are at highest risk for fatal and nonfatal CHD. With pravastatin treatment, this group has the highest absolute risk reduction and the lowest NNT to prevent fatal and nonfatal CHD., (© 2014, Copyright the Authors. Journal compilation © 2014, The American Geriatrics Society.)

Background: Homocysteine is an independent predictor of cardiovascular risk. The mechanisms underlying this link are not fully elucidated. Whereas the role of vascular dysfunction in conduit arteries is extensively studied, the role of the microcirculation in this relationship is largely unexplored. We assessed the relationship between homocysteine levels and microvascular structure and function in a healthy, population-based cohort., Materials and Methods: We cross-sectionally studied 260 participants (aged 42 years, 47% men) of the Amsterdam Growth and Health Longitudinal Study. Nailfold videocapillaroscopy was used to assess capillary density at baseline, during venous occlusion and during peak reactive hyperaemia. The relationship between tertiles of homocysteine and microvascular outcomes was evaluated using linear regression analyses, with adjustment for BMI and blood pressure. Stratified analyses were performed for men and women., Results: In men, we observed a negative, nonlinear relationship between homocysteine and baseline capillary density, showing a lower capillary density in the highest tertile of homocysteine [adjusted B -8.65 capillaries/mm(2) (95%-CI: -16.05 to -1.25); P = 0.02]. In women, no significant associations were found between homocysteine and microvascular outcomes., Conclusions: In men, higher homocysteine levels are associated with a reduction in basal perfusion of skin capillaries. This finding provides a novel potential explanation for how homocysteine influences cardiovascular disease risk., (© 2014 Stichting European Society for Clinical Investigation Journal Foundation.)

Apoptosis of endothelial cells related to homocysteine (Hcy) has been reported in several studies. In this study, we evaluated whether reactive oxygen species (ROS)-producing signaling pathways contribute to Hcy-induced apoptosis induction, with specific emphasis on NADPH oxidases. Human umbilical vein endothelial cells were incubated with 0.01-2.5 mM Hcy. We determined the effect of Hcy on caspase-3 activity, annexin V positivity, intracellular NOX1, NOX2, NOX4, and p47(phox) expression and localization, nuclear nitrotyrosine accumulation, and mitochondrial membrane potential (ΔΨ m). Hcy induced caspase-3 activity and apoptosis; this effect was concentration dependent and maximal after 6-h exposure to 2.5 mM Hcy. It was accompanied by a significant increase in ΔΨ m. Cysteine was inactive on these parameters excluding a reactive thiol group effect. Hcy induced an increase in cellular NOX2, p47(phox), and NOX4, but not that of NOX1. 3D digital imaging microscopy followed by image deconvolution analysis showed nuclear accumulation of NOX2 and p47(phox) in endothelial cells exposed to Hcy, but not in control cells, which coincided with accumulation of nuclear nitrotyrosine residues. Furthermore, Hcy enhanced peri-nuclear localization of NOX4 coinciding with accumulation of peri-nuclear nitrotyrosine residues, a reflection of local ROS production. p47(phox) was also increased in the peri-nuclear region. The Hcy-induced increase in caspase-3 activity was prevented by DPI and apocynin, suggesting involvement of NOX activity. The data presented in this article reveal accumulation of nuclear NOX2 and peri-nuclear NOX4 accumulation as potential source of ROS production in Hcy-induced apoptosis in endothelial cells.

Background: In randomized trails, folic acid (FA) lowered plasma homocysteine, but failed to reduce cardiovascular risk. We hypothesize this is due to a discrepancy between plasma and intracellular effects of FA., Methods: In a double-blind trial, 50 volunteers were randomized to received 500 µg FA daily for 8 weeks, or placebo. Plasma and peripheral blood mononuclear cell (PBMC) concentrations of homocysteine, S-adenosylmethionine (SAM), S-adenosylhomocysteine, methionine, cystathionine and 5-methyltetrahydrofolate (bioactive folate) were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). PBMCs were used as a cellular model since they display the full spectrum of one-carbon (1C) enzymes and reactions., Results: At baseline, plasma concentrations were a poor reflection of intracellular concentrations for most 1C metabolites, except 5-methyltetrahydrofolate (R=0.33, p=0.02), homocysteine (Hcy) (R=0.35, p=0.01), and cystathionine (R=0.45, p=0.001). FA significantly lowered plasma homocysteine (p=0.00), but failed to lower intracellular homocysteine or change the concentrations of any of the other PBMC 1C metabolites. At baseline, PBMC homocysteine concentrations correlated to PBMC SAM. After FA supplementation, PBMC homocysteine no longer correlated with PBMC SAM, suggesting a loss of SAM's regulatory function. In vitro experiments in lymphoblasts confirmed that at higher folate substrate concentrations, physiological concentrations of SAM no longer effectively inhibit the key regulatory enzyme methylenetetrahydrofolate reductase (MTHFR)., Conclusions: FA supplementation does not reduce intracellular concentrations of Hcy or any of its closely related substances. Rather, FA may disturb physiological regulation of intracellular 1C metabolism by interfering with SAM's inhibitory effect on MTHFR activity.

Objective: Homocysteine has been shown to be a more accurate predictor of cardiovascular mortality in very old persons than models based on classical risk factors. Arterial stiffening is a structural abnormality involved in the pathway of cardiovascular disease. We expect this underlying pathophysiology to be a possible explanation for the association between homocysteine and cardiovascular risk, particularly in older populations., Methods: Baseline cross-sectional data of the B-PROOF study were used to determine associations between homocysteine and outcomes of vascular function and structure. The cardiovascular subgroup of the B-PROOF study was included [n = 560, 58% men, age 72.6 ± 5.5 years, median homocysteine level 14.2 μmol/l (IQR 13.0-16.6)]. We assessed carotid distensibility coefficient, carotid compliance coefficient, aortic pulse wave velocity (aPWV), augmentation index (AIx) and aortic pulse pressure (aortic PP). Associations were tested using linear regression analysis and ANCOVA and were adjusted for possible confounders including age, sex, renal function, mean arterial pressure and heart rate., Results: Ln-homocysteine was strongly associated with aPWV [β 0.005 95% confidence interval (0.001-0.009)]. Furthermore, this association was shown to be age-dependent (P = 0.02) and it was most strong in the upper tertile of age (77-98 years). No significant associations with ln-homocysteine were observed for AIx, carotid distensibility coefficient and compliance coefficient and aortic PP. Sex stratification shows the association between ln-homocysteine and aPWV is only significant in men., Conclusion: In older persons, homocysteine is associated with aortic stiffness, predominantly in the oldest old. This suggests that the strong association between homocysteine and cardiovascular mortality in the elderly may be mediated by aortic stiffness.

