Plasma S-adenosylhomocysteine Research Articles

One-carbon metabolism markers are associated with cardiometabolic risk factors

Background and aimsAlterations to one-carbon metabolism, especially elevated plasma homocysteine (Hcy), have been suggested to be both a cause and a consequence of the metabolic syndrome (MS). A deeper understanding of the role of other one-carbon metabolites in MS, including s-adenosylmethionine (SAM), s-adenosylhomocysteine (SAH), and the methylation capacity index (SAM:SAH ratio) is required. Methods and results118 men and women with MS-risk factors were included in this cross-sectional study and cardiometabolic outcomes along with markers of one-carbon metabolism, including fasting plasma SAM, SAH, Hcy and vitamin B12 concentrations, were analysed. Multiple linear regression models were also used to examine the association between plasma one-carbon metabolites and cardiometabolic health features.We found that fasting plasma concentrations of Hcy, SAM and SAH were all positively correlated with markers of adiposity, including BMI (increase in BMI per 1-SD increase in one-carbon metabolite: 0.92 kg/m2 95% CI (0.28; 1.56), p = 0.005; 0.81 (0.15; 1.47), p = 0.02; 0.67 (−0.01; 1.36), p = 0.05, respectively). Hcy, but not SAM, SAH or SAM:SAH ratio was associated with BMI and body fat percentage after mutual adjustments. SAM concentrations were associated with higher fasting insulin (9.5% 95% CI (0.3; 19.5) per SD increase in SAM, p = 0.04), HOMA-IR (10.8% (0.8; 21.9), p = 0.03) and TNF-α (11.8% (5.0; 19.0), p < 0.001). ConclusionWe found little evidence for associations between SAM:SAH ratio and cardiometabolic variables, but higher plasma concentrations of SAM, SAH and Hcy are related to an overall higher risk of metabolic dysfunctions.The studies were registered at www.clinicaltrials.gov (NCT01719913 &NCT01731366).

Higher intake of fish and fat is associated with lower plasma s-adenosylhomocysteine: a cross-sectional study

Several B-vitamins act as co-factors in one-carbon metabolism, a pathway that plays a central role in several chronic diseases. However, there is a lack of knowledge of how diet affects markers in one-carbon metabolism. The aim of this study was to explore dietary patterns and components associated with one-carbon metabolites. We hypothesized that intake of whole-grains and fish would be associated with lower Hcy, and higher SAM:SAH ratio due to their nutrient content. We assessed dietary information using a four-day dietary record in 118 men and women with features of the metabolic syndrome. In addition we assessed whole-blood fatty acid composition and plasma alkylresorcinols. Plasma s-adenosylmethionine (SAM), s-adenosylhomocysteine (SAH), homocysteine (Hcy) and vitamin B12 was included as one-carbon metabolism markers. We used principal component analysis (PCA) to explore dietary patterns and multiple linear regression models to examine associations between dietary factors and one-carbon metabolites. PCA separated subjects based on prudent and unhealthy dietary patterns, but the dietary pattern score was not related to the one-carbon metabolites. Whole grain intake was found to be inversely associated to plasma Hcy (−4.7% (−9.3; 0.0), P=.05) and total grain intake tended to be positively associated with SAM and SAH (2.4% (−0.5; 5.5), P=.08; 5.8% (−0.2; 12.1), P=.06, respectively, per SD increase in cereal intake). Fish intake was inversely associated with plasma Hcy and SAH concentrations (−5.4% (−9.7; −0.8), P=.02 and −7.0% (−12.1; −1.5), P=.01, respectively) and positively associated with the SAM:SAH ratio (6.2% (1.6; 11.0), P=.008). In conclusion, intake and fish and whole-grain appear to be associated with a beneficial one-carbon metabolism profile. This indicates that dietary components could play a role in regulation of one-carbon metabolism with a potential impact on disease prevention.

