Plasma Betaine Research Articles

Comparison of the effects of nicotinic acid and nicotinamide degradation on plasma betaine and choline levels

The present study was to compare the effects of nicotinic acid and nicotinamide on the plasma methyl donors, choline and betaine. Thirty adult subjects were randomly divided into three groups of equal size, and orally received purified water (C group), nicotinic acid (300mg, NA group) or nicotinamide (300mg, NM group). Plasma nicotinamide, N1-methylnicotinamide, homocysteine, betaine and choline levels before and 1.5-h and 3-h post-dosing, plasma normetanephrine and metanephrine concentrations at 3-h post-dosing, and the urinary excretion of N1-methyl-2-pyridone-5-carboxamide during the test period were examined. The level of 3-h plasma nicotinamide, N1-methylnicotinamide, homocysteine, the urinary excretion of N1-methyl-2-pyridone-5-carboxamide and pulse pressure (PP) in the NM group was 221%, 3972%, 61%, 1728% and 21.2% higher than that of the control group (P<0.01, except homocysteine and PP P<0.05), while the 3-h plasma betaine, normetanephrine and metanephrine level in the NM group was 24.4%, 9.4% and 11.7% lower (P<0.05, except betaine P<0.01), without significant difference in choline levels. Similar but less pronounced changes were observed in the NA group, with a lower level of 3-h plasma N1-methylnicotinamide (1.90±0.20μmol/l vs. 3.62±0.27μmol/l, P<0.01) and homocysteine (12.85±1.39μmol/l vs. 18.08±1.02μmol/l, P<0.05) but a higher level of betaine (27.44±0.71μmol/l vs. 23.52±0.61μmol/l, P<0.05) than that of the NM group. The degradation of nicotinamide consumes more betaine than that of nicotinic acid at identical doses. This difference should be taken into consideration in niacin fortification.

Restriction of dietary methyl donors limits methionine availability and affects the partitioning of dietary methionine for creatine and phosphatidylcholine synthesis in the neonatal piglet

Methionine is required for protein synthesis and provides a methyl group for >50 critical transmethylation reactions including creatine and phosphatidylcholine synthesis as well as DNA and protein methylation. However, the availability of methionine depends on dietary sources as well as remethylation of demethylated methionine (i.e., homocysteine) by the dietary methyl donors folate and choline (via betaine). By restricting dietary methyl supply, we aimed to determine the extent that dietary methyl donors contribute to methionine availability for protein synthesis and transmethylation reactions in neonatal piglets. Piglets 4–8 days of age were fed a diet deficient (MD−) (n=8) or sufficient (MS+) (n=7) in folate, choline and betaine. After 5 days, dietary methionine was reduced to 80% of requirement in both groups to elicit a response. On day 8, animals were fed [3H-methyl]methionine for 6h to measure methionine partitioning into hepatic protein, phosphatidylcholine, creatine and DNA. MD− feeding reduced plasma choline, betaine and folate (P<.05) and increased homocysteine ~3-fold (P<.05). With MD− feeding, hepatic phosphatidylcholine synthesis was 60% higher (P<.05) at the expense of creatine synthesis, which was 30% lower during MD− feeding (P<.05); protein synthesis as well as DNA and protein methylation were unchanged. In the liver, ~30% of dietary label was traced to phosphatidylcholine and creatine together, with ~50% traced to methylation of proteins and ~20% incorporated in synthesized protein. Dietary methyl donors are integral to neonatal methionine requirements and can affect methionine availability for transmethylation pathways.

Prospective Associations of Systemic and Urinary Choline Metabolites with Incident Type 2 Diabetes.

