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

摘要

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 delta N-15 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 delta N-15 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. delta C-13, delta N-15, 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 delta N-15 values; extracted samples generally showed an increase in delta N-15 values which varied from 0.0 parts per thousand to 1.7 parts per thousand. Chloroform-methanol extraction resulted in alterations to delta N-15 values greater than the analytical precision for all analyzed tissues. Changes to delta N-15 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.