Dietary l-carnitine produces γBB in a gut microbiota–dependent manner in humans. In prior studies we showed gut microbiota–dependent TMA and TMAO generation following oral l-carnitine ingestion in omnivores, but virtually nonexistent TMA/TMAO formation from oral l-carnitine in long-standing (>1 year) vegans and vegetarians (6). However, we did not look for γBB formation in those studies. Therefore, in initial pilot clinical studies (Figure 1), we first explored (in omnivores) whether γBB could be formed from l-carnitine following oral ingestion of heavy isotope–labeled l-carnitine (d 3 -l-carnitine), and the potential participation of gut microbiota in that reaction. At the initial baseline visit, serial venous sampling performed after oral d 3 -l-carnitine challenge revealed rapid increases in plasma concentrations of d 3 -l-carnitine, and subsequent increases in d 3 -γBB and d 3 -TMAO following a lag phase (Figure 1, A–C, filled circles). After the initial baseline challenge, subjects were placed on a week-long oral regimen of a cocktail of poorly absorbed antibiotics previously shown to effectively suppress intestinal microbiota (6), and then the d 3 -l-carnitine challenge was repeated (Figure 1, open circles). Complete suppression of d 3 -TMAO and almost complete suppression of d 3 -γBB formation were observed (Figure 1). These results are consistent with results observed in mice (26) and strongly support a role for gut microbiota in γBB generation from dietary l-carnitine in humans. Finally, examination of plasma d 3 -l-carnitine concentrations before versus after exposure to the antibiotics cocktail also showed no differences (Figure 1C).

Figure 1 γBB and TMAO production from l-carnitine is a gut microbiota–dependent process in humans. Subjects (n = 5) ingested a capsule containing d 3 -l-carnitine (250 mg; t0), after which serial plasma aliquots were obtained at the times shown (Baseline, filled circles). After a week-long regimen of oral broad-spectrum antibiotics to suppress the intestinal microbiota, the oral l-carnitine challenge was repeated (+ Abx, open circles). Stable isotope dilution LC-MS/MS was used to quantify d 3 -TMAO (A), d 3 -γBB (B), and d 3 -l-carnitine (C) in plasma collected from sequential venous blood draws at the noted times. Time points are represented as mean ± SEM plasma concentrations, and a zero-inflated linear mixed-effects model was used to compare subjects before and after antibiotic exposure.

Ingestion of γBB produces TMAO in a gut microbiota–dependent manner in humans. We next sought to test whether gut microbiota–formed γBB could produce TMAO in humans. Heavy isotope–labeled γBB (d 9 -γBB) was synthesized and used to perform a similar oral “γBB challenge” of subjects (n = 6 omnivores). Following oral ingestion, rapid elevation in plasma concentrations of both d 9 -γBB and d 9 -l-carnitine was observed, followed by d 9 -TMAO generation after a lag phase (Figure 2, A–C, filled circles). Interestingly, after a week-long suppression of the gut microbiota with oral broad-spectrum antibiotics cocktail, a rechallenge with oral d 9 -γBB showed complete suppression of d 9 -TMAO formation in subjects, confirming gut microbiota–dependent conversion of γBB into TMAO (Figure 2A, open circles). The production of d 9 -l-carnitine from oral d 9 -γBB, however, was not suppressed with the oral antibiotics; moreover, on closer inspection, while the peak plasma concentration of d 9 -γBB (2 hours) after oral ingestion significantly decreased by 8 hours (P < 0.05), the peak blood concentrations of d 9 -l-carnitine showed a relative plateau between 2 and 8 hours (Figure 2, B and C). These observations indicate that d 9 -l-carnitine production from oral d 9 -γBB occurs via a mechanism that is not suppressed by antibiotics, and are consistent with expected results from the endogenous l-carnitine biosynthetic pathway (i.e., host conversion of γBB to l-carnitine during carnitine biosynthesis) (32–34). Finally, examination of plasma d 9 -γBB concentrations before versus after exposure to the antibiotics cocktail showed no differences (Figure 2B).