Background: Disturbances in the levels of one-carbon (1C) metabolism metabolites have been associated with a wide variety of neuropsychiatric diseases. Cerebrospinal fluid (CSF) levels of homocysteine (Hcy) and the other 1C metabolites, nor their interrelatedness and putative determinants, have been studied extensively in a healthy population., Methods: Plasma and CSF samples from 100 individuals free from neuropsychiatric diseases were analyzed (55 male, 45 female; age 50±17 years). In blood, we measured plasma Hcy, serum folate and serum vitamin B12. In CSF, we measured total Hcy, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and 5-methyltetrahydrofolate (5-methylTHF). Highly selective analytical methods like liquid chromatography combined with either mass spectrometry or fluorescence detection were used., Results: CSF Hcy was inversely correlated with CSF 5-methylTHF and positively with plasma Hcy, independent of serum folate status. CSF SAH correlated with age, lower CSF 5-methylTHF and higher CSF Hcy. CSF 5-methylTHF showed independent negative correlations with age and positive correlations with serum folate. CSF SAM did not correlate with any of the 1C metabolites., Conclusions: Aging is characterized by a reduction in CSF 5-methylTHF levels and increased CSF levels of the potentially neurotoxic transmethylation inhibitor SAH. CSF 5-methylTHF, which is itself determined in part by systemic folate status, is a powerful independent determinant of CSF levels of Hcy and SAH.

Objective: We have previously shown that homocysteine (Hcy) induces phosphatidylserine (PS) exposure, apoptosis and necrosis in human endothelial cells. Since it has been suggested that S-adenosylhomocysteine (SAH) is the main causative factor in Hcy-induced pathogenesis of cardiovascular disease, we evaluate here whether the cytotoxic Hcy effect in endothelial cells is also SAH dependent., Methods and Results: Human umbilical vein endothelial cells (HUVECs) were exposed to the following conditions: (1) non-treated control (resulting in 2.8 nM intracellular SAH and 3.1 μM extracellular l-Hcy); and incubation with (2) 50 μM adenosine-2,3-dialdehyde (ADA; resulting in 17.7 nM intracellular SAH and 3.1 μM extracellular l-Hcy), (3) 2.5 mM Hcy (resulting in 20.9 nM intracellular SAH and 1.8 mM extracellular l-Hcy), and (4) 1, 10 and 100 μM SAH. We then determined the effect of treatment on annexin V-positivity, caspase-3 activity, cytochrome c release (sub)cellular expression of NOX2, NOX4, p47(phox) and nitrotyrosine, and H(2)O(2). Both Hcy and ADA significantly increased PS exposure (n=5), caspase-3 activity (n=6) and cytochrome c release (n=3). Incubation with extracellular SAH alone did not affect cell viability. Both Hcy and ADA also induced similar increases in nuclear NOX2 and (peri)nuclear NOX4, coinciding with (peri)nuclear p47(phox) expression and local reactive oxygen species (ROS) (n=3). Inhibition of NOX-mediated ROS by the flavoenzyme inhibitor diphenylene iodonium (DPI) significantly decreased apoptosis induction (n=3) and ROS production (n=3)., Conclusion: SAH induces PS exposure and apoptosis in endothelial cells independently of Hcy. Our study therefore shows that Hcy-mediated endothelial dysfunction, as determined in the cell model used, is mainly due to SAH accumulation., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: Growth retardation in utero leading to small-for-gestational-age (SGA) newborns is associated with increased neonatal morbidity and mortality and with lifelong consequences such as poor cognitive function and cardiovascular diseases. Maternal total homocysteine (tHcy) concentrations have been linked to a wide range of adverse pregnancy outcomes and could possibly influence birth weight., Objective: We performed a systematic review of and meta-analysis on the association of maternal tHcy and birth weight., Design: A literature search of English, German, and French publications with the use of the PubMed database (January 1966-July 2010) found 78 abstracts. Search terms were as follows: homocysteine AND (birth weight OR small for gestational age OR intrauterine growth retardation). Studies were eligible if information on maternal tHcy and birth weight and the possible association between maternal tHcy and birth weight was available. Effect size estimates were converted to ORs as estimates of the RR of a woman to deliver SGA offspring when maternal tHcy exceeded the 90th percentile., Results: The search yielded 19 studies for analysis, consisting of 21,326 individuals. Pooled analysis resulted in a crude OR of 1.25 (95% CI: 1.09, 1.44). When this estimate was expressed as a linear effect, it corresponded to a decrease in birth weight of 31 g (95% CI: -13, -51 g) for a 1-SD increase in maternal tHcy., Conclusions: Higher maternal tHcy concentrations are associated with a small increased risk for SGA offspring. The small estimated birth weight difference might be of little clinical relevance for the individual newborn; however, it could be of greater importance at a population level.

Homocysteine concentrations are a read-out of methionine metabolism and have been related to changes in lifespan in animal models. In humans, high homocysteine concentrations are an important predictor of age related disease. We aimed to explore the association of homocysteine with familial longevity by testing whether homocysteine is lower in individuals that are genetically enriched for longevity. We measured concentrations of total homocysteine in 1907 subjects from the Leiden Longevity Study consisting of 1309 offspring of nonagenarian siblings, who are enriched with familial factors promoting longevity, and 598 partners thereof as population controls. We found that homocysteine was related to age, creatinine, folate, vitamin B levels and medical history of hypertension and stroke in both groups (all p<0.001). However, levels of homocysteine did not differ between offspring enriched for longevity and their partners, and no differences in the age-related rise in homocysteine levels were found between groups (p for interaction 0.63). The results suggest that homocysteine metabolism is not likely to predict familial longevity.

