Microbiota-dependent metabolite trimethylamine-N-oxide is associated with disease severity and survival of patients with chronic heart failure.

OBJECTIVES: Recent metabolomic, experimental and clinical studies have demonstrated that trimethylamine-N-oxide (TMAO), a microbiota-dependent metabolite from dietary phosphatidylcholine and carnitine, is a strong predictor of coronary artery disease (CAD). This finding suggests a link between the gut microbiota and atherosclerosis. The potential impact of TMAO in chronic heart failure (HF) is unknown. We hypothesized that TMAO levels would provide prognostic information about adverse outcomes in chronic HF. DESIGN: Prospective, observational study including 155 consecutive patients with chronic HF. In addition, 100 patients with stable CAD without HF and 33 matched healthy individuals were included as controls. Plasma levels of TMAO and its precursors choline and betaine were measured, and associations with symptoms, aetiology and transplant-free survival in the patients with HF were explored. RESULTS: Plasma levels of TMAO (P = 0.01), choline (P < 0.001) and betaine (P < 0.001) were elevated in patients with chronic HF compared to control subjects, with the highest levels in patients with New York Heart Association (NYHA) classes III and IV. Furthermore, TMAO levels were highest in individuals with ischaemic HF, followed by those with stable CAD and nonischaemic HF. TMAO, but not choline or betaine, was associated with reduced transplant-free survival: approximately 50% of patients in the upper tertile of TMAO levels died or received a heart transplant during 5.2 years of follow-up (unadjusted Cox-regression: hazard ratio 2.24, 95% confidence interval 1.28-3.92, P = 0.005). CONCLUSIONS: TMAO levels were elevated in patients with HF and associated with NYHA class, ischaemic aetiology and adverse outcomes. Future studies should focus on gut microbiota, dietary composition and intestinal dysfunction in relation to TMAO levels and clinical outcome in HF.

Oral choline supplementation for postoperative pain.

BACKGROUND: Activation of nicotinic receptors with nicotine has been shown to reduce post-surgical pain in clinical and preclinical studies. Choline is a selective agonist at α7-type nicotinic receptors that does not have addictive or sympathetic activating properties. It is anti-nociceptive in animal studies. We conducted a double-blind randomized trial of oral choline supplementation with lecithin to aid in the treatment of pain after gynaecological surgery. METHODS: Sixty women having open gynaecological surgery were randomly assigned to receive 20 g of lecithin before surgery or placebo. Plasma choline concentration and tumour necrosis factor (TNF) were measured. Pain report was the primary outcome measure. RESULTS: We achieved a small but statistically significant increase in choline after surgery with oral supplementation. Plasma TNF was not decreased and pain report was not different between groups at rest or with movement. There were no adverse effects of treatment. CONCLUSIONS: Oral supplementation with lecithin during the perioperative period resulted in very slow absorption and thus only a small increase in plasma choline was achieved. This concentration was inadequate to reduce TNF as has been shown in other studies. The absence of an anti-inflammatory effect was likely related to our failure to demonstrate efficacy in pain reduction.

Biomarkers in acute coronary syndrome and percutaneous coronary intervention.

The universal definition of myocardial infarction has proved how important the role of biomarkers in the assessment of acute coronary syndrome (ACS) has become. As a result, management of patients with ACS today is more specific and personalized than ever, but there is still a lot of room for improvement. Unmet needs for a faster and more specific rule-in and rule-out of myocardial infarction, for a pronounced risk assessment allowing for standardized guidelines on personalized therapy and for an effective monitoring of our therapeutic efforts to guarantee an optimal risk-benefit turnout still require intensive biomarker research and clinical validation. This review addresses a set of cardiovascular biomarkers with different pathophysiological backgrounds and discusses their diagnostic, prognostic and therapeutic value in the setting of ACS and percutaneous coronary intervention (PCI). The article provides a review of the current knowledge and literature on biomarkers in ACS and PCI, discussing currently used biomarkers like cardiac troponin (cTN), high sensitive cardiac troponin (hscTn), natriuretic peptides (NPs) as well as promising future biomarkers like copeptin, choline and lipoprotein-associated phospholipase A2 (LP-PLA2). The review concentrates on the clinical application of these markers, evaluating not only their diagnostic and prognostic value but also their integrability into routine practice. There are currently a number of new biomarkers and new biomarker assays under investigation which give hope for a much improved diagnostic and risk stratification process. Large diagnostic clinical trials are still needed to evaluate their impact on ACS patient management and subsequent PCI in clinical practice.

