Liquid chromatography – mass spectrometry methods for investigating osmolytes and related one-carbon metabolites in health and disease.
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Organic osmolytes are methylamines, polyols, or amino acids that are essential for cell volume regulation. Some have other important biochemical roles. For example, N,N,N-trimethylglycine (betaine) is an osmolyte that also acts as a methyl group donor in one-carbon metabolism. Previous methods for the analysis of betaine and its metabolite N,N-dimethylglycine (DMG) used derivatization and high performance liquid chromatography with ultra violet detection (HPLC-UV). They required a long sample run time, and often lacked specificity. Liquid chromatography - tandem mass spectrometry (LC-MS/MS) methods were developed with the aim of improving the analysis of betaine and other osmolytes. The objective was to measure the osmolytes: betaine; glycerophosphorylcholine (GPC); taurine; myo-inositol; sorbitol; and trimethylamine-N-oxide (TMAO), as well as N,N-dimethylglycine (DMG), choline, sarcosine, and carnitines. The use of LC-MS/MS improved the analysis of many osmolytes (and related metabolites) compared to previous HPLC- UV methods, and the number of osmolytes that could be analyzed was increased. The analysis of polyols was challenging and no suitable method was found for the analysis of sorbitol; however, amide columns were effective at separating inositol isomers. To demonstrate applications for these methods, samples from overweight people with type 2 diabetes were analyzed to improve knowledge about how osmolytes interact with other metabolites, and their potential use as risk markers in disease. Betaine, DMG, and choline concentrations had low intra-individual variation in the plasma and urine. However, GPC, taurine, and TMAO were more variable. Most subjects with diabetes who were taking bezafibrate had extremely elevated urine betaine. A previously unknown metabolite, N,N-dimethylglycine-N-oxide (DMGO), was identified in plasma and urine. DMGO significantly correlated (p < 0.05) with DMG, betaine, and choline, suggesting that it is derived from the oxidation of DMG. The discovery of DMGO in human samples suggests that a re-evaluation of the widely accepted pathway for choline metabolism may be required.