Biomarkers of the one-carbon pathway in association with congenital diaphragmatic hernia

Homocysteine is an intermediate of the one-carbon (1-C) pathway and increased concentrations have been related to neural crest-related congenital anomalies. The neural crest and the 1-C pathway might be involved also in the etiology of Congenital Diaphragmatic Hernia (CDH). In 22 CDH and 28 control newborns and their mothers, general characteristics were obtained by standardized questionnaires. The 1-C pathway intermediates total homocysteine (tHcy), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) were determined in cord blood. Correlations between maternal and newborn factors and risk estimates were investigated by univariate and multivariable logistic regression analyses. Birth weight (2962 vs. 3418 gram; p < 0.001) was lower and gestational age (270 vs. 277 days; p = 0.006) was shorter in case children. Control mothers were slightly older (32 vs. 35 year; p = 0.05). Other characteristics were comparable between case and control children and mothers. The concentrations of homocysteine, SAM and SAH, and the SAM/SAH ratio were comparable (tHcy: 8.57 vs. 8.56 μmol/l, p = 0.99; SAM: 152.7 vs. 157.3 nmol/l, p = 0.76; SAH: 43.5 vs. 48.9, p = 0.26; ratio: 3.8 vs. 3.5, p = 0.50). Maternal and newborn characteristics were not correlated to the biomarker concentrations. In conclusion, the biomarkers of methylation determined in cord blood are not associated with CDH risk. Maternal and child characteristics could not predict newborn biomarker concentrations of the 1-C pathway.     

Dietary vitamin A intake below the recommended daily intake during pregnancy and the risk of congenital diaphragmatic hernia in the offspring

BACKGROUND

Vitamin A has been related to the etiology of congenital diaphragmatic hernia (CDH). We performed a case‐control study to investigate whether maternal dietary vitamin A intake is related to CDH in the offspring.

METHODS

Thirty‐one pregnancies diagnosed with CDH and 46 control pregnancies were included during the study. After CDH diagnosis and inclusion of controls by risk set sampling, maternal vitamin A intake was investigated with a food frequency questionnaire. Serum retinol and retinol‐binding protein were determined. Univariable and multivariable logistic regression models were used to calculate risk estimates with adjustment for potential confounders.

RESULTS

We found no significant differences in the overall nutrient and vitamin A intake between case and control mothers. After stratification in body mass index (BMI) categories, case mothers with normal weight showed a lower energy adjusted vitamin A intake (685 vs. 843 μg retinol activity equivalents [RAEs] / day; p = 0.04) and a slightly lower serum retinol (1.58 vs. 1.67 μmol/L; p = 0.08) than control mothers. Vitamin A intake <800 μg retinol activity equivalents (recommended daily intake) in normal weight mothers was associated with a significantly increased CDH risk (odds ratio [OR], 7.2; 95% confidence interval [CI], 1.5–34.4; p = 0.01). Associations were not significantly different in underweight and overweight mothers.

CONCLUSIONS

In normal‐weight mothers, dietary vitamin A intake during pregnancy below the recommended daily intake is significantly associated with an increased risk of a child with CDH. This finding supports the retinoid hypothesis in human CDH, but warrants further investigation in larger study populations. Birth Defects Research (Part A), 2013. © 2013 Wiley Periodicals, Inc.     

Serum vitamin B12 not reflecting vitamin B12 status in patients with type 2 diabetes

