Pharmacokinetic properties of a novel formulation of S-adenosyl-L-methionine phytate.

S-adenosyl-L-methionine (SAM), the main endogenous methyl donor, is the adenosyl derivative of the amino acid methionine, which displays many important roles in cellular metabolism. It is widely used as a food supplement and in some countries is also marketed as a drug. Its interesting nutraceutical and pharmacological properties prompted us to evaluate the pharmacokinetics of a new form of SAM, the phytate salt. The product was administered orally to rats and pharmacokinetic parameters were evaluated by comparing the results with that obtained by administering the SAM tosylated form (SAM PTS). It was found that phytate anion protects SAM from degradation, probably because of steric hindrance exerted by the counterion, and that the SAM phytate displayed significant better pharmacokinetic parameters compared to SAM PTS. These results open to the perspective of the use of new salts of SAM endowed with better pharmacokinetic properties.

Moderate Folic Acid Supplementation in Pregnant Mice Results in Altered Methyl Metabolism and in Sex-Specific Placental Transcription Changes.

SCOPE: Many pregnant women have higher folic acid (FA) intake due to food fortification and increased vitamin use. It is reported that diets containing five-fold higher FA than recommended for mice (5xFASD) during pregnancy resulted in methylenetetrahydrofolate reductase (MTHFR) deficiency and altered choline/methyl metabolism, with neurobehavioral abnormalities in newborns. The goal is to determine whether these changes have their origins in the placenta during embryonic development. METHODS AND RESULTS: Female mice are fed control diet or 5xFASD for a month before mating and maintained on these diets until embryonic day 17.5. 5xFASD led to pseudo-MTHFR deficiency in maternal liver and altered choline/methyl metabolites in maternal plasma (increased methyltetrahydrofolate and decreased betaine). Methylation potential (S-adenosylmethionine:S-adenosylhomocysteine ratio) and glycerophosphocholine are decreased in placenta and embryonic liver. Folic acid supplemented diet results in sex-specific transcriptome profiles in placenta, with validation of dietary expression changes of 29 genes involved in angiogenesis, receptor biology or neurodevelopment, and altered methylation of the serotonin receptor 2A gene. CONCLUSION: Moderate increases in folate intake during pregnancy result in placental metabolic and gene expression changes, particularly in angiogenesis, which may contribute to abnormal behavior in pups. These results are relevant for determining a safe upper limit for folate intake during pregnancy.

Hyperhomocysteinemia is key for increased susceptibility to PND in aged mice.

BACKGROUND: Postoperative neurocognitive disorder (PND) is a severe postoperative complication with no effective therapy that affects up to 19-52% of senior patients. Age and surgery type have been identified as risk factors. However, what caused the increased risk in the elderly is poorly understood. METHODS: We utilized a PND model in aged mice undergoing experimental laparotomy with general anesthesia to evaluate the causal relationship between hyperhomocysteinemia and increased PND susceptibility. PND was assessed by Novel Object Tasks, Fear Conditioning Tests, and Barnes Maze Tests. Serum homocysteine (Hcy) as well as vitamin B12 and folate acid levels were tested before, immediately after surgery and from day 1 to day 29 after surgery by ELISA. The effectiveness of preventative strategy including diet supplementation of vitamin B12 + folic acid (Vit B12 + FA) and S-adenosylmethionine (SAM) injection targeting hyperhomocysteinemia were also tested. RESULTS: PND in aged mice lasted for at least 2 weeks after experimental laparotomy, which was not observed in young adult mice. Serum Hcy results indicated a significant correlation between postoperative cognitive performance and perioperative Hcy level. Preoperative supplementation with VB12 and folic acid (FA) in the diet or S-adenosylmethionine (SAM) injection reduced perioperative serum Hcy level and inhibited the development of PND in aged mice. CONCLUSIONS: Serum homocysteine accumulation is a fundamental cause for increased susceptibility of PND in aged mice. Preoperative diet supplementation of VitB12 + FA can effectively reduce PND in aged mice, which may be a promising prophylaxis treatment in clinical settings.

Global methylation in relation to methotrexate-induced oral mucositis in children with acute lymphoblastic leukemia.