Figure 2 TMAO is a gut microbiota–dependent product of γBB in humans. Subjects (n = 6) received oral d 9 -γBB (250 mg; t0), and then serial plasma aliquots were obtained at the noted time points (Baseline, filled circles). After a week-long regimen of oral broad-spectrum antibiotics to suppress the intestinal microbiota, the oral d 9 -γBB challenge was repeated (+ Abx, open circles). Stable isotope dilution LC-MS/MS was used to quantify d 9 -TMAO (A), d 9 -γBB (B), and d 9 -l-carnitine (C) in plasma collected from sequential venous blood draws at the noted times. Time points are mean ± SEM plasma concentrations, and a zero-inflated linear mixed-effects model was used to compare subjects before and after antibiotic exposure.

Omnivores generate significantly more TMAO than vegans/vegetarians from oral L-carnitine because of marked increase in gut microbial conversion of γBB into TMA. We had previously reported that d 3 -l-carnitine challenge in omnivores showed significantly elevated d 3 -TMAO generation in comparison with vegans/vegetarians (6); we first recapitulated these findings and confirmed that omnivores (n = 15) have a much greater capacity to generate d 3 -TMAO from orally ingested d 3 -l-carnitine than vegans and vegetarians (n = 9) (Figure 3A, left panel). To determine the origins of this difference, we next explored each step in the multistep reaction. Notably, despite the dramatic difference in d 3 -TMAO production from oral d 3 -l-carnitine in omnivores versus vegans/vegetarians, the difference observed in the first step of that gut microbiota–mediated transformation, conversion of d 3 -l-carnitine to d 3 -γBB, was not significant, and if anything showed a trend toward a lower conversion rate in the omnivores (P = 0.051; Figure 3A, right panel). Further, plasma concentrations of d 3 -γBB observed following oral d 3 -l-carnitine challenge were approximately 20-fold lower relative to plasma d 3 -TMAO (Figure 3A, left vs. right panel). We next examined fasting endogenous plasma γBB concentrations before versus after 1-week exposure to the oral antibiotics cocktail in subjects (n = 9 omnivores) and noted no significant differences (P = 0.55; Figure 3B). Moreover, while oral d 3 -l-carnitine ingestion produced d 3 -γBB, comparison of endogenous fasting levels of γBB in an expanded number of omnivores (n = 40) versus vegans/vegetarians (n = 32) failed to show any significant differences (P = 0.38; Figure 3C) and was notable also for the relatively low plasma levels of γBB observed (in general, approximately 5- to 10-fold reduced compared with TMAO). Despite the expanded number of subjects examined, no difference in endogenous plasma l-carnitine levels was observed between the vegans/vegetarians (n = 32) and the omnivores (n = 40) (mean ± SEM: omnivores 31.3 ± 2.5 vs. vegans/vegetarians 39.1 ± 4.3 μM; P = 0.18; Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/JCI94601DS1).