This overview addresses homocysteine and folate metabolism. Its functions and complexity are described, leading to explanations why disturbed homocysteine and folate metabolism is implicated in many different diseases, including congenital birth defects like congenital heart disease, cleft lip and palate, late pregnancy complications, different kinds of neurodegenerative and psychiatric diseases, osteoporosis and cancer. In addition, the inborn errors leading to hyperhomocysteinemia and homocystinuria are described. These extreme human hyperhomocysteinemia models provide knowledge about which part of the homocysteine and folate pathways are linked to which disease. For example, the very high risk for arterial and venous occlusive disease in patients with severe hyperhomocysteinemia irrespective of the location of the defect in remethylation or transsulphuration indicates that homocysteine itself or one of its "direct" derivatives is considered toxic for the cardiovascular system. Finally, common diseases associated with elevated homocysteine are discussed with the focus on cardiovascular disease and neural tube defects.

Mild to moderate hyperhomocysteinemia has been identified as a strong predictor of cardiovascular disease, independent from classical atherothrombotic risk factors. In the last decade, a number of large intervention trials using B vitamins have been performed and have shown no benefit of homocysteine-lowering therapy in high-risk patients. In addition, Mendelian randomization studies failed to convincingly demonstrate that a genetic polymorphism commonly associated with higher homocysteine levels (methylenetetrahydrofolate reductase 677 C>T) is a risk factor for cardiovascular disease. Together, these findings have cast doubt on the role of homocysteine in cardiovascular disease pathogenesis, and the homocysteine hypothesis has turned into a homocysteine controversy. In this review, we attempt to find solutions to this controversy. First, we explain that the Mendelian randomization analyses have limitations that preclude final conclusions. Second, several characteristics of intervention trials limit interpretation and generalizability of their results. Finally, the possibility that homocysteine lowering is in itself beneficial but is offset by adverse side effects of B vitamins on atherosclerosis deserves serious attention. As we explain, such side effects may relate to direct adverse effects of the B-vitamin regimen (in particular, the use of high-dose folic acid) or to proinflammatory and proproliferative effects of B vitamins on advanced atherosclerotic lesions.

Objective: We designed a large prospective study to explore the relationship between maternal homocysteine concentrations and related B vitamins and birthweight., Study Design: Blood was sampled from pregnant women at 30-34 weeks of gestation and their newborn infants (n = 366)., Results: Concentrations of all analytes were higher in umbilical cord compared with maternal samples. Birthweight was related negatively to maternal homocysteine (r = -0.12) but not related to maternal cobalamin, methylmalonic acid, and folate (r = 0.02, r = 0.06, and r = 0.04, respectively). Regression analysis revealed smoking (beta = -313; 95% confidence interval [CI], -479 to -149), gestational age (beta = 150; 95% CI, 118-182), female sex (beta = -146; 95% CI, -256 to -35), and parity (beta = 104; 95% CI, 37-171) as strong determinants of birthweight. Maternal homocysteine, cobalamin, methylmalonic acid, and folate were not determinants of birthweight in multivariate analysis., Conclusion: Maternal homocysteine and B vitamins are not related to birthweight in a multivariate model that was adjusted for potential confounders., (Copyright 2010 Mosby, Inc. All rights reserved.)

The risk of nutrient deficiencies increases with age in our modern Western society, and vitamin B(12) deficiency is especially prevalent in the elderly and causes increased homocysteine (Hcy) and methylmalonic acid (MMA) levels. These three factors have been recognized as risk factors for reduced bone mineral density and increased fracture risk, though mechanistic evidence is still lacking. In the present study, we investigated the influence of B(12), Hcy, and MMA on differentiation and activity of bone cells. B(12) deficiency did not affect the onset of osteoblast differentiation, maturation, matrix mineralization, or adipocyte differentiation from human mesenchymal stem cells (hMSCs). B(12) deficiency caused an increase in the secretion of Hcy and MMA into the culture medium by osteoblasts, but Hcy and MMA appeared to have no effect on hMSC osteoblast differentiation. We further studied the effect of B(12), Hcy, and MMA on the formation of multinucleated tartrate-resistant acid phosphatase-positive osteoclasts from mouse bone marrow. We observed that B(12) did not show an effect on osteoclastogenesis. However, Hcy as well as MMA were found to induce osteoclastogenesis in a dose-dependent manner. On the basis of these results, we conclude that B(12) deficiency may lead to decreased bone mass by increased osteoclast formation due to increased MMA and Hcy levels.

Background: Patients with chronic kidney disease (CKD) have an increased risk of cardiovascular disease (CVD). Preliminary evidence suggests a role for global DNA hypomethylation in the pathogenesis of atherosclerotic complications in CKD. The aims of this study in patients with stage 2-4 CKD were (1) to assess the association between renal function and DNA methylation, (2) to assess the association between DNA methylation and two markers of atherosclerosis [common carotid intima-media thickness (CCA-IMT)] and brachial artery endothelium-dependent, flow-mediated dilatation (BA-FMD) and (3) to examine the effect of a multi-step treatment strategy on DNA methylation., Methods: In the Anti-Oxidant Therapy In Chronic Renal Insufficiency study (ATIC-study), 93 patients with stage 2-4 CKD were included. In a randomized, double-blind, placebo-controlled design, the treatment group received pravastatin to which vitamin E was added after 6 months and homocysteine-lowering B-vitamin therapy after another 6 months. DNA methylation was assessed using tandem mass spectrometry. CCA-IMT and BA-FMD were assessed using B-mode ultrasonography., Results: At baseline, global DNA methylation was not associated with the estimated glomerular filtration rate (P = 0.32) or with CCA-IMT (P = 0.62) or BA-FMD (P = 0.51). No effect of the treatment strategy including B-vitamin on global DNA methylation was found either in the total study group or within separate strata of homocysteine concentration and renal function., Conclusion: In patients with stage 2-4 CKD, global DNA methylation is not associated with renal function or with CCA-IMT or BA-FMD. A treatment strategy that includes B-vitamins did not alter global DNA methylation in these patients. These data do not support the role of DNA hypomethylation in CKD-associated vascular disease in patients with stage 2-4 CKD.

