Evaluation of two lipid removal methods for stable carbon and nitrogen isotope analysis in whale tissue

Abstract

Rationale: The presence of lipids in animal tissues can influence the interpretation of stable isotope data, particularly in lipid-rich tissues such as the skin and muscle of marine mammals. The traditionally employed chloroform-methanol delipidation protocol has the potential to alter δ¹⁵ N values in proteinaceous tissues. Our objective was to determine whether the use of cyclohexane could be an alternative extraction method, effectively removing lipids without altering δ¹⁵ N values.

Methods: Kidney, liver, muscle, and skin samples were collected from beach-cast Sowerby's beaked whales (Mesoplodon bidens). Control subsamples were processed without delipidation extraction, and duplicate subsamples were extracted with either chloroform-methanol or cyclohexane. δ¹³ C, δ¹⁵ N, and C:N values were determined by continuous-flow elemental analysis isotope ratio mass spectrometry. Paired Wilcoxon tests were used to evaluate the change in isotope ratios after extraction, and unpaired Wilcoxon tests were used to evaluate differences in isotope ratios between extractions.

Results: Use of cyclohexane is an effective delipidation technique for tissues with low and moderate lipid content. Chemical delipidation influenced δ¹⁵ N values; extracted samples generally showed an increase in δ¹⁵ N values which varied from 0.0‰ to 1.7‰. Chloroform-methanol extraction resulted in alterations to δ¹⁵ N values greater than the analytical precision for all analyzed tissues. Changes to δ¹⁵ N values after cyclohexane extraction were at or near the analytical precision for liver and muscle but greater than the analytical precision for kidney and skin.

Conclusions: We recommend processing duplicate subsamples for stable isotope analysis, one with and one without extraction, in order to obtain accurate values for each isotope ratio. Prolonged chemical extractions are not necessary to effectively remove lipids. When samples are limited, we suggest using cyclohexane for tissues with low or moderate lipid content, and chloroform-methanol for lipid-rich tissues.