BACKGROUND: Children with acute lymphoblastic leukemia (ALL) often suffer from toxicity of chemotherapeutic drugs such as Methotrexate (MTX). Previously, we reported that 20% of patients receiving high-dose MTX developed oral mucositis. MTX inhibits folate metabolism, which is essential for DNA methylation. We hypothesize that MTX inhibits DNA methylation, which results into adverse effects. We studied DNA methylation markers during high-dose methotrexate treatment in pediatric acute lymphoblastic leukemia (ALL) in relation to developing oral mucositis. MATERIALS & METHODS: S-Adenosyl-Methionine (SAM) and S-Adenosyl-Homocysteine (SAH) levels and LINE1 DNA methylation were measured prospectively before and after high-dose methotrexate (HD-MTX 4 x 5g/m2) therapy in 82 children with ALL. Methotrexate-induced oral mucositis was registered prospectively. Oral mucositis (grade ≥ 3 National Cancer Institute Criteria) was used as clinical endpoint. RESULTS: SAM levels decreased significantly during methotrexate therapy (-16.1 nmol/L (-144.0 -+46.0), p<0.001), while SAH levels and the SAM:SAH ratio did not change significantly. LINE1 DNA methylation (+1.4% (-1.1 -+6.5), p<0.001) increased during therapy. SAM and SAH levels were not correlated to LINE1 DNA methylation status. No association was found between DNA methylation markers and developing oral mucositis. CONCLUSIONS: This was the first study that assessed DNA methylation in relation to MTX-induced oral mucositis in children with ALL. Although global methylation markers did change during methotrexate therapy, methylation status was not associated with developing oral mucositis.

Prospective study of telomere length and LINE-1 methylation in peripheral blood cells: the role of B vitamins supplementation.

PURPOSE: Deficiencies of folate, vitamins B12 and D are common age-related conditions. Vitamin B12 and folate are necessary for DNA methylation. Telomeres appear to be regulated by DNA methylation. Here, we study the effect of B vitamins supplementation on telomere length and global DNA methylation in a prospective study. METHODS: In total, 60 elderly subjects were supplemented for 1 year with either vitamin B12, B6, folate, vitamin D and calcium (group A n = 31) or only vitamin D and calcium (group B n = 29). LINE-1 methylation, relative telomere length (T/S), vitamin B12, folate, homocysteine (tHcy) , 5-methyltetrahydrofolate (5-methylTHF), S-adenosylhomocysteine (SAH), S-adenosylmethionine (SAM), cystathionine and vitamin D were quantified before and after supplementation. RESULTS: At baseline, tHcy was high, vitamin D was low, and T/S did not differ between groups A and B. Vitamin supplementation increased LINE-1 methylation in group A at site 317 but reduced LINE-1 methylation in group B at site 327. There was no correlation between T/S and LINE-1 methylation at baseline. Multiple backward regression analysis revealed baseline tHcy and 5-methylTHF are significant predictors of T/S. After supplementation in group B but not in group A, LINE-1 methylation correlated inversely with T/S, and LINE-1 methylation variation was an independent predictor of T/S variation. B vitamins decreased tHcy significantly in group A. Multiple backward regression analysis showed 5-methylTHF in group A and tHcy in group B were significant predictors for LINE-1 methylation. At baseline, the lower LINE-1 methylation observed in subjects with 5-methylTHF >10 nmol/l was in agreement with a reduced methyl group transfer due to a lower SAM formation. In group B, an increase in telomere length was correlated with lower LINE-1 methylation. Subjects with hyperhomocysteinemia >12 µmol/L had compared to those with normal tHcy a reduced LINE-1 methylation accompanied by a higher SAM and SAH (that inhibits demethylation of SAM) as well as lower 5-methylTHF. Additionally, subjects with tHcy > 12 µmol/L had longer telomeres when compared with subjects having tHcy < 12 µmol/L. CONCLUSIONS: The results suggest a possible effect of B vitamins for telomere biology in blood cells. Suboptimal B vitamins status and hyperhomocysteinemia are associated with altered DNA methylation and telomere length. These data have to be confirmed in future studies.

Low-Dose Creatine Supplementation Lowers Plasma Guanidinoacetate, but Not Plasma Homocysteine, in a Double-Blind, Randomized, Placebo-Controlled Trial.