This case-cohort designed study prospectively investigated whether elevated homocysteine levels measured in blood samples drawn before the event and methylenetetrahydrofolate reductase (MTHFR) gene polymorphism (MTHFR C677T) were associated with subsequent first venous thrombosis (VT) in a general population. Between August 1995 and June 1997, blood was collected from 66 140 people in the second Norwegian Health Study of Nord-Trøndelag (HUNT2). During a seven-year follow-up, 505 VT cases were identified. 1458 age- and sex-matched controls were selected from the original cohort. Serum total homocysteine (tHcy) and MTHFR genotype were measured in stored samples that were drawn a median of 33 months before the events. The overall odds ratio (OR) was 1.50 [95% confidence interval (CI) 0.97-2.30] for homocysteine levels above versus below the 95th percentile. There was no graded association with VT over quintiles of homocysteine. In men the OR was 2.17 (95% CI 1.20-3.91) for levels above versus below the 95th percentile, but no association was found in women (OR 1.00). Stratification by age, predisposing risk factors or time to event did not change these results. The MTHFR 677TT genotype was not related to risk for VT. In conclusion, elevated homocysteine levels in the general population predicted subsequent first VT in men but not in women.

A disturbed methylation has been proposed as a mechanism via which homocysteine is associated with diseases like vascular disease, neural tube defects and mental disorders. Catechol-O-methyltransferase (COMT) is involved in the S-adenosylmethionine-dependent methylation of catecholamines and catecholestrogens and in this way contributes to homocysteine synthesis. COMT dysfunction has been related to schizophrenia and breast cancer. We hypothesized that COMT dysfunction by virtue of functional genetic polymorphisms may affect plasma total homocysteine (tHcy). Our primary objective was to study the association between common COMT polymorphisms and tHcy. Secondly, we evaluated these polymorphisms as a risk factor for recurrent venous thrombosis. We obtained genotype data from four polymorphisms in the COMT gene (rs2097603, rs4633, rs4680 [324G>A] and rs174699) from 401 population-based controls. We performed haplotype analysis to investigate the association between common haplotypes and tHcy. In addition, we assessed the rs4680 variant as a genetic risk factor in a case-control study on recurrent venous thrombosis (n = 169). We identified a common haplotype that was significantly associated with tHcy levels. This effect was largely explained by the rs4680 variant, resulting in an increase in tHcy of 10.4% (95% CI 0.01 to 0.21, p = 0.03) for 324AA compared with 324GG subjects. Interestingly, we found that the 324AA genotype was more common in venous thrombosis patients (OR 1.61 [95% CI 0.97 to 2.65], p = 0.06) compared to control subjects. We show that the COMT rs4680 variant modulates tHcy, and might be associated with venous thrombosis risk as well.

The importance of the one-carbon metabolites, choline and homocysteine, to brain function is well known. However, the associations between the one-carbon metabolites choline, betaine, methionine and dimethylglycine with cognition in elderly are unclear. We therefore examined the associations of these metabolites with cognition in a double-blind, placebo-controlled trial. Individuals (n 195) were randomized to receive daily oral capsules with either 1000 microg cobalamin (vitamin B12), or 1000 microg cobalamin plus 400 microg folic acid, or placebo for 24 weeks. Concentrations of homocysteine, methionine, choline, betaine and dimethylglycine were assessed before and after 12 and 24 weeks of treatment. Cognitive function, including domains of attention, construction, sensomotor speed, memory and executive function, was assessed before and after 24 weeks of treatment. At baseline, elevated plasma homocysteine was associated with lower performance of attention, construction, sensomotor speed and executive function. In addition, betaine was positively associated with better performance of construction, sensomotor speed and executive function, whereas elevated concentrations of methionine were positively associated with sensomotor speed. Daily combined supplementation with cobalamin plus folic acid decreased total homocysteine concentrations by 36%, and increased betaine concentrations by 38%. Participants with the largest increases in betaine concentrations showed a borderline significant (P = 0.07) higher memory performance compared to those without it. Although this trial observed associations of homocysteine and betaine with cognitive domains prior to supplementation, decreased concentrations of homocysteine were not related to improved cognitive performance. There was a tendency of participants with the largest increases in betaine concentrations to show the greatest improvement in memory function.

Cobalamin is an essential cofactor for two mammalian enzymes: methionine synthase and methylmalonyl-CoA mutase. Patients with the cobalamin C (CblC) defect have combined methylmalonic aciduria and homocystinuria. Recently, the gene responsible for the CblC type, MMACHC, was identified, which enables molecular diagnostics. In this study, we describe two siblings, a 16-year-old girl and her 11-year-old brother, of a consanguineous family who presented with a very distinct clinical manifestation. The girl presented at the age of 13 years with macrocytic anaemia, cognitive regression and Marfanoid features such as increased arm-span, arachnodactyly, joint hyperlaxity and scoliosis. Her brother presented at the age of 10 months with developmental delay and behavioural abnormalities. Biochemical analysis showed severely increased homocysteine and methylmalonic acid levels in plasma of both siblings. In addition, plasma cysteine levels were decreased in the girl but not in her brother. The diagnosis of CblC defect was confirmed by genomic sequencing of the coding exons of the MMACHC gene. Two heterozygous mutations were identified in both siblings; the common c.271dupA p.Arg91LysfsX14 and a novel mutation, c.1A > G p.Met1?. Therapy consisting of folic acid, vitamin B6, l-carnitine and intramuscular vitamin B12 resulted in a clear improvement of biochemical parameters and, importantly, resulted in amelioration of the Marfanoid features in the girl. These data might suggest that low cysteine levels account for the Marfanoid features observed in the girl and indicate that the CblC type of combined methylmalonic aciduria and homocystinuria should be considered in the differential diagnosis of patients with Marfanoid features.