Contradictory results for concentrations of vitamin B12, holotranscobalamin (holoTC), and methylmalonic acid (MMA) have been reported. We tested the hypothesis that the extracellular vitamin B12 markers are not reflecting the intracellular vitamin B12-dependent biochemical reactions in individuals with type 2 diabetes. The study included 92 patients with diabetes and 72 controls with similar age and sex distribution. We measured vitamin B12 markers [MMA, total serum vitamin B12, holoTC, total homocysteine (tHcy)], red blood cell (RBC)-B12, and the plasma concentrations of the methylation markers [S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH)]. In comparison to controls, diabetic patients showed significantly higher concentrations of plasma SAH (median 15.1 vs. 11.8 nmol/L; p < 0.001) and lower SAM/SAH ratio (9.1 vs. 8.2; p = 0.006). Concentrations of total vitamin B12 and holoTC did not differ significantly between the groups, but plasma MMA concentrations were significantly higher in diabetics (250 vs. 206 nmol/L). However, RBC-B12 was lower in diabetics compared to controls (median 230 vs. 260 pmol/L; p = 0.001). The inverse correlation between MMA and RBC-B12 was stronger in the controls compared to that in the patients (correlation coefficient in controls R = −0.446, p = 0.001; in patients R = −0.289, p = 0.022). Metformin treatment was associated with a lower total serum vitamin B12, but a comparable RBC-B12 and a slightly lower MMA and better methylation index. In conclusion, patients with type 2 diabetes showed normal extracellular vitamin B12, but disturbed intracellular B12-dependent biochemical reactions. Metformin treatment was associated with low serum vitamin B12 and improved intracellular vitamin B12 metabolism despite low serum vitamin B12.     

L-arginine increases plasma homocysteine in apoE-/-/iNOS-/- double knockout mice.

Previous studies have shown that L-arginine (L-Arg) administration to apoE-/-/iNOS-/- double knockout mice (dKO) on a Western diet paradoxically results in an increase in atherosclerotic lesion size. We hypothesized that the potential beneficial effects of L-Arg could be offset, in part, by the byproducts of L-Arg catabolism, especially the atherogenic risk factor, homocysteine. In the kidney, L-Arg is converted to L-ornithine and guanidinoacetate (GAA) by L-arginine-glycine amidinotransferase. The efficient transmethylation of GAA by an S-adenosyl-methionine (SAM)-dependent methyltransferase in liver yields creatine and S-adenosylhomocysteine (SAH), which is readily hydrolyzed to homocysteine and adenosine. We, therefore, measured total plasma homocysteine in the dKO mice and control mice. We found that L-Arg supplementation caused a 37% increase in total plasma homocysteine (tHcy) levels in dKO mice compared to controls not treated with L-Arg (5.2+/-2.2 vs 3.8+/-1.5 microM Hcy, p<0.04). In a liver cell line, HepG2, addition of 10 and 50 microM GAA in the presence of 50 microM L-methionine (L-Met) increased tHcy production by approximately 1.47 (p<0.0001) and 2.3-fold (p<0.0001), respectively. In the presence of additional 100 microM L-Met, baseline homocysteine production was elevated by 20% (p<0.005), and 10 and 50 microM GAA augmented homocysteine production by an additional 1.88- (p<0.0001) and 3.4-fold (p<0.001), respectively, compared with 50 microM L-Met. These data suggest that increased concentrations of a methyl acceptor, such as L-Arg-derived GAA, drives SAM-dependent-methylation and consequent homocysteine formation. Furthermore, L-Met levels can also influence homocysteine production likely by regulating the synthesis of the methyl donor SAM. Epidemiological studies have suggested that homocysteine is a graded risk factor. In animal models, modestelevations of homocysteine can cause endothelial dysfunction and augment atherosclerosis. Our data suggest that L-arginine supplementation may contribute to vascular injury and atherogenesis under some circumstances by elevating homocysteine levels.     

S‐adenosyl‐l‐methionine usage during climacteric ripening of tomato in relation to ethylene and polyamine biosynthesis and transmethylation capacity