Plasma choline and betaine and risks of cardiovascular events and recurrent stroke after ischemic stroke.

BACKGROUND: Choline and betaine have been suggested to play a pivotal role in neurotransmitter synthesis, cell membrane integrity, and methyl-group metabolism, exerting neuroprotective effects in patients with various neurological disorders. However, population-based evidence on choline and betaine with subsequent cardiovascular events after stroke is rare. OBJECTIVES: We aimed to prospectively investigate the relationships of circulating choline and betaine with cardiovascular events and recurrent stroke in patients with ischemic stroke. METHODS: We performed a nested case-control study within the China Antihypertensive Trial in Acute Ischemic Stroke. A total of 323 cardiovascular events (including 264 recurrent strokes) and 323 controls (free of recurrent cardiovascular events) matched for age (±1 y), sex, and treatment group were included. The primary endpoint was a composite of cardiovascular events after ischemic stroke. Plasma choline and betaine were measured at baseline by ultra-high-performance LC-MS/MS. Conditional logistic regression models were applied, and discrimination, reclassification, and calibration of models with choline pathway metabolites were evaluated. RESULTS: Plasma choline and betaine were inversely associated with cardiovascular events and recurrent stroke after ischemic stroke. Specifically, in fully adjusted models, each additional SD of choline and betaine was associated with 35% (95% CI: 20%-48%) and 30% (95% CI: 14%-43%) decreased risks of subsequent cardiovascular events, respectively, and 34% (95% CI: 16%-48%) and 29% (95% CI: 12%-43%) decreased risks of recurrent stroke, respectively. In addition, both choline and betaine offered substantial risk discrimination and reclassification improvement for cardiovascular events and recurrent stroke beyond traditional risk factors, as evidenced by an increase in C statistics, the net reclassification index, and integrated discrimination improvement. CONCLUSIONS: Plasma choline pathway metabolites, including choline and betaine, were associated with decreased risks of cardiovascular events and recurrent stroke and provided incremental value in risk discrimination and stratification in patients with ischemic stroke. This nested case-control study was based on the China Antihypertensive Trial in Acute Ischemic Stroke, which is registered at clinicaltrials.gov as NCT01840072.

Dietary Choline Supplements, but Not Eggs, Raise Fasting TMAO Levels in Participants with Normal Renal Function: A Randomized Clinical Trial.

BACKGROUND: Choline is a dietary precursor to the gut microbial generation of the prothrombotic and proatherogenic metabolite trimethylamine-N-oxide (TMAO). Eggs are rich in choline, yet the impact of habitual egg consumption on TMAO levels and platelet function in human subjects remains unclear. METHODS: Healthy volunteers (41% male, 81% Caucasian, median age 28 years) with normal renal function (estimated glomerular filtration rate >60) were recruited and assigned to 1 of 5 daily interventions for 4 weeks: 1) hardboiled eggs (n = 18); 2) choline bitartrate supplements (n = 20); 3) hardboiled eggs + choline bitartrate supplements (n = 16); 4) egg whites + choline bitartrate supplements (n = 18); 5) phosphatidylcholine supplements (n = 10). Fasting blood and urine samples were collected for quantification of TMAO, its precursors, and platelet aggregometry. RESULTS: Participants' plasma TMAO levels increased significantly in all 3 intervention arms containing choline bitartrate (all P < .0001), but daily ingestion of 4 large eggs (P = .28) or phosphatidylcholine supplements (P = .27) failed to increase plasma TMAO levels. Platelet reactivity also significantly increased in the 3 intervention arms containing choline bitartrate (all P < .01), but not with eggs (P = .10) or phosphatidylcholine supplements (P = .79). CONCLUSIONS: Despite high choline content in egg yolks, healthy participants consuming 4 eggs daily showed no significant increase in TMAO or platelet reactivity. However, choline bitartrate supplements providing comparable total choline raised both TMAO and platelet reactivity, demonstrating that the form and source of dietary choline differentially contributes to systemic TMAO levels and platelet responsiveness.

