Name: determination of total lipid and lipid classes in marine samples
Uploaded: 2021-12-11T13:00:00
Description: Watch this Scientific Journal Video about Determination of Total Lipid and Lipid Classes in Marine Samples at JoVE.com

This research was funded by Natural Sciences and Engineering Research Council of Canada (NSERC) grant number 105379 to C.C. Parrish. Memorial University&#39;s Core Research Equipment &#38; Instrument Training (CREAIT) Network helped fund this publication.

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&#176;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 &#181;m.
<ol>
	<li>Set up the filtration manifold without a fi...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+the+isolation+and+purification+of+total+lipides+from+animal+tissues.">A simple method for the isolation and purification of total lipides from animal tissues.</a> Journal of Biological Chemistry. 226, 497-509 (1957).</li><li>Vaz, F. M., Pras-Raves, M., Bootsma, A. H., van Kampen, A. H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+practice+of+lipidomics.">Principles and practice of lipidomics.</a> Journal of Inherited Metabolic Disease. , (2014).</li><li>Wolf, C., Quinn, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipidomics:+practical+aspects+and+applications.">Lipidomics: practical aspects and applications.</a> Progress in Lipid Research. 47, 15-36 (2008).</li><li>Parrish, C. C., Arts, M. T., ainman, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+total+lipid,+lipid+classes,+and+fatty+acids+in+aquatic+samples.">Determination of total lipid, lipid classes, and fatty acids in aquatic samples.</a> Lipids in Freshwater Ecosystems. , 4-20 (1999).</li><li>J&#252;ttner, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liberation+of+5,8,11,14,17-eicosapentaenoic+acid+and+other+polyunsaturated+fatty+acids+from+lipids+as+a+grazer+defense+reaction+in+epilithic+diatom+biofilms.">Liberation of 5,8,11,14,17-eicosapentaenoic acid and other polyunsaturated fatty acids from lipids as a grazer defense reaction in epilithic diatom biofilms.</a> Journal of Phycology. 37, 744-755 (2001).</li><li>Carre&#243;n-Palau, L., Parrish, C. C., P&#233;rez-Espa&#241;a, H., Agui&#241;iga-Garcia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elemental+ratios+and+lipid+classes+in+a+coral+reef+food+web+under+river+influence.">Elemental ratios and lipid classes in a coral reef food web under river influence.</a> Progress in Oceanography. 164, 1-11 (2018).</li><li>Maciel, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprospecting+of+marine+macrophytes+using+MS-based+lipidomics+as+a+new+approach.">Bioprospecting of marine macrophytes using MS-based lipidomics as a new approach.</a> Marine Drugs. 14, 49 (2016).</li><li>Pernet, F., Tremblay, R., Comeau, L., Guderley, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+adaptation+in+two+bivalve+species+from+different+thermal+habitats:+energetics+and+remodelling+of+membrane+lipids.">Temperature adaptation in two bivalve species from different thermal habitats: energetics and remodelling of membrane lipids.</a> Journal of Experimental Biology. 210, 2999-3014 (2007).</li><li>Bergen, B. J., Quinn, J. G., Parrish, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality-assurance+study+of+marine+lipid-class+determination+using+Chromarod/Iatroscan+thin-layer+chromatography-flame+ionization+detector.">Quality-assurance study of marine lipid-class determination using Chromarod/Iatroscan thin-layer chromatography-flame ionization detector.</a> Environmental Toxicology and Chemistry. 19, 2189-2197 (2000).</li><li>Foroutani, B. M., Parrish, C. C., Wells, J., Taylor, R. G., Rise, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimizing+marine+ingredients+in+diets+of+farmed+Atlantic+salmon+(Salmo+salar):+effects+on+liver+and+head+kidney+lipid+class,+fatty+acid+and+elemental+composition.">Minimizing marine ingredients in diets of farmed Atlantic salmon (Salmo salar): effects on liver and head kidney lipid class, fatty acid and elemental composition.