medicine nature Metabolomics Studies Reveal New Pathways in Cardiovascular Disease with Potential for Diagnostics and Therapeutic Targeting

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1 Metabolomics Studies Reveal New Pathways in Cardiovascular Disease with Potential for Diagnostics and Therapeutic Targeting nature medicine VOLUME 19 NUMBER 5 MAY Stanley L Hazen, MD, PhD Chair, Dept. of Cellular & Molecular Medicine; Section Head, Preventive Cardiology, Cleveland Clinic How red meat promotes atherosclerosis Analysis of brown fat in humans Maturing research on aging

2 Disclosure Information Stanley L. Hazen, MD, PhD Grants/Research Support: All work presented was supported by the NIH Additional research support in the Hazen laboratory: Abbott Laboratories, Cleveland Heart Lab (CHL), Esperion, and LipoScience. Mass spectrometry equipment used is housed in a core facility partially supported by a Center of Innovation Award by AB SCIEX

3 Phase 1: Discovery-based investigations Metabolomics screening and structural identification Phase 2: Clinical validation Replication and demonstration of clinical utility Phase 3: Mechanistic studies Demonstration of causality for a novel pathway

4 Strategy of metabolomics study design for identifying unbiased small molecule profiles predictive of incident risks for major adverse cardiovascular events GeneBank (N=1,) Wang Z et al (211) Nature

5 LDL Plasma analytes with m/z 76, 14, and 118 are associated with CVD, show a doseresponse relationship with MACE (3yr MI stroke or death) and are correlated, suggesting participation in a common pathway Wang Z et al (211) Nature

6 Example data from metabolomics study Intensity, cps 2.6e8 2.4e8 2.2e8 2.e8 1.8e8 1.6e8 1.4e8 1.2e8 1.e8 8.e7 6.e7 4.e7 2.e7 +Q1: 418 MCAscans fromsample 6 (177) of ,TMANOQ1 scan.wiff (Turbo Spray... Mass spectrum of plasma scanned in positive ion mode Max. 2.6e8 cps XICof +Q1: 75.5 to 76.5 amu fromsample 6 (177) of ,TMANOQ1 scan.wiff (Turbo... Max. 8.6e6 cps m/z,amu XICof +Q1: to amu fromsample 6 (177) of ,TMANOQ1 scan.wiff (Tur... Max. 2.9e7 cps. 8.5e6 8.e6 7.5e6 7.e6 6.5e6 6.e6 5.5e6 Intensity, cps 5.e6 4.5e6 4.e6 3.5e6 3.e6 2.5e6 2.e6 1.5e6 1.e6 5.e5. Extracted ion LC chromatogram at m/z= Time,min 2.9e7 2.8e7 Intensity, cps 2.6e7 2.4e7 2.2e7 2.e7 1.8e7 1.6e7 1.4e7 1.2e7 1.e7 8.e6 6.e6 4.e6 2.e Extracted ion LC Chromatogram at m/z= Time,min 5.13

7 Structural identification/validation of plasma analyte at m/z=76 as TMANO (trimethylamine N-oxide) 1 Analyte m/z=76 Plasma Trimethylamine N-oxide 1 (TMANO) CID MS Spectrum MS Deriv of TCEF GC Chromatogram Of Deriv of TCEF m/z Wang Z et al, Nature (211) Deriv-TMANO (standard) M + =219 M Deriv-plasma m/z=76 analyte M Derivatized-plasma analyte m/z=76 1 Derivatized-TMANO (standard) Time (min)

8 What is TMANO? - It is proposed to be a gut floradependent metabolite of dietary lecithin (phosphatidylcholine, PC) Wang Z et al, Nature (211) m/z 76

9 Dietary egg yolk PC produces increases in analytes with m/z 76, 14,and 118 in both human and mouse plasma human 1 m/z=76 m/z=14 m/z=118 Time (min) Male mouse 1 m/z=76 m/z=14 m/z=118 Time (min) Female mouse 1 m/z=76 Time (min) m/z=14 m/z=118