Choline and risk of neural tube defects in a folate-fortified population.

BACKGROUND: Folic acid is known to reduce risk of neural tube defects (NTDs). Even so, NTDs continue to occur despite individual supplementation or population fortification with folic acid. We investigated other nutrients related to one-carbon metabolism that may affect NTD risk. METHODS: This prospective study included data from more than 180,000 pregnant women in California from 2003 through 2005. Midpregnancy serum specimens were linked with delivery information regarding the presence of a NTD, another structural malformation, or no malformation in the fetus. We identified 80 NTD-affected pregnancies (cases) and we randomly selected 409 pregnancy controls. Serum specimens were tested for methylmalonic acid, homocysteine, cysteine, methionine, total choline, betaine, cystathionine, vitamin B6, folate, vitamin B12, riboflavin, and creatinine. RESULTS: We observed elevated NTD risks associated with lower levels of total choline, and reduced risks with higher levels of choline. Specifically, we observed an odds ratio of 2.4 (95% confidence interval = 1.3-4.7) associated with the lowest decile and an odds ratio of 0.14 (0.02-1.0) associated with the highest decile, both relative to the 25th-74th percentiles of the control distribution. These data did not show meaningful differences between cases and controls for any other analytes. CONCLUSIONS: This is the first study to investigate total choline in NTD-affected pregnancies. Our findings for choline, for which low levels were a risk factor and higher levels were a protective factor for NTDs, may offer a useful clue toward understanding the complex etiologies of NTDs in an era of folic acid fortification of the food supply.

Behavioral and hematological responses of broiler chickens administered with betaine and ascorbic acid during hot-dry season.

Heat stress is a major problem in poultry production in tropical regions. Assessing the impact of thermally stressful environmental conditions on the welfare of broiler chickens is of great importance. Behavioral responses in a novel environment and hematology of broiler chickens administered with betaine and/or ascorbic acid (AA) during the hot-dry season were evaluated. Broiler chickens were randomly divided into four groups: Group I (control) was given sterile water, Group II was given betaine, Group III was given AA, and Group IV received betaine + AA orally and daily for 42 days. An open-field test was used to assess behavior. Hematological parameters were obtained using a hematology auto-analyzer. The natural environmental conditions were predominantly outside the thermoneutral zone for broiler chickens. Results demonstrated that treated groups exhibited improved ability to adjust faster to a new environment and better hematological responses than controls, evidenced by enhanced behavioral responses, oxygen-carrying capacity, and immune responses of broiler chickens under unfavorable environmental conditions. Betaine and/or AA administration to broiler chickens improved some behavioral responses, hemoglobin concentrations, packed cell volume, and total leukocyte count during the hot-dry season.

Measurement of beta-endorphin in human plasma.

beta-endorphin has been identified in human plasma by means of gel filtration and a sensitive radioimmunoassay for human beta-endorphin (beta h-endorphin). Mean baseline plasma beta h-endorphin concentration in 5 individuals was 21 +/- 7.3 (SD) pg/ml (6.2 +/- 2.2 (SD) fmole/ml). Following metyrapone stimulation mean plasma concentration increased to 55.4 +/- 10.1 (SD) pg/ml (16.3 +/- 3.1 (SD) fmole/ml). The molar ratio of human beta-lipotropin (beta h-LPH) to beta h-endorphin was 2.2 in baseline plasma and 2.4 after metyrapone stimulation.

Development and validation of a radioimmunoassay for peptides related to beta-melanocyte-stimulating hormone in human plasma: the lipotropins.