</a> Fish Physiology &amp; Biochemistry. 46, 2331-2353 (2020).</li><li>Parrish, C. C., Deibel, D., Thompson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+sinking+spring+phytoplankton+blooms+on+lipid+content+and+composition+in+suprabenthic+and+benthic+invertebrates+in+a+cold+ocean+coastal+environment.">Effect of sinking spring phytoplankton blooms on lipid content and composition in suprabenthic and benthic invertebrates in a cold ocean coastal environment.</a> Marine Ecology Progress Series. 391, 33-51 (2009).</li><li>Sinanoglou, V. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+combined+application+of+Iatroscan+TLC-FID+and+GC-FID+to+identify+total,+neutral,+and+polar+lipids+and+their+fatty+acids+extracted+from+foods.">On the combined application of Iatroscan TLC-FID and GC-FID to identify total, neutral, and polar lipids and their fatty acids extracted from foods.</a> ISRN Chromatography. , 59024 (2013).</li><li>Peters-Didier, J., Sewell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+investment+and+nutrient+utilization+during+early+larval+development+of+the+sea+cucumber+Australostichopus+mollis.">Maternal investment and nutrient utilization during early larval development of the sea cucumber Australostichopus mollis.</a> Marine Biology. 164, 178 (2017).</li><li>Triesch, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concerted+measurements+of+lipids+in+seawater+and+on+submicron+aerosol+particles+at+the+Cape+Verde+Islands:+biogenic+sources,+selective+transfer+and+high+enrichments.">Concerted measurements of lipids in seawater and on submicron aerosol particles at the Cape Verde Islands: biogenic sources, selective transfer and high enrichments.</a> Atmospheric Chemistry and Physics. 21, 4267-4283 (2021).</li><li>Parrish, C. C., Bodennec, G., Gentien, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+glycoglycerolipids+by+Chromarod+thin-layer+chromatography+with+Iatroscan+flame+ionization+detection.">Determination of glycoglycerolipids by Chromarod thin-layer chromatography with Iatroscan flame ionization detection.</a> Journal of Chromatography A. 741, 91-97 (1996).</li><li>Mejri, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bonefish+(Albula+vulpes)+oocyte+lipid+class+and+fatty+acid+composition+related+to+their+development.">Bonefish (Albula vulpes) oocyte lipid class and fatty acid composition related to their development.</a> Environmental Biology of Fishes. 102, 221-232 (2019).</li><li>Sewell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+lipids+during+early+development+of+the+sea+urchin+Evechinus+chloroticus.">Utilization of lipids during early development of the sea urchin Evechinus chloroticus.</a> Marine Ecology Progress Series. 304, 133-142 (2005).</li><li>Parrish, C. C., Bodennec, G., Gentien, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+polyunsaturated+and+saturated+lipids+from+marine+phytoplankton+on+silica+gel+coated+Chromarods.">Separation of polyunsaturated and saturated lipids from marine phytoplankton on silica gel coated Chromarods.</a> Journal of Chromatography A. 607, 97-104 (1992).</li><li>Stevens, C. J., Deibel, D., Parrish, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+bacterial+fatty+acids+and+changes+in+a+wax+ester-based+omnivory+index+during+a+long-term+incubation+experiment+with+Calanus+glacialis+Jaschnov.">Incorporation of bacterial fatty acids and changes in a wax ester-based omnivory index during a long-term incubation experiment with Calanus glacialis Jaschnov.</a> Journal of Experimental Marine Biology and Ecology. 303, 135-156 (2004).</li><li>Goutx, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+term+summer+to+autumn+variability+of+dissolved+lipid+classes+in+the+Ligurian+Sea+(NW+Mediterranean).">Short term summer to autumn variability of dissolved lipid classes in the Ligurian Sea (NW Mediterranean).</a> Biogeosciences. 6, 1229-1246 (2009).</li><li>Conlan, J. A., Rocker, M. 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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...

