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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol is for the determination of lipids in seawater and biological specimens. Lipids in filtrates are extracted with chloroform or mixtures of chloroform and methanol in the case of solids. Lipid classes are measured by rod thin-layer chromatography with flame ionization detection and their sum gives the total lipid content.

Abstract

Lipids are largely composed of carbon and hydrogen and, therefore, provide a greater specific energy than other organic macromolecules in the sea. Being carbon- and hydrogen-rich they are also hydrophobic and can act as a solvent and absorption carrier for organic contaminants and thus can be drivers of pollutant bioaccumulation in marine ecosystems. Their hydrophobic nature facilitates their isolation from seawater or biological specimens: marine lipid analysis begins with sampling and then extraction in non-polar organic solvents, providing a convenient method for their separation from other substances in an aquatic matrix.

If seawater has been sampled, the first step usually involves separation into operationally defined 'dissolved' and 'particulate' factions by filtration. Samples are collected and lipids isolated from the sample matrix typically with chloroform for truly dissolved matter and colloids, and with mixtures of chloroform and methanol for solids and biological specimens. Such extracts may contain several classes from biogenic and anthropogenic sources. At this time, total lipids and lipid classes may be determined. Total lipid can be measured by summing individually determined lipid classes which customarily have been chromatographically separated. Thin-layer chromatography (TLC) with flame ionization detection (FID) is regularly used for the quantitative analysis of lipids from marine samples. TLC-FID furnishes synoptic lipid class information and, by summing classes, a total lipid measurement.

Lipid class information is especially useful when combined with measurements of individual components e.g., fatty acids and/or sterols, after their release from lipid extracts. The wide variety of lipid structures and functions means they are used broadly in ecological and biogeochemical research assessing ecosystem health and the degree of influence by anthropogenic impacts. They have been employed to measure substances of dietary value to marine fauna (e.g., aquafeeds and/or prey), and as an indicator of water quality (e.g., hydrocarbons).

Introduction

The methods described here concern substances that are defined operationally as marine lipids. This definition is based on their amenability to liquid-liquid extraction in non-polar organic solvents, and it provides a convenient method for their separation from other substances in an aquatic matrix. Their hydrophobic nature facilitates their isolation from seawater or biological specimens, as well as their enrichment, and the removal of salts and proteins.

The measurement of lipid content and its composition in marine organisms has been of great interest in food web ecology, aquaculture nutrition, and food science for decades. Lipids are un....

Protocol

NOTE: To clean glassware, instruments and filters for lipid analyses, wash them 3 times with methanol followed by 3 washes with chloroform, or heat them to 450°C for at least 8 hours.

1. Filtration procedure for seawater dissolved and particulate lipids

NOTE: The particular fraction of interest is operationally defined by the filtration procedure. In this case the pore size is 1.2 µm.

  1. Set up the filtration manifold without a filter and rinse the setup with filtered seawater.
  2. Using clean forceps place a 47 mm glass fiber (GF/C) filter, that has been washed, into the....

Results

As the fastest growing food production sector, aquaculture is evolving in terms of technological innovations and adaptations to meet changing requirements. One of these is to reduce the dependence on wild-sourced fishmeal and fish oil, which provide feed ingredients for many aquaculture species. Terrestrial plant oils are being investigated as sustainable and economical replacements for fish oil in aquafeeds, and the liver is a target tissue for analysis because it is the primary site for lipid metabolism

Discussion

The speed with which the TLC-FID system provides synoptic lipid class information from small samples makes TLC-FID an able tool for screening marine samples before undertaking more involved analytical procedures. Such analyses usually require release of component compounds from lipid extracts and derivatization to increase volatility in the case of gas chromatography. TLC-FID combined with GC-FID has been found to be a powerful combination for extracts of seafood and other foodstuffs14. For succes.......

Disclosures

The authors have no competing financial interests.

Acknowledgements

This research was funded by Natural Sciences and Engineering Research Council of Canada (NSERC) grant number 105379 to C.C. Parrish. Memorial University's Core Research Equipment & Instrument Training (CREAIT) Network helped fund this publication.

....

Materials

NameCompanyCatalog NumberComments
15 ml vialsVWR66009-560
1-hexadecanolSigma258741-1G
1-Monopalmitoyl-rac-glycerolSigmaM1640-1g
2 ml vialsVWR46610-722
25 mm glass fibre filtersFisher09 874 32A
2ml pipet bulbsVWR82024-554
47 mm glass fibre filtersFisher09 874 32
5 3/4" pipetsFisher1367820A
9" pipetsFisher1367820C
AcetoneVWRCAAX0116-1
Agilent GC-FID 6890Agilent
Calcium Chloride ANHS 500gmVWRCACX0160-1
Caps for 2 ml vialsVWR46610-712
chloroformVWRCACX1054-1
Cholesteryl palmitateSigmaC6072-1G
Chromarod S5Shell USA3252
DichloromethaneVWRCADX0831-1
DL-a-phosphatidylcholine, dipalmotoylSigmaP5911-1g
Ethyl Ether, ACS grade anhydr 4LVWRCAEX0190-4
Glyceryl tripalmitateSigmaT5888-100MG
Hamilton Syringe 702SNR 25µlSigma58381
HeliumAir LiquideA0492781
HexaneVWRCAHX0296-1
Hydrogen regulatorVWR55850-484
Iatroscan MK6Shell USA
KimwipesFisher066662
Medical AirAir LiquideA0464563
Medium nitrile glovesFisher191301597C
Nitrile gloves LVWRCA82013-782
NitrogenAir LiquideA0464775
Nitrogen RegulatorVWR55850-474
NonadecaneSigma74158-1G
Palmitic acidSigmaP0500-10G
Repeating dispenserSigma20943
Sodium Bicarbonate 1kgVWRCA97062-460
Sodium Sulfate Anhy ACS 500grVWRCA71008-804
Sulfuric acidVWRCASX1244-5
Teflon tapeFisher14610120
tissue master 125 115V w/7mm homogenatorOMNI InternationalTM125-115
TLC development tankShell USA3201
UHP hydrogenAir LiquideA0492788
VWR solvent repippetterVWR82017-766
VWR timer Flashing LED 2 channelVWR89140-196
Zebron ZB-Wax GC columnPhenomenex7HM-G013-11

References

  1. Couturier, L. I. E., et al. State of art and best practices for fatty acid analysis in aquatic sciences. ICES Journal of Marine Science. , (2020).
  2. Parrish, C. C. Lipids in Marine Ecosystems. ISRN Oceanography. , 6040....

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