A radioimmunoassay is described for the measurement of human "beta-melanocyte-stimulating hormone" ("betah-MSH"). Two antisera have been used, one of which cross-reacts with synthetic betah-MSH as well as with the two larger pituitary peptides betah- and gammah-lipotropin (betah- and gammah-LPH) and the other mainly with betah-MSH and gammah-LPH. The sensitivity and reliability of the assay have been improved by employing a simple plasma extraction procedure, and the shelf-life of the iodinated betah-MSH tracer has been increased more than five-fold by storage in a concentrated human serum albumin solution. Using a 5 ml plasma sample the detection limit is 6 pg/ml. The mean resting "betah-MSH" level in normal subjects is 21 pg/ml (range 13-38 pg/ml) at 9 AM and 12 pg/ml (range 6-20 pg/ml) at 9 PM. Levels are considerably elevated (51-12,000 pg/ml) in patients with Addison's disease. Nelson's syndrome, Cushing's disease and the "ectopic" ACTH syndrome. After administration of insulin or pyrogen, the concentration of plasma "betah-MSH" increases in parallel with that of ACTH and they are approximately equivalent on a molar basis. The stability of purified betah- and gammah-LPH and endogenous "betah-MSH" when incubated in vitro in fresh blood or plasma are similar, in contrast to the less stable peptide synthetic betah-MSH. It is suggested that "betah-MSH" immunoreactivity in human plasma is due to betah- and gammah-LPH rather than betah-MSH.

Betaine supplementation decreases plasma homocysteine concentrations but does not affect body weight, body composition, or resting energy expenditure in human subjects.

BACKGROUND: Betaine (trimethylglycine) is found in several tissues in humans. It is involved in homocysteine metabolism as an alternative methyl donor and is used in the treatment of homocystinuria in humans. In pigs, betaine decreases the amount of adipose tissue. OBJECTIVE: The aim of the study was to examine the effect of betaine supplementation on body weight, body composition, plasma homocysteine concentrations, blood pressure, and serum total and lipoprotein lipids. DESIGN: Forty-two obese, white subjects (14 men, 28 women) treated with a hypoenergetic diet were randomly assigned to a betaine-supplemented group (6 g/d) or a control group given placebo for 12 wk. The intervention period was preceded by a 4-wk run-in period with a euenergetic diet. RESULTS: Body weight, resting energy expenditure, and fat mass decreased significantly in both groups with no significant difference between the groups. Plasma homocysteine concentrations decreased in the betaine group ( +/- SD: 8.76 +/- 1.63 micro mol/L at 4 wk, 7.93 +/- 1.52 micro mol/L at 16 wk; P = 0.030 for the interaction of time and treatment). Diastolic blood pressure decreased without a significant difference between the groups. Serum total and LDL-cholesterol concentrations were higher in the betaine group than in the control group (P < 0.05). CONCLUSION: A hypoenergetic diet with betaine supplementation (6 g daily for 12 wk) decreased the plasma homocysteine concentration but did not affect body composition more than a hypoenergetic diet without betaine supplementation did.

Choline deficiency causes reversible hepatic abnormalities in patients receiving parenteral nutrition: proof of a human choline requirement: a placebo-controlled trial.

BACKGROUND: Previous studies have shown that plasma free choline concentrations are significantly decreased in many long-term home total parenteral nutrition (TPN) patients. Furthermore, low choline status has been associated with both hepatic morphologic and hepatic aminotransferase abnormalities. A preliminary pilot study suggested choline-supplemented TPN may be useful in reversal of these hepatic abnormalities. METHODS: Fifteen patients (10 M, 5 F) who had required TPN for > or =80% of their nutritional needs were randomized to receive their usual TPN (n = 8), or TPN to which 2 g choline chloride had been added (n = 7) for 24 weeks. Baseline demographic data were similar between groups. Patients had CT scans of the liver and spleen, and blood for plasma free and phospholipid-bound choline, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, gamma glutamyl transferase (GGT), bilirubin, serum lipids, complete blood count (CBC), and chemistry profile obtained at baseline, and weeks 2, 4, 6, 12, 16, 20, 24, and 34. CT scans were analyzed for Hounsfield unit (HU) densities. RESULTS: There were no significant differences in any measured parameters after 2 weeks. However, at 4 weeks, a significant difference in liver HU between groups was observed (13.3+/-5.0 HU [choline] vs 5.8+/-5.2 HU [placebo], p = .04). This significant trend continued through week 24. Recurrent hepatic steatosis and decreased HU were observed at week 34, 10 weeks after choline supplementation had been discontinued. A significant increase in the liver-spleen differential HU was also observed in the choline group (10.6+/-6.2 HU [choline] vs 1.3+/-3.3 HU [placebo], p = .01). Serum ALT decreased significantly (p = .01 to .05) in the choline group vs placebo at weeks 6,12, 20, and 24. Serum AST was significantly decreased in the choline group by week 24 (p = .02). The serum alkaline phosphatase was significantly reduced in the choline group at weeks 2, 12, 20, 24, and 34 (p = .02 to 0.07). Total bilirubin was normal in these patients and remained unchanged during the study. Serum GGT tended to decrease more in the choline group, but the greater decrease was not statistically significant. CONCLUSIONS: Choline deficiency is a significant contributor to the development of TPN-associated liver disease. The data suggest choline is a required nutrient for long-term home TPN patients.

