Metabolomics studies hold promise for the discovery of pathways linked to disease processes. Cardiovascular disease (CVD) represents the leading cause of death and morbidity worldwide. Here we used a metabolomics approach to generate unbiased small-molecule metabolic profiles in plasma that predict risk for CVD. Three metabolites of the dietary lipid phosphatidylcholine—choline, trimethylamine N-oxide (TMAO) and betaine—were identified and then shown to predict risk for CVD in an independent large clinical cohort. Dietary supplementation of mice with choline, TMAO or betaine promoted upregulation of multiple macrophage scavenger receptors linked to atherosclerosis, and supplementation with choline or TMAO promoted atherosclerosis. Studies using germ-free mice confirmed a critical role for dietary choline and gut flora in TMAO production, augmented macrophage cholesterol accumulation and foam cell formation. Suppression of intestinal microflora in atherosclerosis-prone mice inhibited dietary-choline-enhanced atherosclerosis. Genetic variations controlling expression of flavin monooxygenases, an enzymatic source of TMAO, segregated with atherosclerosis in hyperlipidaemic mice. Discovery of a relationship between gut-flora-dependent metabolism of dietary phosphatidylcholine and CVD pathogenesis provides opportunities for the development of new diagnostic tests and therapeutic approaches for atherosclerotic heart disease.
At a glance
- The role of infection in restenosis and atherosclerosis: focus on cytomegalovirus. Lancet 348 (suppl. 1). S13–S17 (1996) et al.
- Association of Helicobacter pylori and Chlamydia pneumoniae infections with coronary heart disease and cardiovascular risk factors. Br. Med. J. 311, 711–714 (1995) et al.
- Chronic infections and coronary heart disease: is there a link? Lancet 350, 430–436 (1997) , &
- Serological evidence of an association of a novel Chlamydia, TWAR, with chronic coronary heart disease and acute myocardial infarction. Lancet 332, 983–986 (1988) et al.
- Azithromycin for the secondary prevention of coronary heart disease events—the WIZARD study: a randomized controlled trial. J. Am. Med. Assoc. 290, 1459–1466 (2003) et al.
- Antibiotic treatment of Chlamydia pneumoniae after acute coronary syndrome. N. Engl. J. Med. 352, 1646–1654 (2005) et al.
- Effects of antibiotic therapy on outcomes of patients with coronary artery disease: a meta-analysis of randomized controlled trials. J. Am. Med. Assoc. 293, 2641–2647 (2005) , &
- Infectious agents are not necessary for murine atherogenesis. J. Exp. Med. 191, 1437–1442 (2000) et al.
- Host-bacterial mutualism in the human intestine. Science 307, 1915–1920 (2005) , , , &
- An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444, 1027–1031 (2006) et al.
- Metabolic profiling reveals a contribution of gut microbiota to fatty liver phenotype in insulin-resistant mice. Proc. Natl Acad. Sci. USA 103, 12511–12516 (2006) et al.
- Biochemical and clinical aspects of the human flavin-containing monooxygenase form 3 (FMO3) related to trimethylaminuria. Curr. Drug Metab. 4, 151–170 (2003) et al.
- The exogenous origin of trimethylamine in the mouse. Metabolism 41, 135–136 (1992) , , &
- Concentrations of choline-containing compounds and betaine in common foods. J. Nutr. 133, 1302–1307 (2003) , , &
- Isoform specificity of trimethylamine N-oxygenation by human flavin-containing monooxygenase (FMO) and P450 enzymes: selective catalysis by fmo3. Biochem. Pharmacol. 56, 1005–1012 (1998) et al.
- Dietary precursors of trimethylamine in man: a pilot study. Food Chem. Toxicol. 37, 515–520 (1999) , &
- Trimethylaminuria: the fish malodor syndrome. Drug Metab. Dispos. 29, 517–521 (2001) &
- An integrative genomics approach to infer causal associations between gene expression and disease. Nature Genet. 37, 710–717 (2005) et al.