Several compounds in the choline oxidation pathway are associated with insulin resistance and prevalent diabetes; however, prospective data are scarce.We explored the relationships between systemic and urinary choline-related metabolites and incident type 2 diabetes in an observational prospective study among Norwegian patients. We explored risk associations by logistic regression among 3621 nondiabetic individuals with suspected stable angina pectoris, of whom 3242 provided urine samples. Reclassification of patients was investigated according to continuous net reclassification improvement (NRI >0). After median (25th to 75th percentile) follow-up of 7.5 (6.4-8.7) years, 233 patients (6.4%) were registered with incident type 2 diabetes. In models adjusted for age, sex, and fasting status, plasma betaine was inversely related to new-onset disease [odds ratio (OR) per 1 SD, 0.72; 95% CI, 0.62-0.83; P < 0.00001], whereas positive associations were observed for urine betaine (1.25; 1.09-1.43; P = 0.001), dimethylglycine (1.22; 1.06-1.40; P = 0.007), and sarcosine (1.30; 1.13-1.49; P < 0.001). The associations were maintained in a multivariable model adjusting for body mass index, hemoglobin A1c, urine albumin-to-creatinine ratio, estimated glomerular filtration rate, C-reactive protein, HDL cholesterol, and medications. Plasma betaine and urine sarcosine, the indices most strongly related to incident type 2 diabetes, improved reclassification [NRI >0 (95% CI) 0.33 (0.19-0.47) and 0.16 (0.01-0.31), respectively] and showed good within-person reproducibility. Systemic and urinary concentrations of several choline metabolites were associated with risk of incident type 2 diabetes, and relevant biomarkers may improve risk prediction.

Supplementation with Folic Acid, but Not Creatine, Increases Plasma Betaine, Decreases Plasma Dimethylglycine, and Prevents a Decrease in Plasma Choline in Arsenic-Exposed Bangladeshi Adults

Background: Folic acid (FA) supplementation facilitates urinary excretion of arsenic, a human carcinogen. A better understanding of interactions between one-carbon metabolism intermediates may improve the ability to design nutrition interventions that further facilitate arsenic excretion.Objective: The objective was to determine if FA and/or creatine supplementation increase choline and betaine and decrease dimethylglycine (DMG).Methods: We conducted a secondary analysis of the Folic Acid and Creatine Trial, a randomized trial in arsenic-exposed Bangladeshi adults (n = 605, aged 24–55 y, 50.3% male) who received arsenic-removal water filters. We examined treatment effects of FA and/or creatine supplementation on plasma choline, betaine, and DMG concentrations, measured by LC–tandem mass spectrometry at baseline and at week 12. Group comparisons were between 1) 400 and 800 μg FA/d (FA400 and FA800, respectively) compared with placebo, 2) creatine (3 g/d) compared with placebo, and 3) creatine plus FA400 compared with FA400.Results: Choline decreased in the placebo group (−6.6%; 95% CI: −10.2%, −2.9%) but did not change in the FA groups (FA400: 2.5%; 95% CI: −0.9%, 6.1%; FA800: 1.4%; 95% CI: −2.5%, 5.5%; P < 0.05). Betaine did not change in the placebo group (−3.5%; 95% CI: −9.3%, 2.6%) but increased in the FA groups (FA400: 14.1%; 95% CI: 9.4%, 19.0%; FA800: 13.0%; 95% CI: 7.2%, 19.1%; P < 0.01). The decrease in DMG was greater in the FA groups (FA400: −26.7%; 95% CI: −30.9%, −22.2%; FA800: −27.8%; 95% CI: −31.8%, −23.4%) than in the placebo group (−12.3%; 95% CI: −18.1%, −6.2%; P < 0.01). The percentage change in choline, betaine, and DMG did not differ between creatine treatment arms and their respective reference groups.Conclusion: Supplementation for 12 wk with FA, but not creatine, increases plasma betaine, decreases plasma DMG, and prevents a decrease in plasma choline in arsenic-exposed Bangladeshi adults. This trial was registered at clinicaltrials.gov as NCT01050556.

