METHODS Getting to the fat of the matter: models, methods and assumptions for dealing with lipids in stable isotope analyses David M. Post Æ Craig A. Layman Æ D. Albrey Arrington Æ Gaku Takimoto Æ John Quattrochi Æ Carman G. Montan ˜a Received: 21 June 2006 / Accepted: 21 November 2006 Ó Springer-Verlag 2007 Abstract Within an organism, lipids are depleted in 13 C relative to proteins and carbohydrates (more neg- ative d 13 C), and variation in lipid content among organisms or among tissue types has the potential to introduce considerable bias into stable isotope analyses that use d 13 C. Despite the potential for introduced error, there is no consensus on the need to account for lipids in stable isotope analyses. Here we address two questions: (1) If and when is it important to account for the effects of variation in lipid content on d 13 C? (2) If it is important, which method(s) are reliable and robust for dealing with lipid variation? We evaluated the reliability of direct chemical extraction, which physi- cally removes lipids from samples, and mathematical normalization, which uses the carbon-to-nitrogen (C:N) ratio of a sample to normalize d 13 C after analysis by measuring the lipid content, the C:N ratio, and the effect of lipid content on d 13 C(Dd 13 C) of plants and animals with a wide range of lipid contents. For ani- mals, we found strong relationships between C:N and lipid content, between lipid content and Dd 13 C, and between C:N and Dd 13 C. For plants, C:N was not a good predictor of lipid content or Dd 13 C, but we found a strong relationship between carbon content and lipid content, lipid content and Dd 13 C, and between and carbon content and Dd 13 C. Our results indicate that lipid extraction or normalization is most important when lipid content is variable among consumers of interest or between consumers and end members, and when differences in d 13 C between end members is <10–12&. The vast majority of studies using natural variation in d 13 C fall within these criteria. Both direct lipid extraction and mathematical normalization reduce biases in d 13 C, but mathematical normalization simplifies sample preparation and better preserves the integrity of samples for d 15 N analysis. Keywords Stable isotopes  Lipid extraction  Lipid concentration  Mathematical normalization  C:N  Plants  Animals Introduction Stable isotopes have emerged as important tools for addressing questions of plant and animal physiology (Peterson and Fry 1987; Gannes et al. 1997; Dawson et al. 2002), paleoecology (Schwarcz and Schoeninger Communicated by Jay Rosenheim. Electronic supplementary material The online version of this article (doi:10.1007/s00442-006-0630-x) contains supplementary material, which is available to authorized users. D. M. Post (&)  G. Takimoto Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT 06520-8105, USA e-mail: david.post@yale.edu C. A. Layman  J. Quattrochi Marine Science Program, Department of Biological Sciences, Florida International University, 3000 NE 151st St., North Miami, FL 33181, USA D. A. Arrington Loxahatchee River District, 2500 Jupiter Park Drive, Jupiter, FL 33458-8964, USA C. G. Montan ˜a Universidad de Los Llanos Ezequiel Zamora, UNELLEZ, Apartado Postal, Guanare 3310, Venezuela 123 Oecologia DOI 10.1007/s00442-006-0630-x