Background: Hyperhomocysteinemia has been associated with vascular disease in many epidemiologic studies, but only a few have reported on the relation between hyperhomocysteinemia and aneurysms of the abdominal aorta (AAAs). Although these studies showed higher homocysteine concentrations in patients with AAA than in controls, little attention had been given to possible confounding factors. Most patients with AAA are of older age, have an impaired renal function, and have other risk factors for cardiovascular disease. This matched case-control study investigated the relation between homocysteine concentration (before and after methionine loading) and AAA, taking into account possible confounders such as age, sex, and concentrations of creatinine and B vitamins., Methods: Patients with a history of AAA were recruited from the outpatient clinic; 60% had already undergone surgery for their AAA. They were asked to invite a friend or neighbor to participate as a control subject (age-matched and sex-matched). Concentrations of homocysteine, vitamin B6, vitamin B12, folate, and creatinine were determined in the fasting state, and blood was taken for methylenetetrahydrofolate reductase (MTHFR) mutation analysis. Six hours after oral methionine loading, the postmethionine load homocysteine concentration was determined., Results: Univariate analysis showed an odds ratio (OR) of 2.2 (95% confidence interval (CI), 0.9 to 5.5) for the risk of AAA for the highest quartile of homocysteine concentration. After adjustment for creatinine, the OR was markedly reduced to 1.24 (95% CI, 0.42 to 3.66), and this risk further attenuated in the multivariate analysis. Univariate analysis of the B vitamins showed an increased risk of AAA for the bottom quartile of vitamin B6 (OR, 3.75; 95% CI, 1.22 to 11.54), which even increased after adjustments. The relative risk associated with the MTHFR 677TT polymorphism was 2.1 (95% CI, 0.9 to 5.3)., Conclusion: Vitamin B6, but not homocysteine, is an independent risk factor for AAA. The role of vitamin B6 in the pathogenesis of AAA needs to be further elucidated.

Background: Folate is important in purine and thymidylate synthesis and, via homocysteine remethylation, facilitates S-adenosylmethionine-dependent transmethylation. Low folate availability leads to hyperhomocysteinemia, which is a risk factor for arterial vascular disease and venous thrombosis. Genetic variation in folate-metabolizing genes may affect folate availability and hence confer a greater risk of venous thrombosis., Methods: We genotyped the thymidylate synthase (TYMS) 28-bp repeat and 6-bp deletion, and the reduced folate carrier (RFC1) 80G>A and AICAR transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) 346C>G polymorphisms in population-based controls (n=431), and assessed their effect on plasma total homocysteine (tHcy), and serum and red blood cell (RBC) folate. We investigated the associations between these variants and disease risk in a retrospective case-control study on recurrent venous thrombosis (n=173) as well., Results: None of the genotypes, alone or in combination, were associated with major changes in tHcy. However, the TYMS 28-bp repeat was associated with serum and RBC folate levels. We found no evidence that the genetic variants studied were associated with recurrent venous thrombosis risk., Conclusions: The TYMS 28-bp repeat and 6-bp deletion, and RFC1 80G>A and ATIC 346C>G polymorphisms are not associated with tHcy, but we did observe an association between the TYMS 28-bp repeat and serum and RBC folate in a general population. None of the polymorphisms was associated with recurrent venous thrombosis risk.

Background: Hyperhomocysteinemia has been associated with vascular disease in many epidemiological studies. However, the pathophysiology is unclear. It is postulated that increased levels of homocysteine induce an inflammatory response in endothelial cells, mediated by pro-inflammatory cytokines and chemokines. The aim of this study was to investigate whether plasma concentrations of interleukin-6, interleukin-8, C-reactive protein, and monocyte chemoattractant protein-1 are increased with higher plasma homocysteine concentrations and whether decreasing homocysteine by vitamin supplementation decreases the concentration of these markers., Methods: Plasma homocysteine, interleukin-6, interleukin-8, C-reactive protein, and monocyte chemoattractant protein-1 concentrations were measured in 230 volunteers before and after 8 weeks of multivitamin supplementation (folic acid, B(6), and B(12))., Results: At baseline, plasma homocysteine concentration was weakly associated with interleukin-8, but not with interleukin-6, C-reactive protein or monocyte chemoattractant protein-1. Vitamin supplementation resulted in a significant decrease in homocysteine concentration, but no effect on interleukin-6, interleukin-8, C-reactive protein or monocyte chemoattractant protein-1 was observed., Conclusions: At baseline homocysteine was only weakly correlated with interleukin-8, but not with interleukin-6, C-reactive protein or monocyte chemoattractant protein-1. Vitamin supplementation affected homocysteine concentration, but not cytokine levels. The hypothesis that hyperhomocysteinemia increases arteriosclerotic or thrombotic risk through vascular inflammation was not supported by this study.

Disturbances in folate metabolism may increase the risk of certain malignancies, congenital defects and cardiovascular diseases. The gene dihydrofolate reductase (DHFR) is primarily involved in the reduction of dihydrofolate, generated during thymidylate synthesis, to tetrahydrofolate in order to maintain adequate amounts of folate for DNA synthesis and homocysteine remethylation. In order to reveal possible variation that may affect plasma total homocysteine (tHcy), serum folate and red blood cell (RBC) folate levels, we sequenced the DHFR coding region as well as the intron-exon boundaries and DHFR flanking regions from 20 Caucasian individuals. We identified a 9-bp repeat in the 5'-upstream region that partially overlapped with the 5'-untranslated region, and several single-nucleotide polymorphisms, all in non-coding regions. We screened subjects for the 9-bp repeat (n=417), as well as the recently reported 19-bp deletion in intron 1 (n=330), and assessed their associations with plasma tHcy, serum and RBC folate levels. The 19-bp del/del genotype was associated with a lower plasma tHcy (-14.4% [95% confidence interval: -23.5 to -4.5], P=0.006) compared with the wild-type genotype. This may suggest that intracellular folate levels are affected.

Elevated total plasma homocysteine concentrations (tHcy), both fasting and post-methionine load, have been established as risk factors for vascular disease. Recently, we described the association of a 31 bp variable number of tandem repeats (VNTR) in the cystathionine beta-synthase (CBS) gene with both CBS enzyme activity and tHcy concentrations. In the present study, we determined the 31 bp VNTR genotypes in 2598 individuals of the Framingham Offspring Study and studied the association between this genotype and fasting, 2-h post-methionine load and delta (ie increase upon methionine loading) tHcy concentrations in 1416 subjects. We observed a positive association between the number of repeat units of the CBS 31 bp VNTR and both postload and delta tHcy concentrations. Adjustment for possible effect modifying factors like age, sex and vitamin (B6, B12 and folate) status did not change this observation. We hereby confirm the results of our earlier study, in which we found that this 31 bp VNTR is a genetic determinant of post-methionine load tHcy concentrations. Since also post-methionine load tHcy concentrations are found to be associated with an increased risk for cardiovascular disease (CVD), this 31 bp VNTR may be considered a risk factor for CVD.