Choline and its metabolites are differentially associated with cardiometabolic risk and cardio‐ and cerebro‐vascular disease

Considerable attention has recently been given to the potential role of choline in cardiovascular disease. While the main focus has been on the microbial metabolism of phosphatidylcholine and the production of a pro‐atherosclerotic metabolite trimethylamine‐N‐oxide, there are also a number of links between choline and cardiometabolic risk through its role in lipid and one‐carbon metabolism. Phosphatidylcholine, a metabolite of choline, is an essential component of lipoproteins, and its biosynthesis in the liver is critical for the synthesis and secretion of very‐low density lipoproteins. Choline is also a precursor of betaine, which is a methyl donor for the conversion of homocysteine (Hcy) to methionine. Some data suggest differing associations of plasma choline and betaine with components of the metabolic syndrome, with an unfavorable cardiovascular risk profile associated with higher choline and a more favorable one with higher betaine plasma concentrations. Whether this same relationship holds true for cerebrovascular disease has not yet been evaluated. In this study, we've evaluated the associations of plasma choline and choline‐related compounds with cardiometabolic risk factors, history of cardiovascular disease, and cerebrovascular pathology in a cross‐sectional analysis of 293 men and women (mean age = 75 years, sd = 8.6) from the Nutrition, Aging, and Memory in Elders (NAME) cohort who had undergone magnetic resonance imaging of the brain and anthropometric and blood pressure measurement, had archived fasting plasma, and who had been administered a health history questionnaire. Plasma concentrations of choline, phophatidylcholine, and betaine were measured using liquid chromatography‐stable isotope dilution‐multiple reaction monitoring –mass spectrometry. Multiple linear and logistic regression models were used to examine relationships of plasma measures with cardiometabolic risk factors, history of cardiovascular disease, and radiological evidence of cerebral large vessel and small vessel infarcts. We found that plasma concentrations of phosphatidylcholine were positively related to low‐density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol (HDL), and triglycerides (TG) (p&lt;0.0001 for all), while the ratio of plasma betaine to choline concentrations were positively related to HDL (p=0.02) and negatively related to TG and Hcy (p&lt;0.0001 for both). Higher concentrations of both metabolites were associated with lower risk of hypertension (3rd vs 1st tertile, OR=0.42, 95% CI 0.18 – 0.99 for both). Participants in the third tertile of plasma choline concentrations had higher odds of having a history of cardiovascular disease (OR =3.26, 95% CI 1.67 – 6.36) and cerebral large vessel infarct (OR=4.19, 95% CI 1.2 – 13.58), but lower odds of cerebral small vessel infarct (OR=0.48, 95% CI 0.23 – 0.98). Our findings that choline and its metabolites had differential associations with cardiometabolic risk factors and subtypes of vascular disease suggest differing roles in the pathogenesis of cardiovascular and cerebral large vessel disease versus small vessel disease.Support or Funding InformationUSDA agreement No. 58‐1950‐0‐014; NIA AG21790‐01; NHLBIT32‐HL069772

Choline Supplementation With a Structured Lipid in Children With Cystic Fibrosis: A Randomized Placebo-Controlled Trial.

Choline depletion is seen in cystic fibrosis (CF) and pancreatic insufficiency in spite of enzyme treatment and may result in liver, fatty acid, and muscle abnormalities. This study evaluated the efficacy and safety of an easily absorbed choline-rich structured lipid (LYM-X-SORB™ [LXS]) to improve choline status. Children with CF and pancreatic insufficiency were randomized to LXS or placebo in a 12-month double blind trial. Dietary choline intake, plasma cholines, plasma and fecal phospholipids, coefficient of fat absorption, pulmonary function, growth status, body composition, and safety measures were assessed. Magnetic resonance spectroscopy for calf muscle choline and liver fat were assessed in a subgroup and compared with a healthy comparison group matched for age, sex, and body size. A total of 110 subjects were enrolled (age 10.4 ± 3.0 years). Baseline dietary choline, 88% recommended, increased 3-fold in the LXS group. Plasma choline, betaine, and dimethylglycine increased in the LXS but not placebo (P = 0.007). Plasma lysophosphatidylcholine and phosphatidylcholine increased, and fecal phosphatidylcholine/phosphatidylethanolamine ratio decreased (P ≤ 0.05) in LXS only, accompanied by a 6% coefficient of fat absorption increase (P = 0.001). Children with CF had higher liver fat than healthy children and depleted calf muscle choline at baseline. Muscle choline concentration increased in LXS and was associated with improvement in plasma choline status. No relevant changes in safety measures were evident. LXS had improved choline intake, plasma choline status, and muscle choline stores compared with placebo group. The choline-rich supplement was safe, accepted by participants, and improved choline status in children with CF.