S‐adenosyl‐l ‐methionine (SAM) is the major methyl donor in cells and it is also used for the biosynthesis of polyamines and the plant hormone ethylene. During climacteric ripening of tomato (Solanum lycopersicum ‘Bonaparte’), ethylene production rises considerably which makes it an ideal object to study SAM involvement. We examined in ripening fruit how a 1‐MCP treatment affects SAM usage by the three major SAM‐associated pathways. The 1‐MCP treatment inhibited autocatalytic ethylene production but did not affect SAM levels. We also observed that 1‐(malonylamino)cyclopropane‐1‐carboxylic acid formation during ripening is ethylene dependent. SAM decarboxylase expression was also found to be upregulated by ethylene. Nonetheless polyamine content was higher in 1‐MCP‐treated fruit. This leads to the conclusion that the ethylene and polyamine pathway can operate simultaneously. We also observed a higher methylation capacity in 1‐MCP‐treated fruit. During fruit ripening substantial methylation reactions occur which are gradually inhibited by the methylation product S‐adenosyl‐l ‐homocysteine (SAH). SAH accumulation is caused by a drop in adenosine kinase expression, which is not observed in 1‐MCP‐treated fruit. We can conclude that tomato fruit possesses the capability to simultaneously consume SAM during ripening to ensure a high rate of ethylene and polyamine production and transmethylation reactions. SAM usage during ripening requires a complex cellular regulation mechanism in order to control SAM levels.     

Neural tube defects and maternal biomarkers of folate, homocysteine, and glutathione metabolism

BACKGROUND: Alterations in maternal folate and homocysteine metabolism are associated with neural tube defects (NTDs). The role played by specific micronutrients and metabolites in the causal pathway leading to NTDs is not fully understood. METHODS: We conducted a case-control study to investigate the association between NTDs and maternal alterations in plasma micronutrients and metabolites in two metabolic pathways: methionine remethylation and glutathione transsulfuration. Biomarkers were measured in a population-based sample of women who had NTD-affected pregnancies (n = 43) and a control group of women who had a pregnancy unaffected by a birth defect (n = 160). We compared plasma concentrations of folate, vitamin B(12), vitamin B(6), methionine, S-adenosylmethionine (SAM), s-adenosylhomocysteine (SAH), adenosine, homocysteine, cysteine, and reduced and oxidized glutathione between cases and controls after adjusting for lifestyle and sociodemographic factors. RESULTS: Women with NTD-affected pregnancies had significantly higher plasma concentrations of SAH (29.12 vs. 23.13 nmol/liter, P = .0011), adenosine (0.323 vs. 0.255 mumol/liter; P = .0269), homocysteine (9.40 vs. 7.56 micromol/liter; P < .001), and oxidized glutathione (0.379 vs. 0.262 micromol/liter; P = .0001), but lower plasma SAM concentrations (78.99 vs. 83.16 nmol/liter; P = .0172) than controls. This metabolic profile is consistent with reduced methylation capacity and increased oxidative stress in women with affected pregnancies. CONCLUSIONS: Increased maternal oxidative stress and decreased methylation capacity may contribute to the occurrence of NTDs. Further analysis of relevant genetic and environmental factors is required to define the basis for these observed alterations.     

l-NAME has Opposite Effects on the Productions of S-adenosylhomocysteine and S-adenosylmethionine in V12-H-Ras and M-CR3B-Ras Pheochromocytoma Cells

Homocysteine is a sulfur-containing, nonproteinogenic, neurotoxic amino acid biosynthesized during methyl cycles after demethylation of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) and subsequent hydrolysis of SAH into homocysteine and adenosine. Formed homocysteine is either catabolized into cystathionine (transsulfuration pathway) by cystathionine β-synthase, or remethylated into methionine (remethylation pathway) by methionine synthase. To demonstrate the specificity of Ras-elicited effects on the activity of methyl cycles, wild-type pheochromocytoma PC12, mutant oncogenic rasH gene (MVR) expressing PC12 pheochromocytoma and normal c-rasH stably transfected M-CR3B cells were incubated with the N^ω-nitro-l-arginine methyl ester (l-NAME), and manumycin, (inhibitors of nitric oxide synthase and farnesyltransferase, respectively). We have found that l-NAME significantly changes the SAM/SAH ratio in both MCR and MVR cells. Moreover, these alterations have reciprocal character; in the MCR cells, the SAM/SAH ratio was raised, whereas in the MVR cells this ratio was decreased. We conclude that depletion of endogenous NO with l-NAME increased the production of SAH only in cells with mutated oncogenic RasH, possibly through enhancement of production of reactive oxygen species (ROS). Oxidative stress can increase cystathionine β-synthase activity that switches methyl cycles from remethylation into transsulfuration pathway to maintain the intracellular glutathione pool (essential for the redox-regulating capacity of cells) via an adaptive process.     