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 universal components in living organisms, acting as essential molecules in cell membranes, as major sources of bioavailable energy, providing thermal insulation and buoyancy, and serving as signaling molecules. Although procedures for lipid determination in other fields have been described well, their use with marine samples commonly necessitates modification to adapt to field conditions as well as to sample type1.
For seawater samples, the first step usually requires separation into the operationally defined &#39;dissolved&#39; and &#39;particulate&#39; fractions, normally by filtration (Protocol step 1). The particulate fraction is what is retained by the filter, and size of the pores is important in defining the cut-off2. Often when we are sampling particulate matter, we would like to relate lipid concentrations to total mass concentrations, in which case a separate, smaller, sample (e.g., 10 mL) has to be taken for this purpose (Protocol step 1, note). To get an accurate mass determination it is important to add ammonium formate (35 g/L) at the end of the filtration.
The seawater filtrate from the larger sample should amount to between 250 mL and 1 L depending on sample type and is subjected to liquid-liquid extraction in a separatory funnel (Protocol step 2). The hydrophobic nature of lipids means they can be separated from other compounds by extraction in a nonpolar solvent such as chloroform. A two-layer system is created where lipids partition into the organic layer while water soluble components stay in the aqueous layer.
Particulate samples on a filter, or biological specimens are extracted with a modified Folch et al. extraction3, also involving chloroform (Protocol step 3). Again, an organic/aqueous system is created in which lipids partition into the organic phase, while water soluble molecules remain in the aqueous phase, and proteins are precipitated. In fact, for solids, most laboratories use some variation of the Folch et al. extraction3 procedure involving chloroform and methanol. For filters, the first step is to homogenize in 2 mL of chloroform and 1 mL of methanol.
During extraction, care should be taken to protect lipids from chemical or enzymatic modification, by keeping samples and solvents on ice to reduce ester bond hydrolysis or carbon-carbon double bond oxidation. Tissues and cell lipids are quite well protected by natural antioxidants and by compartmentalization4; however, following the homogenization of samples, cell contents are combined rendering lipids more disposed to alteration, chemically or enzymatically. Some lipids, such as most sterols, are very stable, while others, such as those containing polyunsaturated fatty acids, are more susceptible to chemical oxidation. Others, such as sterols with conjugated double bonds, are prone to oxidation catalyzed by light5. Following extractions, lipids are much more susceptible to chemical oxidation, and samples should be stored under an inert gas such as nitrogen. A gentle stream of nitrogen would also be used to concentrate extracts.
After concentration, lipids would then normally be quantified in bulk as they are an important component of marine ecosystems providing a high concentration of energy, more than twice the kJ/g of carbohydrates and proteins. Invariably they would next be quantified as individual components: the comprehensive analysis of lipids generally involves separation into simpler categories, according to their chemical nature. Thus, a full analysis involves measuring total lipids, lipid classes and individual compounds.
Total lipid can be determined by taking the sum of individually measured lipid classes separated by chromatography6. A marine lipid extract may contain more than a dozen classes from biogenic and anthropogenic sources. The wide variety of lipid structures means much information can be gained by determining individual groupings of structures. Lipid classes individually, or in certain groups, have been used to signal presence of certain types of organisms, as well as their physiological status and activity2. They have also been used as an indicator of the origins of organic material, including dissolved organic matter (DOM) as well as hydrophobic contaminants.
Triacylglycerols, phospholipids and sterols are among the more important biogenic lipid classes. The first two are biochemically related as they possess a glycerol backbone to which two or three fatty acids are esterified (Figure 1). Triacylglycerols, together with wax esters are very important storage substances, while other fatty acid-containing lipid classes such as diacylglycerols, free fatty acids, and monoacylglycerols are generally minor constituents. Free fatty acids are present at lower concentrations in living organisms, as the unsaturated ones can be toxic7. Sterols (both in their free and esteri&#64257;ed forms) and fatty alcohols are also included among the less polar lipids, while glycolipids and phospholipids are polar lipids. Polar lipids have a hydrophilic group, which allows for the formation of lipid bilayers found in cell membranes. Free sterols are also membrane structural components, and when taken in ratio to triacylglycerols they provide a condition or nutritional index (TAG : ST) which has been widely used8. When taken in ratio to phospholipids (ST : PL) they can be used to indicate plant sensitivity to salt: higher values maintain structural integrity and decrease membrane permeability9. The inverse of this ratio (PL : ST) has been studied in bivalve tissues during temperature adaptation10.
Marine lipid classes can be separated by thin-layer chromatography (TLC) on silica gel coated rods (Protocol step 4) and then detected and quantified by flame ionization detection (FID) in an automatic FID scanner. TLC/FID has become routinely used for marine samples as it rapidly furnishes synoptic lipid class data from small samples, and by taking the sum of all classes, a value for total lipids. TLC/FID has been subjected to a quality-assurance (QA) assessment and was found to meet standards required for consistent external calibration, low blanks, and precise replicate analysis11. Coefficients of variation (CV) or relative standard deviations are around 10%, and FID scanner total lipid data are normally around 90% of those obtained by gravimetric and other methods2. Gravimetry gives higher total lipids likely because the FID scanner measures only non-volatile compounds, and also as a result of possible inclusion of non-lipid material in gravimetric measurements.
The information provided by lipid class analysis is especially useful when combined with determinations of fatty acids as individuals, or sterols, or the two in combination. The first step towards these analyses involves the release of all component fatty acids together with sterols in the lipid extracts (Protocol step 5). The wide variety of lipid structures and functions means they have seen broad use in ecological and biogeochemical studies assessing ecosystem health and the extent to which they have been influenced by anthropogenic and terrestrial inputs. They have been used to measure biosynthesis of substances of dietary value to marine fauna as well as to indicate the quality of water samples. Measuring lipids in sediment core samples helps show the sensitivity of sediments to changes in human land use near the land-sea margin.
The primary tool for identifying and quantifying individual lipid compounds has traditionally been gas chromatography (GC) with FID. Before analysis however, these compounds are made more volatile by derivatization. Fatty acids are released in the presence of an acidic catalyst (H2SO4) from acyl lipid classes (Figure 1). In organic chemistry, the acyl group (R-C=O) is usually derived from a carboxylic acid (R-COOH). They are then re-esterified to fatty acid methyl esters (FAME) which gives better separations on GC columns (Protocol step 5).