BACKGROUND: Creatine synthesis from guanidinoacetate consumes ~50% of s-adenosylmethionine (SAM)-derived methyl groups, accounting for an equivalent proportion of s-adenosylhomocysteine (SAH) and total homocysteine (tHcys) synthesis. Dietary creatine inhibits the synthesis of guanidinoacetate, thereby lowering plasma tHcys in rats. OBJECTIVE: We tested the hypotheses that creatine supplementation lowers plasma guanidinoacetate, increases blood SAM, lowers blood SAH, and lowers plasma tHcys. METHODS: Bangladeshi adults were randomly assigned to receive 1 of 4 treatments for 12 wk: placebo (n = 101), 3 g/d creatine (Cr; n = 101), 400 µg/d folic acid (FA; n = 153), or 3 g/d creatine plus 400 µg/d folic acid (Cr+FA; n = 103). The outcomes of plasma guanidinoacetate and tHcys, as well as whole blood SAM and SAH, were analyzed at baseline and week 12 by HPLC. Treatment effects of creatine supplementation were examined with the use of the group comparisons of Cr vs. placebo and Cr+FA vs. FA. RESULTS: Plasma guanidinoacetate declined by 10.6% (95% CI: 4.9, 15.9) in the Cr group while increasing nonsignificantly in the placebo group (3.7%; 95% CI: -0.8, 8.5) (Pgroup difference = 0.0002). Similarly, plasma guanidinoacetate declined by 9.0% (95% CI: 3.4, 14.2) in the Cr+FA group while increasing in the FA group (7.0%; 95% CI: 2.0, 12.2) (Pgroup difference < 0.0001). Plasma tHcys declined by 23.4% (95% CI: 19.5, 27.1) and 21.0% (95% CI: 16.4, 25.2) in the FA and Cr+FA groups, respectively (Pgroup difference = 0.41), with no significant changes in the placebo or Cr groups (Pgroup difference = 0.35). A decrease in guanidinoacetate over time was associated with a decrease in tHcys over time in the Cr+FA group (ß = 0.30; 95% CI: 0.17, 0.43; P < 0.0001). CONCLUSIONS: Our findings indicate that whereas creatine supplementation downregulates endogenous creatine synthesis, this may not on average lower plasma tHcys in humans. However, tHcys did decrease in those participants who experienced a decline in plasma guanidinoacetate while receiving creatine plus folic acid supplementation. This trial was registered at clinicaltrials.gov as NCT01050556.

Short-term creatine supplementation does not reduce increased homocysteine concentration induced by acute exercise in humans.

PURPOSE: The purpose of the study is to evaluate the effects of creatine supplementation on homocysteine (Hcy) plasma levels after acute exercise in humans. METHODS: Twenty-three young (under-20) soccer players were divided into 2 groups: creatine (Cr)- and placebo (Pla)-supplemented groups. The supplementation was performed in double-blind controlled manner using creatine or placebo tablets with 0.3 g/kg during 7 days. Before and after 7 days of supplementation, the athletes performed an acute high-intensity sprint exercise (two consecutive running-based anaerobic sprint test protocol consisted in 6 × 35 m sprint with 10 s between them). Blood samples were collected before and after 7 days of supplementation as well as 0 and 1 h after exercise protocol. RESULTS: Homocysteine concentration significant increased (P < 0.05) 1 h after acute exercise (18%). Acute exercise also decreased red blood cell S-adenosylmethionine (SAM) 30% with no changes in SAM/SAH ratio. Seven days of creatine supplementation were able to increase (P < 0.05) plasma creatine concentration (Pla 130.1 ± 21.7 vs Cr 1,557.2 ± 220.3 µmol/L) as well as decrease (P < 0.05) plasma guanidinoacetic acid (33%). Controversially, creatine supplementation did not change Hcy plasma level after 7-day supplementation (Pla 6.9 ± 0.2 vs Cr 7.2 ± 0.2 µmol/L) or after acute exercise (Pla 8.2 ± 0.3 vs Cr 8.4 ± 0.3 µmol/L). No changes in plasma vitamin B12 and folate as well as cysteine and methionine were found. CONCLUSIONS: Seven days of creatine supplementation does not avoid increased plasma Hcy induced by acute sprint exercise in humans.

Plasma sulfur amino acids and stearoyl-CoA desaturase activity in two Caucasian populations.

