Maternal and neonatal one-carbon metabolites and the epigenome-wide infant response.

Maternal prenatal status, as encapsulated by that to which a mother is exposed through diet and environment, is a key determinant of offspring health and disease. Alterations in DNA methylation (DNAm) may be a mechanism through which suboptimal prenatal conditions confer disease risk later in life. One-carbon metabolism (OCM) is critical to both fetal development and in supplying methyl donors needed for DNAm. Plasma concentrations of one-carbon metabolites across maternal first trimester (M1), maternal term (M3), and infant cord blood (CB) at birth were tested for association with DNAm patterns in CB from the Michigan Mother and Infant Pairs (MMIP) pregnancy cohort. The Illumina Infinium MethylationEPIC BeadChip was used to quantitatively evaluate DNAm across the epigenome. Global and single-site DNAm and metabolite models were adjusted for infant sex, estimated cell type proportions, and batch as covariates. Change in mean metabolite concentration across pregnancy (M1 to M3) was significantly different for S-adenosylhomocysteine (SAH), S-adenosylmethionine (SAM), betaine, and choline. Both M1 SAH and CB SAH were significantly associated with the global distribution of DNAm in CB, with indications of a shift toward less methylation. M3 SAH and CB SAH also displayed significant associations with locus-specific DNAm in infant CB (FDR<0.05). Our findings underscore the role of maternal one-carbon metabolites in shifting the global DNAm pattern in CB and emphasizes the need to closely evaluate how dietary status influences cellular methylation potential and ultimately offspring health.

Plasma S-Adenosylmethionine Is Associated with Lung Injury in COVID-19.

OBJECTIVE: S-Adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are indicators of global transmethylation and may play an important role as markers of severity of COVID-19. METHODS: The levels of plasma SAM and SAH were determined in patients admitted with COVID-19 (n = 56, mean age = 61). Lung injury was identified by computed tomography (CT) in accordance with the CT0-4 classification. RESULTS: SAM was found to be a potential marker of lung damage risk in COVID-19 patients (SAM > 80 nM; CT3,4 vs. CT 0-2: relative ratio (RR) was 3.0; p = 0.0029). SAM/SAH > 6.0 was also found to be a marker of lung injury (CT2-4 vs. CT0,1: RR = 3.47, p = 0.0004). There was a negative association between SAM and glutathione level (ρ = -0.343, p = 0.011). Interleukin-6 (IL-6) levels were associated with SAM (ρ = 0.44, p = 0.01) and SAH (ρ = 0.534, p = 0.001) levels. CONCLUSIONS: A high SAM level and high methylation index are associated with the risk of lung injury in patients with COVID-19. The association of SAM with IL-6 and glutathione indicates an important role of transmethylation in the development of cytokine imbalance and oxidative stress in patients with COVID-19.

Association between plasma S-adenosylmethionine and risk of mortality in patients with coronary artery disease: A cohort study.