Figure 3 γBB is a major gut microbiota metabolite of l-carnitine, and TMA formation from γBB is influenced by dietary habits. (A) Plasma d 3 -TMAO and d 3 -γBB concentrations in vegans/vegetarians (d 3 -TMAO, n = 9; d 3 -γBB, n = 9) versus omnivores (d 3 -TMAO, n = 15; d 3 -γBB, n = 12) participating in an oral d 3 -l-carnitine (250 mg) challenge. The left panel illustrates the marked differences in d 3 -TMAO generation previously reported in omnivores versus vegans/vegetarians. The right panel shows a small difference in plasma d 3 -γBB concentration between omnivores and vegans/vegetarians. Data represent mean ± SEM. A Mann-Whitney test was used to compare the AUCs between dietary groups. (B) Box-and-whisker plots of fasting plasma concentrations of γBB from subjects (n = 9) before versus after 1 week of oral broad-spectrum antibiotics to suppress gut microbiota. Boxes represent the 25th, 50th, and 75th percentiles, and whiskers represent the 10th and 90th percentiles. Differences were assessed using a Wilcoxon matched-pairs test. (C) Fasting plasma concentrations of γBB in vegans/vegetarians (n = 32) versus omnivores (n = 40). Boxes represent the 25th, 50th, and 75th percentiles, and whiskers represent the 10th and 90th percentiles. A Mann-Whitney test was used to assess differences between groups. (D) Baseline human fecal metabolite studies in vegans/vegetarians and omnivores (n = 10 each group). Fecal samples were incubated anaerobically with d 3 -l-carnitine, and d 3 -TMA and d 3 -γBB were quantified by LC-MS/MS. Data are expressed as mean ± SEM. A Mann-Whitney test was used to assess differences between groups. (E) Baseline human fecal metabolite studies in vegans/vegetarians (n = 10) versus omnivores (n = 10). Fecal samples were incubated with d 3 -l-carnitine or d 9 -γBB as indicated. Metabolites were quantified by LC-MS/MS. Data are expressed as mean ± SEM. A Mann-Whitney test was used to assess differences between groups.

Given that isotope tracer studies show γBB is clearly formed following oral l-carnitine ingestion, and similarly, isotope-labeled γBB was readily converted into isotope-labeled TMA/TMAO following oral ingestion, we hypothesized that low overall plasma concentrations of gut microbiota–produced γBB in humans may be the result of microbial γBB breakdown into TMA, predominantly occurring in the lower intestinal track (colon) distal to the absorption of most nutrients like l-carnitine and γBB. To explore this possibility, we characterized human fecal polymicrobial metabolism under ex vivo anaerobic conditions using distinct heavy isotope–labeled substrates — i.e., synthetic d 3 -l-carnitine and d 9 -γBB, allowing us to monitor d 3 -TMA (from carnitine) and d 9 -TMA (from γBB) at the same time — from both omnivores (n = 10) and vegans/vegetarians (n = 10) (Figure 3, D and E). Multiple notable findings were observed. First, quantitatively, the generation of d 3 -γBB from d 3 -l-carnitine by fecal microbiota under anaerobic culture was approximately 50-fold higher than d 3 -TMA formation in omnivore and vegan/vegetarian alike (P < 0.001; Figure 3D). These results indicate that γBB is a major gut microbial metabolite formed from oral l-carnitine in humans (for both omnivores and vegans/vegetarians). Second, fecal microbiota from omnivores (vs. vegans/vegetarians) showed a significantly enhanced (P = 0.02) enzymatic capacity to produce TMA from l-carnitine (Figure 3E, left). And yet, third, no difference was observed in the fecal transformation of l-carnitine to γBB in vegans/vegetarians compared with omnivores (P = 0.54; Figure 3E, middle). Finally, results of the fecal polymicrobial culture studies indicate that the marked increased generation of TMA (and TMAO) from oral l-carnitine in omnivores versus vegans/vegetarians appears to be due to significantly (up to 10-fold) greater gut microbial conversion of γBB to TMA in omnivores relative to vegans/vegetarians (i.e., chronic exposure to l-carnitine in an omnivorous diet is associated with an increase in microbial capacity to catalyze the transformation of γBB to TMA; P = 0.04; Figure 3E). Collectively, these data suggest that in the multistep metabolism of dietary l-carnitine into TMAO (i.e., l-carnitine→γBB→TMA→TMAO), gut microbial metabolism of l-carnitine→γBB occurs rapidly in omnivore and vegan/vegetarian alike, and the latter microbial transformation of γBB to TMA is the one markedly enhanced in omnivores over vegans/vegetarians (i.e., influenced by chronic dietary habits; Figure 3E).