In rats, dietary restriction of the cysteine precursor methionine suppresses hepatic stearoyl-CoA desaturase (SCD)-1 expression and activity, whereas cysteine supplementation reverses these effects. In 2 independent cohorts: Hordaland Health Study (HUSK; N=2021, aged 71-74y), Norway, and Hoorn study (N=686, aged 50-87y), Netherlands, we examined the cross-sectional associations of plasma sulfur-containing compounds (SCC; methionine, S-adenosylmethionine, S-adenosylhomocysteine, homocysteine, cystathionine, total cysteine (tCys), glutathione and cysteinylglycine) with SCD-16 index (16:1n-7/16:0), estimated from fatty acid profiles of total plasma or serum lipids. Only tCys was consistently associated with SCD-16 index after adjustments for sex and age (HUSK: partial r=0.14; Hoorn: partial r=0.11, P<0.001 for both), and after further adjustments for other SCC, body fat, diet, exercise and plasma lipids (HUSK: partial r=0.07, P=0.004; Hoorn: partial r=0.12, P=0.013). Together with animal data showing an effect of dietary cysteine on SCD1, our results suggest a role for cysteine in SCD1 regulation in humans.

DNA methylation potential: dietary intake and blood concentrations of one-carbon metabolites and cofactors in rural African women.

BACKGROUND: Animal models show that periconceptional supplementation with folic acid, vitamin B-12, choline, and betaine can induce differences in offspring phenotype mediated by epigenetic changes in DNA. In humans, altered DNA methylation patterns have been observed in offspring whose mothers were exposed to famine or who conceived in the Gambian rainy season. OBJECTIVE: The objective was to understand the seasonality of DNA methylation patterns in rural Gambian women. We studied natural variations in dietary intake of nutrients involved in methyl-donor pathways and their effect on the respective metabolic biomarkers. DESIGN: In 30 women of reproductive age (18-45 y), we monitored diets monthly for 1 y by using 48-h weighed records to measure intakes of choline, betaine, folate, methionine, riboflavin, and vitamins B-6 and B-12. Blood biomarkers of these nutrients, S-adenosylhomocysteine (SAH), S-adenosylmethionine (SAM), homocysteine, cysteine, and dimethylglycine were also assessed monthly. RESULTS: Dietary intakes of riboflavin, folate, choline, and betaine varied significantly by season; the most dramatic variation was seen for betaine. All metabolic biomarkers showed significant seasonality, and vitamin B-6 and folate had the highest fluctuations. Correlations between dietary intakes and blood biomarkers were found for riboflavin, vitamin B-6, active vitamin B-12 (holotranscobalamin), and betaine. We observed a seasonal switch between the betaine and folate pathways and a probable limiting role of riboflavin in these processes and a higher SAM/SAH ratio during the rainy season. CONCLUSIONS: Naturally occurring seasonal variations in food-consumption patterns have a profound effect on methyl-donor biomarker status. The direction of these changes was consistent with previously reported differences in methylation of metastable epialleles. This trial was registered at www.clinicaltrials.gov as NCT01811641.

S-adenosylmethionine and S-adenosylhomocysteine in plasma and cerebrospinal fluid in Rett syndrome and the effect of folinic acid supplementation.

Rett syndrome is a neurodevelopmental disorder characterized by cognitive and locomotor regression and stereotypic hand movements. The disorder is caused by mutations in the X chromosomal MECP2 a gene encoding methyl CpG-binding protein. It has been associated with disturbances of cerebral folate homeostasis, as well as with speculations on a compromised DNA-methylation. Folinic acid is the stable form of folate. Its derived intermediate 5-MTHF supports the conversion of homocysteine to methionine, the precursor of S-adenosylmethionine (SAM). This in turn donates its methyl group to various acceptors, including DNA, thereby being converted to S-adenosylhomocysteine (SAH). The SAM/SAH ratio reflects the methylation potential. The goal of our study was to influence DNA methylation processes and ameliorate the clinical symptoms in Rett syndrome. Therefore we examined the hypothesis that folinic acid supplementation, besides increasing cerebrospinal fluid (CSF) 5-MTHF (p = 0.003), influences SAM and SAH and their ratio. In our randomized, double-blind crossover study on folinic acid supplementation, ten female Rett patients received both folinic acid and placebo for 1 year each. It was shown that both SAM and SAH levels in the CSF remained unchanged following folinic acid administration (p = 0.202 and p = 0.097, respectively) in spite of a rise of plasma SAM and SAH (p = 0.007; p = 0.009). There was no significant change in the SAM/SAH ratio either in plasma or CSF. The apparent inability of Rett patients to upregulate SAM and SAH levels in the CSF may contribute to the biochemical anomalies of the Rett syndrome. Our studies warrant further attempts to promote DNA methylation in the true region of interest, i.e. the brain.

