Direct estimation of metabolic flux by heavy isotope labeling simultaneous with pathway inhibition: metabolic flux inhibition assay

Zhang, T., Labuschagne, C. F., Vousden, K. H. and Maddocks, O. D.K. (2018) Direct estimation of metabolic flux by heavy isotope labeling simultaneous with pathway inhibition: metabolic flux inhibition assay. In: Fendt, S.-M. and Lunt, S. Y. (eds.) Metabolic Signalling: Methods and Protocols. Series: Methods in molecular biology (1862). Humana Press: New York, pp. 109-119. ISBN 9781493987689 (doi:10.1007/978-1-4939-8769-6_8)

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Abstract

Heavy isotope labeled metabolites are readily detected by mass spectrometry and are commonly used to analyze the rates of metabolic reactions in cultured cells. The ability to detect labeled metabolites—and infer fluxes—is influenced by a number of factors that can confound simplistic comparative assays. The accumulation of labeled metabolites is strongly influenced by the pool size of the metabolite of interest and also by changes in downstream reactions, which are not always fully perceived. Here, we describe a method that overcomes some of these limitations and allows simple calculation of reaction rates under low nutrient, rapid reaction rate conditions. Acutely increasing the pool of the metabolite of interest (by adding a pulse of excess unlabeled nutrient to the cells) rapidly increases accumulation of labeled metabolite, facilitating a more accurate assessment of reaction rate.

Item Type:Book Sections
Status:Published
Glasgow Author(s) Enlighten ID:Zhang, Mr Tong and Labuschagne, Dr Christiaan Fred and Maddocks, Dr Oliver and Vousden, Karen
Authors: Zhang, T., Labuschagne, C. F., Vousden, K. H., and Maddocks, O. D.K.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
Publisher:Humana Press
ISBN:9781493987689
Published Online:13 October 2018

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
666241Targeting Tumour Metabolism for Cancer Therapy and Diagnosis.Oliver MaddocksCancer Research UK (CRUK)19702RI CANCER SCIENCES