Table 3.
Preclinical evidence for cardio-protective or neutral effects of TMAO.
| Literature Source | Test System | Treatment | Outcome | Distress Factors | Daily Dosage of TMAO or Precursors * | Plasma TMAO Level * (or Control Level) |
|---|---|---|---|---|---|---|
| Mayr 2005 [114] | Male and female apoE−/− and apoE+/+mice on normal chow diet | Proteomics and metabolomics to identify protein and metabolite changes in vessels |
No significant difference in TMAO concentration in the aortas of 18-month-old apoE−/− and ApoE+/+mice. Lesion formation in apoE−/− mice due to increase in oxidative stress, not to increased TMAO. |
apoE−/− mice | none | Females 0.06 Males 0.25 (units not stated, controls not stated) |
| Martin 2009 [202] | Male hamsters | Hyperlipidemic diet (normal diet plus 100 g/kg fat for 5 weeks + 200 g/kg for 12 weeks; fat as anhydrous butter or cheese) or controls; (1)H NMR-based metabonomics | VLDL lipids, cholesterol, and N-acetylglycoproteins had the best correlation to onset of atherosclerosis. TMAO was found to be negatively associated with atherogenesis. |
High-fat diet | none | Absolute concentrations not determined (only relative ones; personal communication) |
| Gao 2014 [203] | Male mice | Control, diet with 25% fat +/− 0.2% TMAO for 4 weeks | Dietary TMAO increased fasting insulin levels and insulin resistance and exacerbated impaired glucose tolerance and MCP-1 mRNA (pro-inflammatory cytokine) in HFD-fed mice. IL-10 mRNA (anti-inflammatory cytokine) in adipose tissue was (more than already by the high-fat diet) decreased by TMAO. However, the increase in atherosclerosis associated with a high-fat diet was prevented by TMAO, suggesting a protective effect of TMAO with regard to atherosclerosis. |
High-fat diet; TMAO supplementation |
320 mg TMAO/kg ** | 17.5 µM (normal chow 11.9 µM; high-fat chow 12 µM) |
| Shih 2015 [118] | Male mice, transgenic FMO3 overexpression | Transgenic compared to control mice, supplemented with water containing 1.3% choline chloride for 6 weeks | FMO3 overexpression caused a 75% increase in plasma TMAO levels and increased hepatic and plasma lipids. | FMO3 overexpression; high choline supplementation | FMO3 overexpression; 2080 mg choline/kg ** |
16 µM (9 µM for control transgene) |
| Shih 2015 [29,118] | Male hyperlipidemic mouse “E3L Tg” with transgenic FMO3 overexpression |
Transgenic compared to control mice; low-fat or high-fat/1% cholesterol chow for 16 weeks | Increased plasma TG, VLDL/IDL/LDL, and unesterified cholesterol with both diets, increased glucose and insulin levels, increased levels of TG, TC, and phosphatidylcholine in the VLDL plasma fractions with high-fat diet. Small increase (20%) in atherosclerotic lesion size compared to knockdown mouse (see above); it is implausible to explain the effect with the slight change in TMAO levels. |
Hyperlipidemic mouse with transgenic FMO3 overexpression; high-fat/high-cholesterol chow |
High-fat diet | w/high-fat/cholesterol: 2.6 µM (2.2 µM for control transgene) (difference = trend) |
| Collins 2016 [185] | Male apoE−/− mice expressing human cholesteryl ester transfer protein (hCETEP) | 12 week treatment with L-carnitine (87 mg/kg and 352 mg/kg; equivalent to 500 and 2000 mg/day in humans); (and/or methimazole =FMO inhibitor) |
High doses of L-carnitine resulted in a significant increase in plasma L-carnitine and TMAO levels. Plasma lipid and lipoprotein levels did not change. Aortic root analysis showed significant decrease in lesion size with L-carnitine treatment and high TMAO compared to the control. Significantly lower levels of lesion were found with elevated plasma TMAO (>0.05 ppm) compared to the low plasma TMAO (<0.05 ppm). TMAO may be protective against atherosclerosis development. |
apoE−/− mice (plus hCETEP) | 352 mg L-carnitine/kg | 0.2 ppm = 2.7 µM (0.08 ppm = 1.07 µM) |
| Empl 2015 [204] | Male Fischer 344 rats | Daily 0, 0.1, 0.2 or 0.5 g/L L-carnitine (0; 70; 141; 352 mg/kg) via drinking water for one year | L-carnitine did not cause any preneoplastic, atherosclerotic, or other lesions. | None | 352 mg L-carnitine/kg (highest dosage) | See below |
| Weinert 2017 [205] | Male Fischer 344 rats from study [204] | Daily 0, 0.1, 0.2 or 0.5 g/L L-carnitine (0; 70; 141; 352 mg/kg) via drinking water for one year | High dose L-carnitine resulted in tenfold higher plasma TMAO concentration compared to the control (25.0 μM). Supplementation did not cause changes in the plasma metabolome. |
None | 352 mg L-carnitine/kg (highest dosage) | 25.0 μM (2.5 µM for control) |