Dietary Betaine Supplementation Increases Fgf21 Levels to Improve Glucose Homeostasis and Reduce Hepatic Lipid Accumulation in Mice.

Identifying markers of human insulin resistance may permit development of new approaches for treatment and prevention of type 2 diabetes. To this end, we analyzed the fasting plasma metabolome in metabolically characterized human volunteers across a spectrum of insulin resistance. We demonstrate that plasma betaine levels are reduced in insulin-resistant humans and correlate closely with insulin sensitivity. Moreover, betaine administration to mice with diet-induced obesity prevents the development of impaired glucose homeostasis, reduces hepatic lipid accumulation, increases white adipose oxidative capacity, and enhances whole-body energy expenditure. In parallel with these beneficial metabolic effects, betaine supplementation robustly increased hepatic and circulating fibroblast growth factor (Fgf)21 levels. Betaine administration failed to improve glucose homeostasis and liver fat content in Fgf21(-/-) mice, demonstrating that Fgf21 is necessary for betaine's beneficial effects. Together, these data indicate that dietary betaine increases Fgf21 levels to improve metabolic health in mice and suggest that betaine supplementation merits further investigation as a supplement for treatment or prevention of type 2 diabetes in humans.

Plasma Concentrations of Trimethylamine-N-oxide Are Directly Associated with Dairy Food Consumption and Low-Grade Inflammation in a German Adult Population

Background: Trimethylamine-N-oxide (TMAO) is a metabolite of carnitine, choline, and phosphatidylcholine, which is inversely associated with survival of cardiovascular disease (CVD) patients.Objective: We examined the associations of diet with plasma concentrations of TMAO, choline, and betaine and the associations of TMAO with plasma concentrations of various cytokines.Methods: Plasma TMAO, choline, and betaine concentrations were measured using LC-high resolution mass spectrometry in 271 participants, ≥18 y old, of the Second Bavarian Food Consumption Survey, conducted in 2002 and 2003. Food consumption was assessed using at least two 24-h dietary recalls. Cytokines were measured in plasma with enzyme-linked immunosorbent assays. Geometric mean concentrations of TMAO, choline, and betaine by categories of meat, dairy food, egg, and fish consumption were computed, adjusted for sex, age, and BMI. Multivariable-adjusted geometric mean concentrations of cytokines [tumor necrosis factor-α (TNF-α), soluble TNF receptors (sTNF-R) p55, sTNF-R p75, interleukin-6 (IL-6), and C-reactive protein (CRP)] were computed by quartiles of TMAO concentration using general linear models.Results: Meat, egg, or fish consumption was not associated with TMAO, choline, or betaine concentrations (all P-trend ≥ 0.05). With increases in milk and other dairy food consumption, the plasma TMAO concentration increased [geometric mean bottom quartile of milk consumption: 2.08 μM (95% CI: 1.69, 2.57 μM); compared with top quartile: 3.13 μM (95% CI: 2.56, 3.84 μM); P-trend = 0.008]. Participants in the top TMAO quartile had higher plasma concentrations of TNF-α, sTNF-R p55, and sTNF-R p75 than participants in the bottom quartile (percentage difference ranging between 14.4% and 17.3%; all P-trend < 0.05), but there were no differences in plasma concentrations of CRP and IL-6 (all P-trend ≥ 0.05).Conclusions: Results of this study conducted among healthy adults from the general population do not indicate a strong effect of diet on plasma concentrations of TMAO, choline, or betaine, with the exception of a positive association between dairy food consumption and plasma TMAO concentrations. Also, plasma TMAO concentrations were positively associated with inflammation. Whether habitual diet is strongly linked to the plasma TMAO concentration, a potential marker of CVD risk, needs to be determined in further studies.

Genome-wide association study and targeted metabolomics identifies sex-specific association of CPS1 with coronary artery disease.