BACKGROUND: S-adenosylmethionine (SAM) as methyl donors participates in methylation and is converted into S-adenosylhomocysteine (SAH), which is a precursor of homocysteine. Increased plasma SAH and homocysteine are associated with increased risk of cardiovascular disease. However, the relation of plasma SAM with cardiovascular risk is still unclear. OBJECTIVES: To determine the relation between plasma SAM and risk of mortality among patients with coronary artery disease (CAD). METHODS: Baseline plasma SAM concentrations were measured in 1553 patients with CAD from the Guangdong Coronary Artery Disease Cohort between October 2008 and December 2011. Proportional hazards Cox analyses were performed to ascertain associations between SAM and risk of all-cause and cardiovascular mortality. RESULTS: After a median follow-up of 9.2 (IQR: 8.5-10.2) y, of 1553 participants, 321 had died, including 227 deaths from cardiovascular diseases. Patients in the lowest quartile of SAM concentrations had a higher risk of all-cause death (HR, 1.59; 95% CI: 1.14, 2.21) and cardiovascular death (HR, 2.14; 95% CI: 1.41, 3.27) than those in the highest quartile in multivariable adjusted analysis. Each 1-SD decrease in the SAM concentration remained associated with a 42% greater risk of total death (HR, 1.42; 95% CI: 1.23, 1.64) and a 66% higher risk of cardiovascular death (HR, 1.66; 95% CI: 1.37, 2.01) after fully adjusting for other cardiovascular risk factors. Furthermore, each 1-SD decrease in plasma SAM/SAH ratio, as the methylation index, was also inversely associated with the risk of all-cause (HR, 1.80; 95% CI: 1.42, 2.29) and cardiovascular mortality (HR, 1.68; 95% CI: 1.29, 2.19) in fully adjusted analyses. CONCLUSIONS: Our data show a significant inverse relation between plasma SAM and risk of mortality in patients with CAD after adjustment for homocysteine, SAH, and other cardiovascular disease risk factors.

Associations of methyl donor and methylation inhibitor levels during anti-oxidant therapy in heart failure.

Redox balance and methylation are crucial to homeostasis and are linked by the methionine-homocysteine cycle. We examined whether differences in methylation potential, measured as plasma levels of S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH), occur at baseline and during anti-oxidant therapy with the xanthine oxidase inhibitor allopurinol in patients with heart failure with reduced ejection fraction. We analyzed plasma samples collected at baseline and 24 weeks in the Xanthine Oxidase Inhibition for Hyperuricemic Heart Failure Patients (EXACT-HF) study, which randomized patients with heart failure with reduced ejection fraction to allopurinol or placebo. Associations between plasma levels of SAM, SAH, SAM/SAH ratio, and outcomes, including laboratory markers and clinical events, were assessed. Despite randomization, median SAM levels were significantly lower at baseline in the allopurinol group. SAH levels at 24 weeks, and change in SAM from baseline to week 24, were significantly higher in the group of patients randomized to allopurinol compared to the placebo group. A significant correlation was observed between change in SAH levels and change in plasma uric acid (baseline to 24-week changes) in the allopurinol group. There were no significant associations between levels of SAM, SAH, and SAM/SAH ratio and clinical outcomes. Our results demonstrate significant biological variability in SAM and SAH levels at baseline and during treatment with an anti-oxidant and suggest a potential mechanism for the lack of efficacy observed in trials of anti-oxidant therapy. These data also highlight the need to explore personalized therapy for heart failure.

Alterations in Sulfur Amino Acids as Biomarkers of Disease.

Homocysteine (Hcy) is methylated by methionine synthase to form methionine with methyl-cobalamin as a cofactor. The reaction demethylates 5-methyltetrahydrofolate to tetrahydrofolate, which is required for DNA and RNA synthesis. Deficiency of either of the cobalamin (Cbl) and/or folate cofactors results in elevated Hcy and megaloblastic anemia. Elevated Hcy is a sensitive biomarker of Cbl and/or folate status and more specific than serum vitamin assays. Elevated Hcy normalizes when the correct vitamin is given. Elevated Hcy is associated with alcohol use disorder and drugs that target folate or Cbl metabolism, and is a risk factor for thrombotic vascular disease. Elevated methionine and cystathionine are associated with liver disease. Elevated Hcy, cystathionine, and cysteine, but not methionine, are common in patients with chronic renal failure. Higher cysteine predicts obesity and future weight gain. Serum S-adenosylhomocysteine (AdoHcy) is elevated in Cbl deficiency and chronic renal failure. Drugs that require methylation for catabolism may deplete liver S-adenosylmethionine and raise AdoHcy and Hcy. Deficiency of Cbl or folate or perturbations of their metabolism cause major changes in sulfur amino acids.

Determination of S-adenosylmethionine and S-adenosylhomocysteine in blood plasma by UPLC with fluorescence detection.