Effects of methionine synthase and methylenetetrahydrofolate reductase gene polymorphisms on markers of one-carbon metabolism

Genetic and nutritional factors play a role in determining the functionality of the one-carbon (1C) metabolism cycle, a network of biochemical reactions critical to intracellular processes. Genes encoding enzymes for methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MTR) may determine biomarkers of the cycle including homocysteine (HCY), S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). MTHFR C677T is an established genetic determinant of HCY but less is known of its effect on SAM and SAH. Conversely, the relationship between MTR A2756G and HCY remains inconclusive, and its effect on SAM and SAH has only been previously investigated in a female-specific population. Folate and vitamin B₁₂ are essential substrate and cofactor of 1C metabolism; thus, consideration of gene–nutrient interactions may clarify the role of genetic determinants of HCY, SAM and SAH. This cross-sectional study included 570 healthy volunteers from Kingston, Ontario, Ottawa, Ontario and Halifax, Nova Scotia, Canada. Least squares regression was used to examine the effects of MTR and MTHFR polymorphisms on plasma HCY, SAM and SAH concentrations; gene–gene and gene–nutrient interactions were considered with the inclusion of cross-products in the model. Main effects of MTR and MTHFR polymorphisms on HCY concentrations were observed; however, no gene–gene or gene–nutrient interactions were found. No association was observed for SAM. For SAH, interactions between MTR and MTHFR polymorphisms, and MTHFR polymorphism and serum folate were found. The findings of this research provide evidence that HCY and SAH, biomarkers of 1C metabolism, are influenced by genetic and nutritional factors and their interactions.     

Effect of folate deficiency on placental DNA methylation in hyperhomocysteinemic rats

We report that the maternal folate status can influence folate-mediated one-carbon metabolism and DNA methylation in the placenta. Thirty-six female Sprague-Dawley rats were divided into the following three dietary groups: folate-supplemented (FS; 8 mg/kg folic acid, n=12), homocystine- and folate-supplemented (HFS; 0.3% homocystine and 8 mg/kg folic acid, n=12) and homocystine-supplemented and folate-deficient (HFD; 0.3% homocystine and no folic acid, n=12). The animals were fed their experimental diets from 4 weeks prior to mating until Day 20 of pregnancy (n=7-9 per group). The HFS diet increased the plasma homocysteine and placental DNA methylation but did not affect plasma folate, vitamin B-12, S-adenosyl methionine (SAM) or S-adenosyl homocysteine (SAH) levels, or the SAM/SAH ratio in the liver and placenta compared with the FS diet. The HFD diet induced severely low plasma folate concentrations, with plasma homocysteine levels increasing up to 100 micromol/L, and increased hepatic SAH and decreased placental SAM levels and SAM/SAH ratio in both tissues, with a concomitant decrease in placental DNA methylation. Placental DNA methylation was significantly correlated with placental (gamma=0.819), hepatic (gamma=0.7) and plasma (gamma=0.752) folate levels; plasma homocysteine level (gamma=-0.688); hepatic SAH level (gamma=-0.662) and hepatic SAM/SAH ratio (gamma=0.494). These results suggest that the maternal folate status in hyperhomocysteinemic rats influences the homeostasis of folate-mediated one-carbon metabolism and the methyl pool, which would, in turn, affect placental DNA methylation by altering the methylation potential of the liver.     