Metabolites derived from dietary choline and L-carnitine, such as trimethylamine N-oxide and betaine, have recently been identified as novel risk factors for atherosclerosis in mice and humans. We sought to identify genetic factors associated with plasma betaine levels and determine their effect on risk of coronary artery disease (CAD). A two-stage genome-wide association study (GWAS) identified two significantly associated loci on chromosomes 2q34 and 5q14.1. The lead variant on 2q24 (rs715) localizes to carbamoyl-phosphate synthase 1 (CPS1), which encodes a mitochondrial enzyme that catalyses the first committed reaction and rate-limiting step in the urea cycle. Rs715 is also significantly associated with decreased levels of urea cycle metabolites and increased plasma glycine levels. Notably, rs715 yield a strikingly significant and protective association with decreased risk of CAD in only women. These results suggest that glycine metabolism and/or the urea cycle represent potentially novel sex-specific mechanisms for the development of atherosclerosis.

Plasma 1-carbon metabolites and academic achievement in 15-yr-old adolescents.

Academic achievement in adolescents is correlated with 1-carbon metabolism (1-CM), as folate intake is positively related and total plasma homocysteine (tHcy) negatively related to academic success. Because another 1-CM nutrient, choline is essential for fetal neurocognitive development, we hypothesized that choline and betaine could also be positively related to academic achievement in adolescents. In a sample of 15-yr-old children (n= 324), we measured plasma concentrations of homocysteine, choline, and betaine and genotyped them for 2 polymorphisms with effects on 1-CM, methylenetetrahydrofolate reductase (MTHFR) 677C>T, rs1801133, and phosphatidylethanolamineN-methyltransferase (PEMT), rs12325817 (G>C). The sum of school grades in 17 major subjects was used as an outcome measure for academic achievement. Lifestyle and family socioeconomic status (SES) data were obtained from questionnaires. Plasma choline was significantly and positively associated with academic achievement independent of SES factors (paternal education and income, maternal education and income, smoking, school) and of folate intake (P= 0.009,R(2)= 0.285). With the addition of thePEMTrs12325817 polymorphism, the association value was only marginally changed. Plasma betaine concentration, tHcy, and theMTHFR677C>T polymorphism did not affect academic achievement in any tested model involving choline. Dietary intake of choline is marginal in many adolescents and may be a public health concern.-Nilsson, T. K., Hurtig-Wennlöf, A., Sjöström, M., Herrmann, W., Obeid, R., Owen, J. R., Zeisel, S. Plasma 1-carbon metabolites and academic achievement in 15-yr-old adolescents.

Effect of Dietary Betaine on Metabolic Syndrome Risk Factors in Asian

Objective: Betaine (trimethylglycine) is a naturally-occurring osmolyte and methyl donor with reported effects on liver metabolism. The aim of this study was to evaluate the safety and the effects of betaine administration to metabolic risk factors in Asian males. Methodology: Twenty Japanese males (30-59 years) with mild fatty liver were enrolled in this blinded, placebocontrolled, randomized, parallel trial to assess the effects of 3 g of daily betaine for 12 weeks on safety and metabolic biomarkers. Selection criteria included fatty liver disease, as assessed by an abdominal ultrasonogram (US), the practice of moderate alcohol consumption, liver performance status and body mass index (≤27). Abdominal computed tomography (CT) as well as blood and urine sampling were performed before and after the intervention period. Results: No differences in the degree of hepatic steatosis or in the occurrence of adverse events were observed between the groups. Compared to the placebo group, the betaine group showed a significant increase in plasma betaine (P<0.0001) and high-density lipoprotein (HDL)-cholesterol (P<0.05) concentrations. Conclusion: Low-dose betaine supplementation to subjects with mild fatty liver disease is safe and well tolerated. Betaine increased plasma HDL which may reflect the favorable effects of betaine on liver function-related metabolic metabolism in Asian population.

Maternal Betaine Supplementation during Gestation Enhances Expression of mtDNA-Encoded Genes through D-Loop DNA Hypomethylation in the Skeletal Muscle of Newborn Piglets.