A validated approach to determine various methionine cycle metabolites (S-adenosylmethionine, S-adenosylhomocysteine, and methylthioadenosine) in human blood plasma is offered. The approach is based on solid-phase extraction (with grafted phenylboronic acid) and derivatization with chloroacetaldehyde followed by ultra-performance liquid chromatography with fluorescence detection. We used a 100 × 2.1 mm × 1.8 µm C18 column for the selective separation of analytes. Chromatographic separation was achieved with gradient elution of acetonitrile (flow rate 0.2 mL/min) from 2 to 20%. The eluent was initially composed of 10 mM KH2PO4 with 10 mM acetic acid and 25 µM heptafluorobutyric acid. The total analysis time was 11 min. Validation of the method included detection of the limit of detection (2 nM), limit of quantification (5 nM), accuracy (97.2-101%), and intra- and interday precision (2.2-9.0%). Analysis of plasma samples from healthy volunteers revealed that the average levels of S-adenosylmethionine, S-adenosylhomocysteine, and methylthioadenosine were 93.6, 20.9 and 14.8 nM, respectively.

Inhibition of S-Adenosylhomocysteine Hydrolase Induces Endothelial Dysfunction via Epigenetic Regulation of p66shc-Mediated Oxidative Stress Pathway.

BACKGROUND: Elevated levels of S-adenosylhomocysteine (SAH), the precursor of homocysteine, are positively associated with the risk of cardiovascular disease and with the development and progression of atherosclerosis. However, the role of SAH in endothelial dysfunction is unclear. METHODS: Apolipoprotein E-deficient ( apoE-/-) mice received dietary supplementation with the SAH hydrolase (SAHH) inhibitor adenosine dialdehyde or were intravenously injected with a retrovirus expressing SAHH shRNA. These 2 approaches, along with the heterozygous SAHH gene knockout ( SAHH+/-) mouse model, were used to elevate plasma SAH levels and to examine the role of SAH in aortic endothelial dysfunction. The relationship between plasma SAH levels and endothelial dysfunction was also investigated in human patients with coronary artery disease and healthy control subjects. RESULTS: Plasma SAH levels were increased in SAHH+/- mice and in apoE-/- mice after dietary administration of adenosine dialdehyde or intravenous injection with SAHH shRNA. SAHH+/- mice or apoE-/- mice with SAHH inhibition showed impaired endothelium-dependent vascular relaxation and decreased nitric oxide bioavailability after treatment with acetylcholine; this was completely abolished by the administration of the endothelial nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester. Furthermore, SAHH inhibition induced production of reactive oxygen species and p66shc expression in the mouse aorta and human aortic endothelial cells. Antioxidants and p66shc siRNA prevented SAHH inhibition-induced generation of reactive oxygen species and attenuated the impaired endothelial vasomotor responses in high-SAH mice. Moreover, inhibition of SAHH induced hypomethylation in the p66shc gene promoter and inhibited expression of DNA methyltransferase 1. Overexpression of DNA methyltransferase 1, induced by transduction of an adenovirus, was sufficient to abrogate SAHH inhibition-induced upregulation of p66shc expression. Finally, plasma SAH levels were inversely associated with flow-mediated dilation and hypomethylation of the p66shc gene promoter and positively associated with oxidative stress levels in patients with coronary artery disease and healthy control subjects. CONCLUSIONS: Our findings indicate that inhibition of SAHH results in elevated plasma SAH levels and induces endothelial dysfunction via epigenetic upregulation of the p66shc-mediated oxidative stress pathway. Our study provides novel molecular insight into mechanisms of SAH-associated endothelial injury that may contribute to the development of atherosclerosis. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov . Unique identifier: NCT03345927.

Fisher discriminant analysis for classification of autism spectrum disorders based on folate-related metabolism markers.