The relationship between S-adenosylhomocysteine and coronary artery lesions: A case control study

The role of homocysteine (Hcy) in the pathogenesis of coronary artery disease (CAD) is controversial, as decreased Hcy levels have not demonstrated consistent clinical benefits. Recent studies propose that S-adenosylhomocysteine (SAH), and not Hcy, plays a role in cardiovascular disease (CVD). We aimed to assess the relationship between plasma SAH and coronary artery lesions. Participants (n=160; aged 40–80years) with chest pain and suspected CAD underwent coronary angiography (CAG) for assessment of coronary artery stenosis, and were assigned to either the atherosclerosis (AS) or CAD group. Plasma SAH and S-adenosylmethionine (SAM) concentrations were measured and the association between coronary artery lesions and SAH was assessed. SAH levels were significantly higher in the CAD group (23.09±2.4nmol/L) than in the AS group (19.2±1.5nmol/L). While the AS group had higher values for SAM/SAH (5.1±0.7 vs. 4.1±1.1), levels of SAM, Hcy, folate, and vitamin B12 were similar in the two groups. Coronary artery lesions were associated with SAH (β=11.8 [95% CI: 5.88, 17.7, P<0.05]. Plasma SAH concentrations are independently associated with coronary artery lesions among patients undergoing coronary angiography. Plasma SAH might be a novel biomarker for the early clinical identification of CVD.

Methionine Metabolites in Patients With Sepsis.

Sepsis is characterized by microvascular dysfunction and thrombophilia. Several methionine metabolites may be relevant to this sepsis pathophysiology. S-adenosylmethionine (SAM) serves as the methyl donor for trans-methylation reactions. S-adenosylhomocysteine (SAH) is the by-product of these reactions and serves as the precursor to homocysteine. Relationships between plasma total homocysteine concentrations (tHcy) and vascular disease and thrombosis are firmly established. We hypothesized that SAM, SAH, and tHcy levels are elevated in patients with sepsis and associated with mortality. This was a combined case-control and prospective cohort study consisting of 109 patients with sepsis and 50 control participants without acute illness. The study was conducted in the medical and surgical intensive care units of the University of Rochester Medical Center. Methionine, SAM, SAH, and tHcy concentrations were compared in patients with sepsis versus control participants and in sepsis survivors versus nonsurvivors. Patients with sepsis had significantly higher plasma SAM and SAH concentrations than control participants (SAM: 164 [107-227] vs73 [59-87 nM], P < .001; SAH: 99 [60-165] vs 35 [28-45] nM, P < .001). In contrast, plasma tHcy concentrations were lower in sepsis patients compared to healthy control participants (4 [2-6]) vs 7 [5-9] μM; P = .04). In multivariable analysis, quartiles of SAM, SAH, and tHcy were independently associated with sepsis ( P = .006, P = .05, and P < .001, respectively). Sepsis nonsurvivors had significantly higher plasma SAM and SAH concentrations than survivors (SAM: 223 [125-260] vs 136 [96-187] nM; P = .01; SAH: 139 [81-197] vs 86 [55-130] nM, P = .006). Plasma tHcy levels were similar in survivors vs nonsurvivors. The associations between SAM or SAH and hospital mortality were no longer significant after adjusting for renal dysfunction. Methionine metabolite concentrations are abnormal in sepsis and linked with clinical outcomes. Further study is required to determine whether these abnormalities have pathophysiologic significance.

Plasma Amyloid Beta 1-42 and DNA Methylation Pattern Predict Accelerated Aging in Young Subjects with Down Syndrome.