AXIN2 and CDH1 polymorphisms, tooth agenesis, and oral clefts

BACKGROUND: AXIN2 and CDH1 genes play important roles during craniofacial morphogenesis. Mutations in these genes have been described in families presenting colorectal cancer and tooth agenesis, and gastric cancer and cleft lip/palate (CL/P). Oral clefts have been associated with tooth agenesis. We investigated if AXIN2 and CDH1 polymorphisms were associated with clefts or with any associated dental subphenotypes. METHODS: Markers in AXIN2 and CDH1 were genotyped using Taqman chemistry in a sample cohort comprised of 500 cleft individuals and 500 unrelated controls. RESULTS: Comparison between cleft and control groups showed a trend for association for AXIN2 with incomplete cleft palate (p = .006) and CDH1 with unilateral CL/P (p = .03 for left CL/P and p = .04 for right CL/P). Comparison of cleft subphenotypes with tooth agenesis and controls revealed borderline associations for CDH1 (p = .008) and AXIN2 (p = .01) with unilateral right CL/P with tooth agenesis. CONCLUSIONS: We observed only borderline results for the association of AXIN2 and CDH1 with CL/P with and without tooth agenesis. Nevertheless, implication of these genes in the simultaneous occurrence of CL/P and cancer, and in tooth agenesis and cancer, is rather intriguing and warrants further investigations with other geographic and ethnic populations. Birth Defects Research (Part A), 2009. © 2008 Wiley‐Liss, Inc.     

The maternal folate hydrolase gene polymorphism is associated with neural tube defects in a high-risk Chinese population

Folate hydrolase 1 (FOLH1) gene encodes intestinal folate hydrolase, which regulates intestinal absorption of dietary folate. Previous studies on the association between polymorphisms rs202676 and rs61886492 and the risk of neural tube defects (NTDs) were inconclusive. A case–control study of women with NTD-affected pregnancies (n = 160) and controls (n = 320) was conducted in the Chinese population of Lvliang, a high-risk area for NTDs. We genotyped the polymorphic sites rs202676 and rs61886492 and assessed maternal plasma folate and total homocysteine (tHcy). Our results showed that in case group, plasma folate concentrations were 18 % lower compared with those of control group (8.32 vs. 6.79 nmol/L, p = 0.033) and tHcy concentrations were 17 % higher (10.47 vs. 12.65 μmol/L, p = 0.047). Almost all samples had the rs61886492 GG genotype (99.78 %). The result showed that the frequency of GG genotype in rs202676 was significantly higher in group with multiple NTDs than in controls (p = 0.030, OR = 2.157, 95 % CI, 1.06–4.38). The multiple-NTD group showed higher maternal plasma concentrations of tHcy (10.47 vs. 13.96 μmol/L, p = 0.024). The GG genotype of rs202676 had a lower maternal folate and higher tHcy concentrations than other genotypes with no significant differences. The result of structural prediction indicated that this variation might change the spatial structure of the protein. These results suggested that the maternal polymorphism rs202676 was a potential risk factor for multiple NTDs in this Chinese population. The allele G might affect maternal plasma folate and tHcy concentration.     

Relationships among biomarkers of one-carbon metabolism

One-carbon metabolism is a network of metabolic pathways, disruption of which has been associated with cancer and other pathological conditions. Biomarkers of these pathways include homocysteine (HCY), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH). A better understanding of the relationships between these biomarkers is needed for their utilization in research. This study investigated the relationships between fasting concentrations of plasma HCY, SAM, SAH and the ratio of SAM:SAH, and serum folate, vitamin B(12) and creatinine in a healthy adult population. A cross-sectional study recruited 678 volunteers; only subjects with complete data (n = 581) were included in this analysis. Correlations were used to examine bivariate relationships among the biomarkers and multivariate linear regression determined independent relationships with HCY, SAM and SAH treated as dependent variables in separate models. Multivariate logistic regression examined determinants of a low SAM:SAH ratio (defined as having a SAM:SAH ratio in the bottom quartile and SAH value in the top quartile). HCY correlated inversely with folate and vitamin B(12) and weakly correlated with SAH and creatinine. Both SAM and SAH correlated with creatinine but were independent of serum folate and vitamin B(12). In multivariate analyses, folate, vitamin B(12), creatinine, sex and age were associated with HCY; age and creatinine were determinants of SAM, and sex and creatinine determinants of SAH. Finally, male sex and increasing creatinine levels were associated with having a low SAM:SAH ratio. Findings suggest that HCY, SAM and SAH are relatively independent parameters and reflect distinct aspects of one-carbon metabolism.     

