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A LC-MS–based workflow for measurement of branched fatty acid esters of hydroxy fatty acids

Nature Protocols volume 11, pages 747–763 (2016)Cite this article

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Abstract

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of endogenous mammalian lipids with antidiabetic and anti-inflammatory effects. We previously identified 16 different FAHFA families, such as branched palmitic acid esters of hydroxy stearic acids (PAHSAs); each family consists of multiple isomers in which the branched ester is at different positions (e.g., 5- and 9-PAHSA). We anticipate increased need for PAHSA measurements as markers of metabolic and inflammatory health. In this protocol, we provide a detailed description of the extraction of FAHFAs from human or mouse tissues, their enrichment by solid-phase extraction and subsequent analysis by LC-MS. For a sample size of 6–12, the time frame is 2–3 d.

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Figure 1: PAHSA isomer detection by MRM.
Figure 2
Figure 3: Lipid extraction from serum and tissues.
Figure 4: SPE.
Figure 5: PAHSA isomer distribution in WT mouse sera and tissues.
Figure 6: FAHFA background.
Figure 7: Contaminant identification.

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Acknowledgements

We thank J. Belcovson for photography of glassware in Figure 3a. This work is supported by a Lilly Fellowship (T.Z.); Chapman Foundation Fellowship (M.J.K.); by grants from the US National Institutes of Health (NIH) (R37 DK43051, P30 DK57521 and R01 DK098002 (B.B.K.)) and the JPB foundation (B.B.K.); by the National Cancer Institute, Cancer Center Support Grant P30 CA014195 MASS core (A.S.); by The Leona M. and Harry B. Helmsley Charitable Trust grant (no. 2012-PG-MED002; A.S.) and the Dr. Frederick Paulsen Chair/Ferring Pharmaceuticals (A.S.); by The Swedish Research Council (J.B. and U.S.); by The Novo Nordisk Foundation (U.S.) and The Torsten Soderberg Foundation (U.S.); and by the Heart-Lung Foundation (J.B.), Sahlgrenska University Hospital ALF funds (J.B.) and the EU Seventh Framework Program RESOLVE (J.B.).

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Authors and Affiliations

  1. Salk Institute for Biological Studies, La Jolla, California, USA

    Tejia Zhang, Shili Chen, Matthew J Kolar, Qian Chu & Alan Saghatelian

  2. Division of Endocrinology, Department of Medicine, Diabetes & Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA

    Ismail Syed & Barbara B Kahn

  3. Department of Molecular and Clinical Medicine, Wallenberg Laboratory, University of Gothenburg, Gothenburg, Sweden

    Marcus Ståhlman & Jan Borén

  4. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA

    Edwin A Homan

  5. Department of Molecular and Clinical Medicine, Lundberg Laboratory for Diabetes Research, University of Gothenburg, Gothenburg, Sweden

    Ulf Smith

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  1. Tejia Zhang
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Contributions

T.Z. developed the experimental design strategy (FAHFA extraction, SPE and LC-MS) and the quality control strategy, performed sample measurements and wrote the manuscript. S.C. performed ongoing optimizations on the first-generation LC-MS method and sample measurements. I.S. performed sample measurements. M.S. assisted with FAHFA enrichment. M.J.K performed sample measurements and experiments to demonstrate the shift in retention times with some of the commercially available deuterated standards. Q.C. performed in vitro activity assays to examine possible exogenous standard incorporation into FAHFAs during lipid extraction. E.A.H. performed initial global lipidomics and tandem MS. A.S., B.B.K., J.B. and U.S. conceived of, designed and supervised the experimental plan and interpreted experiments. A.S., B.B.K., T.Z., S.C., I.S. and M.S. edited the manuscript.

Corresponding authors

Correspondence to Barbara B Kahn or Alan Saghatelian.

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Competing interests

A.S., E.A.H., I.S. and B.B.K. are inventors on patents (patent nos. WO2013166431, US20150133551, and EP2844303A1) related to these novel lipids. The other authors declare no competing financial interests.

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Zhang, T., Chen, S., Syed, I. et al. A LC-MS–based workflow for measurement of branched fatty acid esters of hydroxy fatty acids. Nat Protoc 11, 747–763 (2016). https://doi.org/10.1038/nprot.2016.040

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