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Comparison of tissue harvest protocols for the quantitation of acylcarnitines in mouse heart and liver by mass spectrometry

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Abstract

Profiling of acylcarnitines (ACs) in tissues and biological fluids by mass spectrometry is a powerful approach to examine the impact of genetic, pharmacological, and environmental factors on intermediary metabolism. The AC pool exhibits rapid changes in composition and abundance in response to altered cellular fuel catabolism. However, the mercurial nature of the AC pool makes it prone to spurious influences arising from experimental variables related to tissue harvesting and processing. We evaluated the impact of various strategies to anesthetize, sedate, or euthanize (A/S/E) mice which included Nembutal, Beuthanasia, isoflurane, ketamine/xylazine (Ket/Xy) and CO2/cervical dislocation (CD) on the tissue AC profiles. ACs extracted from heart and liver were derivatized to methyl esters and quantitated by mass spectrometry. Marked differences were seen in the tissue AC profiles, especially in heart, depending upon the choice of the A/S/E strategy. Most importantly, a uniform A/S/E protocol must be employed. While tissue AC profiles in situ cannot be unambiguously defined, use of Nembutal appears to be superior to other A/S/E strategies especially when assessing the AC levels in the heart. We also showed that it is preferable to expeditiously harvest and flash-freeze tissues to avoid procedure-related perturbation of the AC profile. A more protracted tissue harvest, when recovering numerous tissues from the same animal, can alter the AC pool. In conclusion, this investigation provides key guidance for harvesting heart and liver from mice in order to minimize the procedure-associated change of the AC pool which can mask the influence of the intended experimental variables.

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Abbreviations

AC:

Acylcarnitine

CD:

Cervical dislocation

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Acknowledgments

The authors thank Drs. Robert Stevens and Brett Wenner from the Sarah W. Stedman Nutrition and Metabolism Center at Duke University School of Medicine for their assistance in validating the acylcarnitine assay. We also thank Jessica M. Nigro of the Sanford-Burnham Medical Research Institute for harvesting tissues from mice.

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Authors and Affiliations

  1. Metabolomics Core, Sanford-Burnham Medical Research Institute, 6400 Sanger Rd., Orlando, FL, 32827, USA

    Christopher Petucci, Stella Rojas-Betancourt & Stephen J. Gardell

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  1. Christopher Petucci
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  2. Stella Rojas-Betancourt
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  3. Stephen J. Gardell
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Correspondence to Christopher Petucci.

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Petucci, C., Rojas-Betancourt, S. & Gardell, S.J. Comparison of tissue harvest protocols for the quantitation of acylcarnitines in mouse heart and liver by mass spectrometry. Metabolomics 8, 784–792 (2012). https://doi.org/10.1007/s11306-011-0370-8

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