Oral choline tolerance test as a novel noninvasive method for predicting nonalcoholic steatohepatitis.

BACKGROUND: Although therapeutic intervention for nonalcoholic steatohepatitis (NASH) at an early stage is important owing to the progressive nature of the disease, diagnosis using noninvasive methods remains difficult. We previously demonstrated NASH specific impairment of choline metabolism and the use of fasting plasma free choline (fCh) levels for NASH diagnosis. Here, we investigated the utility of an oral choline tolerance test (OCTT), based on disordered choline metabolism, as a novel noninvasive method for NASH diagnosis. METHODS: Sixty-five patients with biopsy proven nonalcoholic fatty liver disease (NAFLD) and 17 healthy controls were enrolled. Blood samples were obtained from all subjects five times during the OCTT (before and 1, 2, 3, and 4 h after oral loading with 260 mg choline). RESULTS: Four-hour fCh levels after oral loading choline were markedly increased in NASH patients, compared with non-NASH subjects. For detecting NASH, compared with non-NASH subjects, the area under the curve for 4-h fCh levels was 0.829 on receiver operating characteristic (ROC) analysis. The cut-off level for NASH diagnosis was ≥0.16 mg/dL, and the sensitivity, specificity, positive predictive value, and negative predictive value were 80.1, 82.6, 78.4, and 84.4 %, respectively. Moreover, 4-h fCh levels were significantly associated with the disease activity based on NAFLD activity score in patients with NAFLD. CONCLUSIONS: Four-hour fCh levels obtained by an OCTT reflect a NASH specific disorder of choline metabolism, suggesting that the OCTT is a novel and useful noninvasive method for diagnosing NASH at an early stage with sufficient accuracy for clinical practice.

Fibroblast growth factor 21 and thyroid hormone show mutual regulatory dependency but have independent actions in vivo.

Thyroid hormone (TH) regulates fibroblast growth factor 21 (FGF21) levels in the liver and in the adipose tissue. In contrast, peripheral FGF21 administration leads to decreased circulating levels of TH. These data suggest that FGF21 and TH could interact to regulate metabolism. In the present study, we confirmed that TH regulates adipose and hepatic FGF21 expression and serum levels in mice. We next investigated the influence of TH administration on key serum metabolites, gene expression in the liver and brown adipose tissue, and energy expenditure in FGF21 knockout mice. Surprisingly, we did not observe any significant differences in the effects of TH on FGF21 knockout mice compared with those in wild-type animals, indicating that TH acts independently of FGF21 for the specific outcomes studied. Furthermore, exogenous FGF21 administration to hypothyroid mice led to similar changes in serum and liver lipid metabolites and gene expression in both hypothyroid and euthyroid mice. Thus, it appears that FGF21 and TH have similar actions to decrease serum and liver lipids despite having some divergent regulatory effects. Whereas TH leads to up-regulation in the liver and down-regulation in brown adipose tissue of genes involved in the lipid synthesis pathway (eg, fatty acid synthase (FASN) and SPOT14), FGF21 leads to the opposite changes in expression of these genes. In conclusion, TH and FGF21 act independently on the outcomes studied, despite their ability to regulate each other's circulating levels. Thus, TH and FGF21 may modulate the availability of each other in critical metabolic states.

