We next studied the plasma analyte with m/z 118. We hypothesized that the analyte was either betaine or one of several potential methyltransferase metabolites of choline (see Supplementary Fig. 5a for structures and strategy for discrimination amongst these isomers). To both distinguish amongst these species, and explore a role for intestinal generation of the various metabolites, different isotopically labeled choline precursors were administered to mice either via oral (gavage) vs. parenteral (intraperitoneal, i.p.) route. The observed m/z of new isotopically labeled analytes at the appropriate retention times identified in plasma following these isotope tracer studies are sumarized in Fig. 2a. Oral administration of non-labeled choline resulted in time-dependent increases in plasma levels of analytes with m/z 76, 104 and 118, consistent with TMAO, choline and either betaine or a methylated choline species (Supplementary Fig. 6a). Use of selectively deuterated choline species at either the trimethylamine moiety (d9-isotopomer) or the ethyl moiety (d4-isotopomer) unambigously confirmed the m/z 118 analyte as betaine (Figs. 2a, Supplementary Fig. 6b). Further confirmation was acquired by observing the same retention time in LC/MS and an identical CID mass spectrum (Supplementary Fig. 5b). Moreover, supplementation of PC or choline isotopomers via gavage or i.p. injection showed an absolute requirement for oral route in TMAO production, whereas betaine production from PC or choline was formed via both oral and i.p. routes (Fig. 2, Supplementary Fig. 7a).