Evaluation of two lipid removal methods for stable carbon and nitrogen isotope analysis in whale tissue
Evaluation of two lipid removal methods for stable carbon and nitrogen isotope analysis in whale tissue
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 δ15N 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 δ15N 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. δ13C, δ15N, 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 δ15N values; extracted samples generally showed an increase in δ15N values which varied from 0.0‰ to 1.7‰. Chloroform–methanol extraction resulted in alterations to δ15N values greater than the analytical precision for all analyzed tissues. Changes to δ15N 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.
e8851
Smith, Kerri J.
87363319-9dc8-44fd-80f9-7218035cccd1
Trueman, Clive N.
d00d3bd6-a47b-4d47-89ae-841c3d506205
France, Christine A.m.
15898d3f-d7f7-4339-b785-63e93d57f59f
Peterson, Markus J.
3afcb5c3-9c5b-448f-b4af-4625d5c0a259
30 September 2020
Smith, Kerri J.
87363319-9dc8-44fd-80f9-7218035cccd1
Trueman, Clive N.
d00d3bd6-a47b-4d47-89ae-841c3d506205
France, Christine A.m.
15898d3f-d7f7-4339-b785-63e93d57f59f
Peterson, Markus J.
3afcb5c3-9c5b-448f-b4af-4625d5c0a259
Smith, Kerri J., Trueman, Clive N., France, Christine A.m. and Peterson, Markus J.
(2020)
Evaluation of two lipid removal methods for stable carbon and nitrogen isotope analysis in whale tissue.
Rapid Communications in Mass Spectrometry, 34 (18), , [e8851].
(doi:10.1002/rcm.8851).
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 δ15N 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 δ15N 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. δ13C, δ15N, 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 δ15N values; extracted samples generally showed an increase in δ15N values which varied from 0.0‰ to 1.7‰. Chloroform–methanol extraction resulted in alterations to δ15N values greater than the analytical precision for all analyzed tissues. Changes to δ15N 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.
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Smith Kerri J Treatment Methods revised CT
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Accepted/In Press date: 2 June 2020
e-pub ahead of print date: 3 June 2020
Published date: 30 September 2020
Additional Information:
© 2020 John Wiley & Sons, Ltd.
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Local EPrints ID: 443682
URI: http://eprints.soton.ac.uk/id/eprint/443682
ISSN: 0951-4198
PURE UUID: ed6a4219-65f7-4d97-8569-5d6d5224099a
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Date deposited: 09 Sep 2020 16:30
Last modified: 17 Mar 2024 05:53
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Author:
Kerri J. Smith
Author:
Christine A.m. France
Author:
Markus J. Peterson
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