| Huc 2018 [206] | Male spontaneously hypertensive rats (SHR) with pressure-overloaded hearts, 2 age groups | TMAO 333 mg/L with drinking water, water controls and normotensive rat controls for 9 weeks; metabolic cage for 2 days at end of study | Chronic, low-dose trimethylamine oxide (TMAO) treatment increased plasma TMAO by 4 to 5-fold and reduced plasma NH2-terminal pro-B-type natriuretic peptide and vasopressin, left ventricular end-diastolic pressure, and cardiac fibrosis. TMAO may be beneficial for reduction of hypertension. | Hypertension | 37 (16 weeks)/32 mg (56 weeks) TMAO/kg (personal communication) | 16 weeks: 37.3 µM (SHR control: 8.8 µM) (normal control: 6.3 µM) 56 weeks: 40.9 µM (SHR control: 8.1 µM) (normal control: 5.2 µM) |
| Lindskog Jonsson 2018 [207] | Male germ-free or conventionally raised apoE−/− mice | Western diet alone or supplemented with 1.2% choline for 12 weeks | Conventionally raised mice had smaller aortic lesions and lower plasma cholesterol levels on a chow diet compared to a Western diet; choline supplementation increased plasma TMAO levels in conventionally raised mice but not in germ-free mice. However, choline supplementation did not affect the size of aortic lesions or plasma cholesterol levels; increased plasma TMAO levels observed in conventionally raised apoE−/− mice fed a choline-supplemented diet did not correlate with aortic lesion size. The microbiota was required for TMAO production from dietary choline, but this process could not be linked to increased atherosclerosis. Choline supplementation reduced body weight and epididymal fat weight in conventionally but not germ-free mice when fed Western diet. |
Western diet/germ-free existence; apoE−/− mice | 1920 mg choline/kg ** | Conventionally raised: Western diet +choline: 8 µM Chow +choline: 21 µM (Western diet: 0.5 µM Chow: 1 µM) No TMAO in germ-free mice |
| Zhao 2019 [208] | Male rats with steatohepatitis induced by high-fat high-cholesterol diet | 16-wk high-fat high-cholesterol (HFHC) diet feeding; daily TMAO (120 mg/kg/day) by oral gavage for 8 weeks | Hepatic and serum levels of cholesterol were both decreased by TMAO treatment in rats on HFHC diet. TMAO treatment also downregulated cholesterol influx-related Niemann-Pick C1-like 1 (intestinal cholesterol absorption transporter) and upregulated cholesterol efflux-related ABCG5/8 in the small intestine; thus, TMAO inhibits intestinal cholesterol absorption. Gut microbiota analysis showed that TMAO could alter the gut microbial profile and restore the diversity of gut flora. TMAO also ameliorated hepatic ER stress and cell death under cholesterol overload, thereby attenuating HFHC diet-induced steatohepatitis in rats. |
Steatohepatitis induced by high-fat high-cholesterol diet | 120 mg TMAO/kg | Not determined (personal communication) |
| Aldana-Hernandez 2019 [209] | Male Ldlr-/- mice and male apoE−/− mice |
40% high-fat diet for atherosclerosis induction; control (0.1% choline) or supplemented with 1% choline or 0.9% betaine or 0.2% TMAO; for 8 or 16 weeks control diet (0.1% choline) or diet for ≤28 weeks supplemented with 1% choline or 0.9% betaine or 0.12% TMAO for 12 or 28 weeks |
In LDLr-/- mice, dietary supplementation for 8 wk with choline or TMAO increased plasma TMAO concentrations by 1.6-and 4-fold, respectively. After 16 wk, there was a 2-fold increase in plasma TMAO after dietary TMAO supplementation. In apoe-/- mice, dietary supplementation with choline, betaine, or TMAO for 12 wk did not increase plasma TMAO concentrations. However, choline and TMAO supplementation for 28 wk significantly increased plasma TMAO concentrations by 1.8-and 1.5-fold, respectively. Atherosclerotic lesion size was not altered by any of the dietary interventions, irrespective of mouse model. |
Ldlr-/- mice (40% high-fat diet) apoE−/− mice |
1600 mg choline/kg (1%) ** 1440 mg betaine/kg (0.9%) ** 320 mg TMAO/kg (high TMAO dosage of 0.2%) ** 192 mg TMAO/kg (low TMAO dosage of 0.12%) ** |
LDLr-/- mice (8 weeks/16 weeks): w/choline: 1.3 µM/1.8 µM w/betaine: 0.9 µM/1.1 µM w/TMAO: 2.5 µM/1.1 µM (0.5-1.1 µM for controls) apoE−/− mice (12 weeks/28 weeks): w/choline: 1.3µM/1.6 µM w/betaine: 0.8 µM/0.8 µM w/TMAO: 0.9 µM/1.3 µM (1.0 µM/0.8 µM for controls) |
* Plasma TMAO level consecutive to the indicated intervention. In the case of multiple dosing levels, it corresponds to the maximum dosage for beneficial/neutral effects and means that they are dosages that result in significant, relevant effects. Several concentrations are approximations derived from graphical illustrations, e.g., bar graphs (numerical values were not always published). Some baseline values are rather high, possibly indicating alternate methodology (see also section on methodology). ** See footnote ** of Table 2 for calculation.