Effect of 1 year B and D vitamin supplementation on LINE-1 repetitive element methylation in older subjects.

BACKGROUND: Disturbed DNA methylation is causally related to chronic diseases like cancer and atherosclerosis. B vitamins are cofactors required for methyl group synthesis and may therefore affect DNA methylation. Vitamin D has epigenetic effects. We tested if B and D vitamin supplementation has an effect on genomic long interspersed nuclear element-1 (LINE-1) methylation and the metabolites S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). METHODS: Fifty subjects (median age 68.0 years) were supplemented with a daily oral dose of B vitamins (500 µg folic acid, 500 µg vitamin B12 and 50 mg vitamin B6), 1200 IU vitamin D and 456 mg calcium. Fasting blood samples were collected before and after 1 year of supplementation. LINE-1 methylation was determined in genomic DNA from blood cells as a surrogate for whole genome methylation. In addition, SAM, SAH and total homocysteine (tHcy) were measured in plasma samples. RESULTS: Plasma homocysteine decreased significantly after supplementation (12.8 vs. 9.1 µmol/L; p<0.05), whereas SAM, SAH, the SAM/SAH ratio and LINE-1 methylation did not change significantly. LINE-1 methylation was not significantly correlated with SAH, homocysteine or B vitamins. CONCLUSIONS: Long-term vitamin B supplementation had no effect on LINE-1 methylation in blood cells nor on plasma levels of SAM and SAH. Vitamin B and D supplementation seems to have no effect on DNA methylation, especially in cases where no severe deficiency exists.

One year B and D vitamins supplementation improves metabolic bone markers.

BACKGROUND: Vitamin D and vitamin B deficiency are common in elderly subjects and are important risk factors for osteoporosis and age-related diseases. Supplementation with these vitamins is a promising preventative strategy. The objective of this study was to evaluate the effects of vitamins D3 and B supplementation on bone turnover and metabolism in elderly people. METHODS: Healthy subjects (n=93; >54 years) were randomly assigned to receive either daily vitamin D3 (1200 IU), folic acid (0.5 mg), vitamin B12 (0.5 mg), vitamin B6 (50 mg), and calcium carbonate (456 mg) (group A) or only vitamin D3 plus calcium carbonate (group B) in a double blind trial. We measured at baseline and after 6 and 12 months of supplementation vitamins, metabolites, and bone turnover markers. RESULTS: At baseline mean plasma 25-hydroxy vitamin D [25(OH)D] was low (40 or 30 nmol/L) and parathormone was high (63.7 or 77.9 pg/mL). 25(OH)D and parathormone correlated inversely. S-Adenosyl homocysteine and S-adenosyl methionine correlated with bone alkaline phosphatase, sclerostin, and parathormone. One year vitamin D3 or D3 and B supplementation increased plasma 25(OH)D by median 87.6% (group A) and 133.3% (group B). Parathormone was lowered by median 28.3% (A) and 41.2% (B), bone alkaline phosphatase decreased by 2.8% (A) and 16.2% (B), osteocalin by 37.5% (A) and 49.4% (B), and tartrate-resistant-acid-phosphatase 5b by 6.1% (A) and 36.0% (B). Median total homocysteine (tHcy) was high at baseline (group A: 12.6, group B: 12.3 µmol/L) and decreased by B vitamins (group A) to 8.9 µmol/L (29.4%). tHcy lowering had no additional effect on bone turnover. CONCLUSIONS: One year vitamin D3 supplementation with or without B vitamins decreased the bone turnover significantly. Vitamin D3 lowered parathormone. The additional application of B vitamins did not further improve bone turnover. The marked tHcy lowering by B vitamins may modulate the osteoporotic risk.

The beneficial effect of folate-enriched egg on the folate and homocysteine levels in rats fed a folate- and choline-deficient diet.

