Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis

Abstract

Intestinal microbiota metabolism of choline and phosphatidylcholine produces trimethylamine (TMA), which is further metabolized to a proatherogenic species, trimethylamine-N-oxide (TMAO). We demonstrate here that metabolism by intestinal microbiota of dietary L-carnitine, a trimethylamine abundant in red meat, also produces TMAO and accelerates atherosclerosis in mice. Omnivorous human subjects produced more TMAO than did vegans or vegetarians following ingestion of L-carnitine through a microbiota-dependent mechanism. The presence of specific bacterial taxa in human feces was associated with both plasma TMAO concentration and dietary status. Plasma L-carnitine levels in subjects undergoing cardiac evaluation (n = 2,595) predicted increased risks for both prevalent cardiovascular disease (CVD) and incident major adverse cardiac events (myocardial infarction, stroke or death), but only among subjects with concurrently high TMAO levels. Chronic dietary L-carnitine supplementation in mice altered cecal microbial composition, markedly enhanced synthesis of TMA and TMAO, and increased atherosclerosis, but this did not occur if intestinal microbiota was concurrently suppressed. In mice with an intact intestinal microbiota, dietary supplementation with TMAO or either carnitine or choline reduced in vivo reverse cholesterol transport. Intestinal microbiota may thus contribute to the well-established link between high levels of red meat consumption and CVD risk.

Figure 1. TMAO production from carnitine is a microbiota dependent process in humans

(a) Structure of carnitine and scheme of carnitine and choline metabolism to TMAO. L-Carnitine and choline (are both dietary trimethylamines that can be metabolized by microbiota to TMA. TMA is then further oxidized to TMAO by flavin monooxygenases (FMOs). (b) Scheme of human carnitine challenge test. After a 12 hour overnight fast, subjects received a capsule of d3-carnitine (250 mg) alone, or in some cases (as in data for subject shown) also an 8 ounce steak (estimated 180 mg L-carnitine), whereupon serial plasma and 24h urine collection was obtained for TMA and TMAO analyses. After a weeklong regimen of oral broad spectrum antibiotics to suppress the intestinal microbiota, the challenge was repeated (Visit 2), and then again a final third time after a ≥ three week period to permit repopulation of intestinal microbiota (Visit 3). Data shown in (panels c-e) are from a representative female omnivorous subject who underwent carnitine challenge. Data is organized to vertically correspond with the indicated visit schedule above (Visit 1, 2 or 3). (c,d) LC/MS/MS chromatograms of plasma TMAO or d3-TMAO in omnivorous subject using specific precursor → product ion transitions indicated at T = 8 hour time point for each respective visit. (e) Stable isotope dilution LC/MS/MS time course measurements stable isotope (d3) labeled TMAO and carnitine, in plasma collected from sequential venous blood draws at noted times.

Figure 2. The formation of TMAO from ingested L-carnitine is negligible in vegans, and fecal microbiota composition associates with plasma TMAO concentrations

(a-b) Data from a male vegan subject in the carnitine challenge consisting of co-administration of both 250 mg d3-carnitine and an 8 ounce sirloin steak, and for comparison, a representative female omnivore with frequent red meat consumption. (a) Plasma TMAO and d3-TMAO were quantified post carnitine challenge, and in a (b) 24 hour urine collection. (c) Baseline fasting plasma concentrations of from male and female (n = 26) vegans and vegetarians and (n = 51) omnivores. Boxes represent the 25^th, 50^th, and 75^th percentile and whiskers represent the 5^th and 95^th percentile. (d) Plasma d3-TMAO levels in male and female (n = 5) vegan and vegetarian versus (n = 5) omnivores participating in a d3-carnitine (250 mg) challenge without concomitant steak consumption. P value shown is for comparison between area under the curve (AUC) of groups using Wilcoxon non-parametric test. (e) Baseline plasma concentrations of TMAO associates with Enterotype 2 (Prevotella) in male and female subjects with a characterized gut microbiome enterotype. (f) Plasma TMAO concentrations (plotted on x axes) and the proportion of taxonomic operational units (OTUs, plotted on Y axes) were determined as described in Supplementary Methods. Subjects were grouped by dietary status as either vegan and vegetarian (n = 23) or omnivore (n = 30). P value shown is for comparisons between dietary groups using a robust Hotelling T² test.