- Missense mutation in flavin-containing mono-oxygenase 3 gene, FMO3, underlies fish-odour syndrome. Nature Genet. 17, 491–494 (1997) , , , &
- Identification of pathways for atherosclerosis in mice: integration of quantitative trait locus analysis and global gene expression data. Circ. Res. 101, e11–e30 (2007) et al.
- The freeze-avoidance response of smelt Osmerus mordax: initiation and subsequent suppression of glycerol, trimethylamine oxide and urea accumulation. J. Exp. Biol. 205, 1419–1427 (2002) , , , &
- Prevention of polymerization of M and Z α1-Antitrypsin (α1-AT) with trimethylamine N-oxide. Implications for the treatment of α1-AT deficiency. Am. J. Respir. Cell Mol. Biol. 24, 727–732 (2001) , , , &
- Natural osmolyte trimethylamine N-oxide corrects assembly defects of mutant branched-chain α-ketoacid decarboxylase in maple syrup urine disease. J. Biol. Chem. 276, 40241–40246 (2001) &
- Accumulation of trimethylamine and trimethylamine-N-oxide in end-stage renal disease patients undergoing haemodialysis. Nephrol. Dial. Transplant. 21, 1300–1304 (2006) , , , &
- DNA methylation and atherosclerosis. J. Nutr. 132, 2406S–2409S (2002) , &
- Nutrition and aberrant DNA methylation patterns in atherosclerosis: more than just hyperhomocysteinemia? J. Nutr. 135, 5–8 (2005) , &
- Cardiovascular disease in choline-deficient rats. Effects of choline deficiency, nature and level of dietary lipids and proteins, and duration of feeding on plasma and liver lipid values and cardiovascular lesions. Arch. Pathol. 73, 190–209 (1962) &
- Whole blood choline and plasma choline in acute coronary syndromes: prognostic and pathophysiological implications. Clin. Chim. Acta 383, 103–109 (2007) , , , &
- Usefulness of elevations in serum choline and free F2-isoprostane to predict 30-day cardiovascular outcomes in patients with acute coronary syndrome. Am. J. Cardiol. 104, 638–643 (2009) et al.
- Usual choline and betaine dietary intake and incident coronary heart disease: the Atherosclerosis Risk in Communities (ARIC) study. BMC Cardiovasc. Disord. 7, 20 (2007) , , , &
- Diversity of the human intestinal microbial flora. Science 308, 1635–1638 (2005) et al.
- Microbial ecology: human gut microbes associated with obesity. Nature 444, 1022–1023 (2006) , , &
- Symbiotic gut microbes modulate human metabolic phenotypes. Proc. Natl Acad. Sci. USA 105, 2117–2122 (2008) et al.
- Regulation of serum amyloid A3 (SAA3) in mouse colonic epithelium and adipose tissue by the intestinal microbiota. PLoS ONE 4, e5842 (2009) , , &
- Probiotic modulation of symbiotic gut microbial–host metabolic interactions in a humanized microbiome mouse model. Mol. Syst. Biol. 4, 157 (2008) et al.
- 2008) Statistical Computing with R (Chapman & Hall/CRC,
- Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 118, 229–241 (2004) , , , &
- Genetic and genomic analysis of a fat mass trait with complex inheritance reveals marked sex specificity. PLoS Genet. 2, e15 (2006) et al.
- Quantitative assay for mouse atherosclerosis in the aortic root. Methods Mol. Med. 129, 83–95 (2006) &
- A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226, 497–509 (1957) , &
- A simple and sensitive enzymatic method for cholesterol quantification in macrophages and foam cells. J. Lipid Res. 51, 3364–3369 (2010) , , &
- Genetic factors for resistance to diet-induced obesity and associated metabolic traits on mouse chromosome 17. Mamm. Genome 20, 71–82 (2009) et al.
- PicoGreen quantitation of DNA: effective evaluation of samples pre- or post-PCR. Nucleic Acids Res. 24, 2623–2625 (1996) , &
- Protein carbamylation links inflammation, smoking, uremia and atherogenesis. Nature Med. 13, 1176–1184 (2007) et al.