Differential effects of dietary selenium (Se) and folate on methyl metabolism in liver and colon of rats

A previous study compared the effects of folate on methyl metabolism in colon and liver of rats fed a selenium-deficient die (<3 μg Se/kg) to those of rats fed a diet containing supranutritional Se (2 mg selenite/kg). The purpose of this study was to investigate the effects of folate and adequate Se (0.2 mg/kg) on methyl metabolism in colon and liver. Weanling, Fischer-344 rats (n=8/diet) were fed diets containing 0 or 0.2 mg selenium (as selenite)/kg and 0 or 2 mg folic acid/kg in a 2×2 design. After 70 d, plasma homocysteine was increased (p<0.0001) by folate deficiency; this increase was markedly, attenuated (p<0.0001) in rats fed the selenium-deficient diet compared to those fed 0.2 mg Se/kg. The activity of hepatic glycine N-methyltransferase (GNMT), an enzyme involved in the regulation of tissue S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), was increased by folate deficiency (p<0.006) and decreased by selenium deprivation, (p<0.0003). Colon and liver SAH were highest (p<0.006) in rats fed deficient folate and adequate selenium. Although folate deficiency decreased liver SAM (p<0.001), it had no effect on colon SAM. Global DNA methylation was decreased (p<0.04) by selenium deficiency in colon but not liver; folate had no effect. Selenium, deficiency did not affect DNA methyltransferase (Dnmt) activity in liver but tended to decrease (p<0.06) the activity of the enzyme in the colon. Dietary folate did not affect liver or colon Dnmt. These results in rats fed adequate selenium are similar to previous results found in rats fed supranutritional selenium. This suggests that selenium deficiency appears to be a more important modifier of methyl metabolism than either adequate or supplemental selenium. The U.S. Department of Agriculture, Agriculture Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination.     

Interaction of PDGFRA promoter haplotypes and maternal environmental exposures in the risk of spina bifida

BACKGROUND: Neural tube defects are multifactorial malformations involving both environmental exposures, such as maternal nutrition, and genetic factors. Aberrant expression of the platelet‐derived growth factor alpha‐receptor (PDGFRA) gene has been implicated in neural‐tube‐defect etiology in both mice and humans. METHODS: We investigated possible interactions between the PDGFRA promoter haplotype of mother and child, as well as maternal glucose, myo‐inositol, and zinc levels, in relation to spina bifida offspring. Distributions were determined of the PDGFRA promoter haplotypes H1 and H2 in a Dutch cohort, consisting of 88 spina bifida children with 56 of their mothers, and 74 control children with 72 of their mothers, as well as maternal plasma glucose, myo‐inositol, and red blood cell zinc concentrations. RESULTS: A significantly higher frequency of H1 was observed in children with spina bifida than in controls (30.1 vs. 20.3%; OR = 1.69, 95% CI 1.02–2.83). High maternal body mass index (BMI) and glucose were significant risk factors for both H1 and H2 children, whereas low myo‐inositol and zinc were risk factors for H2 but not for H1 children. Stepwise multiple logistic regression analysis showed that high maternal glucose and low myo‐inositol are the main risk factors for H2 spina bifida children, whereas for H1 spina bifida children, maternal BMI was the main risk factor. Interestingly, H1 mothers (median 165.5 cm) showed a significantly lower body height than H2 mothers (median 169.1 cm; p = 0.003). CONCLUSIONS: These data suggest that the child's PDGFRA promoter haplotype is differentially sensitive for periconceptional exposure to glucose, myo‐inositol, and zinc in the risk of spina bifida. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Rate of T alleles and TT genotype at MTHFR 677C‐>T locus or C alleles and CC genotype at MTHFR 1298A‐>C locus among healthy subjects in Turkey: impact on homocysteine and folic acid status and reference intervals