Chronic dietary l-carnitine supplementation enhances gut microbiota–dependent generation of TMAO. The significant differences noted in overall metabolism of oral l-carnitine→TMA/TMAO in omnivores and, to a lower extent, in vegans/vegetarians are a striking finding. We therefore sought to further explore the impact of chronic daily dietary l-carnitine exposure on these differences. Both vegans/vegetarians (n = 7) and omnivores (n = 10) gave consent and were instructed to continue with their typical diets but with the addition of supplemental l-carnitine (500 mg l-carnitine tartrate per day, provided in a Vegicap). Subjects were monitored at baseline, at 1 month, and after 2–3 months of continuous daily supplemental l-carnitine ingestion by examination of the rate of plasma appearance of both d 3 -TMAO (Figure 4A) and d 3 -γBB (Figure 4C) following oral d 3 -l-carnitine challenge. In addition, fasting plasma levels of endogenous TMAO (Figure 4B) and γBB (Supplemental Figure 2) were monitored. At baseline, vegans/vegetarians showed minimal synthetic capacity to produce d 3 -TMAO following ingestion of d 3 -l-carnitine, whereas omnivores readily generated d 3 -TMAO (Figure 4A). After 1 month of daily l-carnitine supplementation, enhancement in the formation of d 3 -TMAO following d 3 -l-carnitine ingestion was observed in vegans/vegetarians and omnivores alike (Figure 4A). Continuation of daily l-carnitine supplementation for at least an additional month resulted in no further increase in d 3 -l-carnitine→d 3 -TMAO transformation in omnivores, but continued to increase d 3 -TMAO generation in vegans/vegetarians following oral d 3 -l-carnitine challenge (Figure 4A). Examination of individual plots of plasma d 3 -TMAO production from oral d 3 -l-carnitine challenges among vegans/vegetarians (n = 7) demonstrated that the mean increase observed was driven by only a subset (n = 3) of subjects, with over half (n = 4) of the vegans/vegetarians demonstrating essentially no metabolic capacity to convert oral d 3 -l-carnitine into d 3 -TMAO even after months of l-carnitine supplementation (Supplemental Figure 3). Fasting plasma concentrations of TMAO in both omnivores and vegans/vegetarians increased upon chronic (1 month) l-carnitine supplementation, but did not significantly further increase with another month of supplementation (Figure 4B). Interestingly, in both vegans/vegetarians and omnivores, chronic l-carnitine supplementation induced no significant differences in the rates of d 3 -γBB formed following oral d 3 -l-carnitine challenge or in fasting plasma γBB concentrations (Figure 4C and Supplemental Figure 2).

Figure 4 l-Carnitine supplementation enhances the synthetic capacity of gut microbiota to form TMAO. (A) Plasma d 3 -TMAO concentrations in sequential venous blood draws after oral d 3 -l-carnitine challenge in vegans (n = 7) and omnivores (n = 8) at baseline, visit 1 (V1, 1 month), and visit 2 (V2, 2–3 months). Data represent mean ± SEM. A zero-inflated linear mixed-effects model reveals that plasma d 3 -TMAO is significantly higher after l-carnitine supplementation. (B) Plasma TMAO concentrations in vegans (n = 7) and omnivores (n =10) at baseline and following daily l-carnitine supplementation at visit 2 (V1, 1 month), and visit 3 (V2, 2 months). Boxes represent the 25th, 50th, and 75th percentiles, and whiskers represent the 10th and 90th percentiles. A repeated-measures 1-way ANOVA test was used to assess differences between baseline visits. (C) Plasma d 3 -γBB concentrations in sequential venous blood draws after oral d 3 -l-carnitine challenge in vegans/vegetarians (n = 7) and omnivores (n = 5) at baseline, visit 1 (V1, 1 month), and visit 2 (V2, 2 months). Data represent mean ± SEM. A zero-inflated linear mixed-effects model reveals that plasma d 3 -γBB production is not significantly higher after l-carnitine supplementation in vegans/vegetarians or omnivores.