We investigated the effects of folate-enriched egg yolk powder on folate and homocysteine levels in plasma and liver of rats fed the folate- and choline-deficient diet to determine bioavailability in vivo. Three-wk-old Wistar rats were fed (1) the pteroylglutamate (PteGlu), (2) the choline, (3) the PteGlu and choline, (4) the folate-enriched egg yolk powder diet for 4 wk after having been fed the folate- and choline-deficient diet. The hepatic folate level in the folate-enriched egg yolk powder group was significantly higher than that in the folate- and choline-deficient or the control groups. The homocysteine concentration in plasma and liver of the folate-enriched egg yolk powder group was significantly lower than that of the folate- and choline-deficient or the PteGlu groups. The S-adenosyl-methionine (SAM)/S-adenosyl-homocysteine (SAH) ratio in the folate-enriched egg yolk powder group was significantly higher than that in the folate- and choline-deficient group. These effects were similar in the PteGlu and choline, but not the PteGlu or the choline groups. These data suggest that the intake of folate-enriched eggs, as well as of both folate and choline, induced the beneficial effects on folate and homocysteine metabolism. Thus, folate-enriched eggs could be used as beneficial source of folate with a high bioavailability.

Bioavailability of S-adenosyl methionine and impact on response in a randomized, double-blind, placebo-controlled trial in major depressive disorder.

OBJECTIVE: To characterize the impact of S-adenosyl methionine (SAMe) on homocysteine and potential risk of adverse cardiovascular effects by examining plasma levels of SAMe, S-adenosyl homocysteine (SAH), total homocysteine (tHCY), methionine (MET), and 5-methyltetrahydrofolate (5-MTHF) in 35 of 73 patients from a 6-week randomized double-blind, placebo-controlled trial of SAMe augmentation in serotonin reuptake inhibitor partial responders with DSM-IV major depressive disorder (MDD), published in 2010. METHOD: Subjects were randomized from June 4, 2004, until August 8, 2008, to adjunctive placebo or SAMe 800-1600 mg/d for 6 weeks. Primary outcome measures included changes in one-carbon cycle intermediates within each treatment arm (by paired t test) and between treatment arms (by independent samples t test). Univariate analysis of variance and Fisher Protected Least Significant Difference were carried out to compare posttreatment levels of each one-carbon cycle intermediate. Secondary outcome measures included associations between clinical improvement and change in plasma intermediate levels, examined by linear regression (for change in Hamilton Depression Rating Scale scores) and logistic regression (for response or remission). RESULTS: We found significant differences in pretreatment plasma levels of tHCY (P = .03) between the SAMe and placebo arms. Following 6 weeks of treatment, plasma SAMe (P = .002) and SAH (P < .0001) levels increased significantly in the SAMe arm; no intermediates in the placebo group changed significantly. Posttreatment plasma SAMe (P = .0035), SAH (P < .0001), and tHCY (P = .0016) levels differed significantly between the SAMe and placebo groups. No significant associations were found between plasma intermediate levels and clinical improvement, response, or remission. CONCLUSIONS: Despite concerns about the impact that SAMe therapy may have on homocysteine levels and risk of adverse cardiovascular effects, the lack of significant increase in tHCY levels after treatment suggests that no toxic effects from SAMe should be expected. Our findings, however, have some significant limitations and should be interpreted with caution. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00093847.

Folic acid effects on s-adenosylmethionine, s-adenosylhomocysteine, and DNA methylation in patients with intermediate hyperhomocysteinemia.

OBJECTIVE: Folic acid (FA) supplementation decreases homocysteine (tHcy) levels. However, little is known about the effects of FA treatment on DNA methylation or plasma S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) concentrations. The purpose of this study was to investigate the effects of FA supplementation on AdoMet, AdoHcy, and genomic DNA methylation in hyperhomocysteinemic subjects without end-stage renal disease. METHODS: To evaluate the effects of 5 mg FA/d for 8 weeks, we recruited 7 hyperhomocysteinemic MTHFR677TT patients (tHcy >30 µmol/L) with normal renal function. RESULTS: FA supplementation induced a decrease in tHcy (from 51.1 ± 21 at baseline to 26.1 ± 27 µmol/L after folate supplementation; p < 0.01). A parallel increase was seen in plasma AdoMet concentrations and the AdoMet/AdoHcy ratio (p < 0.05). However, FA supplementation had no effect on global DNA methylation levels in the present study. CONCLUSIONS: Supraphysiologic FA supplementation can modulate biochemical markers in one-carbon metabolism such as tHcy, AdoMet, and the AdoMet/AdoHcy ratio in hyperhomocysteinemic subjects. However, the reduction in homocysteinemia and the increased availability of methyl compounds provided by vitamin supplementation may not be sufficient to affect genomic DNA methylation.

Concentrations of unmetabolized folic acid and primary folate forms in plasma after folic acid treatment in older adults.