Gene methylation is an age-related dynamic process that influences diseases. Premature aging and disturbed methylation are components of Down syndrome (DS). We studied blood biomarkers and DNA methylation (DNAm) of three CpG sites (ASPA, ITGA2B, and PDE4C) in 60 elderly subjects (mean age=68years), 31 subjects with DS (12.1years) and 44 controls (12.8years). Plasma concentrations of amyloid beta (Aβ) 1-42 and biomarkers of methylation were measured in the young groups. Subjects with DS had significantly higher concentrations of plasma S-adenosylhomocysteine (SAH) and Aβ and reduced S-adenosylmethionine/SAH ratio compared with the controls. Methylations (%) of ASPA and ITGA2B were lower in DS [mean difference; 95% confidence intervals=-2.2 (-4.5, 0.1) for ASPA and -5.0 (-8.9, -1.1) for ITGA2B]. Methylation of PDE4C did not differ between the groups. The sum of z-scores for methylations of ASPA and ITGA2B, both of which declined with age, was significantly lower in DS [-1.01 (-1.93, -0.20), p=0.017]. Subjects with DS were found to be 3.1 (1.5-4.6) years older than their predicted age based on a regression model of the controls. Elevated SAH levels predicted lower DNAm of ASPA and ITGA2B in stepwise regression analysis. Therefore, methylation of three CpGs combined with plasma Aβ has shown a 3-year accelerated aging in subjects with DS at the age of 12years. Disorders in the methylation cycle explained pathoepigenetic modifications in subjects with DS. The influence of modifications in the methylation cycle on epigenetic markers of aging warrants further investigations.

Plasma S-adenosylhomocysteine: A potential risk marker for cerebral venous thrombosis

BackgroundDespite a plethora of studies suggesting that hyperhomocysteinemia is associated with an increased risk for arterial and venous thrombosis, there is paucity of data on the role of the S-adenosylhomocysteine (SAH), the metabolic precursor of homocysteine (Hcy) as a risk predictor for cerebral venous thrombosis (CVT). MethodWe estimated fasting plasma concentrations of total homocysteine (tHcy), SAH and S-adenosylmethionine (SAM), in 185 CVT patients and 248 healthy controls, by reverse-phase high performance liquid chromatography coupled with coulometric electrochemical detection. ResultsFasting tHcy, SAH and SAM were significantly higher in patients compared with controls. Increased tHcy and SAH concentrations were associated with 4.54-fold (95% CI, 2.74–7.53) and 35.77-fold (95% CI, 19.45–65.79) increase in risk for CVT, respectively. Receiver operating characteristic (ROC) curve analysis showed that the area under curve, sensitivity and specificity was higher for SAH compared to tHcy. Further, discriminant analysis to distinguish between tHcy and SAH showed that SAH had a significantly higher percentage classification, with lower Wilk's lambda and higher χ2, compared to tHcy. ConclusionIncreased plasma SAH may be a more sensitive risk marker for CVT than plasma tHcy.

Simultaneous detection of five one-carbon metabolites in plasma using stable isotope dilution liquid chromatography tandem mass spectrometry.

Disturbance in one-carbon (1-C) cycle occurs due to nutritional deficiencies (vitamin B12/folate) or specific genetic polymorphisms. This leads to altered levels of key 1-C metabolites such as SAM (s-adenosyl methionine), SAH (s-adenosyl homocysteine), methionine, homocysteine and MMA (methyl malonic acid). These 1-C metabolites are determinants of cellular methylation potential and epigenetic modifications of DNA which impairs metabolic pathways in several pathological diseases and developmental programming. Though methods were able to measure these analytes only independently, none of the methods detect simultaneously. Therefore we developed a method to measure these five 1-C metabolites in a single run using liquid chromatography tandem mass spectrometry (LC-MS/MS). We used stable isotopes dilution LC-MS/MS to measure the 1-C metabolites in human plasma. Blood samples were collected from pregnant women (n=30) at early gestation in the ongoing, multicentre, prospective PRiDE study. Linearity exhibited across the calibration range for all the analytes with the limit of detection (LOD) of 1.005nmol/l for SAM, 0.081nmol/l for SAH, 0.002μmol/l for methionine, 0.046μmol/l for homocysteine and 3.920nmol/l for MMA. The average recovery for SAM was 108%, SAH-110%, methionine-97%, homocysteine-91% and MMA-102%. The inter-assay CV for SAM was 7.3, SAH-5.6%, methionine-3.5%, homocysteine-7.0% and MMA-4.0%. The intra-assay CV for SAM was 8.7%, SAH-4.7%, methionine-5.4%, homocysteine-8.1% and MMA-6.1%. Pregnant women at early gestation with low B12 levels had significantly higher homocysteine, MMA, lower levels of methionine, SAM and SAM:SAH ratio and higher triglycerides. We developed a simple and rapid method to simultaneously quantify 1-C metabolites such as SAM, SAH, methionine, homocysteine and MMA in plasma by stable isotope dilution LC-MS/MS which would be useful to elucidate the epigenetic mechanisms related in the gene-nutrient interactions.