Betaine has been widely used in animal and human nutrition to promote muscle growth and performance, yet it remains unknown whether maternal betaine supplementation during gestation affects the metabolic characteristics of neonatal skeletal muscles. In the present study, feeding sows with betaine-supplemented diets throughout gestation significantly upregulated the expression of mtDNA-encoded OXPHOS genes (p < 0.05), including COX1, COX2, and ND5, in the muscle of newborn piglets, which was associated with enhanced mitochondrial COX enzyme activity (p < 0.05). Concurrently, maternal betaine supplementation increased the plasma betaine concentration and muscle expression of methyl transfer enzymes (p < 0.05), BHMT and GNMT, in offspring piglets. Nevertheless, Dnmt3a was downregulated at the level of both mRNA and protein, which was associated with a hypomethylated mtDNA D-loop region (p < 0.05). These results suggest that maternal betaine supplementation during gestation enhances expression of mtDNA-encoded genes through D-loop DNA hypomethylation in the skeletal muscle of newborn piglets.

Probiotic supplementation and trimethylamine-N-oxide production following a high-fat diet.

The objective of this study was to test the hypothesis that the multi-strain probiotic VSL#3 would attenuate the increase in fasting plasma concentrations of trimethylamine-N-oxide (TMAO) following a high-fat diet. Nineteen healthy, non-obese males (18-30 years) participated in the present study. Following a 2-week eucaloric control diet, subjects were randomized to either VSL#3 (900 billion live bacteria) or placebo (cornstarch) during the consumption of a hypercaloric (+1,000 kcalday(-1) ), high-fat diet (55% fat) for 4 weeks. Plasma TMAO, L-carnitine, choline, and betaine (UPLC-MS/MS) were measured at baseline and following a high-fat diet. Plasma TMAO significantly increased 89% ± 66% vs. 115% ± 61% in both the VSL#3 and placebo groups, respectively; however, the magnitude of change in plasma TMAO was not different (P > 0.05) between them. Plasma L-carnitine, choline, and betaine concentrations did not increase following the high-fat diet in either group. A high-fat diet increases plasma TMAO in healthy, normal-weight, young males. However, VSL#3 treatment does not appear to influence plasma TMAO concentrations following a high-fat diet. Future studies are needed to determine whether other therapeutic strategies can attenuate the production of TMAO.

Betaine is as effective as folate at re-synthesizing methionine for protein synthesis during moderate methionine deficiency in piglets

Both folate and betaine (synthesized from choline) are nutrients used to methylate homocysteine to reform the amino acid methionine following donation of its methyl group; however, it is unclear whether both remethylation pathways are of equal importance during the neonatal period when remethylation rates are high. Methionine is an indispensable amino acid that is in high demand in neonates not only for protein synthesis, but is also particularly important for transmethylation reactions, such as creatine and phosphatidylcholine synthesis. The objective of this study was to determine whether supplementation with folate, betaine, or a combination of both can equally re-synthesize methionine for protein synthesis when dietary methionine is limiting. Piglets were fed a low methionine diet devoid of folate, choline, and betaine, and on day 6, piglets were supplemented with either folate, betaine, or folate+betaine (n=6 per treatment) until day 10. [1-13C]-phenylalanine oxidation was measured as an indicator of methionine availability for protein synthesis both before and after 2days of supplementation. Prior to supplementation, piglets had lower concentrations of plasma folate, betaine, and choline compared to baseline with no change in homocysteine. Post-supplementation, phenylalanine oxidation levels were 20-46% lower with any methyl donor supplementation (P=0.006) with no difference among different supplementation groups. Furthermore, both methyl donors led to similarly lower concentrations of homocysteine following supplementation (P<0.05). These data demonstrate an equal capacity for betaine and folate to remethylate methionine for protein synthesis, as indicated by lower phenylalanine oxidation.

High homocysteine induces betaine depletion.