We next sought to biochemically characterize microbial transformation activities in feces of subjects before versus after l-carnitine supplementation. A subset of subjects (n = 7 omnivores, n = 6 vegans/vegetarians) consented to provide feces at both baseline and the end of the study following at least 2 months of l-carnitine supplementation for analyses (Figure 5). At baseline, comparisons between omnivores and vegans/vegetarians showed no significant differences in any of the fecal microbial metabolic transformations monitored (d 9 -l-carnitine→d 9 -TMA, d 9 -l-carnitine→d 9 -γBB, and d 9 -γBB→d 9 -TMA; P = 0.52, P = 0.63, P = 0.35, respectively) (Figure 5, A–C). However, following chronic l-carnitine supplementation, fecal samples recovered from omnivores showed significantly enhanced generation of d 9 -TMA from either d 9 -l-carnitine or d 9 -γBB relative to vegan/vegetarian fecal samples (P < 0.01 and P = 0.01, respectively; Figure 5, A and C). Further, while dietary l-carnitine supplementation induced no differences in fecal transformation of the first step of the l-carnitine→γBB→TMA/TMAO metaorganismal pathway in either omnivores or vegans/vegetarians, dietary l-carnitine provision induced enhanced fecal microbial transformation of the second microbial step in the overall pathway (i.e., d 9 -γBB→d 9 -TMA) in omnivores (P = 0.02), but not vegans/vegetarians (P = 0.69) (Figure 5C).

Figure 5 l-Carnitine supplementation does not enhance the synthetic capacity of gut microbiota to produce γBB from l-carnitine, but does enhance gut microbiota–dependent transformation of γBB into TMA. At baseline and after l-carnitine supplementation (l-Carn; 2–3 months), human fecal metabolite studies (n = 6 omnivores and n = 7 vegans/vegetarians) in the conversion of d 9 -l-carn to d 9 -TMA (A), d 9 -l-carn to γBB (B), or d 9 -γBB to d 9 -TMA (C). Fecal samples were incubated with either d 9 -l-carnitine or d 9 -γBB as described in Methods, and d 9 -TMA and d 9 -γBB were quantified by LC-MS/MS. A Wilcoxon matched-pairs test was used to assess for differences between groups.

Multiple organisms are involved in the gut microbial production of TMA from l-carnitine. Since l-carnitine catabolism is mediated by microbial enzymes, we sought to identify the organisms involved in anaerobic l-carnitine degradation in fecal samples from a healthy omnivore donor (Figure 6A). In previous studies, single gastrointestinal microbial strains were reported to be unable to catabolize l-carnitine into TMA (35). We therefore hypothesized that multiple organisms are needed for catabolism of l-carnitine to TMA presumably with γBB as an intermediate. Therefore, we isolated not only single microbial colonies from feces but also subcommunities of 2–3 and 4–5 colonies, and tested the ability of each single isolate or subcommunity to produce TMA from l-carnitine. Though no single isolate produced TMA, 2 subcommunities out of 768 did produce TMA, and 1 of those subcommunities was further fractionated using a similar strategy as before (i.e., single, 2–3, and 4–5 colonies; Figure 6A). A noticeable enrichment in the number of TMA-producing subcommunities was observed, but again no individual isolate could perform the l-carnitine→TMA transformation. The subcommunity producing the most TMA was spread and plated once more, and 100 colonies of the community were preliminarily identified by proteomics analyses using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (36). The colonies were pooled based on best-match organism into 5 species pools (SP1 through SP5; Supplemental Table 1). Individual pools and combinations were evaluated for l-carnitine→TMA activity using a fractional factorial design (Figure 6B). Absence of either SP2 or, to a lesser extent, SP5 abolished the l-carnitine→TMA activity, whereas maximum production of TMA was attained only when both SP2 and SP5 were present. Each species pool (SP2 and SP5) was further subfractionated into individual colonies, and all members were evaluated alone or in combination with the members of the other pool. Only the combination of SP5-56 or SP5-62 with SP2-71 yielded maximum TMA production (Figure 6C). These data confirm our hypothesis and demonstrate that at least 2 different organisms are required for the anaerobic catabolism of l-carnitine to TMA. Both SP5-56 and SP5-62 were identified as isolates of Eggerthella lenta by 16S-rRNA gene sequencing. Further characterization of SP2-71 indicated that it was not a single strain, but a combination of 4 microbes (Supplemental Figure 4A). Of the 4 microbes, 1 had not been reported at the time of isolation, but showed 99% 16S-rRNA gene sequence identity (Supplemental Figure 4B) with an organism recently isolated by others and classified as Emergencia timonensis (37). Interestingly, E. timonensis (SP2-71.3) produced TMA in the absence of molecular oxygen, suggesting a novel microbial pathway for l-carnitine catabolism independent of the CntA/B oxygenase (25, 26).

