High-Speed Tracer Analysis of Metabolism (HS-TrAM) [version 2; referees: 2 approved, 2 approved with reservations]

Abstract

Tracing the fate of stable isotopically-enriched nutrients is a sophisticated method of describing and quantifying the activity of metabolic pathways. Nuclear Magnetic Resonance (NMR) spectroscopy offers high resolution data in terms of resolving metabolic pathway utilisation. Despite this, NMR spectroscopy is under-utilised due to length of time required to collect the data, quantification requiring multiple samples and complicated analysis. Here we present two techniques, quantitative spectral filters and enhancement of the splitting of 13C signals due to homonuclear 13C,13C or heteronuclear 13C,15N J-coupling in 1H,13C-HSQC NMR spectra. Together, these allow the rapid collection of NMR spectroscopy data in a quantitative manner on a single sample. The reduced duration of HSQC spectra data acquisition opens up the possibility of real-time tracing of metabolism including the study of metabolic pathways in vivo. We show how these techniques can be used to trace the fate of labelled nutrients in a whole organ model of kidney preservation prior to transplantation using a porcine kidney as a model organ. In addition, we show how the use of multiple nutrients, differentially labelled with 13C and 15N, can be used to provide additional information with which to profile metabolic pathways.