Folate deficiency can cause age-related disease. Folic acid (FA) has been used in studies aiming at disease prevention. Recently, unmetabolized FA in plasma raised public health concerns; but numerous studies used FA for disease prevention. Concentrations of the folate forms FA, 5-methyltetrahydrofolate (5-MTHF), and tetrahydrofolate (THF) were measured before and after 3-week placebo or FA 5 mg, vitamin B6 40 mg, and cyanocobalamin 2 mg per day administrated to 74 older adults (median age, 82 years). Concentrations of 5-MTHF and total homocysteine (tHcy) (r = -0.392) and S-adenosylmethionine (r = 0.329) were correlated at baseline. Twenty-six percent of the elderly subjects had unmetabolized FA in plasma at the start, and concentrations of FA were increased after 3 weeks of FA treatment (median FA = 0.08 nmol/L at baseline and 15.3 nmol/L at the end of the treatment in the vitamin group). Folic acid caused a 10- and a 5-fold increase in 5-MTHF and THF, respectively, and lowered tHcy (median tHcy = 17.2 µmol/L at baseline vs 9.0 µmol/L after treatment). Concentrations of unmetabolized FA were positively related to those of 5-MTHF and THF. People showed wide variations in folate forms at baseline, but these were reduced after FA treatment. Folic acid given to older adults is mostly converted to THF and 5-MTHF and lowered concentrations of tHcy, but caused a substantial increase in unmetabolized FA in the plasma.

Blood folate status and expression of proteins involved in immune function, inflammation, and coagulation: biochemical and proteomic changes in the plasma of humans in response to long-term synthetic folic acid supplementation.

We used plasma proteomics to identify human proteins responsive to folate status. Plasma was collected from subjects treated with placebo or 1.2 mg of folic acid daily for 12 weeks in a randomized controlled trial. Homocysteine and folate were measured by immunoassay and uracil misincorporation by electrophoresis. The plasma proteome was assessed by 2-D gel electrophoresis, and proteins were identified by LC MS/MS. 5-methylTHF increased 5-fold (P = 0.000003) in response to intervention. Red cell folate doubled (P = 0.013), and lymphocyte folate increased 44% (P = 0.0001). Hcy and uracil dropped 22% (P = 0.0005) and 25% (P = 0.05), respectively. ApoE A-1, alpha-1-antichymotrypsin, antithrombin, and serum amyloid P were downregulated, while albumin, IgM C, and complement C3 were upregulated (P < 0.05). More than 60 proteins were significantly associated with folate pre- and postintervention (P < 0.01). These were categorized into metabolic pathways related to complement fixation (e.g., C1, C3, C4, Factor H, Factor 1, Factor B, clusterin), coagulation (e.g., antithrombin, alpha-1-antitrypsin, kininogen) and mineral transport (e.g., transthyretin, haptoglobin, ceruloplasmin). Low folate status pre- and post-treatment were associated with lower levels of proteins involved in activation and regulation of immune function and coagulation. Supplementation with synthetic folic acid increased expression of these proteins but did not substantially disrupt the balance of these pathways.

Homocysteine-lowering vitamins do not lower plasma S-adenosylhomocysteine in older people with elevated homocysteine concentrations.

Elevated plasma total homocysteine (tHcy) is a risk factor for vascular disease but lowering tHcy with B-vitamins, including folate, has generally not reduced vascular events in secondary prevention trials. Elevated plasma S-adenosylhomocysteine (AdoHcy) concentration may be a more sensitive indicator of vascular disease than plasma tHcy. However, unlike tHcy, plasma AdoHcy did not correlate with folate concentration in one study indicating that folate supplementation may not lower AdoHcy. Our aim was to determine whether providing B-vitamin supplements to healthy older people with elevated tHcy (>13 micromol/l) affects plasma AdoHcy and S-adenosylmethionine (AdoMet) concentrations. Healthy older participants (n 276; > or = 65 years) were randomised to receive a daily supplement containing folate (1 mg), vitamin B12 (500 microg) and vitamin B6 (10 mg), or placebo, for 2 years. Of these participants, we selected the first fifty participants in each treatment group and measured plasma AdoHcy and AdoMet. Plasma tHcy was 4.4 (95 % CI 3.2, 5.6; P < 0.001) micromol/l lower at 2 years in the vitamins group compared with the placebo group. At 2 years, there were no significant differences in plasma AdoMet (+4 % (95 % CI - 2, 11); P = 0.19), AdoHcy ( - 1 % (95 % CI - 10, 8); P = 0.61) or the AdoMet:AdoHcy ratio (0.22 (95 % CI - 0.04, 0.49); P = 0.10) between the two groups. In conclusion, B-vitamin supplementation of older people lowered plasma tHcy but had no effect on plasma AdoMet or AdoHcy concentration. If elevated plasma AdoHcy is detrimental, this may explain why B-vitamins have generally failed to reduce vascular events in clinical trials.