Neural-tube defects (NTD) are common congenital malformations that can lead to severe disability or even death. Periconceptional supplementation with the B-vitamin folic acid has been demonstrated to prevent 50-70% of NTD cases. Since the identification of the first genetic risk factor of NTD, the C677T single-nucleotide polymorphism (SNP) in the methylenetetrahydrofolate reductase (MTHFR) gene, and the observation that elevated plasma homocysteine levels are associated with NTD, research has focused on genetic variation in genes encoding for enzymes of folate metabolism and the closely-related homocysteine metabolism. In the present review relevant SNP in genes that code for enzymes involved in folate transport and uptake, the folate cycles and homocysteine metabolism are summarised and the importance of these SNP discussed in relation to NTD risk.

Periconceptional folic acid supplementation can reduce the occurrence of neural tube defects. A low folate status will result in reduced remethylation of homocysteine (Hcy) to methionine and, subsequently, in a rise of Hcy levels. Indeed, elevated Hcy concentrations have been reported in mothers of children with neural tube defects. In our previous study, we showed that treatment of chick embryos with Hcy resulted in a delay of neural tube closure in an in vitro model. In the present study, we examined whether this effect of Hcy is due to inhibition of transmethylation via elevation of S-adenosylhomocysteine (AdoHcy). Transmethylation involves methylation of DNA, RNA and proteins by donation of a methyl group from S-adenosylmethionine (AdoMet). After application of inhibitors of S-adenosylhomocysteine hydrolase and of methionine adenosyltransferase, a delay of anterior neuropore closure, comparable to that observed after Hcy treatment, was observed. The changes in AdoMet and AdoHcy concentrations confirmed the inhibition of S-adenosylhomocysteine hydrolase or methionine adenosyltransferase, respectively, and the AdoMet/AdoHcy ratio was decreased in all cases, indicating reduced transmethylation. Moreover, the inhibition of methionine adenosyltransferase was prevented by pretreatment with methionine. This study, therefore, indicates that the Hcy-induced delay of the neural tube closure is caused by the inhibition of transmethylation via elevation of AdoHcy levels and a reduction of the AdoMet/AdoHcy ratio.

Hyperhomocysteinemia (Hhcy) is an established risk factor for various pathologies including arterial vascular disease and venous thrombosis, congenital malformations and other pregnancy complications, and dementia. Homocysteine remethylation, transsulfuration, and export to the blood/extracellular compartment determine homocysteine concentrations. Any disturbance in these routes may lead to Hhcy and potentially increase risk of disease. In this report, we aim to review all known polymorphisms involved in homocysteine and B-vitamin metabolism that have been assessed for their effect on tHcy. In the last section, we summarize the polymorphisms, for which the obtained data provides evidence for their involvement in Hhcy at the population level, and discuss how to continue our search for genetic determinants of tHcy.

Background: Vitamin status, methylenentetrahydrofolate reductase (MTHFR) genotype, age, sex, and lifestyle factors are all predictors of total homocysteine (tHcy) concentrations in adults. Limited data are available about the influence of these factors on tHcy in children., Objective: The objective was to describe tHcy and its predictors in Dutch children., Design: A sample of 234 white children aged 0-19 y was analyzed cross-sectionally., Results: The geometric mean tHcy concentrations were 5.1 (95% CI: 4.6, 5.6), 4.6 (4.2, 5.1), 6.2 (5.6, 6.9), 7.3 (6.7, 8.0), and 8.7 (7.9, 9.6) micromol/L in the 0-1, 2-5, 6-10, 11-14, and 15-19 y groups, respectively. Plasma folate and vitamin B-12 concentrations decreased markedly with age. The inverse association between tHcy and plasma folate seen at all ages was stronger than that between tHcy and plasma vitamin B-12. A negative association of plasma folate with tHcy was confined to folate concentrations <20 nmol/L. Homozygosity for the MTHFR 677C-->T polymorphism was identified in 8.2% of the children. The homocysteine concentration did not differ significantly between the MTHFR genotypes., Conclusions: This study provided age-specific data regarding tHcy concentrations and their predictors in the whole range of childhood. The tHcy concentration increased as a function of age in both sexes. Plasma folate was a concentration-dependent predictor of tHcy. The MTHFR 677C-->T polymorphism played a minor role in determining tHcy concentrations in children.

Objective: Two published studies have demonstrated that betaine in the circulation is a determinant of plasma total homocysteine, but none had sufficient power to investigate the possible effect modification by folate status., Methods and Results: We measured homocysteine, betaine, folate, vitamin B(6), and related compounds in serum/plasma from 500 healthy men and women aged 34 to 69 years before (fasting levels) and 6 hours after a standard methionine loading test. Choline, dimethylglycine, and folate were determinants of plasma betaine in a multiple regression model adjusting for age and sex. The increase in homocysteine after loading showed a strong inverse association with plasma betaine and a weaker inverse association with folate and vitamin B(6). Fasting homocysteine showed a strong inverse relation to folate, a weak relation to plasma betaine, and no relation to vitamin B(6). Notably, adjusted (for age and sex) dose-response curves for the postmethionine increase in homocysteine or fasting homocysteine versus betaine showed that the inverse associations were most pronounced at low serum folate, an observation that was confirmed by analyses of interaction., Conclusions: Collectively, these results show that plasma betaine is a strong determinant of increase in homocysteine after methionine loading, particularly in subjects with low folate status. In 500 healthy subjects, postmethionine load increase in tHcy showed a stronger inverse relation to betaine than to folate and vitamin B6, whereas for fasting tHcy, betaine was a weaker determinant than folate. For both tHcy modalities, the association with betaine was most pronounced in subjects with low folate status.

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. 16.5 micromol/L in healthy controls), and not in dementia patients. Homocysteine was elevated in patients with progressive multiple sclerosis (15.0 micromol/L, n=39, compared to 12.0 micromol/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.

