Lipid Extraction Techniques for Stable Isotope Analysis and Ecological Assays

Abstract

Lipid extraction is an important component of many ecological and ecotoxicological measurements. For instance, percent lipid is often used as a measure of body condition, under the assumption that those individuals with higher lipid reserves are healthier. Likewise, lipids are depleted in C compared with protein, and it is consequently a routine to remove lipids prior to measuring carbon isotopes in ecological studies so that variation in lipid content does not obscure variation in diet. We provide detailed methods for two different protocols for lipid extraction: Soxhlet apparatus and manual distillation. We also provide methods for polar and nonpolar solvents. Neutral (nonpolar) solvents remove some lipids but few non-lipid compounds, whereas polar solvents remove most lipids but also many non-lipid compounds. We discuss each of the methods and provide guidelines for best practices. We recommend that, for stable isotope analysis, researchers test for a relationship between the change in carbon stable isotope ratio and the amount of lipid extracted to see if the degree of extraction has an impact on isotope ratios. Stable isotope analysis is widely used by ecologists, and we provide a detailed methodology that minimizes known biases.

Diet–tissue discrimination factors (δ15N and δ13C values) for blood components in Magellanic (Spheniscus magellanicus) and southern rockhopper (Eudyptes chrysocome) penguins

“…Indeed, even after delipidation, the seabird plasma C:N values of alcids and shearwaters were >4.0 (common murre Uria aalge, 4.04 ± 0.02; Atlantic puffin Fratercula arctica, 4.37 ± 0.21 16 ; great shearwater Ardenna gravis, 4.34 ± 0.13; and sooty shearwater Ardenna grisea, 4.30 ± 0.23 32 ). In our study, however, the C:N values for plasma for both species after delipidation were 3.9, similar to those found by Cherel et al, 25 suggesting that most lipids were removed prior to stable isotope analysis. As the other published studies do not report the C:N values of penguin blood (Table 2), it is difficult to assess whether our C:N values are common and, thus, we recommend that future studies provide this information.…”

“…Blood components, whole fish, and fish muscle samples were freeze‐dried and homogenized. As high lipid content can influence the δ 13 C values of tissues, the bird plasma and fish samples went through chemical lipid extraction using petroleum ether within a Soxhlet apparatus for 8 h, with samples being subsequently air‐dried . The CC samples did not require lipid extraction due to their low lipid content, as demonstrated by C:N < 3.5 (Cherel et al and our study).…”

“…As high lipid content can influence the δ 13 C values of tissues, 24 the bird plasma and fish samples went through chemical lipid extraction using petroleum ether within a Soxhlet apparatus for 8 h, with samples being subsequently air-dried. 25 The CC samples did not require lipid extraction due to their low lipid content, as demonstrated by C:N < 3.5 (Cherel et al 26 The precision, based on the standard deviation of replicate analyses (a triplicate was run for every tenth sample), was ≤0.16‰ for δ 15 N values and ≤0.12‰ for δ 13 C values.…”

Isotopic niche differs between seal and fish‐eating killer whales (Orcinus orca) in northern Norway

“…Skin samples from killer whales, and muscle from lumpfish, herring, and seal were freeze‐dried and ground individually with an agate mortar and pestle to a fine powder. An aliquot was rinsed three times in a 2:1 chloroform: methanol solution to remove lipids, following the method developed by Folch, Lees, and Stanley (1957) and modified by Elliott, Roth, and Crook (2017). An aliquot from the bulk tissue was not treated with any chloroform: methanol solution.…”

Isotopic turnover rates and diet-tissue discrimination depend on feeding habits of freshwater snails

“…We extracted lipids from the snail and diet samples because lipids have different carbon isotopic composition than other components, which could lead to high variation in δ 13 C measurement for samples with different amounts of lipids [ 36 ]. Lipids were removed from samples with petroleum ether for 16 hours using a Soxhlet apparatus, and then oven dried at 60°C for 24 hours [ 37 ]. Low-protein (lettuce) samples were placed in a drying oven at 60°C for 48 hours and then ball-milled to a fine powder.…”