Use of dietary phytochemicals for inhibition of trimethylamine N-oxide formation

doi:10.1016/j.jnutbio.2021.108600

The Journal of Nutritional Biochemistry

Volume 91, May 2021, 108600

https://doi.org/10.1016/j.jnutbio.2021.108600 Get rights and content

Abstract

Trimethylamine-N-oxide (TMAO) has been reported as a risk factor for atherosclerosis development, as well as for other cardiovascular disease (CVD) pathologies. The objective of this review is to provide a useful summary on the use of phytochemicals as TMAO-reducing agents. This review discusses the main mechanisms by which TMAO promotes CVD, including the modulation of lipid and bile acid metabolism, and the promotion of endothelial dysfunction and oxidative stress. Current knowledge on the available strategies to reduce TMAO formation are discussed, highlighting the effect and potential of phytochemicals. Overall, phytochemicals (i.e., phenolic compounds or glucosinolates) reduce TMAO formation by modulating gut microbiota composition and/or function, inhibiting host's capacity to metabolize TMA to TMAO, or a combination of both. Perspectives for design of future studies involving phytochemicals as TMAO-reducing agents are discussed. Overall, the information provided by this review outlines the current state of the art of the role of phytochemicals as TMAO reducing agents, providing valuable insight to further advance in this field of study.

Section snippets

Microbial metabolism of TMAO precursors

TMAO is formed via meta-organismal metabolism of dietary substrates containing a trimethylamine group (Fig. 1): The first step includes the oxidation of dietary substrates or moieties (i.e., choline and L-carnitine) by gut bacteria to trimethylamine (TMA) [11,12]. TMA can then be absorbed by the host and oxidized into TMAO [13]; this can amount to up to 40%–60% of the dose ingested [14]. Thus, TMAO is an oxidized product of gut-microbiota generated TMA. TMAO can also be found per se in the diet

Relationship of TMAO and CVD

TMAO has a wide range of molecular and physiological effects associated with the development of atherosclerosis and other CVD-related pathologies [64]. TMAO levels have been recently demonstrated to improve the prediction of CVD beyond traditional risk factors, including systolic blood pressure, body mass index, high-density lipoprotein-cholesterol (HDL-c), total triglycerides (TG), and diabetes mellitus [65]. However, several studies associate TMAO and atherosclerosis development in different

Strategies to reduce in vivo TMAO formation

Given the emerging associations between TMAO levels and atherosclerosis, there is great interest in developing effective strategies to reduce levels of this risk factor in efforts to mitigate atherosclerosis risk. Several known strategies can be followed to reduce TMAO formation in vivo (Fig. 2), which have been tested in human, rat and mice models (Supplementary Table 5). So far, there does not exist a specific approved drug to lower TMAO, but some drugs do report TMAO-lowering effects,

Phytochemicals with TMAO-lowering bioactivities

Phytochemicals are plant secondary metabolites that are generally non-essential for humans and do not report clinical manifestations due to their deficiency (major exceptions: carotenoids and tocopherols/tocotrienols), but that may be relevant to provide added health benefits during adulthood and aging [167]. Some phytochemicals such as phenolic compounds, carotenoids and phytosterols have reported a wide and diverse range of bioactivities to prevent/manage CVD development [5], [6], [7].

Research opportunities in the use of phytochemicals as TMAO-lowering bioactive components

There has been great interest in strategies for the reduction of TMAO formation. However, there still exists lack of safe, clearly effective, non-pharmacological ways to reliably control TMAO levels beyond substrate reduction. Overall, future studies not only should focus on the efficacy of a compound/treatment to reduce TMAO formation, but also on elucidating the mechanisms by which this is achieved. Indeed, the upstream molecular mechanisms remain poorly understood, which makes developments

Concluding remarks

TMAO, an oxidized product of microbiota-derived TMA, is linked to the development of CVD, especially atherosclerosis. Strategies to reduce TMAO formation include dietary changes, modulation of gut microbiota composition (pre- and pro-biotics) and/or function (gene copy numbers and expression of TMA-lyase, and TMA-lyase inhibition), and inhibition of host FMO3. Phytochemicals are emerging as a potential strategy to reduce TMAO levels. To date, promising phytochemicals include phenolic compounds

Author contributions

Lisard Iglesias-Carres: Conceptualization, Writing - original draft

Michael D. Hughes: Writing - review & editing, Cortney N. Steele: Writing - review & editing, Monica A. Ponder: Writing - review & editing, Kevin P. Davy: Writing - review & editing, Andrew P. Neilson: Conceptualization, Writing - review & editing

Declaration of competing interests

The authors declare no conflict of interest.