Hyperhomocysteinemia is an independent and graded risk factor for arterial vascular disease and venous thrombosis. It is still debated via which mechanism homocysteine (Hcy) causes vascular disease. S-adenosylhomocysteine hydrolase (AHCY) catalyses the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) to Hcy. As an increase in AdoHcy, a strong inhibitor of many methyltransferases, is observed in hyperhomocysteinemic individuals, AdoHcy may play a role in the development of cardiovascular diseases by inhibiting transmethylation reactions. We sequenced the entire coding region and parts of the untranslated regions (UTRs) of the AHCY gene of 20 patients with recurrent venous thrombosis in order to identify genetic variation within this gene. We identified three sequence variants in the AHCY gene: a C > T transition in the 5' UTR (-34 bp C > T), a missense mutation in exon 2, which mandates an amino-acid conversion at codon 38 (112 C > T; Arg38Trp) and a silent mutation in exon 4 (390 C > T; Asp130Asp). We studied the effect of the first two variants on total plasma Hcy and venous thrombosis risk in a case-control study on recurrent venous thrombosis. The two polymorphisms under study seem to have no evident effect on tHcy. The adjusted relative risk of venous thrombosis associated with the 112CT genotype compared with 112CC individuals was 1.27 (95% CI 0.55-2.94), whereas the -34CT genotype confers a risk of 1.25 (95% CI 0.44-3.52) compared with the wild-type genotype at this locus. However, the wide confidence intervals do not allow firm conclusions to be drawn.

Hyperhomocysteinemia is a risk factor for arterial vascular disease and venous thrombosis. The pathophysiology of this relation is unclear, but several studies suggest that hyperhomocysteinemia impairs endothelial function. We examined the effect of homocysteine lowering by B-vitamin supplementation on tissue plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI) and von Willebrand factor (vWf)--markers of endothelial dysfunction--in hyperhomocysteinemic and normohomocysteinemic volunteers. A total of 123 healthy volunteers were randomized to placebo or B-vitamins (5 mg folic acid, 0.4 mg hydroxycobalamin and 50 mg pyridoxine) daily for 8 weeks. Before and after the intervention period, blood samples were taken for measurements of homocysteine, tPA, PAI and vWf. There was no evident association between homocysteine concentration and concentrations of markers of endothelial dysfunction at baseline. The mean reduction of homocysteine concentration was 31% (95%CI 22.7 to 39.1) in the B-vitamin group compared to 3% reduction in the placebo group. Concentrations of tPA, PAI and vWf did not change after supplementation of B-vitamins. In conclusion, the results of our study show that homocysteine reduction by B-vitamin supplementation has no effect on markers of endothelial dysfunction in healthy volunteers.

Background: Before dietary folate is absorbed, polyglutamate folates are deconjugated to monoglutamates by folylpoly-gamma-glutamyl carboxypeptidase in the small intestine. The 1561T allele of the glutamate carboxypeptidase II gene (GCPII), which codes for folylpoly-gamma-glutamyl carboxypeptidase, may impair intestinal absorption of dietary folates., Objective: Our aim was to study the bioavailability of polyglutamyl folic acid relative to that of monoglutamyl folic acid across GCPII 1561 genotypes., Design: In a randomized study, 180 healthy adults aged 50-75 y received 323 nmol monoglutamyl folic acid/d (n = 59), 262 nmol heptaglutamyl folic acid/d (n = 61), or placebo (n = 60) for 12 wk. Genotypes were assessed after the intervention. The bioavailability of heptaglutamyl folic acid relative to that of monoglutamyl folic acid was calculated by using the changes in serum folate concentration in the treatment groups, after correction for changes in the placebo group and for the administered dose., Results: No subjects with the TT genotype were encountered. At baseline, serum and erythrocyte folate concentrations were higher (P < 0.05) in subjects with the CT genotype [16.3 nmol/L (geometric x; 95% CI: 13.7, 19.3 nmol/L) and 863 nmol/L (735, 1012 nmol/L), respectively; n = 19] than in subjects with the CC genotype [13.7 (13.1, 14.3) and 685 (652, 721) nmol/L, respectively; n = 161]. Baseline homocysteine concentrations were not significantly different between genotypes. The bioavailability of heptaglutamyl folic acid relative to that of monoglutamyl folic acid was not significantly different between subjects with the CC (64%; 52%, 76%) and CT genotypes (70%; 49%, 91%)., Conclusions: The 1561T allele of the GCPII gene does not impair the bioavailability of polyglutamyl folic acid. However, the allele is associated with higher folate status. This association may be explained by yet unidentified factors controlling the expression of the GCPII gene.

Objective: This study was undertaken to investigate the involvement of maternal and infant B vitamins and homocysteine as risk factors for orofacial clefting., Study Design: Venous blood samples were taken from 96 infants with nonsyndromic orofacial clefts and 88 infants without a congenital malformation and from their mothers at approximately 14 months after the index pregnancy. Red blood cell and serum folate, serum vitamin B(12), whole blood vitamin B(6) as pyridoxal-5'-phosphate (PLP), and plasma homocysteine concentrations were measured., Results: A vitamin B(12) concentration of 185 pmol/L or less and a PLP concentration of 44 nmol/L or less in mothers increased the risk of having a child with an orofacial cleft (odds ratio [OR]=3.1; 95% CI: 1.3-7.4, OR=2.9; 95% CI: 1.2-7.1, respectively). Infants with orofacial clefts had a 15% lower serum folate concentration compared with controls (P=.06)., Conclusion: A low vitamin B(12) and PLP concentration in mothers increased the risk of orofacial clefts in the offspring. A possible role of the infant's folate status is suggested.

Background: The pathogenic mechanism of homocysteine's effect on cardiovascular risk is poorly understood. Recent studies show that DNA hypomethylation induced by increases in S-adenosylhomocysteine (AdoHcy), an intermediate of Hcy metabolism and a potent inhibitor of methyltransferases, may be involved in homocysteine-related pathology., Methods: We measured fasting plasma total Hcy (tHcy), AdoHcy, and S-adenosylmethionine (AdoMet) and methylation in leukocytes in 17 patients with vascular disease and in 15 healthy, age- and sex-matched controls., Results: Patient with vascular disease had significantly higher plasma tHcy and AdoHcy concentrations and significantly lower plasma AdoMet/AdoHcy ratios and genomic DNA methylation. AdoMet concentrations were not significantly different between the two groups. More than 50% of the patients fell into the highest quartiles of plasma tHcy, AdoHcy, and [(3)H]dCTP incorporation/ micro g of DNA (meaning the lowest quartile of DNA methylation status) and into the lowest quartile of the AdoMet/AdoHcy ratios of the control group. Plasma tHcy was significantly correlated with plasma AdoHcy and AdoMet/AdoHcy ratios (n = 32; P < 0.001). DNA methylation status was significantly correlated with plasma tHcy and AdoHcy (n = 32; P < 0.01) but not with plasma AdoMet/AdoHcy ratios., Conclusion: Global DNA methylation may be altered in vascular disease, with a concomitant increase in plasma tHcy and AdoHcy.

