Untargeted metabolomics identifies trimethyllysine, a TMAO-producing nutrient precursor, as a predictor of incident cardiovascular disease risk

Abstract

Using an untargeted metabolomics approach in initial (N = 99 subjects) and replication cohorts (N = 1,162), we discovered and structurally identified a plasma metabolite associated with cardiovascular disease (CVD) risks, N6,N6,N6-trimethyl-L-lysine (trimethyllysine, TML). Stable-isotope-dilution tandem mass spectrometry analyses of an independent validation cohort (N = 2,140) confirmed TML levels are independently associated with incident (3-year) major adverse cardiovascular event risks (hazards ratio [HR], 2.4; 95% CI, 1.7-3.4) and incident (5-year) mortality risk (HR, 2.9; 95% CI, 2.0-4.2). Genome-wide association studies identified several suggestive loci for TML levels, but none reached genome-wide significance; and d9(trimethyl)-TML isotope tracer studies confirmed TML can serve as a nutrient precursor for gut microbiota-dependent generation of trimethylamine (TMA) and the atherogenic metabolite trimethylamine N-oxide (TMAO). Although TML was shown to be abundant in both plant- and animal-derived foods, mouse and human fecal cultures (omnivores and vegans) showed slow conversion of TML to TMA. Furthermore, unlike chronic dietary choline, TML supplementation in mice failed to elevate plasma TMAO or heighten thrombosis potential in vivo. Thus, TML is identified as a strong predictor of incident CVD risks in subjects and to serve as a dietary precursor for gut microbiota-dependent generation of TMAO; however, TML does not appear to be a major microbial source for TMAO generation in vivo.

Keywords: Atherosclerosis; Cardiology; Cardiovascular disease; Cholesterol; Vascular Biology.

Figure 1. Untargeted metabolomics studies discover candidate compound with m/z of 189.1598 is associated with CVD and is trimethyllysine.

(A) Forest plot indicating plasma metabolite of unknown structure with m/z of 189.1 is associated with risk for cardiovascular disease (CVD) according to relative peak area intensity ranked by tertiles among subjects (N = 99) in discovery cohort 1. (B) Forest plot indicating plasma metabolite of unknown structure with high-resolution m/z of 189.1598 is associated with the risk for incident CVD and mortality risks according to relative peak intensity from untargeted mass spectrometry analyses of subjects (N = 1,162; discovery cohort 2). MACE, major adverse cardiac events, including myocardial infarction, stroke, or death. The analyses were performed using R 3.4.1. (C) Collision-induced dissociation (CID) spectrum in positive-ion mode of the metabolite m/z of 189.1598 in plasma. (D) CID spectra in positive mode of synthetic trimethyllysine (TML) standard. (E) Demonstration of cochromatography of multiple unique parent→daughter ion transitions for plasma analyte m/z 189.1598 and synthetic d9-TML.

Figure 2. Stable-isotope-dilution LC-MS/MS analyses verify systemic levels of TML are associated with incident cardiovascular disease risks independent of TMAO.

Kaplan-Meier estimates and 3-year risks (HR [95% CI]) for (A) major adverse cardiac events (MACE, including myocardial infarction, stroke or death); and (B) all-cause mortality (5 years) ranked by trimethyllysine (TML) quartiles in the validation cohort (N = 2,140). (C) Forest plots indicate the HR (95% CI) for incident (3-year) risks for MACE and all-cause mortality (5-year) according to TML quartiles. HR (unadjusted, open circles) and multivariable Cox model 1 adjusted (filled black circles; adjusted for age, sex, high-density lipoprotein [HDL], low-density lipoprotein [LDL], smoking, diabetes mellitus, hypertension, C-reactive protein level), or model 2 adjusted (filled red squares, adjusted for model 1 plus trimethylamine N-oxide [TMAO]). The 5%–95% confidence interval is indicated by line length. The analyses were performed using R 3.4.1. HR, hazard ratio.

Figure 3. Relationship between plasma TML and both CVD risks and TMAO.