10 Dietary phosphatidylcholine enhances levels of the 3 analytes, indicating they are metabolites of PC Female mouse, 1.5 mg PC gavage m/z=76 m/z=14 m/z= Time (hr) Time (hr) Time (hr)

11 Maybe choline is the plasma analyte associated with CVD with m/z 14 Wang Z et al, Nature (211) m/z 14 m/z 76

12 Plasma analyte m/z=14 choline 2-amino-3-methyl- 1-butanol 3-aminoisobutyric acid 2-aminobutyric acid biuret N,N-dimethyl glycine benzonitrile diethylenetriamine ethyl-n-hydroxyl acetimidate 2-isopropylaminoethanol 2-propylaminoethanol 1-dimethyl-amino- 2-propanol MH Candidate plasma analytes linked to CVD risks with m/z 14 Identity as Choline was confirmed by: LC-MS n GC/MS/MS NMR Isotope tracer studies: d9-choline and d4-choline

13 Strategy to determine the analyte at m/z=118 by choline deuterated isotopologue feeding study Betaine D Choline m/z= D D m/z=14 18 Methyl transferase? D D 1- methylcholine m/z= O D D 2- methylcholine m/z= D D choline methyl ether m/z=

14 Isotope challenge studies confirm the identities of TMAO, choline and betaine as the plasma analytes predicting CVD risk Wang Z et al, Nature (211)

15 Choline, betaine and trimethylamine-n-oxide are plasma analytes associated with CVD (TMAO) m/z 14 Identities confirmed by: LC-MS n, 1 H, 13 C, 15 N NMR GC/MS/MS, Isotope tracer studies Wang Z et al (211) Nature Betaine m/z 118 (TMAO) m/z 76

16 Dietary Lipid What is the role of gut flora? Wang Z et al, Nature (211)

17 Intestinal Microbial Organisms Play an Obligatory Role in TMAO Generation from Dietary Egg Yolk PC in Mice 2 weeks TMAO Wang Z et al, Nature (211)

18 TMAO is a gut flora dependent metabolite in humans : Ion extracted LC chromatograms in positive MRM MS in human plasma and urine after oral taking d9-dppc and two hard eggs PC challenge - Oral d9-pc and 2 hard boiled eggs at each visit 6 h post PC challenge Pre-antibiotics Antibiotics (visit 1) Suppression Suspension of gut flora Plasma a 1 b 1 Post-antibiotics (visit 2) c Acquisition of gut flora 1 visit 3 d 5 1 TMAO Time (min) e 5 1 TMAO Time (min) f 5 1 TMAO Time (min) 5 d9-tmao d9-tmao d9-tmao Time (min) Time (min) Time (min) 24 h post PC challenge Urine g 1 h 1 i 1 j 5 1 TMAO k 5 1 TMAO l 5 1 TMAO Tang, Wang et al, NEJM (213) 5 d9-tmao Time (min) 5 d9-tmao Time (min) 5 d9-tmao Time (min)

19 Phase 1: Discovery-based investigations Metabolomics screening and structural identification Phase 2: Clinical validation Replication and demonstration of clinical prognostic utility Phase 3: Mechanistic studies Demonstration of causality for a novel pathway

20 Development of stable isotope dilution LC/MS/MS assays for choline, TMAO and betaine using d9(trimethyl) isotopologues as internal standards (controls) Intraday CV Interday CV TMAO (low) (high) Betaine (low) (high) Choline (low) (high)

21 Prospective Cohort: N=1865 Sequential Cardiology Patients Plasma choline, TMAO and betaine levels predict CVD risks (N=1865) Odds ratio (95%CI) adjusted for age, sex, DM, HTN, smoking, LDL, HDL, TG, CRP, egfr Z Wang et al, Nature (211)