Quantitation of S-Adenosylmethionine and S-Adenosylhomocysteine in Plasma Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry.

We describe a simple stable isotope dilution method for accurate determination of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in plasma as a diagnostic test. SAM and SAH are key metabolic intermediates of methionine metabolism and the methylation cycle. Determination of SAM and SAH in plasma was performed by high performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Calibrators (SAM and SAH) and internal standards ((2)H3-SAM and (2)H4-SAH) were included in each analytical run for calibration. Sample preparation involved combining 20 μL sample with 180 μL of internal standard solution consisting of heavy isotope labeled internal standards in mobile phase A and filtering by ultracentrifugation through a 10 kd MW cutoff membrane. Sample filtrate (3 μL) was injected by a Shimadzu Nexera LC System interfaced with a 5500 QTRAP(®) (AB Sciex). Chromatographic separation was achieved on a 250 mm × 2.0 mm EA:faast column from Phenomenex. Samples were eluted at a flow rate of 0.20 mL/min with a binary gradient with a total run time of 10 min. The source operated in positive ion mode at an ion spray voltage of +5000 V. SAM and SAH resolved by a gradient to 100 % methanol with retention times of 6.0 and 5.7 min, respectively. The observed m/z values of the fragment ions were m/z 399 → 250 for SAM, m/z 385 → 136 for SAH, m/z 402 → 250 for (2)H3-SAM, m/z 203 → 46. The calibration curve was linear over the ranges of 12.5-5000 nmol/L for SAM and SAH.

Adenosine kinase deficiency: expanding the clinical spectrum and evaluating therapeutic options.

Adenosine kinase deficiency is a recently described defect affecting methionine metabolism with a severe clinical phenotype comprising mainly neurological and hepatic impairment and dysmorphism. Clinical data of 11 additional patients from eight families with adenosine kinase deficiency were gathered through a retrospective questionnaire. Two liver biopsies of one patient were systematically evaluated. The main clinical symptoms are mild to severe liver dysfunction with neonatal onset, muscular hypotonia, global developmental retardation and dysmorphism (especially frontal bossing). Hepatic involvement is not a constant finding. Most patients have epilepsy and recurrent hypoglycemia due to hyperinsulinism. Major biochemical findings are intermittent hypermethioninemia, increased S-adenosylmethionine and S-adenosylhomocysteine in plasma and increased adenosine in urine. S-adenosylmethionine and S-adenosylhomocysteine are the most reliable biochemical markers. The major histological finding was pronounced microvesicular hepatic steatosis. Therapeutic trials with a methionine restricted diet indicate a potential beneficial effect on biochemical and clinical parameters in four patients and hyperinsulinism was responsive to diazoxide in two patients. Adenosine kinase deficiency is a severe inborn error at the cross-road of methionine and adenosine metabolism that mainly causes dysmorphism, brain and liver symptoms, but also recurrent hypoglycemia. The clinical phenotype varies from an exclusively neurological to a multi-organ manifestation. Methionine-restricted diet should be considered as a therapeutic option.