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

Abstract 311: Network Analysis of Pathways Associated with Genetic Regulation of Trimethylamine-N-Oxide

Common complex diseases, such as atherosclerosis, diabetes and obesity, are the result of genetic, environmental and gene/environmental interactions. Genetic reference panels are powerful tools to interrogate how the environment interacts with the underlying genetic architecture to influence disease susceptibility. Recently 2 genetic reference panels have been created: the Diversity Outbred (DO) mouse population and Collaborative Cross (CC). The mice comprising the CC/DO are mosaics of C57BL6/J, A/J, NOD/ShiLtJ, NZO/HiLtJ, WSB/EiJ, CAST/EiJ, PWK/PhJ, and 129S1/SvImJ. Both the DO and CC populations have tremendous genetic diversity, containing approximately 45 million segregating single-nucleotide polymorphisms (SNPs). Our primary goal was to characterize the genetic regulation of risk factors for atherosclerosis susceptibility in the 8 founder strains of the DO/CC reference panels. Using a systems genetic approach, we perform integrative analysis of clinical traits, targeted metabolomics and RNA expression. We quantitated genome wide mRNA levels using RNA sequencing followed by network analysis. The network was then related to clinical traits and a targeted panel of 5 metabolites (trimethylamine-N-oxide (TMAO), Betaine, Choline and Creatinine) to identify key groups of genes (called modules). We performed weighted gene network co-expression analysis (WGCNA) using 17,122 genes across 46 mice (N = 5-8 mice per strain). The network contains 47 independent co-expression modules and associated these modules with plasma lipoproteins, liver triglyceride levels and the novel plasma risk factors TMAO and Betaine. For example, the turquoise module was most strongly associated with TMAO levels (p &lt; 2 x 10-8) and the black module was associated with plasma Betaine concentrations. Both modules contain several candidate genes and enrichment analysis using Database for Annotation, Visualization and Integrated Discovery (DAVID ) v6.7 Studies are ongoing to validate the key molecular drivers of the network with a particular interest in the transcriptional network regulating plasma TMAO and Betaine concentrations.

Choline Alleviates Parenteral Nutrition-Associated Duodenal Motility Disorder in Infant Rats.

Parenteral nutrition (PN) has been found to influence duodenal motility in animals. Choline is an essential nutrient, and its deficiency is related to PN-associated organ diseases. Therefore, this study was aimed to investigate the role of choline supplementation in an infant rat model of PN-associated duodenal motility disorder. Three-week-old Sprague-Dawley male rats were fed chow and water (controls), PN solution (PN), or PN plus intravenous choline (600 mg/kg) (PN + choline). Rats underwent jugular vein cannulation for infusion of PN solution or 0.9% saline (controls) for 7 days. Duodenal oxidative stress status, concentrations of plasma choline, phosphocholine, and betaine and serum tumor necrosis factor (TNF)-α were assayed. The messenger RNA (mRNA) and protein expression of c-Kit proto-oncogene protein (c-Kit) and membrane-bound stem cell factor (mSCF) together with the electrophysiological features of slow waves in the duodenum were also evaluated. Rats on PN showed increased reactive oxygen species; decreased total antioxidant capacity in the duodenum; reduced plasma choline, phosphocholine, and betaine; and enhanced serum TNF-α concentrations, which were reversed by choline intervention. In addition, PN reduced mRNA and protein expression of mSCF and c-Kit, which were inversed under choline administration. Moreover, choline attenuated depolarized resting membrane potential and declined the frequency and amplitude of slow waves in duodenal smooth muscles of infant rats induced by PN, respectively. The addition of choline to PN may alleviate the progression of duodenal motor disorder through protecting smooth muscle cells from injury, promoting mSCF/c-Kit signaling, and attenuating impairment of interstitial cells of Cajal in the duodenum during PN feeding.

Distinct difference in absorption pattern in pigs of betaine provided as a supplement or present naturally in cereal dietary fiber.

The net absorption of betaine and choline was determined for 4 h after the first meal of the day in three experiments with porto-arterial catheterized pigs in which betaine was added as a supplement to a low-betaine diet (n=4 pigs) and compared to the net absorption of betaine and choline from high-fiber breads differing in amount and source of dietary fiber (two experiments, n=6 pigs each). Plasma betaine peaked after 30 min when betaine was fed as a supplement, whereas it peaked after 120-180 min when high-fiber breads were fed. Plasma betaine showed no diet×time interaction after feeding with high-fiber breads, indicating that the absorption kinetic did not differ between fiber sources. The net absorption of choline was not affected by the experimental diets. In conclusion, betaine in cereal sources has to be liberated from the matrix prior to absorption, causing delayed absorption.