- Lipoprotein (a) levels and long-term cardiovascular risk in the contemporary era of statin therapy. J. Lipid Res. 51, 3055–3061 (2010) et al.
- MDRD equation estimates of glomerular filtration rate in potential living kidney donors and renal transplant recipients with impaired graft function. Nephrol. Dial. Transplant. 17, 2036–2037 (2002) , , , &
- Appropriateness of cholesterol management in primary care by sex and level of cardiovascular risk. Prev. Cardiol. 12, 95–101 (2009) et al.
- The measurement of dimethylamine, trimethylamine, and trimethylamine N-oxide using capillary gas chromatography-mass spectrometry. Anal. Biochem. 187, 234–239 (1990) , &
- Lymphatic endothelium-specific hyaluronan receptor LYVE-1 is expressed by stabilin-1+, F4/80+, CD11b+ macrophages in malignant tumours and wound healing tissue in vivo and in bone marrow cultures in vitro: implications for the assessment of lymphangiogenesis. J. Pathol. 209, 67–77 (2006) et al.
- Conditional macrophage ablation demonstrates that resident macrophages initiate acute peritoneal inflammation. J. Immunol. 174, 2336–2342 (2005) et al.
- Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages. J. Biol. Chem. 277, 49982–49988 (2002) et al.
- Tissue-specific expression and regulation of sexually dimorphic genes in mice. Genome Res. 16, 995–1004 (2006) et al.
- Activation of the unfolded protein response occurs at all stages of atherosclerotic lesion development in apolipoprotein E-deficient mice. Circulation 111, 1814–1821 (2005) , , &
- Dietary fish oil reduces intercellular adhesion molecule 1 and scavenger receptor expression on murine macrophages. Atherosclerosis 152, 43–50 (2000) , &
- Hypochlorite-oxidized low-density lipoprotein upregulates CD36 and PPARγ mRNA expression and modulates SR-BI gene expression in murine macrophages. Mol. Cell. Biochem. 277, 143–152 (2005) , &
- Dietary trans 10, cis 12-conjugated linoleic acid reduces the expression of fatty acid oxidation and drug detoxification enzymes in mouse liver. Br. J. Nutr. 97, 58–66 (2007) , , &
- Quantitative analysis of FMO gene mRNA levels in human tissues. Drug Metab. Dispos. 34, 19–26 (2006) &
- Myeloid blasts are the mouse bone marrow cells prone to differentiate into osteoclasts. J. Leukoc. Biol. 85, 919–927 (2009) , , , &
- A new method of quatenizing amines and its use in amino acid and peptide chemistry. Can. J. Chem. 54, 3310–3311 (1976) &
- Multiple isotopic labels for quantitative mass spectrometry. Anal. Chem. 80, 9298–9309 (2008) , &
- The lipid whisker model of the structure of oxidized cell membranes. J. Biol. Chem. 283, 2385–2396 (2008) et al.
- Student’s t-test. Nutrition 9, 386 (1993) &
- Mann–Whitney/Wilcoxon’s nonparametric cumulative probability distribution. Comput. Methods Programs Biomed. 63, 21–28 (2000) &
- Introduction to biostatistics: part 6, correlation and regression. Ann. Emerg. Med. 19, 1462–1468 (1990) &
- S100-β, melanoma-inhibiting activity, and lactate dehydrogenase discriminate progressive from nonprogressive American Joint Committee on Cancer stage IV melanoma. J. Clin. Oncol. 17, 1891–1896 (1999) et al.
- Comparative sensitivity of survival-adjusted chi-square and normal statistics for the mutagenesis fluctuation assay. J. Appl. Toxicol. 6, 95–100 (1986) , &
- Logistic regression vs other generalized linear models to estimate prevalence rate ratios. Rev. Epidemiol. Sante Publique 47, 593–604 (1999) , , &
- Test for linear trend in 2 × K ordered tables with open-ended categories. Biometrics 53, 1163–1169 (1997)
- Supplementary Information (2M)
This file contains Supplementary Tables 1-8 and Supplementary Figures 1-24 with legends.