Comparison of Three Extraction Methods at Room Temperature for 1H-NMR-Based Metabolomics of Eastern Oyster (Crassostrea virginica) Adductor Muscle
IAAAM 2016
Scott H. Hammer1*+; Jennifer N. Niemuth1,2,3; Michael K. Stoskopf1,2,3
1Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA; 2Fisheries, Wildlife, & Conservation Biology Program, College of Natural Resources, North Carolina State University, Raleigh, NC, USA; 3Environmental Medicine Consortium, North Carolina State University, Raleigh, NC, USA


Metabolomics is an emerging diagnostic modality that uses mass spectroscopy and/or proton nuclear magnetic resonance spectroscopy (1H-NMR) to generate a profile of the metabolites of a tissue. The eastern oyster (Crassostrea virginica) and other bivalves have been the subject of numerous 1H-NMR-based metabolomics studies in the recent literature, particularly because of their importance as environmental sentinels.1,2,3,4 The most common protocols for this technique have included immediate freezing of tissue samples in liquid nitrogen during sample collection followed by various forms of extraction.1,2,5 This study evaluated use of three different extraction solutions and their relative ability to obtain quantitative extractions of key components of Crassostrea metabolism at room temperature: 100% methanol (MeOH), 100% chloroform (CHCl3), and a 50:50 (volume:volume) ratio of methanol and chloroform (M:C). The results of this study established that each extraction solvent protocol provided spectra that were optimal for different regions of the NMR chemical shift. MeOH extraction yielded a similar suite of metabolites (i.e., amino acids and simple carbohydrates) to those reported in the literature in the chemical shift range of 1.0 to 4.2 ppm (Hz/MHz). CHCl3 extraction consistently resolved the metabolites involved with osmotic regulation (e.g., betaine and taurine). M:C spectra extracted additional metabolites observed in the 7.9 to 9.1 ppm region. These techniques show substantial potential to benefit field studies, provided that the solvent chosen coincides with the targeted region of interest in the NMR spectrum.


The authors thank Drs Stasia Bembeneck-Bailey and Andrey Tikunov for assistance processing samples, and the faculty and staff at the Center for Marine Science and Technology (CMAST) at North Carolina State University for their assistance with sample collection. Additionally, the authors thank The Environmental Medicine Consortium for the financial support of this project.

* Presenting author
+ Student presenter

Literature Cited

1.  Tikunov AP, Johnson CB, Lee H, Stoskopf MK, Macdonald JM. Metabolomic investigations of American oysters Using 1H-NMR spectroscopy. Mar Drugs. 2010;8(10):2578–2596.

2.  Tikunov AP, Stoskopf MK, Macdonald JM. Fluxomics of the Eastern oyster for environmental stress studies. Metabolites. 2014;4(1):53–70.

3.  Lannig G, Eilers S, Portner HO, Sokolova IM, Bock C. Impact of ocean acidification on energy metabolism of oyster, Crassostrea gigas - changes in metabolic pathways and thermal response. Mar Drugs. 2010;8(8):2318–2339.

4.  Martínez-Gómez C, Robinson CD, Burgeot T, Gubbins M, Halldorsson HP, Albentosa M, Bignell JP, Hylland K, Vethaak AD. Biomarkers of general stress in mussels as common indicators for marine biomonitoring programmes in Europe: The ICON experience. 2015. In press. doi:10.1016/j.marenvres.2015.10.012.

5.  Hurley-Sanders JL, Levine JF, Nelson SAC, Law JM, Showers WJ, Stoskopf MK. Key metabolites in tissue extracts of Elliptio complanata identified using 1H nuclear magnetic resonance spectroscopy. Conserv Physiol. 2015;3:1–10.


Speaker Information
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Scott H. Hammer, Student
Department of Clinical Sciences
College of Veterinary Medicine
North Carolina State University
Raleigh, NC, USA

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