Autism spectrum disorders (ASDs) are neurodevelopmental disorders with an increasing prevalence but lack reliable biomarkers for early diagnosis. The present study investigated 13 serological metabolites and 2 genetic variants related to folate metabolism in a total of 89 ASD cases and 89 matched controls. Fisher discriminant analysis was used to establish the classification model to recognize ASD cases and controls. Ten metabolites were significantly different between the groups, of which six metabolites were used as predictors to determine the discriminant prediction model: vitamin B12, 5-methylene-tetrahydrofolate, methonine, the ratio of S-adenosylmethionine/S-adenosylhomocysteine, methionine synthase and transcobalamin II. The model had statistical significance (lambda=0.520, χ2=113.103, df=6, P<.001) and correctly identified 84.3% of ASD and normal cohorts. The area under the receiver operating characteristic curve was 0.913, with a sensitivity of 86.5% and a specificity of 85.4%. Overall, the results indicated that folate-related metabolism contributed to predisposition of ASD and the combined detection of folate-related metabolism biomarkers could be effective in distinguishing ASD from healthy controls, and provide new insights for the early diagnosis of ASD in the future.

Serum concentrations of folate vitamers in patients with a newly diagnosed prostate cancer or hyperplasia.

BACKGROUND: Folate is required for synthesis of methyl groups and DNA in growing cells. The association between folate and prostate cancer (PCa) is not conclusive. METHODS: We investigated concentrations of folate vitamers, S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) in blood of men with PCa (n = 129) or benign prostatic hyperplasia (BPH) (n = 73) who were recruited just after the first diagnosis. RESULTS: In younger subjects <65 years, concentrations of (6S)-5-CH3-H4folate (15.3 vs. 17.7 nmol/L) or total folate (UPLC-MS/MS) (18.7 vs. 23.0 nmol/L) did not differ between men with BPH and those with PCa, while SAM was higher in the controls (128 vs. 116 nmol/L). Younger patients with low- and high grade cancer did not differ in (6S)-5-CH3-H4folate (17.8 vs. 17.3 nmol/L) or total folate (UPLC-MS/MS) (22.9 vs. 23.3 nmol/L), but SAM was lower in patients with low grade PCa (111 vs. 126 nmol/L). In the older group ≥65 years, (6S)-5-CH3-H4folate (18.4 vs. 18.2 nmol/L) and total folate (UPLC-MS/MS) (22.5 vs. 22.1 nmol/L) did not differ between BPH and PCa. Older patients with advanced tumors had lower (6S)-5-CH3-H4folate compared with those with low grade tumor (12.8 vs. 20.0 nmol/L: p = 0.013). Plasma SAM was not different between older patients and controls and was not related to PCa grade. CONCLUSIONS: Lowered serum methyl folate measured at the time of diagnosis in older patients with advanced PCa, and lowered plasma SAM in younger patients with low grade PCa suggest differential folate metabolism that may have mechanistic, prognostic or predictive values.

One-carbon metabolism markers are associated with cardiometabolic risk factors.

BACKGROUND AND AIMS: Alterations 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 RESULTS: 118 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). CONCLUSION: We 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 (ß=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.

Characterization and Interrelations of One-Carbon Metabolites in Tissues, Erythrocytes, and Plasma in Mice with Dietary Induced Folate Deficiency.