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			<td>15 ml vials</td>
			<td>VWR</td>
			<td>66009-560</td>
			<td></td>
		</tr>
		<tr>
			<td>1-hexadecanol</td>
			<td>Sigma</td>
			<td>258741-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Monopalmitoyl-rac-glycerol</td>
			<td>Sigma</td>
			<td>M1640-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>2 ml vials</td>
			<td>VWR</td>
			<td>46610-722</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mm glass fibre filters</td>
			<td>Fisher</td>
			<td>09 874 32A</td>
			<td></td>
		</tr>
		<tr>
			<td>2ml pipet bulbs</td>
			<td>VWR</td>
			<td>82024-554</td>
			<td></td>
		</tr>
		<tr>
			<td>47 mm glass fibre filters</td>
			<td>Fisher</td>
			<td>09 874 32</td>
			<td></td>
		</tr>
		<tr>
			<td>5 3/4&#34; pipets</td>
			<td>Fisher</td>
			<td>1367820A</td>
			<td></td>
		</tr>
		<tr>
			<td>9&#34; pipets</td>
			<td>Fisher</td>
			<td>1367820C</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>VWR</td>
			<td>CAAX0116-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent GC-FID 6890</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride ANHS 500gm</td>
			<td>VWR</td>
			<td>CACX0160-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Caps for 2 ml vials</td>
			<td>VWR</td>
			<td>46610-712</td>
			<td></td>
		</tr>
		<tr>
			<td>chloroform</td>
			<td>VWR</td>
			<td>CACX1054-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesteryl palmitate</td>
			<td>Sigma</td>
			<td>C6072-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromarod S5</td>
			<td>Shell USA</td>
			<td>3252</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>VWR</td>
			<td>CADX0831-1</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-a-phosphatidylcholine, dipalmotoyl</td>
			<td>Sigma</td>
			<td>P5911-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Ether, ACS grade anhydr 4L</td>
			<td>VWR</td>
			<td>CAEX0190-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glyceryl tripalmitate</td>
			<td>Sigma</td>
			<td>T5888-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton Syringe 702SNR 25&#181;l</td>
			<td>Sigma</td>
			<td>58381</td>
			<td></td>
		</tr>
		<tr>
			<td>Helium</td>
			<td>Air Liquide</td>
			<td>A0492781</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>VWR</td>
			<td>CAHX0296-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen regulator</td>
			<td>VWR</td>
			<td>55850-484</td>
			<td></td>
		</tr>
		<tr>
			<td>Iatroscan MK6</td>
			<td>Shell USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Fisher</td>
			<td>066662</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical Air</td>
			<td>Air Liquide</td>
			<td>A0464563</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium nitrile gloves</td>
			<td>Fisher</td>
			<td>191301597C</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrile gloves L</td>
			<td>VWR</td>
			<td>CA82013-782</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen</td>
			<td>Air Liquide</td>
			<td>A0464775</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen Regulator</td>
			<td>VWR</td>
			<td>55850-474</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonadecane</td>
			<td>Sigma</td>
			<td>74158-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Palmitic acid</td>
			<td>Sigma</td>
			<td>P0500-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Repeating dispenser</td>
			<td>Sigma</td>
			<td>20943</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate 1kg</td>
			<td>VWR</td>
			<td>CA97062-460</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Sulfate Anhy ACS 500gr</td>
			<td>VWR</td>
			<td>CA71008-804</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>VWR</td>
			<td>CASX1244-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon tape</td>
			<td>Fisher</td>
			<td>14610120</td>
			<td></td>
		</tr>
		<tr>
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determination of total lipid and lipid classes in marine samples

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 &#39;dissolved&#39; and &#39;particulate&#39; 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).

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<sup class="xref...

Watch this Scientific Journal Video about Determination of Total Lipid and Lipid Classes in Marine Samples at JoVE.com

Determination of Total Lipid and Lipid Classes in Marine Samples

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 ...

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