Quantitative analysis of fatty acid precursors in marine samples

To apply fatty acid analyses to the study of foraging ecology and diet determination, all compounds that may be deposited as fatty acids in a predator must be quantified in the prey. These compounds include the usual fatty acids in acyl lipids, but also the alcohols of wax esters and the vinyl ethers of plasmalogens. In routine fatty acid analysis, samples are extracted and transesterified (methylated), resulting in the formation of fatty acid methyl esters (FAMEs); however, fatty alcohols and dimethylacetals (DMAs) are also generated if wax esters or plasmalogens are present. Here, we present a new method using a modified Jones' reagent to oxidize these alcohols and DMAs to free fatty acids (FFAs). These FFAs are then easily methylated and quantitatively recombined with FAMEs from the same sample. This generates a fatty acid signature of prey that is equivalent to that which the predator has available for deposition upon digestion of that prey.This method is validated with alcohol and DMA standards. Its application to typical marine samples is also presented, demonstrating the change in effective fatty acid signature after inclusion of fatty acids derived from wax esters and plasmalogens. To apply fatty acid analyses to the study of foraging ecology and diet determination, all compounds that may be deposited as fatty acids in a predator must be quantified in the prey. These compounds include the usual fatty acids in acyl lipids, but also the alcohols of wax esters and the vinyl ethers of plasmalogens. In routine fatty acid analysis, samples are extracted and transesterified (methylated), resulting in the formation of fatty acid methyl esters (FAMEs); however, fatty alcohols and dimethylacetals (DMAs) are also generated if wax esters or plasmalogens are present. Here, we present a new method using a modified Jones' reagent to oxidize these alcohols and DMAs to free fatty acids (FFAs). These FFAs are then easily methylated and quantitatively recombined with FAMEs from the same sample. This generates a fatty acid signature of prey that is equivalent to that which the predator has available for deposition upon digestion of that prey. This method is validated with alcohol and DMA standards. Its application to typical marine samples is also presented, demonstrating the change in effective fatty acid signature after inclusion of fatty acids derived from wax esters and plasmalogens. In the past three decades, the application of fatty acid signatures (1Iverson S.J. Milk secretion in marine mammals in relation to foraging: can milk fatty acids predict diet?.Symp. Zool. Soc. Lond. 1993; 66: 263-291Google Scholar) has developed from a potential tool for delineating marine food webs (2Ackman R.G. Eaton C.A. Lipids of the fin whale (Balaenoptera physalus) from north Atlantic waters. III. Occurrence of eicosenoic and docosenoic fatty acids in the zooplankter Meganyctiphanes norvegica (M. Sars) and their effect on whale oil composition.Can. J. Biochem. 1966; 44: 1561-1566Crossref Google Scholar) into a powerful technique for quantitative diet assessment of predators (S. J. Iverson, C. Field, W. D. Bowen, and W. Blanchard, unpublished observations). A variety of studies using lipid techniques between these two extremes have been conducted by comparing the fatty acids found in predator fat stores with those found in the prey (3Sargent J.R. Parkes R.J. Mueller-Harvey I. Henderson R.J. Lipid biomarkers in marine ecology.in: Sleigh M.A. Microbes in the Sea. Wiley and Sons, New York1987: 119-138Google Scholar, 4St. John M.A. Lund T. Lipid biomarkers: linking the utilization of frontal plankton biomass to enhanced condition of juvenile North Sea cod.Mar. Ecol. Prog. Ser. 1996; 131: 75-85Crossref Scopus (115) Google Scholar, 5Raclot T. Groscolas R. Cherel Y. Fatty acid evidence for the importance of myctophid fishes in the diet of king penguins, Aptenodytes patagonicus.Mar. Biol. 1998; 132: 523-533Crossref Scopus (108) Google Scholar, 6Dahl T.M. Lydersen C. Kovacs K.M. Falk-Petersen S. Sargent J. Gjertz I. Guilliksen B. Fatty acid composition of the blubber in white whales (Delphinapterus leucas).Polar Biol. 2000; 23: 401-409Crossref Scopus (89) Google Scholar), allowing qualitative, and potentially quantitative, statements to be made about diets and trophic interactions. These types of studies are possible because the fatty acid signatures of prey items consumed are deposited largely unaltered in most predators, providing an integrated record of diet. However, a potential complication is that some types of prey may contain fatty acid precursors. These are compounds that do not have an acid structure but are metabolized in the predator to form fatty acids that are, in turn, deposited in adipose tissue. This, of course, means that any such compound must be accurately quantified. Without determining these compounds, one may be comparing the fatty acid signature of the predator's fat store with an incomplete and possibly erroneous prey fatty acid signature. The two most common lipid classes that present these problems in marine ecosystems are wax esters (WEs) and plasmalogens. WEs consist of a fatty acid