Increased plasma S-adenosylhomocysteine-accelerated atherosclerosis is associated with epigenetic regulation of endoplasmic reticulum stress in apoE-/- mice.

S-Adenosylhomocysteine (SAH) is a better predictor of cardiovascular disease than homocysteine is, and it has been implicated in mediating the pathogenicity of hyperhomocysteinemia in atherosclerosis via an epigenetic mechanism. However, the underlying mechanism remains unclear. Here, we tested the hypothesis whether the effect of SAH on atherosclerosis is involved in epigenetic regulation of endoplasmic reticulum stress. A total of 48 apolipoprotein E-deficient mice at 8 weeks were randomly divided into 4 groups (n=12 for each group). The control group was fed a conventional diet, the adenosine dialdehyde group was fed a diet that was supplemented with the SAH hydrolase inhibitor adenosine dialdehyde, and the other 2 groups were intravenously injected with a retrovirus that expressed either SAH hydrolase short hairpin RNA or scrambled short hairpin RNA semiweekly for 16 weeks. Plasma SAH levels and atherosclerotic lesion size were significantly increased in adenosine dialdehyde and SAH hydrolase short hairpin RNA groups when compared with control group. Expression of endoplasmic reticulum stress markers glucose-regulated protein-78 and CEBP-homologous protein was significantly increased in the mice with elevated plasma SAH levels. Moreover, plasma SAH was negatively associated with a decrease in the expression of trimethylated histone H3 lysine 9 and histone methyltransferases. Chromatin immunoprecipitation assays showed a significant decrease in trimethylated histone H3 lysine 9 occupancy at the glucose-regulated protein-78 and CEBP-homologous protein promoters in mice treated with adenosine dialdehyde and SAH hydrolase short hairpin RNA when compared with control mice. Our results suggest that elevated plasma SAH levels-accelerated atherosclerosis was associated with the activation of endoplasmic reticulum stress via modulation of histone methylation.

Elevated S-Adenosylhomocysteine Alters Adipocyte Functionality With Corresponding Changes in Gene Expression and Associated Epigenetic Marks

Maternal deficiencies in micronutrients affecting one-carbon metabolism before and during pregnancy can influence metabolic status and the degree of insulin resistance and obesity of the progeny in adulthood. Notably, maternal and progeny plasma S-adenosylhomocysteine (SAH) levels are both elevated after vitamin deficiency in pregnancy. Therefore, we investigated whether this key one-carbon cycle intermediate directly affects adipocyte differentiation and function. We found that expansion and differentiation of murine 3T3-L1 preadipocytes in the presence of SAH impaired both basal and induced glucose uptake as well as lipolysis compared with untreated controls. SAH did not alter preadipocyte factor 1 (Dlk1) or peroxisome proliferator-activated receptor-γ 2 (Pparγ2) but significantly reduced expression of CAAT enhancer-binding protein-α (Cebpα), Cebpβ, and retinoid x receptor-α (Rxrα) compared with untreated adipocytes. SAH increased Rxrα methylation on a CpG unit (chr2:27,521,057+, chr2:27,521,049+) and CpG residue (chr2:27,521,080+), but not Cebpβ methylation, relative to untreated adipocytes. Trimethylated histone H3-Lys27 occupancy was significantly increased on Cebpα and Rxrα promoters in SAH-treated adipocytes, consistent with the reduction in gene expression. In conclusion, SAH did not affect adipogenesis per se but altered adipocyte functionality through epigenetic mechanisms, such that they exhibited altered glucose disposal and lipolysis. Our findings implicate micronutrient imbalance in subsequent modulation of adipocyte function.