Studies on one-carbon metabolism for the assessment of folate deficiency have focused on either metabolites of folate metabolism or methionine cycle. To bridge the gap between deficiency markers in these pathways we designed a dietary induced folate deficiency study using male C57BL/6N mice. After weaning (3 weeks) mice were fed a defined control diet (1 week) before being fed a folate deficient diet (n = 6 mice) and the control diet (n = 6 mice) for 12 additional weeks. Thereafter, we determined total homocysteine in plasma and folate in erythrocytes as well as S-adenosylmethionine, S-adenosylhomocysteine, and six folate vitamers in tissues including 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, 5,10-methenyltetrahydrofolate, tetrahydrofolate, 10-formylfolic acid, and folic acid by means of stable isotope dilution assays coupled with liquid chromatography tandem mass spectrometry. In all organs, except heart (mainly 5-mehtyltetrahydrofolate), tetrahydrofolate constitutes the main vitamer. Moreover, in liver tetrahydrofolate was most abundant followed by 5-methyltetrahydrofolate (heart: tetrahydrofolate), 5-formyltetrahydrofolate, and 5,10-methenyltetrahydrofolate. Because of the significant decrease (p < 0.05) of folate status and S-adenosylmethionine/S-adenosylhomocysteine ratio accompanied with increasing S-adenosylhomocysteine (p < 0.05), hepatocytes are most susceptible to folate deficiency. To the best of our knowledge, we herein present the first method for simultaneous quantitation of eight metabolites for both folate and methionine cycle in one tissue sample, tHcy in plasma, and erythrocyte folate to shed light on physiological interrelations of one-carbon metabolism.

Ratio of S-adenosylmethionine to S-adenosylhomocysteine as a sensitive indicator of atherosclerosis.

The present study aimed to confirm whether the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) is a sensitive indicator, and whether it can be used as a biomarker for the clinical diagnosis of atherosclerosis. Apolipoprotein E (ApoE)-/- mice were randomly divided into four groups and fed with a high methionine diet for 15 weeks. Serum levels of homocysteine (Hcy) were measured using an automatic biochemistry analyzer. The concentrations of SAM and SAH were determined using high­performance liquid chromatography. The methylation levels of B1 repetitive elements, adipocyte fatty acid binding protein (FABP4), monocyte chemoattractant protein-1 (MCP-1) and extracellular superoxide dismutase (EC­SOD) were analyzed using nested touchdown-methylation-specific-polymerase chain reaction analysis. After 15 weeks, compared with the normal control group, serum concentrations of Hcy were significantly increased by 1.15­, 2.54­ and 1.17­fold (P<0.05) in the ApoE­/­ control group, Meth group and Meth­F group, respectively. The sizes of the atherosclerotic lesions were increased in the ApoE­/­ control group, Meth group and Meth­F group, by up to 1.44­, 2.40­ and 1.45­fold, respectively, compared with the normal control group (P<0.05). The concentrations of SAM were significantly increased by 3.02­, 3.42­ and 2.46­fold in the ApoE­/­ control group, Meth group and Meth­F group, respectively (P<0.05). The ratios of SAM/SAH were increased by 1.67­ and 2.75­fold in the in ApoE­/­ control group and Meth group, respectively, compared with the normal control group. The methylation levels of B1 repetitive elements, FABP4, MCP­1 and EC­SOD were decreased and exhibited hypomethylation. The methylation statuses of these genes were correlated with the ratio of the serum levels of SAM and SAH. These findings suggested that the SAM/SAH ratio is a biomarker and may provide a sensitive indicator for the clinical diagnosis of atherosclerosis.

Metabolite Profiles Predict Acute Kidney Injury and Mortality in Patients Undergoing Transcatheter Aortic Valve Replacement.