Figure 3. The metabolism of carnitine to TMAO is an inducible trait and associates with microbiota composition

(a) d3-Carnitine challenge of mice on either a carnitine supplemented diet (1.3%) at 10 weeks and age versus age-matched normal chow controls. Plasma d3-TMA and d3-TMAO were measured at the indicated times following d3-carnitine administration by oral gavage using stable isotope dilution LC/MS/MS. Data points represents mean ± SE of 4 replicates per group. (b) Correlation heat map demonstrating the association between the indicated microbiota taxonomic genera and TMA and TMAO levels (all reported as mean ±SEM in μM) of mice grouped by dietary status (chow, n = 10 (TMA,1.3±0.4; TMAO, 17±1.9); and carnitine, n = 11 (TMA, 50±16; TMAO, 114±16). Red denotes a positive association, blue a negative association, and white no association. A single asterisk indicates a significant false discovery rate adjusted (FDR) association of P ≤ 0.1 and a double asterisk indicates a significant FDR adjusted association of P ≤ 0.01. (c) Plasma TMAO and TMA concentrations were determined by stable isotope dilution LC/MS/MS (plotted on x axes) and the proportion of taxonomic operational units (OTUs, plotted on Y axes) were determined. Statistical and laboratory analyses were performed as described in Supplementary Methods.

Figure 4. Relation between plasma carnitine and CVD risks

(a-c) Forrest plots of odds ratio of CAD, PAD, and CVD and quartiles of carnitine before (closed circles) and after (open circles) logistic regression adjustments with traditional cardiovascular risk factors including age, sex, history of diabetes mellitus, smoking, systolic blood pressure, low density lipoprotein cholesterol, and high density lipoprotein cholesterol. Bars represent 95% confidence intervals. (d) Relationship of fasting plasma carnitine levels and angiographic evidence of CAD. Boxes represent the 25^th, 50^th, and 75^th percentile of plasma carnitine and the whiskers represent the 10^th and 90^th percentile. The Kruskal-Wallis test was used to assess the degree of coronary vessel disease on L-carnitine levels. (e) Forrest plot of hazard ratio of MACE (death, non fatal-MI, stroke, and revascularization) and quartiles of carnitine unadjusted (closed circles), and after adjusting for traditional cardiovascular risk factors (open circles), or traditional cardiac risk factors plus creatinine clearance, history of MI, history of CAD, burden of CAD (one, two, or three vessel disease), left ventricular ejection fraction, baseline medications (ACE inhibitors, statins, β-blockers, and aspirin) and TMAO levels (open squares). Bars represent 95% confidence intervals. (f) Kaplan Meier plot (graph) and hazard ratios with 95% confidence intervals for unadjusted model, or following adjustments for traditional risk factors as in panel e. Median levels of carnitine (46.8 μM) and TMAO (4.6 μM) within the cohort were used to stratify subjects as ‘high’ (≥ median) or ‘low’ (< median) concentrations.

Figure 5. Dietary carnitine accelerates atherosclerosis and inhibits reverse cholesterol transport in a microbiota dependent fashion

(a) Representative Oil-red-O stained (counterstained with hematoxylin) aortic roots of 19 week old C57BL/6J, Apoe^−/− female mice on the indicated diets in the presence versus absence of antibiotics (ABS) as described under Methods. (b) Quantification of mouse aortic root plaque lesion area. 19 week-old C57BL/6J, Apoe^−/− female mice were started on the indicated diets at the time of weaning (4 weeks of age) before sacrifice, and lesion area was quantified as described under Methods. (c) Carnitine, TMA, and TMAO were determined using stable isotope dilution LC/MS/MS analysis of plasma recovered from mice at time of sacrifice. (d) Reverse cholesterol transport (RCT) (72 hour stool collection) in adult female (> 8 weeks of age) C57BL/6J, Apoe^−/− mice on normal chow versus diet supplemented with either carnitine or choline, as well as following suppression of microbiota using cocktail of antibiotics (+ ABS). Also shown are RCT (72 hour stool collection) results in adult female (> 8 weeks of age) C57BL/6J, Apoe^−/− mice on normal chow versus diet supplemented with TMAO. (e,f) Relative mRNA levels (to β-actin) of mouse liver candidate genes involved in bile acid synthesis or transport. Ephx1, epoxide hydrolase 1, microsomal.

Figure 6. Effect of TMAO on cholesterol and sterol metabolism

Measurement of (a) total bile acid pool size and composition, as well as (b) cholesterol absorption in adult female (> 8 weeks of age) C57BL/6J, Apoe^−/− mice on normal chow diet versus diet supplemented with TMAO for 4 weeks. (c) Summary scheme outlining pathway for microbiota participation in atherosclerosis via metabolism of dietary carnitine and choline forming TMA and TMAO, as well as the impact of TMAO on cholesterol and sterol metabolism in macrophages, liver and intestines. FMOs, flavin monooxygenases; TMA, trimethylamine; TMAO, trimethylamine-N-oxide; OST-α, solute carrier family 51, alpha subunit; ASBT, solute carrier family 10, member 2.