Serum Trimethylamine-N-Oxide is Elevated in CKD and Correlates with Coronary Atherosclerosis Burden

Trimethlyamine-N-oxide (TMAO) was recently identified as a promoter of atherosclerosis. Patients with CKD exhibit accelerated development of atherosclerosis; however, no studies have explored the relationship between TMAO and atherosclerosis formation in this group. This study measured serum concentrations and urinary excretion of TMAO in a CKD cohort (n=104), identified the effect of renal transplant on serum TMAO concentration in a subset of these patients (n=6), and explored the cross-sectional relationship between serum TMAO and coronary atherosclerosis burden in a separate CKD cohort (n=220) undergoing coronary angiography. Additional exploratory analyses examined the relationship between baseline serum TMAO and long-term survival after coronary angiography. Serum TMAO concentrations demonstrated a strong inverse association with eGFR (r(2)=0.31, P<0.001). TMAO concentrations were markedly higher in patients receiving dialysis (median [interquartile range], 94.4 μM [54.8–133.0 μM] for dialysis-dependent patients versus 3.3 μM [3.1–6.0 μM] for healthy controls; P<0.001); whereas renal transplantation resulted in substantial reductions in TMAO concentrations (median [min–max] 71.2 μM [29.2–189.7 μM] pretransplant versus 11.4 μM [8.9–20.2 μM] post-transplant; P=0.03). TMAO concentration was an independent predictor for coronary atherosclerosis burden (P=0.02) and predicted long-term mortality independent of traditional cardiac risk factors (hazard ratio, 1.26 per 10 μM increment in TMAO concentration; 95% confidence interval, 1.13 to 1.40; P<0.001). In conclusion, serum TMAO concentrations substantially increase with decrements in kidney function, and this effect is reversed by renal transplantation. Increased TMAO concentrations correlate with coronary atherosclerosis burden and may associate with long-term mortality in patients with CKD undergoing coronary angiography

Development and validation of a simple LC-MS/MS method for the simultaneous quantitative determination of trimethylamine-N-oxide and branched chain amino acids in human serum

Serum branched chain amino acids and trimethylamine-N-oxide are monitored as potential indicators of diabetes and cardiovascular health respectively. A rapid method for their simultaneous determination using liquid chromatography and tandem mass spectrometry is described here. Branched chain amino acids and trimethylamine-N-oxide were quantified based on their specific MS/MS fragments using a selected reaction monitoring approach. A number of columns were tested for their ability to separate the analytes. A C18-PFP column separated the analytes in just 4 minutes, and resulted in excellent peak shape and retention time repeatability, and was therefore chosen as the optimal column. A second column, the Intrada Amino Acid column, was chosen for comparison and validation experiments as it provided an orthogonal separation mechanism. The intra-day and inter-day precision and accuracy were less than 12% for trimethylamine-N-oxide and less than 6% for the branched chain amino acids. Recoveries, where serum was spiked with three different concentrations of the analytes, ranged from 97 to 113%. The LODs and LOQs for trimethylamine-N-oxide were 1 and 6 ng/mL, for leucine and isoleucine were 4 and 8 ng/mL, and for valine were 5 and 15 ng/mL, respectively. The C18-PFP column method was validated using the Intrada Amino Acid column method and percentage agreement for all four analytes was within 10%. Sample preparation was minimal, and use of labelled internal standards accounted for matrix effects. The method was successfully applied to human plasma samples