It is inconclusive whether trimethylamine N-oxide (TMAO) and choline and related metabolites, namely trimethylamine (TMA), l-carnitine, betaine and dimethylglycine (DMG), are associated with non-alcoholic fatty liver disease (NAFLD). Our objective was to investigate these potential associations. Additionally, we sought to determine the mediating role of TMAO. In this 1:1 age- and sex-matched case–control study, a total of 150 pairs comprising NAFLD cases and healthy controls were identified. According to the fully adjusted model, after the highest tertile was compared with the lowest tertile, the plasma TMAO concentration (OR = 2·02 (95 % CI 1·04, 3·92); P trend = 0·003), l-carnitine concentration (OR = 1·79 (1·01, 3·17); P trend = 0·020) and DMG concentration (OR = 1·81 (1·00, 3·28); P trend = 0·014) were significantly positively associated with NAFLD incidence. However, a significantly negative association was found for plasma betaine (OR = 0. 50 (0·28, 0·88); P trend = 0·001). The restricted cubic splines model consistently indicated positive dose–response relationships between exposure to TMAO, l-carnitine, and DMG and NAFLD risk, with a negative association being observed for betaine. The corresponding AUC increased significantly from 0·685 (0·626, 0·745) in the traditional risk factor model to 0·769 (0·716, 0·822) when TMAO and its precursors were included (l-carnitine, betaine and choline) (P = 0·032). Mediation analyses revealed that 14·7 and 18·6 % of the excess NAFLD risk associated with l-carnitine and DMG, respectively, was mediated by TMAO (the P values for the mediating effects were 0·021 and 0·036, respectively). These results suggest that a higher concentration of TMAO is associated with increased NAFLD risk among Chinese adults and provide evidence of the possible mediating role of TMAO.

Elevated plasma concentrations of several one-carbon metabolites are associated with increased CVD risk. Both diet-induced regulation and dietary content of one-carbon metabolites can influence circulating concentrations of these markers. We cross-sectionally analysed 1928 patients with suspected stable angina pectoris (geometric mean age 61), representing elevated CVD risk, to assess associations between dietary macronutrient composition (FFQ) and plasma one-carbon metabolites and related B-vitamin status markers (GC–MS/MS, LC–MS/MS or microbiological assay). Diet-metabolite associations were modelled on the continuous scale, adjusted for age, sex, BMI, smoking, alcohol and total energy intake. Average (geometric mean (95 % prediction interval)) intake was forty-nine (38, 63) energy percent (E%) from carbohydrate, thirty-one (22, 45) E% from fat and seventeen (12, 22) E% from protein. The strongest associations were seen for higher protein intake, i.e. with higher plasma pyridoxal 5’-phosphate (PLP) (% change (95 % CI) 3·1 (2·1, 4·1)), cobalamin (2·9 (2·1, 3·7)), riboflavin (2·4 (1·1, 3·7)) and folate (2·1 (1·2, 3·1)) and lower total homocysteine (tHcy) (–1·4 (–1·9, −0·9)) and methylmalonic acid (MMA) (–1·4 (–2·0, −0·8)). Substitution analyses replacing MUFA or PUFA with SFA demonstrated higher plasma concentrations of riboflavin (5·0 (0·9, 9·3) and 3·3 (1·1, 5·6)), tHcy (2·3 (0·7, 3·8) and 1·3 (0·5, 2·2)) and MMA (2·0 (0·2, 3·9) and 1·7 (0·7, 2·7)) and lower PLP (–2·5 (–5·3, 0·3) and −2·7 (–4·2, −1·2)). In conclusion, a higher protein intake and replacing saturated with MUFA and PUFA were associated with a more favourable metabolic phenotype regarding metabolites associated with CVD risk.

Methionine, an essential sulphur-containing amino acid (SAA), plays an integral role in many metabolic processes. Evidence for the methionine requirements of adult dogs is limited, and we employed the indicator amino acid oxidation (IAAO) method to estimate dietary methionine requirements in Labrador retrievers (n 21). Using semi-purified diets, the mean requirement was 0·55 (95 % CI 0·41, 0·71) g/4184 kJ. In a subsequent parallel design study, three groups of adult Labrador retrievers (n 52) were fed semi-purified diets with 0·55 g/4184 kJ (test diet 1), 0·71 g/4184 kJ (test diet 2) or 1·37 g/4184 kJ (control diet) of methionine for 32 weeks to assess the long-term consequences of feeding. The total SAA content (2·68 g/4184 kJ) was maintained through dietary supplementation of cystine. Plasma methionine did not decrease in test group and increased significantly on test diet 1 in weeks 8 and 16 compared with control. Reducing dietary methionine did not have a significant effect on whole blood, plasma or urinary taurine or plasma N-terminal pro B-type natriuretic peptide. Significant effects in both test diets were observed for cholesterol, betaine and dimethylglycine. In conclusion, feeding methionine at the IAAO-estimated mean was sufficient to maintain plasma methionine over 32 weeks when total SAA was maintained. However, choline oxidation may have increased to support plasma methionine and have additional consequences for lipid metabolism. While the IAAO can be employed to assess essential amino acid requirements, such as methionine in the dog using semi-purified diets, further work is required to establish safe levels for commercial diet formats.

