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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Environment

Determination of Total Lipid and Lipid Classes in Marine Samples

Published: December 11th, 2021

DOI:

10.3791/62315

1Department of Ocean Sciences and CREAIT Network, Memorial University of Newfoundland

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.

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).

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....

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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 fi.......

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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

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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.......

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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.

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Name Company Catalog Number Comments
15 ml vials VWR 66009-560
1-hexadecanol Sigma 258741-1G
1-Monopalmitoyl-rac-glycerol Sigma M1640-1g
2 ml vials VWR 46610-722
25 mm glass fibre filters Fisher 09 874 32A
2ml pipet bulbs VWR 82024-554
47 mm glass fibre filters Fisher 09 874 32
5 3/4" pipets Fisher 1367820A
9" pipets Fisher 1367820C
Acetone VWR CAAX0116-1
Agilent GC-FID 6890 Agilent
Calcium Chloride ANHS 500gm VWR CACX0160-1
Caps for 2 ml vials VWR 46610-712
chloroform VWR CACX1054-1
Cholesteryl palmitate Sigma C6072-1G
Chromarod S5 Shell USA 3252
Dichloromethane VWR CADX0831-1
DL-a-phosphatidylcholine, dipalmotoyl Sigma P5911-1g
Ethyl Ether, ACS grade anhydr 4L VWR CAEX0190-4
Glyceryl tripalmitate Sigma T5888-100MG
Hamilton Syringe 702SNR 25µl Sigma 58381
Helium Air Liquide A0492781
Hexane VWR CAHX0296-1
Hydrogen regulator VWR 55850-484
Iatroscan MK6 Shell USA
Kimwipes Fisher 066662
Medical Air Air Liquide A0464563
Medium nitrile gloves Fisher 191301597C
Nitrile gloves L VWR CA82013-782
Nitrogen Air Liquide A0464775
Nitrogen Regulator VWR 55850-474
Nonadecane Sigma 74158-1G
Palmitic acid Sigma P0500-10G
Repeating dispenser Sigma 20943
Sodium Bicarbonate 1kg VWR CA97062-460
Sodium Sulfate Anhy ACS 500gr VWR CA71008-804
Sulfuric acid VWR CASX1244-5
Teflon tape Fisher 14610120
tissue master 125 115V w/7mm homogenator OMNI International TM125-115
TLC development tank Shell USA 3201
UHP hydrogen Air Liquide A0492788
VWR solvent repippetter VWR 82017-766
VWR timer Flashing LED 2 channel VWR 89140-196
Zebron ZB-Wax GC column Phenomenex 7HM-G013-11

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