Figure 6 l-Carnitine catabolism to TMA involves multiple microorganisms. (A) l-Carnitine–enriched fecal communities were plated on solid media. Single microbial colonies and subcommunities of up to 3 and 4+ colonies were picked and tested for their l-carnitine→TMA activity. The top producing community was refractionated to select the highest TMA-producing subcommunity. (B) Species pools (SP1 through SP5) were evaluated alone or in combination for l-carnitine→TMA activity in n = 2 replicate values. Data are expressed as the mean. (C) Combinations of individual members of SP2 and SP5 were evaluated for TMA production. Concentrations of d 9 -TMA were determined by stable isotope dilution LC-MS/MS.

Human commensal utilization of l-carnitine is decoupled from TMA production and generates γBB as intermediate. We next studied which of the isolated human commensals were necessary and sufficient to metabolize l-carnitine to TMA. Each of the microbes contained in species pool SP2-71 was evaluated alone or in combination with E. lenta (species pool SP5-62; Figure 7A). l-Carnitine was always consumed in the presence of E. lenta (SP5-62), regardless of whether other microbes were present. However, TMA production was decoupled from l-carnitine utilization and was only produced when both E. lenta (SP5-62) and SP2-71.3 (or the entire SP2-71 pool) were present, which suggests that E. lenta (SP5-62) is associated with l-carnitine consumption and SP2-71.3 is responsible for TMA production (Figure 7A). We mined the genome of E. lenta (SP5-62) for genes likely to be associated with l-carnitine utilization and noticed genes of the caiTABCDE gene operon that encode for a crotonobetaine reductase, l-carnitine-CoA transferase, and l-carnitine-CoA ligase, among other genes (Figure 7B). Reasoning that the enzymes encoded by these genes could be responsible for the utilization of l-carnitine and its conversion to γBB (Figure 7C), we used comparative genomics tools to identify 3 other organisms that contain the caiTABCDE gene operon (Supplemental Figure 5), and assessed their ability to consume l-carnitine and produce γBB (Figure 8). Although none of the 3 microbes, Escherichia fergusonii, Edwardsiella tarda, and Proteus penneri, produced TMA, all 3 consumed l-carnitine while producing γBB, suggesting that the utilization of l-carnitine is associated with the presence of the caiTABCDE genes. The combination of any of the 3 microbes with E. timonensis (SP2-71.3) led to anaerobic, oxygen-independent, TMA production from l-carnitine and consumption of γBB (Figure 8). The E. timonensis type strain SN18 (37) performed very similarly to E. timonensis SP2-71.3 in this study (data not shown).