Methylenetetrahydrofolate reductase (MTHFR) is important for folate and homocysteine (Hcy) metabolism. MTHFR 677C‐>T and 1298A‐>C MTHFR are two most common mutations which can affect folate and total homocysteine (tHcy) status. This study was designed to determine the rate of MTHFR 677C‐>T and 1298A‐>C mutations, and their influence on serum folate, Hcy and vitamin B12 status and the reference intervals in 402 healthy Turkish adults. The rate of MTHFR 677C‐>T or 1298A‐>C mutations was 50.7% or 54.7%, respectively. The MTHFR 677C‐>T mutation‐specific reference intervals for serum folate and tHcy were characterized by marked shifts in their upper limits. In homozygote subjects for MTHFR 677C‐>T serum folate concentration was lower and serum tHcy concentration was higher than those in the wild genotype; all subjects had lower serum folate and 54% of the subjects had higher tHcy concentrations than the cutoff values of ≤10 nmol/L and ≥12 µmol/L, respectively. Serum vitamin B12 status was similar in all genotypes. Serum tHcy concentrations were inversely correlated with serum folate and vitamin B12 concentrations in all genotypes. These data show that the rate of MTHFR 677C‐>T and 1298A‐>C mutations is very high in Turks and serum folate and tHcy status are impaired by these mutations. Copyright © 2009 John Wiley & Sons, Ltd.     

Global DNA hypomethylation is associated with NTD‐affected pregnancy: A case‐control study

BACKGROUND: Neural tube defects are severe, common birth defects that result from failure of neural tube closure. They are considered to be a multifactorial disorder, and our knowledge of causal mechanisms remains limited. We hypothesized that abnormal DNA methylation occurs in NTD‐affected fetuses. The correlations of global DNA methylation levels with complexity of NTDs and known risk factors of NTDs, MTHFR genotype and fever, were analyzed. METHODS: A hospital‐based case‐control study was performed. Epidemiologic data, pathologic diagnosis, and methylenetetrahydrofolate reductase (MTHFR) genotype analysis were completed. Array comparative genomic hybridization was used to exclude cytogenetic abnormalities. Global DNA methylation statuses were determined for both brain and skin tissue. RESULTS: Sixty‐five NTD‐affected fetuses and 65 normal controls matched for gestational and maternal ages were collected. In brain tissue, global DNA methylation levels were significantly decreased in cases compared with controls (4.12 vs. 4.99%; p < 0.001). DNA hypomethylation (<4.35%) resulted in a significant 5.736‐fold increased risk for NTDs (95% confidence interval, 1.731–19.009; p = 0.004). Nonisolated NTDs had lower levels of global DNA methylation than did isolated NTDs (3.77 vs. 4.70%; p = 0.022). After stratifying subjects by MTHFR genotype, we observed a skewed distribution of global DNA methylation levels. For genotype C/C, global DNA methylation status was the same in the two groups (4.51 vs. 4.72%; p = 0.687). For T/T, cases had significantly lower global methylation levels than did controls (5.23 vs. 3.79%; p < 0.001). CONCLUSIONS: Global DNA hypomethylation in fetal brain tissue was associated with NTD‐affected pregnancy. DNA methylation levels were correlated with NTD complexity. The MTHFR genotype contributed to global DNA hypomethylation. Birth Defects Research (Part A), 2010. © 2010 Wiley‐Liss, Inc.     

Impaired glucose tolerance (IGT) is not associated with disturbed homocysteine metabolism

Summary. Elevated plasma total homocysteine (tHcy) has been suggested to be an additional risk factor for cardiovascular disease in subjects with impaired glucose tolerance (IGT) and Type 2 diabetes (T2D). In order to investigate whether an insulin resistant/chronic hyperinsulinemic situation in male diabetic and prediabetic subjects directly influences the tHcy metabolism, fasting tHcy and post-methionine load tHcy plasma levels (PML-tHcy) were determined in 15 men with IGT, 13 men with newly dia-gnosed T2D, and 16 normoglycemic controls (NGT). Fasting tHcy (IGT, 13.1 ± 4.6; T2D, 12.8 ± 4.0; NGT, 10.7 ± 4.4 μmol/L) and PML-tHcy (IGT, 46.5 ± 17.39; T2D, 41.1 ± 6.8; NGT, 38.0 ± 9.7 μmol/L) showed no differences between the groups. Fasting tHcy and PML-tHcy correlated with fasting proinsulin (r = 0.395, p < 0.05; r = 0.386, p< 0.05) and creatinine (r = 0.489, p < 0.01; r = 0.339, p < 0.05), resp. Multiple regression analysis showed only a relationship between fasting tHcy and creatinine. No relationships have been found between fasting tHcy and PML-tHcy, resp., and indicators of an insulin resistant state, e.g., insulin and proinsulin, as well as serum cobalamin and folate concentrations. In conclusion, our data suggest that the degree of glucose intolerance has no direct impact on the metabolism of homocysteine. However, tHcy levels tend to be elevated with the development of nephropathy, indicating an association between tHcy and renal function in these subjects. Received May 11, 1999     