Maternal one-carbon metabolism during pregnancy is crucial for fetal development and programming by DNA methylation. However, evidence on one-carbon biomarkers other than folate is lacking. We, therefore, investigated whether maternal plasma methyl donors, that is, choline, betaine and methionine, are associated with birth outcomes. Blood samples were obtained from 115 women during gestation (median 26·3 weeks, 90 % range 22·7–33·0 weeks). Plasma choline, betaine, methionine and dimethylglycine were measured using HPLC-tandem MS. Multivariate linear and logistic regression models were used to estimate the association between plasma biomarkers and birth weight, birth length, the risk of small-for-gestational-age and large-for-gestational-age (LGA). Higher level of maternal betaine was associated with lower birth weight (–130·3 (95 % CI –244·8, –15·9) per 1 sd increment for log-transformed betaine). Higher maternal methionine was associated with lower risk of LGA, and adjusted OR, with 95 % CI for 1 sd increase in methionine concentration was 0·44 (95 % CI 0·21, 0·89). Stratified analyses according to infant sex or maternal plasma homocysteine status showed that reduction in birth weight in relation to maternal betaine was only limited to male infants or to who had higher maternal homocysteine status (≥5·1 µmol/l). Higher maternal betaine status was associated with reduced birth weight. Maternal methionine was inversely associated with LGA risk. These findings are needed to be replicated in future larger studies.

N, N-dimethylglycine (DMG) is a tertiary amino acid that naturally occurs as an intermediate metabolite in choline-to-glycine metabolism. The objective of the present trial was to evaluate tolerance, safety and bioaccumulation of dietary DMG in broilers when supplemented at 1 g and 10 g Na-DMG/kg. A feeding trial was conducted using 480 1-d-old broiler chicks that were randomly allocated to twenty-four pens and fed one of three test diets added with 0, 1 or 10 g Na-DMG/kg during a 39 d growth period. Production performance was recorded to assess tolerance and efficacy of the supplement. At the end of the trial, toxicity was evaluated by means of haematology, plasma biochemistry and histopathology of liver, kidney and heart (n 12), whereas bioaccumulation was assessed on breast meat, liver, blood, kidney and adipose tissue (n 8). Carcass traits were similar between the control and 1 g Na-DMG/kg feed groups (P>0·05), but the feed:gain ratio was significantly improved at 1 g Na-DMG/kg feed compared with the control or the 10-fold dose (P = 0·008). Histological examinations showed no pathological effects and results of haematology and plasma biochemistry revealed similar values between the test groups (P>0·05). Bioaccumulation occurred at the 10-fold dose, but the resulting DMG content in breast meat was comparable with, for instance, wheat bran and much lower than uncooked spinach. In conclusion, DMG at 1 g Na-DMG/kg improved the feed:gain ratio in broilers without DMG being accumulated in consumer parts. Furthermore, dietary supplementation with DMG up to 10 g Na-DMG/kg did not induce toxicity or impaired performance in broilers.

The importance of the one-carbon metabolites, choline and homocysteine, to brain function is well known. However, the associations between the one-carbon metabolites choline, betaine, methionine and dimethylglycine with cognition in elderly are unclear. We therefore examined the associations of these metabolites with cognition in a double-blind, placebo-controlled trial. Individuals (n 195) were randomized to receive daily oral capsules with either 1000 μg cobalamin (vitamin B12), or 1000 μg cobalamin plus 400 μg folic acid, or placebo for 24 weeks. Concentrations of homocysteine, methionine, choline, betaine and dimethylglycine were assessed before and after 12 and 24 weeks of treatment. Cognitive function, including domains of attention, construction, sensomotor speed, memory and executive function, was assessed before and after 24 weeks of treatment. At baseline, elevated plasma homocysteine was associated with lower performance of attention, construction, sensomotor speed and executive function. In addition, betaine was positively associated with better performance of construction, sensomotor speed and executive function, whereas elevated concentrations of methionine were positively associated with sensomotor speed. Daily combined supplementation with cobalamin plus folic acid decreased total homocysteine concentrations by 36 %, and increased betaine concentrations by 38 %. Participants with the largest increases in betaine concentrations showed a borderline significant (P = 0·07) higher memory performance compared to those without it. Although this trial observed associations of homocysteine and betaine with cognitive domains prior to supplementation, decreased concentrations of homocysteine were not related to improved cognitive performance. There was a tendency of participants with the largest increases in betaine concentrations to show the greatest improvement in memory function.