Diserens, Gaëlle; Hertig, Damian; Eggimann, Sandra; Vermathen, Martina; Furrer, Julien; Nuoffer, Jean-Marc; Vermathen, Peter (27 June 2016). Improving the metabolic stability of cultured cells during extended HR-MAS NMR measurements by prior heating (Unpublished). In: 12th Annual Conference of the Metabolomics Society. Dublin, Ireland. 27.-30.06.2016.
Introduction:
1HHRMAS NMR is an established tool for metabolic characterization of biological samples.
However, the accuracy of biomarker detection depends on the sample stability over
measurement time. Previously, Duarte et al. found enhanced metabolite visibility and different lipid profiles in lysed compared to frozen cells [1]. Here we investigated effects of shorttime heating prior to HRMAS measurements on metabolite stability of cells. We hypothesized that cell heating has only minor effects on initial metabolite contents (i.e. initially similar spectra with and without heating) and results in increased temporal metabolite stability due to reduced enzymatic activity.
Methods:
The metabolite content of six lysed nonheated (LFB) and six lysed heated fibroblastsamples (LHFB) was measured as a function of time. Additionally, one sample each of nonheated
and heated lysed adrenal cells (LAD & LHAD), which are less robust than fibroblasts, were measured. After lysis according to [1], half of the samples were heated (70°C) for 20min. 1HPROJECT [2] spectra were obtained on a Bruker AvanceII spectrometer (500.13MHz, 277K, MAS=3kHz) at different times over 9 hours. Individual peak analysis was performed investigating temporal changes. For fibroblasts, statistical methods were applied including PCA.
Results and Discussion:
PCA results and individual peak analysis clearly confirmed our hypothesis: The temporal
metabolite stability for LHFB was largely maintained in contrast to LFB. Average temporal
variation of all peaks was 14.4% for LHFB compared to 43.1% for LFB. Additionally, the
PCA plot demonstrated close clustering for LHFB, whereas LFB were spreading out over
time. Also LHAD showed temporal metabolic stability, while LAD exhibited strong changes.
Our results suggest using cell lysis in combination with heating for extended longterm
HRMAS NMR measurements, in order to minimize metabolite content modifications over
measurement time.
References:
1. Duarte IF, et al. Anal. Chem. 2009;81:5023.
2. Aguilar JA, et al. Chemical Communications 2012;48:811.
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