The effects of 5‐azacytidine and cadmium on global 5‐methylcytosine content and secondary metabolites in the freshwater microalgae Chlamydomonas reinhardtii and Scenedesmus quadricauda

Epigenetic changes are important mechanisms in the regulation of chromatin structure and gene expression. Cytosine methylation is one of the major epigenetic modifications, mediated by DNA methyltransferases, which transfer methyl groups from S‐adenosyl‐L‐methionine (SAM) to the fifth carbon of cytosine. Various external environmental conditions can change the global hypo/hypermethylation pattern of DNA. These alterations may affect the organism's response to stress conditions. In this study, for the first time, we investigated the effects of 5‐azacytidine, a DNA methyltransferase inhibitor, and cadmium, a toxic metal and environmental pollutant, on the growth, biosynthesis of secondary metabolites (phenols, flavonoids, carotenoids), SAM, S‐adenosylhomocysteine, 5′‐methylthioadenosine and global 5‐methylcytosine (5‐mC) in the green microalgae Chlamydomonas reinhardtii and Scenedesmus quadricauda. The studied species showed major differences in 5‐mC content, secondary metabolite content, and antioxidant activity. Cadmium increased GSH (glutathione) content in C. reinhardtii by 60% whereas 5‐azacytidine did not affect GSH. The biosynthesis of GSH in S. quadricauda in response to the stressors was the opposite. Global 5‐mC content of C. reinhardtii was 1%–1.5%, and the content in S. quadricauda was 3.5%. Amount of some investigated methionine cycle metabolites (SAM, S‐adenosyl homocysteine [SAH], methionine) in S. quadricauda distinctly exceeded C. reinhardtii as well. However, chlorophylls a and b, carotenoids, total phenolic content, total flavonoid content and, antioxidant activity were significantly higher in C. reinhardtii than S. quadricauda. Therefore, in further studies it would be advisable to verify whether methylation of cytosine affects the expression of genes encoding certain secondary metabolites.     

Correlation between maternal plasma homocysteine and zinc levels in preeclamptic women

Women with preeclampsia have been shown to have elevated blood levels of the metabolite homocysteine, and alterations in blood levels of zinc and copper have also been reported. This study measured plasma levels of zinc, copper, and homocysteine in women with preeclampsia and in women with healthy, normotensive pregnancies. For the patients with preeclampsia compared with controls, significantly higher mean plasma levels were found of homocysteine (16.39 vs 9.45 nmol/mL; p≤0.001), zinc (15.53 vs 11.93 μg/g protein; p < 0.05), and copper (47.90 vs 31.60 μg/g protein; p=0.001). The ratio of plasma Cu/Zn levels tended to be higher in preeclamptic women and could be taken as an index of inflammatory reaction, but the difference was not significant. Homocysteine concentrations correlated positively with plasma zinc concentrations in women with preeclampsia (r=0.588, p=0.003) but not in women with healthy pregnancies. No correlations were observed between plasma levels of homocysteine and copper. Thus, the present study found evidence that preeclampsia might be associated with hyperhomocysteinemia and elevated blood levels of zinc and copper. Furthermore, elevated blood levels of zinc were significantly associated with hyperhomocysteinemia in preeclampsia. More studies are warranted to investigate further any relationship between altered homocysteine metabolism and levels of zinc and copper in preeclampsia.