Large-scale human metabolic phenotyping and molecular epidemiological studies via ¹H NMR spectroscopy of urine: Investigation of borate preservation

Borate is an antibacterial preservative widely used in clinical and large-scale epidemiological studies involving urine sample analysis. Since it readily forms covalent adducts and reversible complexes with hydroxyl and carboxylate groups, the effects of borate preservation in ¹H NMR-spectroscopy-based metabolic profiling of human urine samples have been assessed. Effects of various concentrations of borate (range 0-30 mM) on ¹H NMR spectra of urine were observed at sequential time points over a 12 month period. Consistent with known borate chemistry, the principal alterations in the ¹H resonance metabolite patterns were observed for compounds such as mannitol, citrate, and α-hydroxyisobutyrate and confirmed by ESI-MS analysis. These included line-broadening, T₁ and T₂ relaxation, and chemical shift changes consistent with complex formation and chemical exchange processes. To further investigate complexation behavior in the urinary metabolite profiles, a new tool for visualization of multi-component relaxation variations in which the spectra were color-coded according to the T₁ and T₂ proton relaxation times respectively ( T₁ or T₂ ordered projection spectroscop y, TOPSY) was also developed and applied. Addition of borate caused a general decrease in ¹H T₁ values, consistent with nonspecific effects such as solution viscosity changes. Minor changes in proton T ₂ relaxation rates were observed for the most strongly complexing metabolites. From a molecular phenotyping and epidemiologic viewpoint, typical interpersonal biological variation was shown to be vastly greater than any variation introduced by the borate complexation, which had a negligible effect on the metabolic mapping and classification of samples. While caution is indicated in the assignment of biomarker signals where metabolites have diol groupings or where there are adjacent hydroxyl and carboxylate functions, it is concluded that borate preservation is "fit-forpurpose" for ¹H NMR-based epidemiological studies, since the essential biochemical classification features of the samples are robustly maintained.