Figure 7 Anaerobic microbial l-carnitine consumption is associated with the presence of the microbial caiTABCDE genes. (A) Individual SP2-71 microbes were evaluated alone or in combination with E. lenta SP5-62 for l-carnitine→TMA activity. Data are expressed as the mean of the d 9 -l-carnitine peak ratio and d 9 -TMA concentration in n = 2 replicate values. (B) E. lenta genome mining revealed the presence of the caiTABCDE gene operon. Data are expressed as the mean. (C) l-Carnitine→γBB pathway involving enzymes encoded by the caiTABCDE gene operon. Crotono, crotonobetaine.

Figure 8 CaiTABCDE gene–expressing microbes consume l-carnitine, but only make TMA in the presence of E. timonensis. Concentrations of l-carnitine and TMA were determined by stable isotope dilution LC-MS/MS in n = 3 replicate values. Data are expressed as mean ± SEM.

During l-carnitine catabolism by human gut commensals and fecal polymicrobial anaerobic cultures, γBB accumulates as an intermediate before it is anaerobically converted to TMA. We then determined the kinetics of consumption of l-carnitine and production of γBB and TMA by P. penneri (ATCC 35198), one of the human commensals shown to produce γBB, alone or in combination with E. timonensis (SP2-71.3) (Figure 9A). Addition of l-carnitine to an anaerobic culture of P. penneri alone (in M9 minimal medium) resulted in quantitative transformation of l-carnitine to γBB within 4 hours with no TMA production. E. timonensis alone did not consume l-carnitine or produce TMA from l-carnitine. However, the combination of P. penneri and E. timonensis led to a transient accumulation of γBB that was subsequently consumed with concomitant production of TMA (Figure 9A, top). When M9 minimal medium was instead supplemented with γBB, P. penneri alone was unable to consume γBB or produce TMA, whereas only E. timonensis was sufficient to produce TMA from γBB in the presence or absence of P. penneri (Figure 9A, bottom). To our knowledge, this is the first report of a single microbial strain capable of producing TMA from one of the metabolites of the l-carnitine pathway under anaerobic conditions. Moreover, kinetic analyses reveal that the production of γBB precedes production of TMA, consistent with γBB being needed to induce the γBB→TMA phenotype.

Figure 9 Anaerobic microbial l-carnitine catabolism generates γBB as an intermediate. (A) Kinetic changes of l-carnitine, γBB, and TMA were determined in cultures of P. penneri alone or in combination with E. timonensis, SP2-71.3, supplemented with l-carnitine (top panel) or γBB (bottom panel). Concentrations were determined in n = 2 replicate values. Data are expressed as the mean. (B) Selected human fecal communities from 4 different donors were studied for their l-carnitine→γBB→TMA activity sampling (n = 1) every 4 hours for 32 hours. Concentrations of l-carnitine, γBB, and TMA were determined by stable isotope dilution LC-MS/MS. All human subjects are displayed in Supplemental Figure 6.

To assess whether the transient buildup of γBB in vitro with only 2 microbial strains represents the biochemical transformations that occur in the presence of gastrointestinal polymicrobial communities, the anaerobic metabolism of l-carnitine by fecal communities from 12 human healthy donors (all omnivores) was studied. For illustrative purposes, biochemical transformations characteristic of omnivores following carnitine supplementation (subjects 1 and 7) and vegans or vegetarians (i.e., little TMA generation: subjects 10 and 12) are shown in Figure 9B, while data from all 12 subjects are shown in Supplemental Figure 6. The communities of all 12 subjects converted the majority of l-carnitine to γBB within 12 hours of incubation, although not all subjects produced TMA from γBB by 32 hours (Figure 9B). Moreover, γBB formation (before TMA generation) was quantitatively associated with l-carnitine consumption, and when produced, TMA formation was quantitatively associated with γBB consumption. Thus, the demonstrated kinetics of generation and decay of the metabolites monitored followed the anticipated precursor→product relationships, and were entirely consistent with the initial microbial conversion of l-carnitine to γBB, followed by transformation of γBB to TMA, reaction kinetics akin to the dynamics of l-carnitine catabolism observed with the human commensals P. penneri and E. timonensis.