title: Quantification of Metabolites by NMR Spectroscopy in the Presence of Protein.
abstract: The high reliability of NMR spectroscopy makes it an ideal tool for large-scale metabolomic studies. However, the complexity of biofluids and, in particular, the presence of macromolecules poses a significant challenge. Ultrafiltration and protein precipitation are established means of deproteinization and recovery of free or total metabolite content, but neither is ever complete. In addition, aside from cost and labor, all deproteinization methods constitute an additional source of experimental variation. The Carr-Purcell-Meiboom-Gill (CPMG) echo-train acquisition of NMR spectra obviates the need for prior deproteinization by attenuating signals from macromolecules, but concentration values of metabolites measured in blood plasma will not necessarily reflect total or free metabolite content. Here, in contrast to approaches that propose the determination of individual T1and T2relaxation times for the computation of correction factors, we demonstrate their determination by spike-in experiments with known amounts of metabolites in pooled samples of the matrix of interest to facilitate the measurement of total metabolite content. Provided that the protein content does not vary too much among individual samples, accurate quantitation of metabolites is feasible. Moreover, samples with significantly deviating protein content may be readily recognized by inclusion of a standard that shows moderate protein binding. It is also shown that urinary proteins when present in high concentrations may effect detection of common urinary metabolites prone to strong protein binding such as tryptophan.
authors.affiliation: Institute of Functional Genomics, University of Regensburg , Am Biopark 9, 93053 Regensburg, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Wallmeier
authors.affiliation: Institute of Functional Genomics, University of Regensburg , Am Biopark 9, 93053 Regensburg, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Samol
authors.affiliation: Institute of Functional Genomics, University of Regensburg , Am Biopark 9, 93053 Regensburg, Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: Ellmann
authors.affiliation: Institute of Functional Genomics, University of Regensburg , Am Biopark 9, 93053 Regensburg, Germany.
authors.forename: Helena U
authors.initials: HU
authors.lastname: Zacharias
authors.affiliation: Institute of Functional Genomics, University of Regensburg , Am Biopark 9, 93053 Regensburg, Germany.
authors.forename: Franziska C
authors.initials: FC
authors.lastname: Vogl
authors.affiliation: Institute of Functional Genomics, University of Regensburg , Am Biopark 9, 93053 Regensburg, Germany.
authors.forename: Muriel
authors.initials: M
authors.lastname: Garcia
authors.affiliation: Institute of Functional Genomics, University of Regensburg , Am Biopark 9, 93053 Regensburg, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Dettmer
authors.affiliation: Institute of Functional Genomics, University of Regensburg , Am Biopark 9, 93053 Regensburg, Germany.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Oefner
authors.affiliation: Institute of Functional Genomics, University of Regensburg , Am Biopark 9, 93053 Regensburg, Germany.
authors.forename: Wolfram
authors.identifier: 0000-0003-3646-0060
authors.initials: W
authors.lastname: Gronwald
mesh_terms: D001798:Blood Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002853:Chromatography, Liquid; D009682:Magnetic Resonance Spectroscopy; D055442:Metabolome / Q000235:genetics*; D055432:Metabolomics; D011485:Protein Binding; D053719:Tandem Mass Spectrometry
chemical_list: D001798:Blood Proteins
keywords: 1D NMR; CPMG; correction factors; plasma; quantification