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The effects of supplementation with L-arginine (L-arg), the precursor of nitric oxide (NO), on vascular and cardiometabolic health have largely been explored. Whether other mechanisms of the action of L-arg exist remains unknown, as arginine metabolism is complicated.
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In a randomized, double-blind, crossover study, 32 healthy overweight adults (mean age 45 y) with cardiometabolic risk (fasting plasma triglycerides >150 mg/dL; waist circumference >94 cm [male] or >80 cm [female]) were treated with 1.5 g sustained-release L-arg 3 times/d (4.5 g/d) or placebo for 4 wk. On the last day of treatment, volunteers consumed a high-fat meal challenge (900 kcal, 80% as fat, 13% as carbohydrate, and 7% as protein). Plasma was collected at fasting, 2, 4, and 6 h after the challenge, and the metabolome was analyzed by high-resolution liquid chromatography–mass spectrometry. Metabolic profiles were analyzed using linear mixed models–principal component analysis.
The challenge meal explained most of the changes in the metabolome. The overall effect of L-arg supplementation significantly explained 0.5% of the total variance, irrespective of the response to the challenge meal (P < 0.05). Among the metabolites that explain most of the L-arg effect, we found many amino acids, including branched-chain amino acids, that were decreased by L-arg supplementation. L-arg also decreased trimethylamine N-oxide (TMAO). Other changes suggest that L-arg increased methyl demand.
Analysis of the effect of 4 wk of L-arg supplementation on the metabolome reveals important effects on methyl balance and gut microbiota activity, such as a decrease in TMAO. Further studies are needed to investigate those mechanisms and the implications of these changes for long-term health.
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Dietary phenolics and their microbial metabolites are poor inhibitors of trimethylamine oxidation to trimethylamine N-oxide by hepatic flavin monooxygenase 3
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High circulating levels of trimethylamine N-oxide (TMAO) have been associated with cardiovascular disease risk. TMAO is formed through a microbiome-host pathway utilizing primarily dietary choline as a substrate. Specific gut microbiota transform choline into trimethylamine (TMA), and, when absorbed, host hepatic flavin-containing monooxygenase 3 (FMO3) oxidizes TMA into TMAO. Chlorogenic acid and its metabolites reduce microbial TMA production in vitro. However, little is known regarding the potential for chlorogenic acid and its bioavailable metabolites to inhibit the last step: hepatic conversion of TMA to TMAO. We developed a screening methodology to study FMO3-catalyzed production of TMAO from TMA. HepG2 cells were unable to oxidize TMA into TMAO due to their lack of FMO3 expression. Although Hepa-1 cells did express FMO3 when pretreated with TMA and NADPH, they lacked enzymatic activity to produce TMAO. Rat hepatic microsomes contained active FMO3. Optimal reaction conditions were: 50 µM TMA, 0.2 mM NADPH, and 33 µL microsomes/mL reaction. Methimazole (a known FMO3 competitive substrate) at 200 µM effectively reduced FMO3-catalyzed conversion of TMA to TMAO. However, bioavailable chlorogenic acid metabolites did not generally inhibit FMO3 at physiological (1 µM) nor supra-physiological (50 µM) doses. Thus, the effects of chlorogenic acid in regulating TMAO levels in vivo are unlikely to occur through direct FMO3 enzyme inhibition. Potential effects on FMO3 expression remain unknown. Intestinal inhibition of TMA production and/or absorption are thus likely their primary mechanisms of action.
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The chemical composition and in vitro biological activities of two globe artichoke varieties, Romanesco Siciliano (RS) and Spinoso Sardo (SS), were studied for their antioxidant activity and potential effects against age-related diseases. The globe artichoke extracts were subjected to three antioxidant activity tests, DPPH, ABTS and FRAP, and to the acetylcholinesterase neuro-related assay. The potential artichoke cardioprotective capacity was investigated by evaluating the inhibition of choline-d₉ conversion into proatherogenic trimethylamine-d₉ (TMA-d₉), in an ex vivo-in vitro fecal fermentation model. In both globe artichoke varieties, the polyphenols profile was similar with chlorogenic and di-caffeoylquinic acids as the main polyphenols identified, although the amount changed with the variety (RS: 108.5; SS: 64.8 mg polyphenols/g DW). The RS sample gave the highest values of antioxidant and anti-acetylcholinesterase activities and mostly inhibited choline-d₉ utilization (+28.0 % choline-d₉ Area Under the Curve (AUC) compared to control), and TMA-d₉ production (-31.9 % TMA-d₉ AUC compared to control). All the reported results demonstrated that globe artichoke may provide potential cardioprotective, neuroprotective and antioxidant effects.
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The cytotoxicity of TMAO on chondrocytes was assayed. Piezo1 expression was measured after TMAO intervention. Pathological mechanical loading or Yoda1 (a specific Piezo1 channel activator) was administered in chondrocytes. The calcium levels and cytoskeleton in chondrocytes were observed by fluorescence microscopy. Flow cytometry, western blotting, and mitochondrial membrane potential assays were utilized to evaluate apoptosis. A rat OA model was constructed by anterior cruciate ligament transection. Hematoxylin-eosin staining, Safranin-O/Fast Green staining, immunochemistry, and TUNEL were applied to estimate OA severity.
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Use of dietary phytochemicals for inhibition of trimethylamine N-oxide formation