Validation of a food-frequency questionnaire assessment of methyl-group donors using estimated diet records and plasma biomarkers: the method of triads

In this study, the intake of methyl-group donors (methionine, folate, betaine, and choline) among Flemish women of reproductive age (n = 30) assessed by a 7-d estimated diet record (7 d EDR) and food-frequency questionnaire (FFQ) was compared with plasma S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and SAH:SAM ratio. Pearson correlation coefficients were calculated between each of the dietary methods and the validity coefficient was calculated using the method of triads. Correlations were higher between intake assessed by the FFQ and biomarkers than between 7 d EDR and biomarkers. The validity coefficients of the FFQ, when using SAH as a biomarker, were high (0.86 for methionine to 0.94 for folate), when the SAH:SAM ratio was used as a biomarker the validity coefficients ranged from 0.63 to 1.00. These data indicate that the FFQ is a reliable tool to estimate the intake of the methyl-group donors in women of reproductive age.

Abstract 18002: S-adenosylhomocysteine Levels May Better Differentiate Between Normal Control, Subjects With Ckd Stages 3 and 4, and Subjects on Hemodialysis

Patients on hemodialysis have higher levels of plasma total homocysteine (tHcy) and are at higher risk for atherosclerotic cardiovascular and vascular events, including death. However, secondary prevention trials to date have not shown any reduction in recurrent atherosclerotic events with B vitamin supplementation that lowers tHcy levels. Because high tHcy levels are still a good predictor of CV events, we investigated whether other metabolites related to tHcy metabolism and the methylation cycle may be altered in subjects at various stages of chronic kidney disease. Methods: We measured 6 intermediates of the methylation cycle in plasma using liquid chromatography mass spectroscopy: tHcy, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), cystathionine, asymmetric dimethylarginine (ADMA), and symmetric dimethylargine (SDMA). Subjects included 92 subjects enrolled in longitudinal studies of healthy aging and dementia (Controls), 21 subjects with chronic kidney disease stages 3 and 4 (CKD), and 25 subjects on hemodialysis (Dialysis). All samples were collected between 2004 and 2012. Results: T-test analysis (corrected for multiplicity using Bonferoni correction) results show that all 6 metabolites were statistically significantly higher (p-values &lt;0.05) in the CKD subjects than the Controls, and again statistically significantly higher (p-values &lt;0.05) in Dialysis subjects than in CKD subjects (see Figure). While ADMA may be an independent risk factor for CAD, both tHcy and SAH levels were the most discriminatory: tHcy levels were 2.8 fold higher in CKD and 4 fold higher in Dialysis; SAH levels were 2.9 fold higher in CKD and 8.7 fold higher in Dialysis. Conclusion: SAH is a potent inhibitor of methylation reactions and has been implicated in causing vascular pathology. Elevated levels of SAH in plasma may provide greater discrimination in assessing risk of vascular disease in patients with chronic kidney disease.

Plasma homocysteine, methionine and S-adenosylhomocysteine levels following high-dose methotrexate treatment in pediatric patients with acute lymphoblastic leukemia or Burkitt lymphoma: association with hepatotoxicity

This study aimed to investigate: (i) changes of plasma homocysteine, methionine and S-adenosylhomocysteine levels following high-dose methotrexate (HD-MTX) treatment and (ii) the correlation of these sulfur-containing amino acids with MTX-induced hepatotoxicity. Fifteen pediatric patients with acute lymphoblastic leukemia and one patient with Burkitt lymphoma, with a total of 26 treatment courses of HD-MTX, were enrolled. Homocysteine levels increased at 24 h after HD-MTX treatment, and showed marginal decreases at 48 and 72 h. Methionine levels showed a biphasic pattern, i.e. an initial decrease at 24 h followed by increases at 48 and 72 h. S-adenosylhomocysteine exhibited a marginal decrease at 24 h. Changes of homocysteine exhibited significant correlation only with a maximum increase of alanine aminotransferase or total bilirubin from baseline. This study has demonstrated, for the first time, simultaneous changes of plasma homocysteine, methionine and S-adenosylhomocysteine following HD-MTX. The potential of homocysteine as a marker of hepatotoxicity is also presented.

