Abstract
The fruit fly Drosophila melanogaster has emerged as an ideal system in which to study 2-hydroxyglutarate (2HG) metabolism. Unlike many mammalian tissues and cell lines, which primarily accumulate d- or l-2HG as the result of genetic mutations or metabolic stress, Drosophila larvae accumulate high concentrations of l-2HG during normal larval growth. As a result, flies represent one of the few model systems that allows for studies of endogenous l-2HG metabolism. Moreover, the Drosophila genome not only encodes key enzymes involved in the synthesis and degradation of d-2HG, but the fly has also been used as to investigate the in vivo effects of oncogenic isocitrate dehydrogenase 1 and 2 (IDH1/2) mutations. All of these studies, however, rely on mass spectrometry-based methods to distinguish between the d- and l-2HG enantiomers. While such approaches are common among labs studying mammalian cell culture, few Drosophila studies have attempted to resolve and measure the individual 2HG enantiomers. Here we describe a highly reproducible gas chromatography-mass spectrometry (GC-MS)-based protocol that allows for quantitative measurements of both 2HG enantiomers in Drosophila homogenates.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Losman JA, Kaelin WG Jr (2013) What a difference a hydroxyl makes: mutant IDH, (R)-2-hydroxyglutarate, and cancer. Genes Dev 27(8):836–852
Ye D, Guan KL, Xiong Y (2018) Metabolism, activity, and targeting of D- and L-2-hydroxyglutarates. Trends Cancer 4(2):151–165
Mishra P et al (2018) ADHFE1 is a breast cancer oncogene and induces metabolic reprogramming. J Clin Invest 128(1):323–340
Oldham WM et al (2015) Hypoxia-mediated increases in l-2-hydroxyglutarate coordinate the metabolic response to reductive stress. Cell Metab 22(2):291–303
Intlekofer AM et al (2015) Hypoxia induces production of L-2-hydroxyglutarate. Cell Metab 22(2):304–311
Tyrakis PA et al (2016) S-2-hydroxyglutarate regulates CD8+ T-lymphocyte fate. Nature 540(7632):236–241
Fan J et al (2015) Human phosphoglycerate dehydrogenase produces the oncometabolite D-2-hydroxyglutarate. ACS Chem Biol 10(2):510–516
Teng X et al (2016) Lactate dehydrogenase C produces S-2-hydroxyglutarate in mouse testis. ACS Chem Biol 11(9):2420–2427
Struys EA et al (2005) Kinetic characterization of human hydroxyacid-oxoacid transhydrogenase: relevance to D-2-hydroxyglutaric and gamma-hydroxybutyric acidurias. J Inherit Metab Dis 28(6):921–930
Becker-Kettern J et al (2016) Saccharomyces cerevisiae Forms D-2-hydroxyglutarate and couples its degradation to D-lactate formation via a cytosolic transhydrogenase. J Biol Chem 291(12):6036–6058
Li H et al (2017) Drosophila larvae synthesize the putative oncometabolite L-2-hydroxyglutarate during normal developmental growth. Proc Natl Acad Sci U S A 114(6):1353–1358
Reinecke CJ et al (2012) Metabolomics of urinary organic acids in respiratory chain deficiencies in children. Metabolomics 8(2):264–283
Nadtochiy SM et al (2016) Acidic pH Is a metabolic switch for 2-hydroxyglutarate generation and signaling. J Biol Chem 291(38):20188–20197
Reitman ZJ et al (2015) Genetic dissection of leukemia-associated IDH1 and IDH2 mutants and D-2-hydroxyglutarate in Drosophila. Blood 125(2):336–345
Gibson KM et al (1993) Stable-isotope dilution analysis of D- and L-2-hydroxyglutaric acid: application to the detection and prenatal diagnosis of D- and L-2-hydroxyglutaric acidemias. Pediatr Res 34(3):277–280
Ashburner M, Bownes M, Abrahamssen N, Gilman C (2005) Drosophila: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York, pp 121–184
Li H, Tennessen JM (2017) Methods for studying the metabolic basis of Drosophila development. Wiley Interdiscip Rev Dev Biol 6(5):e280
Acknowledgment
J.M.T. is supported by a R35 Maximizing Investigators’ Research Award (MIRA; 1R35GM119557) from the National Institute of General Medical Sciences of the National Institutes of Health.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Li, H., Tennessen, J.M. (2019). Quantification of d- and l-2-Hydroxyglutarate in Drosophila melanogaster Tissue Samples Using Gas Chromatography-Mass Spectrometry. In: D'Alessandro, A. (eds) High-Throughput Metabolomics. Methods in Molecular Biology, vol 1978. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9236-2_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9236-2_10
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9235-5
Online ISBN: 978-1-4939-9236-2
eBook Packages: Springer Protocols