Postprandial plasma betaine and other methyl donor-related responses after consumption of minimally processed wheat bran or wheat aleurone, or wheat aleurone incorporated into bread.

The bran and particularly the aleurone fraction of wheat are high in betaine and other physiological methyl donors, which may exert beneficial physiological effects. We conducted two randomised, controlled, cross-over postprandial studies to assess and compare plasma betaine and other methyl donor-related responses following the consumption of minimally processed bran and aleurone fractions (study A) and aleurone bread (study B). For both studies, standard pharmacokinetic parameters were derived for betaine, choline, folate, dimethylglycine (DMG), total homocysteine and methionine from plasma samples taken at 0, 0·5, 1, 2 and 3h. In study A (n 14), plasma betaine concentrations were significantly and substantially elevated from 0·5 to 3h following the consumption of both bran and aleurone compared with the control; however, aleurone gave significantly higher responses than bran. Small, but significant, increases were also observed in DMG measures; however, no significant responses were observed in other analytes. In study B (n 13), plasma betaine concentrations were significantly and substantially higher following consumption of the aleurone bread compared with the control bread; small, but significant, increases were also observed in DMG and folate measures in response to consumption of the aleurone bread; however, no significant responses were observed in other analytes. Peak plasma betaine concentrations, which were 1·7-1·8 times the baseline levels, were attained earlier following the consumption of minimally processed aleurone compared with the aleurone bread (time taken to reach peak concentration 1·2 v. 2·1h). These results showed that the consumption of minimally processed wheat bran, and particularly the aleurone fraction, yielded substantial postprandial increases in plasma betaine concentrations. Furthermore, these effects appear to be maintained when aleurone was incorporated into bread.

Plasma choline metabolites and colorectal cancer risk in the Women's Health Initiative Observational Study.

Few studies have examined associations between plasma choline metabolites and risk of colorectal cancer. Therefore, we investigated associations between plasma biomarkers of choline metabolism [choline, betaine, dimethylglycine, and trimethylamine N-oxide (TMAO)] and colorectal cancer risk among postmenopausal women in a case-control study nested within the Women's Health Initiative Observational Study. We selected 835 matched case-control pairs, and cases were further stratified by tumor site (proximal, distal, or rectal) and stage (local/regional or metastatic). Colorectal cancer was assessed by self-report and confirmed by medical records over the mean of 5.2 years of follow-up. Baseline plasma choline metabolites were measured by LC/MS-MS. In multivariable-adjusted conditional logistic regression models, plasma choline tended to be positively associated with rectal cancer risk [OR (95% confidence interval, CI)(highest vs. lowest quartile) = 2.44 (0.93-6.40); P trend = 0.08], whereas plasma betaine was inversely associated with colorectal cancer overall [0.68 (0.47-0.99); P trend = 0.01] and with local/regional tumors [0.64 (0.42-0.99); P trend = 0.009]. Notably, the plasma betaine:choline ratio was inversely associated with colorectal cancer overall [0.56 (0.39-0.82); P trend = 0.004] as well as with proximal [0.66 (0.41-1.06); P trend = 0.049], rectal [0.27 (0.10-0.78); P trend = 0.02], and local/regional [0.50 (0.33-0.76); P trend = 0.001] tumors. Finally, plasma TMAO, an oxidative derivative of choline produced by intestinal bacteria, was positively associated with rectal cancer [3.38 (1.25-9.16); P trend = 0.02] and with overall colorectal cancer risk among women with lower (vs. higher) plasma vitamin B12 levels (P interaction = 0.003). Collectively, these data suggest that alterations in choline metabolism, which may arise early in disease development, may be associated with higher risk of colorectal cancer. The positive association between plasma TMAO and colorectal cancer risk is consistent with an involvement of the gut microbiome in colorectal cancer pathogenesis.