Plasma S-adenosylhomocysteine is associated with the risk of cardiovascular events in patients undergoing coronary angiography: a cohort study

Although cross-sectional studies have shown that plasma S-adenosylhomocysteine (SAH), the metabolic precursor of homocysteine, is associated with cardiovascular disease, the prospective relation between plasma SAH and cardiovascular disease risk is unknown. The aim of this study was to prospectively evaluate the association between plasma SAH and cardiovascular disease risk in coronary angiography patients. Baseline plasma SAH and homocysteine concentrations were measured in 1003 patients aged between 21 and 87 y who underwent coronary angiography. Cox proportional hazards models were used to analyze the association between SAH and homocysteine and the risk of cardiovascular events, including fatal cardiovascular diseases, nonfatal myocardial infarction, and stroke. During the median follow-up period of 3.0 y, 93 participants developed cardiovascular events (32.7/1000 person-years). The age- and sex-adjusted hazard ratio of cardiovascular events was 3.38 (95% CI: 2.12, 5.39) for each 1-SD increase in the natural log-transformed SAH concentration. The age- and sex-adjusted hazard ratios of cardiovascular events across quartiles of SAH concentrations were 1.0, 2.25, 2.72, and 3.40 (P-trend = 0.007). Further adjustment for other cardiovascular disease risk factors and plasma homocysteine affected the results only slightly. This positive association between SAH and cardiovascular disease risk did not change when participants were stratified by age group, sex, and other baseline covariates. The results among a subset of participants with significant coronary stenosis were similar. Higher concentrations of plasma SAH are independently associated with an increased risk of cardiovascular events among patients undergoing coronary angiography. This trial was registered at www.chictr.org as ChiCTR-RNRC-08000270.

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.

PP4.6 – 1784 S-adenosylmethionine and S-adenosylhomocysteine in plasma and cerebrospinal fluid in Rett syndrome and the effect of folinic acid supplementation

PP4.6 – 1784 S-adenosylmethionine and S-adenosylhomocysteine in plasma and cerebrospinal fluid in Rett syndrome and the effect of folinic acid supplementation

Development and validation of an LC-MS/MS assay for the quantification of the trans-methylation pathway intermediates S-adenosylmethionine and S-adenosylhomocysteine in human plasma

BackgroundAlthough increased levels of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) have been implicated as markers for renal and vascular dysfunction, until now there have been no studies investigating their association with clinical post-transplant events such as organ rejection and immunosuppressant nephrotoxicity. MethodsA newly developed and validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for the quantification of SAM and SAH in human EDTA plasma was used for a clinical proof-of-concept pilot study. Retrospective analysis was performed using samples from a longitudinal clinical study following de novo kidney transplant patients for the first year (n=16). ResultsThe ranges of reliable response were 8 to 1024nmol/l for SAM and 16 to 1024nmol/l for SAH. The inter-day accuracies were 96.7–103.9% and 97.9–99.3% for SAM and SAH, respectively. Inter-day imprecisions were 8.1–9.1% and 8.4–9.8%. The total assay run time was 5min.SAM and SAH concentrations were significantly elevated in renal transplant patients preceding documented acute rejection and nephrotoxicity events when compared to healthy controls (n=8) as well as transplant patients void of allograft dysfunction (n=8). ConclusionThe LC-MS/MS assay will provide the basis for further large-scale clinical studies to explore these thiol metabolites as molecular markers for the management of renal transplant patients.

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.

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. 11.8nmol/L; p<0.001) and lower SAM/SAH ratio (9.1 vs. 8.2; p=0.006). Concentrations of total vitamin B12 andholoTC did not differ significantly between the groups, but plasma MMA concentrations were significantly higher in diabetics (250 vs. 206nmol/L). However, RBC-B12 was lower in diabetics compared tocontrols (median 230 vs. 260pmol/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.