Evidence for a circulating ergostatic factor: studies on parabiotic rats.

The effect of electrolytic ventromedial hypothalamic lesions or forced overfeeding through gastric cannulas was studied in parabiotic rats. In one experiment a ventromedial hypothalamic lesion in one member of the parabiotic union was associated with an increased fatness in the lesioned rat and a decrease in fat pad weight and a rise in body density (loss of fat) of the unlesioned animal. In the other two experiments a high fat or high carbohydrate diet was tube fed to one member of a parabiotic union. The average preinfusion intake per rat was 56 kcal/day. When 90 kcal/day were given through the intragastric cannula, oral intake of calories for the two animals fell to approximately 44 kcal/day. Body fat of the non-tube-fed animal was less than in the tube-fed controls. These studies suggest the presence of an ergostatic (energy stabilizing) factor that enters and is transported in the blood with passage from one animal to another.

Potentiation of the classic ovine corticotrophin releasing hormone stimulation test by the combined administration of small doses of lysine vasopressin.

OBJECTIVE: To assess the corticotrophic response to ovine corticotrophin releasing hormone (CRH) with the lowest dose of lysine vasopressin able to induce both the greatest stimulation and the lowest degree of side-effects. SUBJECTS: Fourteen healthy young adult males. DESIGN: Increasing intravenous doses (either 0, 0.03, 0.1, 0.3, or 1 IU) of lysine vasopressin, infused over 20 minutes, combined with a bolus of 100 micrograms ovine CRH. MEASUREMENT: Radioimmunoassay of plasma ACTH, lipotrophin hormones and cortisol levels. RESULTS: (1) Responses to stimulation tests were evaluated as the area under the curves of plasma levels versus sample times, from 0 to 120 minutes after injection or start of perfusion (six subjects). The lowest dose of lysine vasopressin that induced an additional stimulation in the CRH-stimulated ACTH response was 0.3 IU. The combination of 1 IU lysine vasopressin with CRH doubled values of the area under the curve for the ACTH. Lysine vasopressin alone (0.3 and 1 IU) failed to stimulate ACTH responses. (2) The combined test (100 micrograms CRH and 1 IU lysine vasopressin) was carried out on eight additional control subjects. From a mean basal level of 23 +/- 5.6 (SEM), plasma ACTH peaked to 104.5 +/- 8 ng/l (23.0 +/- 1.8 pmol/l) as early as 20-30 minutes after the start of injection. When repeated after a two-week interval, the combined test induced identical stimulation in a given subject. Results of lipotrophin hormone determinations roughly paralleled those of ACTH. However the effects on cortisol levels were less clear. Subjects injected with CRH experienced slight facial flush. Following the 1 IU lysine vasopressin dosage, side-effects were reduced to skin pallor. No changes in heart-rate or blood-pressure were observed. CONCLUSIONS: Under these conditions, the combination of 100 micrograms CRH with 1 IU lysine vasopressin constitutes a powerful test for direct assessment of the pituitary reserve and therefore can be employed as a routine investigational tool.

Betaine reduces hepatic lipidosis induced by carbon tetrachloride in Sprague-Dawley rats.

Carbon tetrachloride-injected rats were given liquid diets with and without betaine for 7 d. Hepatic lipidosis was induced by 4 daily injections of carbon tetrachloride (CCl4). Animals were killed and their livers and blood taken for analysis of betaine, S-adenosylmethionine (SAM), betaine homocysteine methyltransferase (BHMT), triglyceride, alanine aminotransferase and aspartate aminotransferase. Liver samples were also processed and stained for histological examination. Supplemental betaine reduced triglyceride in the liver and centrilobular hepatic lipidosis induced by the CCl4 injections. In both the control and experimental groups receiving betaine, liver betaine, BHMT and SAM were significantly higher than in their respective groups not receiving betaine. This study provides evidence that betaine protects the liver against CCl4-induced lipidosis and may be a useful therapeutic and prophylactic agent in ameliorating the harmful effects of CCl4.