Introduction: Most studies showed that mothers of children with NTD have elevated homocysteine levels pointing to a disturbed homocysteine metabolism as a risk factor for NTD. Folate lowers homocysteine levels by remethylation of homocysteine to methionine. Homocysteine can be irreversibly converted to cystathionine by the vitamin B6-dependent enzyme CBS. Recently, our group showed that a 31 bp VNTR in the CBS gene was associated with decreased CBS activity and increased tHcy levels after methionine loading in a CVD population., Aim: The aim of our study was to investigate whether this VNTR influences tHcy levels and risk for NTD. In addition, we assessed the role of vitamin B6 as an effect modifier in this possible interaction. We examined possible gene-gene interaction with the MTHFR 677C > T polymorphism. We screened genomic DNA of 88 NBD patients, 100 mothers, 88 fathers, and 505 controls for this CBS 31 bp VNTR., Results: In this study population five different alleles with 16,17, 18, 19, and 21 times the 31 bp repeat were observed that constituted 10 different genotypes. The most common 18/18 VNTR genotype was associated with higher tHcy levels compared with the 17/18 and 18/19 VNTR genotypes. Vitamin B6 levels did not influence this association. In addition, no association with risk for NTD was found. Combination of the CBS VNTR with the MTHFR 677C > T polymorphism revealed an additional increase in homocysteine levels in 18-18 individuals compared with 17-18 peers within subjects homozygous mutant for the MTHFR 677C > T polymorphism., Conclusions: The present study indicates that the number of 31 bp repeat elements in the CBS gene influences tHcy levels. This VNTR seems not to be associated with an increased risk for NTD., (Copyright 2003 Elsevier Science (USA))

Background: Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate and homocysteine metabolism. The common MTHFR 677C-->T polymorphism decreases the enzyme's activity., Objective: The objective of the study was to assess the effect of the polymorphism on the relations among folate intake, plasma folate concentration, and total plasma homocysteine (tHcy) concentration., Design: The design was a cross-sectional analysis in a random sample (n = 2051) of a Dutch cohort (aged 20-65 y)., Results: At a low folate intake (166 micro g/d), folate concentrations differed significantly among the genotypes (7.1, 6.2, and 5.4 nmol/L for the CC, CT, and TT genotypes, respectively; P for all comparisons < 0.05). At a high folate intake (250 microg/d), folate concentrations in CT and CC subjects did not differ significantly (8.3 and 8.6 nmol/L, respectively, but were significantly higher (P = 0.2) than those in TT subjects (7.3 nmol/L; P = 0.04). At a low folate concentration (4.6 nmol/L), TT subjects had a significantly higher (P = 0.0001) tHcy concentration than did CC and CT subjects (20.3 compared with 15.0 and 14.1 micromol/L, respectively), whereas at a high folate concentration (11.9 nmol/L), the tHcy concentration did not differ significantly between genotypes (P > 0.2; <13.1 for all genotypes). The relation between folate intake and tHcy concentration had a pattern similar to that of the relation between plasma folate and tHcy concentrations., Conclusions: At any folate intake level, TT subjects have lower plasma folate concentrations than do CT and CC subjects. Yet, at high plasma folate concentrations, tHcy concentrations in TT subjects are as low as those in CT and CC subjects.

The results of prospective studies on the relations between the plasma concentration of total homocysteine (tHcy) and B-vitamins, on the one hand, and coronary heart disease (CHD) mortality, on the other hand, are inconclusive and scarce considering the relation with B-vitamins. We prospectively determined these relations in a case-cohort study. The full-cohort existed in approximately 36,000 Dutch adults aged 20-59 years at baseline. The statistical analyses were done with a random sample from the cohort (n=630) complemented with all subjects who died of CHD (n=102) during a mean follow-up of 10.3 years. All subjects reported the absence of cardiovascular diseases (CVDs) at baseline. The plasma concentrations of tHcy, folate, PLP, and vitamin B12 were determined in samples obtained at baseline. Men with a tHcy concentration in the highest tertile (T3) compared with men in the lowest tertile (T1) had a relative risk (RR) of 1.14 for CHD (95% confidence interval (CI): 0.50, 2.61) after adjusting for age, study center, hypertension, HDL and total cholesterol, smoking, and creatinine. For women, this RR was 2.04 (95% CI: 0.48, 8.62). For each 5 micromol/l increase in tHcy, the RR of CHD was 1.03 (95% CI: 0.83-1.29) for men and women combined. In women only, high folate levels were associated with a statistically significant protection of fatal CHD (T3 versus T1; RR: 0.22, 95% CI: 0.06, 0.87). Plasma PLP (B6) and vitamin B12 concentrations were not associated with CHD risk. We conclude that elevated tHcy concentrations do not seem to be a risk factor for CHD mortality in these relatively young healthy Dutch subjects free of baseline CVD. Higher folate concentrations may be protective of CHD, but this needs confirmation.

Homocysteine, a sulphur amino acid, is a branch-point intermediate of methionine metabolism. It can be degraded in the transsulphuration pathway to cystathionine, or remethylated to methionine via the remethylation pathway. In both pathways, major genetic defects that cause enzyme deficiencies are associated with very high plasma homocysteine concentrations and excretion of homocystine into the urine. Mildly elevated plasma homocysteine concentrations are thought to be an independent and graded risk factor for both arterial occlusive disease and venous thrombosis. Genetic defects in genes encoding enzymes involved in homocysteine metabolism, or depletion of important cofactors or (co)substrates for those enzymes, including folate, vitamin B(12) and vitamin B(6), may result in elevated plasma homocysteine concentrations. Plasma homocysteine concentrations are also influenced by dietary and lifestyle factors. In the last decade, several studies have been conducted to elucidate the genetic determinants of hyperhomocysteinaemia in patients with cardiovascular disease. We report on both environmental and genetic determinants of hyperhomocysteinaemia and give a detailed overview of all the genetic determinants that have been reported to date.