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Unless provided in the caption above, the following copyright applies to the content of this slide: Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Eur Heart J, Volume 40, Issue 7, 10 December 2018, Pages 583–594, https://doi.org/10.1093/eurheartj/ehy799 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 1 Gut microbial and host pathways in trimethylamine N-oxide metabolism and study design. (A) Trimethylamine ... Figure 1 Gut microbial and host pathways in trimethylamine N-oxide metabolism and study design. (A) Trimethylamine containing nutrients that can generate trimethylamine N-oxide via an initial gut microbiota-dependent step, followed by host hepatic flavin monooxygenase conversion to generate trimethylamine N-oxide. Arrows in black represent transformations performed by the host, and arrows in red represent reactions performed by gut microbes. (B) Overall study design. After consumption of a 2-week baseline diet, subjects were randomly assigned to either a high-fat or low-fat arm. Subjects in each arm underwent in cross-over design three sequential 4-week isocaloric investigational diet where protein source was derived from either red meat, white meat, or non-meat sources, with 2-week wash-out diets between each intervention dietary period, as described under Methods section. During the 4th week of each diet challenge (typically towards end), blood was collected on two separate days (Visit A and Visit B). Participants within each experimental diet arm were randomly assigned to either high- or low-saturated fat containing meal plans, as described in the text. Unless provided in the caption above, the following copyright applies to the content of this slide: Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Eur Heart J, Volume 40, Issue 7, 10 December 2018, Pages 583–594, https://doi.org/10.1093/eurheartj/ehy799 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 2 Differences in plasma trimethylamine N-oxide and other trimethylamine containing compounds following a 4-week ... Figure 2 Differences in plasma trimethylamine N-oxide and other trimethylamine containing compounds following a 4-week exposure to different diets. (A) Box–whisker plots of trimethylamine N-oxide, choline, betaine, carnitine, γ-butyrobetaine, and crotonobetaine levels after 4 week consumption of the red meat, white meat, or non-meat diets. (B) Changes in plasma trimethylamine N-oxide in subjects at completion of the 4-week red meat investigational diet period, upon switching to either the 4-week non-meat or white meat investigational diets. (C) After completion of the 2-week run-in baseline diet, subjects with the top (red) and bottom (blue) 10 percentile levels of trimethylamine N-oxide were identified, and their trimethylamine N-oxide levels plotted following completion of the indicated 4-week interventional dietary period. Except where indicated, all metabolite levels were from blood drawn on Visit B. Unless provided in the caption above, the following copyright applies to the content of this slide: Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Eur Heart J, Volume 40, Issue 7, 10 December 2018, Pages 583–594, https://doi.org/10.1093/eurheartj/ehy799 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 3 Differences in urine trimethylamine N-oxide and other trimethylamine containing compounds following a 4-week ... Figure 3 Differences in urine trimethylamine N-oxide and other trimethylamine containing compounds following a 4-week exposure to different diets. Unless provided in the caption above, the following copyright applies to the content of this slide: Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Eur Heart J, Volume 40, Issue 7, 10 December 2018, Pages 583–594, https://doi.org/10.1093/eurheartj/ehy799 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 4 Impact of high-saturated fat on plasma trimethylamine N-oxide and other trimethylamine-related metabolites. ... Figure 4 Impact of high-saturated fat on plasma trimethylamine N-oxide and other trimethylamine-related metabolites. Comparison of plasma levels of the indicated metabolites in subjects on the low-saturated fat vs. high-saturated fat diet arms, stratified by dietary protein source (A-F). Values reported for base-line diet are those for participants later randomized to low vs. high saturated fat in their first intervention (protein source) diet. P-values were calculated by unpaired t-test. Unless provided in the caption above, the following copyright applies to the content of this slide: Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Eur Heart J, Volume 40, Issue 7, 10 December 2018, Pages 583–594, https://doi.org/10.1093/eurheartj/ehy799 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 5 Impact of diet on fractional renal excretion of trimethylamine N-oxide and both choline and carnitine related ... Figure 5 Impact of diet on fractional renal excretion of trimethylamine N-oxide and both choline and carnitine related metabolites (N = 113 subjects) based on paired plasma and urine levels of concurrently measured creatinine (A–F). Fractional renal excretion was also calculated by replacing plasma and urine creatinine concentrations with paired plasma and urine concentrations of alternative metabolites (concurrently measured in the same samples) whose concentrations are highly correlated with measures of renal function (C-mannosyl-tryptophan, pseudouridine, or symmetric dimethylarginine) (G–I). Fractional renal excretion of the indicated metabolites were determined following 4-weeks of the indicated diet. SDMA, symmetric dimethylarginine. Unless provided in the caption above, the following copyright applies to the content of this slide: Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Eur Heart J, Volume 40, Issue 7, 10 December 2018, Pages 583–594, https://doi.org/10.1093/eurheartj/ehy799 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 6 Urine analyses (24 h) following ingestion of d6-choline isotope tracer. During the last week of each ... Figure 6 Urine analyses (24 h) following ingestion of d6-choline isotope tracer. During the last week of each interventional dietary period (red meat, white meat, or non-meat), subjects (N=13) underwent an oral d6-choline challenge and 24 h urine output of the indicated d6-labelled metabolites (A-D) was collected and analyzed as described (Methods section). Unless provided in the caption above, the following copyright applies to the content of this slide: Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Eur Heart J, Volume 40, Issue 7, 10 December 2018, Pages 583–594, https://doi.org/10.1093/eurheartj/ehy799 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 7 Urine analyses (24 h) following ingestion of d3-carnitine isotope tracer. During the last week of each ... Figure 7 Urine analyses (24 h) following ingestion of d3-carnitine isotope tracer. During the last week of each interventional dietary period (red meat, white meat, or non-meat), subjects (N=13) underwent an oral d3-carnitine challenge and 24 h urine output of the indicated d3-labelled metabolites (A-E) was collected and analyzed as described (Methods section). Unless provided in the caption above, the following copyright applies to the content of this slide: Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Eur Heart J, Volume 40, Issue 7, 10 December 2018, Pages 583–594, https://doi.org/10.1093/eurheartj/ehy799 The content of this slide may be subject to copyright: please see the slide notes for details.

Take home figure Summary scheme: effect of a red meat containing diet on the metaorganismal trimethylamine N-oxide pathway. Take home figure Summary scheme: effect of a red meat containing diet on the metaorganismal trimethylamine N-oxide pathway. Unless provided in the caption above, the following copyright applies to the content of this slide: Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Eur Heart J, Volume 40, Issue 7, 10 December 2018, Pages 583–594, https://doi.org/10.1093/eurheartj/ehy799 The content of this slide may be subject to copyright: please see the slide notes for details.