Enhanced one-carbon flux towards DNA methylation: Effect of dietary methyl supplements against gamma-radiation-induced epigenetic modifications.

Radiation exposure poses a major risk for workers in the nuclear power plants and other radiation related industry. In this context, we demonstrate that gamma-radiation is an efficient DNA demethylating agent and its injurious effect can be minimized by dietary methyl supplements (folate, choline and vitamin B12). To elucidate the possible underlying mechanism(s), male Swiss mice were maintained on normal control diet (NCD) and methyl-supplemented diet (MSD). After 2 weeks of NCD and MSD dietary regimen, we exposed the animals to gamma-radiation (2, 4 and 6Gy) and investigated the profile of downstream metabolites and activity levels of one-carbon (C(1)) flux generating enzymes. In MSD fed and irradiated animals, hepatic folate levels increased (P<0.01), while hepatic homocysteine levels decreased (P<0.01) compared to NCD fed and irradiated animals. Although hepatic folate level increased significantly in MSD fed animals (P<0.01), it showed a decrease in response to high doses of gamma-irradiation. Under these conditions, a marked suppression of S-adenosylmethionine (SAM) levels occurred in NCD fed and irradiated animals, suggesting reduced conversion of homocysteine to SAM. Concomitant with decline in liver SAM Pool, activities of DNA methyltransferase (Dnmt, that methylates DNA) and methionine synthase (MSase, that regenerates methionine from homocysteine) were both decreased in NCD fed and irradiated mice. However, in MSD fed and irradiated mice, they were increased. These results strongly indicated that increased levels of dnmt and MSase may enhance C(1) flux towards DNA methylation reactions in MSD fed animals. These results were confirmed and further substantiated by measuring genomic DNA methylation levels, which were maintained at normal levels in MSD fed and irradiated mice compared to NCD fed and irradiated animals (P<0.01). In conclusion, our results suggest that maintenance of genomic DNA methylation under gamma-radiation stress might be a very dynamic, progressive diet dependent process that could involve increased one-carbon flux through various C(1) metabolites.

Body mass index is an important determinant of methylation biomarkers in women of reproductive ages.

B vitamin deficiencies lead to moderate hyperhomocysteinemia, which has been associated with health and disease. However, concomitant derangements in cellular methylation, reflected by altered plasma S-adenosylmethionine (SAM) or S-adenosylhomocysteine (SAH) concentrations, may be the primary cause. Therefore, we identified determinants of homocysteine, SAM, and SAH concentrations in 336 women, aged 20-48 y, as part of a large study focusing on risk factors for reproductive disorders. Blood was obtained to determine plasma SAM, SAH, and total homocysteine (tHcy), serum vitamin B-12 and folate, RBC folate concentrations, and the related single nucleotide polymorphisms 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C > T and 1298A > C, methionine synthase reductase (MTRR) 66A > G, and nicotinamide N-methyltransferase IVS1-151G > A. Questionnaires provided information on demographics, lifestyles, and nutrient intakes. Correlation coefficients were calculated and multivariable associations were assessed with a general linear model. Serum folate was positively correlated with SAM concentrations (r = 0.159; P = 0.004). Folate and vitamin B-12 were not correlated with SAH concentrations or the SAM:SAH ratio but were inversely correlated with tHcy concentrations (serum folate r = -0.324; RBC folate r = -0.294; vitamin B-12 r = -0.307; P < 0.01). From the multivariable analysis, BMI was the strongest determinant of SAM (standardized beta = 19.145; P < 0.001) and SAH concentrations (standardized beta = 3.241; P = 0.010). MTHFR 677TT (standardized beta = 0.195; P = 0.001), B vitamin supplement use (standardized beta = -0.156; P < 0.001) and dietary protein intake (standardized beta = -0.011; P < 0.001) were the strongest determinants of tHcy concentrations. Thus, the determinants of SAM and SAH differ from those of tHcy concentrations. Given that BMI was a strong determinant of SAM concentrations, it should be included in future studies on cellular methylation.