22 Plasma levels of the gut flora dependent metabolite TMAO predict incident (3 year) CVD risks New Independent Cohort: N=47 Sequential Subjects TMAO TMANO TMAO 1 Q1 Adjusted for age, sex, DM, HTN, smoking, LDL, HDL, TG, CRP, egfr Tang, Wang et al, NEJM (213) Log-Rank p<.1 Q2 Q3 Q Time (days)

23 Phase 1: Discovery-based investigations Metabolomics screening and structural identification Phase 2: Clinical validation Replication and demonstration of clinical utility Phase 3: Mechanistic studies Demonstration of causality for a novel pathway

24 Suppression of gut flora inhibits TMAO formation and dietary choline induced atherosclerosis Intact flora Gut flora suppression Wang Z et al, Nature (211) TMAO: 8 77

25 Cholesterol metabolism in cells of the artery wall: MPO Forward Cholesterol Transport LDL-R Reverse Cholesterol Transport (RCT) Spherical HDL Nascent discoidal HDL Macrophage Foam cell

26 TMAO alters cholesterol and sterol metabolism in multiple compartments - net effect - increased atherosclerosis Koeth, et al, Nature Med., 213

27 Carnitine, an abundant nutrient in red meat, is pro-atherogenic too Robert Koeth et al, Nature Medicine (213)

28 Carnitine supplementation accelerates atherosclerosis in apoe-/- mice, but not with suppression of intestinal flora (and suppression of TMA/TMAO formation) 2-fold Robert Koeth et al, Nature Medicine (213)

29 Human carnitine tolerance study: There is an obligatory role for gut flora in TMAO production from oral carnitine 12h post challenge Robert Koeth et al, Nature Medicine (213)

30 Hypothesis: Dietary patterns alter the composition of the gut microbial community N=3 N=23 Omnivore and Vegans/Vegetarians Stool Collected Blood Collected Gut Microbiota Composition TMAO measured by mass spectrometry

31 TMAO is formed from dietary carnitine in omnivors, but minimally in vegans Carnitine challenge: 8oz tenderloin +d3(methyl)-carnitine Carnitine challenge: d3(methyl)-carnitine Robert Koeth et al, Nature Med (213)

32 Specific microbiota taxa are associated with longterm dietary patterns and plasma TMAO levels Omnivores Vegan/ Vegetarian Robert Koeth et al, Nature Medicine (213)

33 Plasma levels of carnitine in subjects predict cardiovascular risks Sequential subjects (N=2595) undergoing cardiac evaluation at the Cleveland Clinic Preventive Cardiology Clinic CAD PAD MACE (3yr) (MI, CVA, death) Unadjusted Adjusted for age, gender, DM, SBP, DBP, smoking, hscrp, LDL, HDL, TG, ecrcl, hscrp Robert Koeth et al, Nature Med (213)

34 Plasma levels of carnitine in subjects predict cardiovascular risks - only if TMAO is high Sequential subjects (N=2595) undergoing cardiac evaluation at the Cleveland Clinic Preventive Cardiology Clinic Carnitine High Low TMAO Low Low Low High High High Robert Koeth et al, Nature Med (213)

35 carnitine carnitine Metabolomics studies are a powerful tool for discovery of new diagnostic and therapeutic targets (TMAO) Gut flora contributes to atherosclerotic heart disease Wang Z et al (211) Nature Bennett B et al (213) Cell Metab Koeth RA et al (213) Nature Medicine Tang WHW et al (213) NEJM Wang Z et al (214) Euro Heart J (in press) Tang WHW et al (214) JACC (in press)

36 The Cleveland Clinic Zeneng Wang Beth Klipfell Robert Koeth Bruce Levison Jonathan Smith Wilson Tang Joe DiDonato UCLA Brian Bennett Jake Lusis Elin Org U Penn Rick Bushman Jun Chen Gary Wu James Lewis Hongzhe Li USC Hooman Allayee Acknowledgments