Abstract

Section snippets

Microbial metabolism of TMAO precursors

Relationship of TMAO and CVD

Strategies to reduce in vivo TMAO formation

Phytochemicals with TMAO-lowering bioactivities

Research opportunities in the use of phytochemicals as TMAO-lowering bioactive components

Concluding remarks

Author contributions

Declaration of competing interests

Food Res Int

J Funct Foods

J Funct Foods

Biomed Pharmacother

J Food Compos Anal

J Nutr

J Biol Chem

Cell

Cell Metab

Food Chem

FEMS Microbiol Lett

Food Chem

Food Chem Toxicol

Cell

Biochim Biophys Acta

Biochem Pharmacol

Biochem Pharmacol

Biochem Pharmacol

Biochem Pharmacol

Biochem Pharmacol

Arch Biochem Biophys

J Nutr Biochem

Fitoterapia

Biochem Pharmacol

J Funct Foods

Atherosclerosis

Biomed Pharmacother

J Biol Chem

Autoimmun Rev

Free Radic Biol Med

Int Immunopharmacol

Am J Cardiol

Food Chem Toxicol

Chem Biol Interact

Lab Investig

J Thromb Haemost

Can J Cardiol

Atherosclerosis

J Nutr

Resveratrol attenuates trimethylamine-N-oxide (TMAO)-induced atherosclerosis by regulating TMAO synthesis and bile acid metabolism via remodeling of the gut microbiota

MBio

Grape seed proanthocyanidins influence gut microbiota and enteroendocrine secretions in female rats

Food Funct

Phytosterols in the treatment of hypercholesterolemia and prevention of cardiovascular diseases

Arq Bras Cardiol

Phenolic compounds in fruits and beverages consumed as part of the mediterranean diet: their role in prevention of chronic diseases

Phytochem Rev

Effects of grape pomace polyphenolic extract (Taurisolo®) in reducing TMAO serum levels in humans: Preliminary results from a randomized, placebo-controlled, cross-over study

Nutrients

Effect of grape pomace polyphenols with or without pectin on TMAO serum levels assessed by LC/MS-based assay: a preliminary clinical study on overweight/obese subjects

Front Pharmacol

Development of a gut microbe–targeted nonlethal therapeutic to inhibit thrombosis potential

Nat Med

Microbiology meets big data: the case of gut microbiota–derived trimethylamine

Annu Rev Microbiol

Metabolic retroconversion of trimethylamine N-oxide and the gut microbiota

BioRxiv

Disclosure of the metabolic retroversion of trimethylamine N-oxide in humans: a pharmacogenetic approach

Clin Pharmacol Ther

Trimethylamine-N-oxide (TMAO) response to animal source foods varies among healthy young men and is influenced by their gut microbiota composition: a randomized controlled trial

Mol Nutr Food Res