BACKGROUND: Acute kidney injury (AKI) occurs commonly after transcatheter aortic valve replacement (TAVR) and is associated with markedly increased postoperative mortality. We previously identified plasma metabolites predictive of incident chronic kidney disease, but whether metabolite profiles can identify those at risk of AKI is unknown. METHODS AND RESULTS: We performed liquid chromatography-mass spectrometry-based metabolite profiling on plasma from patients undergoing TAVR and subjects from the community-based Framingham Heart Study (N=2164). AKI was defined by using the Valve Academic Research Consortium-2 criteria. Of 44 patients (mean age 82±9 years, 52% female) undergoing TAVR, 22 (50%) had chronic kidney disease and 9 (20%) developed AKI. Of 85 metabolites profiled, we detected markedly concordant cross-sectional metabolic changes associated with chronic kidney disease in the hospital-based TAVR and Framingham Heart Study cohorts. Baseline levels of 5-adenosylhomocysteine predicted AKI after TAVR, despite adjustment for baseline glomerular filtration rate (odds ratio per 1-SD increase 5.97, 95% CI 1.62-22.0; P=0.007). Of the patients who had AKI, 6 (66.7%) subsequently died, compared with 3 (8.6%) deaths among those patients who did not develop AKI (P=0.0008) over a median follow-up of 7.8 months. 5-adenosylhomocysteine was predictive of all-cause mortality after TAVR (hazard ratio per 1-SD increase 2.96, 95% CI 1.33-6.58; P=0.008), independent of baseline glomerular filtration rate. CONCLUSIONS: In an elderly population with severe aortic stenosis undergoing TAVR, metabolite profiling improves the prediction of AKI. Given the multifactorial nature of AKI after TAVR, metabolite profiles may identify those patients with reduced renal reserve.

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

BACKGROUND: Despite 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). METHOD: We 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. RESULTS: Fasting 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 χ(2), compared to tHcy. CONCLUSION: Increased plasma SAH may be a more sensitive risk marker for CVT than plasma tHcy.

Plasma trimethylamine N-oxide concentration is associated with choline, phospholipids, and methyl metabolism.

BACKGROUND: Elevated plasma concentrations of the gut bacteria choline metabolite trimethylamine N-oxide (TMAO) are associated with atherosclerosis. However, the determinants of TMAO in humans require additional assessment. OBJECTIVE: We examined cardiometabolic risk factors and pathways associated with TMAO concentrations in humans. DESIGN: A total of 283 individuals (mean ± SD age: 66.7 ± 9.0 y) were included in this observational study. Plasma concentrations of trimethylamine, TMAO, choline, lipids, phospholipids, and methyl metabolites were measured. RESULTS: Study participants were divided into 4 groups by median concentrations of TMAO and choline (4.36 and 9.7 µmol/L, respectively). Compared with the group with TMAO and choline concentrations that were less than the median (n = 82), the group with TMAO and choline concentrations that were at least the median (n = 83) was older and had lower high-density lipoprotein (HDL) cholesterol, phospholipids, and methylation potential, higher creatinine, betaine, S-adenosylhomocysteine (SAH), and S-adenosylmethionine (SAM), and higher percentages of men and subjects with diabetes. The difference in plasma TMAO concentrations between men and women (7.3 ± 10.0 compared with 5.4 ± 5.6 µmol/L, respectively) was NS after adjustment for age and creatinine (P = 0.455). The TMAO:trimethylamine ratio was higher in men (P < 0.001). Diabetes was associated with significantly higher plasma TMAO concentration (8.6 ± 12.2 compared with 5.4 ± 5.2 µmol/L) even after adjustments. Sex and diabetes showed an interactive effect on trimethylamine concentrations (P = 0.010) but not on TMAO concentrations (P = 0.950). Positive determinants of TMAO in a stepwise regression model that applied to the whole group were SAH, trimethylamine, choline, and female sex, whereas plasma phosphatidylcholine was a negative determinant. CONCLUSIONS: High TMAO and choline concentrations are associated with an advanced cardiometabolic risk profile. Diabetes is related to higher plasma TMAO concentrations but also to alterations in interrelated pathways such as lipids, phospholipids, and methylation. Elevated plasma TMAO concentrations likely reflect a specific metabolic pattern characterized by low HDL and phospholipids in addition to hypomethylation. This trial was registered at clinicaltrials.gov as NCT02586181 and NCT02588898.

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.

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.

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

BACKGROUND: Adenosine kinase deficiency is a recently described defect affecting methionine metabolism with a severe clinical phenotype comprising mainly neurological and hepatic impairment and dysmorphism. METHODS: 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. RESULTS: 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. CONCLUSION: 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.