DISCUSSION
Using a targeted metabolomics approach aimed at identifying plasma metabolites whose levels predict risk of CVD in subjects, we have identified a novel pathway linking dietary lipid intake, intestinal microflora and atherosclerosis (Fig. 6). The pathway identified (dietary PC/choline -> gut flora-formed TMA -> hepatic FMO-formed TMAO) represents a unique additional nutritional contribution to the pathogenesis of CVD that involves choline metabolism, an obligate role for the intestinal microbial community, and regulation of surface expression levels of macrophage scavenger receptors known to participate in the atherosclerotic process. The pro-atherogenic gut flora-generated metabolite, TMAO, is formed in a two-step process initiated by gut flora-dependent cleavage of a trimethylamine species (e.g. PC, choline, betaine) generating the precursor TMA, and subsequent oxidation by FMO3 and possibly other FMOs (Fig. 6). PC is by far the most abundant dietary source of choline in most humans. The present results indicate that both environmental exposure (dietary lipid) and microbial flora participate in TMAO production and the atherogenic macrophage phenotype. While the present genetic studies also suggest a role for hepatic expression levels of one or more FMOs in both enhanced atherosclerotic plaque and decreased HDL levels in mice, the participation of FMOs in human atherosclerosis and HDL cholesterol levels remains to be established. Strong associations between systemic TMAO levels and both angiographic measures of coronary artery atherosclerotic burden and cardiac risks were observed among subjects; however, no correlation was observed between plasma TMAO levels and HDL cholesterol levels in subjects. It remains to be determined whether genetic impairment in FMO3 alone or in combination with other FMOs, is protective for CVD. No phenotype other than the objectionable odor accompanying this disorder is known. In fact, individuals with trimethylaminuria often become vegans, since reducing ingestion of dietary lipids decreases TMA production and the associated noxious odor. Little is also known about the biologic functions of TMAO in humans. TMAO apparently serves as an osmolite in the freeze-avoidance response of some species21. In vitro it can function as a small molecule chaperone, affecting the folding and functioning of some proteins22–23. In addition, TMAO and TMA accumulate in plasma of subjects on maintenance hemodialysis24, suggesting that TMAO might contribute to the well-established enhanced CVD risk noted among subjects with end-stage renal disease.
Choline is an essential nutrient that is usually grouped within the vitamin B complex. Choline and its metabolite, betaine, are methyl donors, along with folate, and are metabolically linked to transmethylation pathways including synthesis of the CVD risk factor homocysteine. Deficiency in both choline and betaine are suggested to produce epigenetic changes in genes linked to atherosclerosis25–26, and acute choline and methionine deficiency in rodent models causes lipid accumulation in liver (steatohepatitis), heart and arterial tissues27. Alternatively, some studies have reported an association between increased whole blood levels of total choline and cardiovascular disease28–29. Few clinical studies have examined the relationship between choline intake and CVD30, probably because accurate measures of the choline content of most foods has only recently become available14 (http://www.nal.usda.gov/fnic/foodcomp/Data/Choline/Choln02.pdf). The association between dietary choline (and alternative trimethyl amine containing species) and atherosclerosis is complex and will be influenced by the composition of the intestinal microflora.
The human intestinal microbial community is an enormous and diverse ecosystem with known functions in nutrition, gut epithelial cell health, and innate immunity31. Intestinal flora also has recently been implicated in development of some metabolic phenotypes such as obesity and insulin resistance, as well as alterations in immune responses11,32–34. As far as we know, the present studies are the first to identify a direct link between intestinal microflora, dietary PC and CVD risk. These results suggest that an appropriately designed probiotic intervention may serve as a therapeutic strategy for CVD. Interestingly, production of TMAO can be altered by probiotic administration35. Thus, in addition to the current clinical recommendation for general reduction in dietary lipids, manipulation of commensal microbial composition may be a novel therapeutic approach for the prevention and treatment of atherosclerotic heart disease and its complications. Finally, the present studies suggest an additional novel treatment for atherosclerosis - blocking the presumed pathogenic biochemical pathway at the level of the gut flora through use of a non-systemically absorbed inhibitor.