Choline/Betaine Prognostics Depend on Trimethylamine-N-Oxide
Choline/Betaine Prognostics Depend on Trimethylamine-N-Oxide
We prospectively recruited sequential stable subjects (n = 3903) undergoing elective, non-urgent coronary angiography at the Cleveland Clinic without any evidence of acute coronary syndrome (cardiac troponin I ≤0.03 ng/mL). All subjects were followed over a period of 3 years from time of enrollment including adjudication of MACE(which includes the future development of all-cause mortality, non-fatal myocardial infarction, or non-fatal stroke). Fasting blood glucose, high-sensitivity (hs) C-reactive protein, and lipid profiles were measured on the Abbott Architect ci8200 platform (Abbott Diagnostics, Abbott Park, IL, USA).
Plasma was immediately prepared from venous blood drawn into ethylenediaminetetraacetic acid tubes and then stored at −80°C until analysis. Trimethylamine-N-oxide, choline, and betaine levels in plasma were determined using stable isotope dilution high-performance liquid chromatography with on line electrospray ionization tandem mass spectrometry (LC/MS/MS) on either an AB SCIEX 5000 or AB SCIEX 5500 triple quadrupole mass spectrometer using d9-(trimethyl)-labelled internal standards as described previously.
Whole blood (50 μL) was collected via the saphenous vein from C57BL/6J mice prior to gavage for the measurement of baseline analyte levels. Mice were administered an oral dose of stable isotope-labelled betaine or choline. The gavage consisted of 150 μL of either 150 mM betaine-N,N,N-trimethyl-d9 (d9-betaine, CDN Isotopes) or 150 mM choline-N,N,N-trimethyl-d9 (d9-choline, Cambridge Isotope Lab) followed by post-gavage blood collection at the indicated times. Where indicated, gut microbiota were suppressed in mice as previously described by placement of a cocktail of antibiotics in drinking water for 3 weeks prior to the d9-betaine challenge. Ethylenediaminetetraacetic acid plasma was generated following centrifugation at 1000 g for 20 min at 4°C, and d9-labelled parent compounds and metabolites were measured by LC/MS/MS using choline-1,1,2,2-d4 (d4-choline, Cambridge Isotope Lab) as an internal standard. The chromatographic gradient employed was as previously reported and specific parent to daughter transitions in positive ion multiple reaction monitoring mode were 85→66 for d9-TMAO, 113→69 for d9-choline, 127→68 for d9-betaine, and 108→60 for d4-choline. In additional studies, the role of gut flora in TMAO formation from dietary betaine was determined in C57BL/6J mice by performance of oral (gavage) d9-betaine challenge. Mice were housed in conventional cages in the absence or presence of a cocktail of broad spectrum antibiotics added to the drinking water previously shown to suppress gut flora.
Student's t-test or Wilcoxon-rank sum test for continuous variables and χ test for categorical variables were used to examine the difference between groups. Kaplan–Meier analysis with Cox proportional hazards regression was used for time-to-event analysis to determine hazard ratio (HR) and 95% confidence intervals (95% CI) for MACE. Stratification between median levels of choline, betaine, and TMAO levels was performed. Adjustments were made for individual traditional risk factors including age, sex, systolic blood pressure, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, smoking, diabetes mellitus, and hs C-reactive protein to predict incident 3-year MACE risks. All analyses performed used R 2.8.0 (Vienna, Austria). P < 0.05 was considered statistically significant.
Methods
Study Population
We prospectively recruited sequential stable subjects (n = 3903) undergoing elective, non-urgent coronary angiography at the Cleveland Clinic without any evidence of acute coronary syndrome (cardiac troponin I ≤0.03 ng/mL). All subjects were followed over a period of 3 years from time of enrollment including adjudication of MACE(which includes the future development of all-cause mortality, non-fatal myocardial infarction, or non-fatal stroke). Fasting blood glucose, high-sensitivity (hs) C-reactive protein, and lipid profiles were measured on the Abbott Architect ci8200 platform (Abbott Diagnostics, Abbott Park, IL, USA).
Mass Spectrometry Analyses of Trimethylamine-N-Oxide, Choline, and Betaine
Plasma was immediately prepared from venous blood drawn into ethylenediaminetetraacetic acid tubes and then stored at −80°C until analysis. Trimethylamine-N-oxide, choline, and betaine levels in plasma were determined using stable isotope dilution high-performance liquid chromatography with on line electrospray ionization tandem mass spectrometry (LC/MS/MS) on either an AB SCIEX 5000 or AB SCIEX 5500 triple quadrupole mass spectrometer using d9-(trimethyl)-labelled internal standards as described previously.
Metabolic Challenges in Mice
Whole blood (50 μL) was collected via the saphenous vein from C57BL/6J mice prior to gavage for the measurement of baseline analyte levels. Mice were administered an oral dose of stable isotope-labelled betaine or choline. The gavage consisted of 150 μL of either 150 mM betaine-N,N,N-trimethyl-d9 (d9-betaine, CDN Isotopes) or 150 mM choline-N,N,N-trimethyl-d9 (d9-choline, Cambridge Isotope Lab) followed by post-gavage blood collection at the indicated times. Where indicated, gut microbiota were suppressed in mice as previously described by placement of a cocktail of antibiotics in drinking water for 3 weeks prior to the d9-betaine challenge. Ethylenediaminetetraacetic acid plasma was generated following centrifugation at 1000 g for 20 min at 4°C, and d9-labelled parent compounds and metabolites were measured by LC/MS/MS using choline-1,1,2,2-d4 (d4-choline, Cambridge Isotope Lab) as an internal standard. The chromatographic gradient employed was as previously reported and specific parent to daughter transitions in positive ion multiple reaction monitoring mode were 85→66 for d9-TMAO, 113→69 for d9-choline, 127→68 for d9-betaine, and 108→60 for d4-choline. In additional studies, the role of gut flora in TMAO formation from dietary betaine was determined in C57BL/6J mice by performance of oral (gavage) d9-betaine challenge. Mice were housed in conventional cages in the absence or presence of a cocktail of broad spectrum antibiotics added to the drinking water previously shown to suppress gut flora.
Statistical Analysis
Student's t-test or Wilcoxon-rank sum test for continuous variables and χ test for categorical variables were used to examine the difference between groups. Kaplan–Meier analysis with Cox proportional hazards regression was used for time-to-event analysis to determine hazard ratio (HR) and 95% confidence intervals (95% CI) for MACE. Stratification between median levels of choline, betaine, and TMAO levels was performed. Adjustments were made for individual traditional risk factors including age, sex, systolic blood pressure, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, smoking, diabetes mellitus, and hs C-reactive protein to predict incident 3-year MACE risks. All analyses performed used R 2.8.0 (Vienna, Austria). P < 0.05 was considered statistically significant.