(A) Correlation between plasma levels of trimethyllysine (TML) and trimethylamine N-oxide (TMAO) in the validation cohort (N = 2,140). (B) Kaplan–Meier plot illustrating the relationship between plasma TML and risk of incident (3-year) major adverse cardiac events (MACE); and (C) incident (5-year) mortality risk according to TML and TMAO levels where each marker is categorized above versus below the median level in the validation cohort (N = 2,140). Also shown are hazard ratio (HR [95% CI]) for the indicated TML and TMAO grouping using either an unadjusted model, or following adjustments for traditional cardiovascular disease (CVD) risk factors (age, sex, HDL, LDL, smoking, diabetes mellitus, hypertension), high-sensitivity C-reactive protein level, estimated glomerular filtration rate (eGFR ), history of CAD and medications. Median plasma concentration of TML (0.53 μM) and TMAO (3.69 μM) within the cohort was used to stratify subjects as high (≥median) or low (<median) values. (D) Plot of HR for incident 3-year MACE risk stratified by indicated low, intermediate, and high levels of TMAO (cutoff values of 2.5 and 6 μM) and TML (cutoff values of 0.5 and 0.8 μM). *P < 0.05, **P < 0.01, ***P < 0.001 relative to low/low TMAO/TML group. (E) Comparison of plasma levels of TML in free form versus protein-bound TML levels in random samples from both healthy subjects and subjects with CVD, as indicated. The analyses were performed using R 3.4.1.

Figure 4. Results of a GWAS for plasma TML levels in the GeneBank cohort.

The Manhattan plot for plasma TML levels (N = 1,297 subjects) shows 6 suggestively associated loci on chromosomes 1q44, 3p24.1, 5p15.33, 6p24.1, 6q23.3, and 8p21.2. The genome-wide thresholds for significant (P = 5.0 × 10^–8) and suggestive (P = 5.0 × 10^–7) association are indicated by the horizontal red and blue lines, respectively. P values were obtained using linear regression with natural-log-transformed values and adjustment for age and sex.

Figure 5. d9-TML and d9-choline oral isotope tracer studies.

Synthetic d9-TML or d9-choline was administered by gastric gavage to the indicated numbers of C57BL/6J mice and serial plasma levels of the indicated isotope-labeled compounds were quantified by LC-MS/MS, as described under Methods. (A–C) Plasma levels of d9-trimethylamine (d9-TMA), d9-trimethylamine N-oxide (d9-TMAO), and d9-trimethyllysine (d9-TML) are shown at the indicated times following oral challenge with time of d9-TML gavage designated as T = 0. (D–F) Plasma d9-TMA, d9-TMAO, or d9-choline at the indicated times following oral d9-choline challenge. (G) Plasma concentrations of d9-TMAO were also quantified by LC-MS/MS in C57BL/6J female mice at the indicated times following challenge with d9-TML via gastric gavage, either before or following 3-week administration of a cocktail of broad-spectrum poorly absorbed antibiotics, as described under Methods. All data are presented as mean ± standard error.

Figure 6. Characterization of microbial TMA formation from TML versus alternative TMA-generating nutrients in mouse intestines, human fecal cultures, and cloned microbial TMA lyases.

(A) Intestines from conventionally reared C57BL/6J mice (N = 7) were sectioned as indicated, incubated with either d9-trimethyllysine (d9-TML), d9-carnitine, or d9-choline anaerobically, and then production of d9-trimethylamine (d9-TMA) quantified as described in Methods. (B) Human feces from vegans (N = 8) and omnivores (N = 10–15 as indicated) were incubated with either d9-TML, d9-carnitine, or d9-choline anaerobically, and then production of d9-TMA quantified as described in Methods. Student’s t test (2 tailed) was used to examine the difference between groups. All data are presented as mean ± standard error. (C) Recombinant microbial TMA lyases were cloned and expressed, and then activity (production of d9-TMA) with the indicated d9-labeled substrates determined as described under Methods.

Figure 7. Impact of TML- versus choline-supplemented diet on plasma levels of TML and TMAO, and in vivo thrombosis potential.

Groups of mice (N = 15) were placed on chemically defined diets supplemented with either 1% trimethyllysine (TML) (A and B) or 1% choline (C) for 11 days as described under Methods. At the indicated times, plasma levels of trimethylamine N-oxide (TMAO) (A and C) or TML (B) were quantified by stable-isotope-dilution LC-MS/MS. (D) In addition, after 11 days of the indicated diet (N = 10), the impact of supplemental dietary TML versus choline on both TMAO levels and in vivo thrombosis potential, as monitored using the FeCl₃ carotid artery injury model, was determined as described under Methods. Plasma levels of metabolites after 11 days of the indicated diets were as follows: for 1% TML group, TMAO =4.5 ± 0.7 μM, TML = 37.0 ± 4.9 μM. For 1% choline group, TMAO = 90.6 ± 19.5 μM, TML = 0.8 ± 0.1 μM. For chemically defined chow group, TMAO= 2.6 ± 0.4 μM; TML = 0.9 ± 0.2 μM. Student’s t test (2 tailed) was used to examine the difference between groups. All data are presented as mean ± standard error.