esterified to a fatty alcohol. Upon digestion by the predator, the WE is hydrolyzed to give one molecule of each of those lipids. The fatty acid enters the pool of fatty acids available for deposition, while the alcohol is oxidized to the corresponding fatty acid, which is then also available for incorporation into fat stores (7Sargent J.R. The structure, metabolism and function of lipids in marine organisms.in: Malins D.C. Sargent J.R. Biochemical and Biophysical Perspectives in Marine Biology. Vol. 3. Academic Press, London1976: 149-212Google Scholar). Plasmalogens are a common type of phospholipid that contain a vinyl-ether linked alkyl chain, in addition to an esterified fatty acid and a polar phosphate group. During digestion, the vinyl ether-linked alkyl chain is first oxidized to an aldehyde, then immediately to an alcohol (8Snyder F. The enzymic pathways of ether-linked lipids and their precursors.in: Snyder F. Ether Lipids: Chemistry and Biology. Academic Press, New York1972: 122-156Crossref Google Scholar). The alcohol is then processed as described above, also entering the predator's fatty acid pool. Typical sample preparation for fatty acid analysis by gas chromatography (GC) involves the transesterification (methylation) of acyl lipids (usually triacylglycerols and phospholipids) and free fatty acids (FFAs) to form fatty acid methyl esters (FAMEs) using an acid-catalyzed reaction. However, exposure of a lipid extract containing WEs or plasmalogens to this acidic environment will generate several products that are not FAMEs. With WEs, the result is similar to that of digestion, where FAMEs and a fatty alcohols are produced. Depending on their concentration and the GC column employed, these alcohols may appear on the chromatogram simply as a rising baseline or, in the worst case, as peaks, usually broad and unresolved, on which FAMEs elute. With plasmalogens, the vinyl-ether linkage is broken in the presence of acid to generate an aldehyde, which immediately reacts further with the acid to produce fatty dimethylacetals (DMAs). DMAs are a particular problem because the common 16:0 and 18:0 DMA coelute with i-16:0 and i-18:0 FAME during GC analysis using a typical FAME (polyethylene glycol) column (9Ackman R.G. Comparison of lipids in marine and freshwater organisms.in: Arts M.T. Wainman B.C. Lipids in Freshwater Ecosystems. Springer-Verlag, New York1999: 263-298Crossref Google Scholar). Although transesterification can be accomplished using base-catalyzed procedures that do not produce DMAs, these procedures are generally less reliable, they do not esterify FFAs, and their imprudent use can cause alterations to fatty acids (10Christie W.W. Lipid Analysis. 2nd edition. Pergamon Press, Oxford1982: 54Google Scholar). Thus, since alcohols and DMAs cannot be simultaneously determined with FAMEs, a more complicated procedure must be carried out. Typically, FAME, alcohol, and DMA bands are first isolated by TLC, then extracted and filtered. Each separate type of compound is then determined individually by GC. Quantification of alcohols is particularly inconvenient because it may require a separate derivatization reaction (11Zweig G. Sherma J. CRC Handbook of Chromatography. Vol. 2. CRC Press, Cleveland, OH1972: 196Google Scholar). Through the use of internal standards, one must then add back the amounts of corresponding structures of alcohols and DMAs to determine the new effective FAME composition. Here we report an alternative to this procedure. We propose the separation of FAMEs from both alcohols and DMA by TLC, followed by simply oxidizing any alcohol or DMAs to the appropriate FFA, which can then be directly methylated and recombined with the FAME fraction for GC analysis. This produces a fatty acid signature of the prey that is equivalent to the signature that the predator has available for deposition from that prey item. We demonstrate the use of this technique on alcohol and DMA standards, as well as on a variety of marine samples containing these compounds. FAME and alcohol standards (C16 to C24) were supplied from Nu-Chek Prep (Elysian, MN). A solution consisting of 5.58 mg of saturated FAMEs (20% each of five components) and 6.0 mg of monounsaturated alcohols with the same carbon number as the FAME series (20% each of five components) in hexane was evaporated to dryness. To this was added 2 ml of acetone and 10 drops (∼0.25 ml) of Jones' reagent (12Bowden K. Heilbron I.M. Jones E.R.H. Weedon B.C. Researches on acetylenic compounds. Part I. The preparation of acetylenic ketones by oxidation of acetylenic carbinols and glycols.J. Chem. Soc. 1946; : 39-45Crossref Scopus (983) Google Scholar), which we modified to: 13.5 g CrO3 and 6.4 ml concentrated H2SO4 made up to 50 ml with distilled H2O. This modified reagent contained half of the volume of H2SO4 employed in the original Jones' reagent. While use of the original concentrations of reagents did effectively oxidize alcohols and DMAs, it also generated artifacts in the chromatogram. These artifacts were absent when the modified reagent was employed. The mixture of standards, acetone, and Jones' reagent was vortexed for 1 min, allowed to sit for 30 min, and then vortexed again for 1 min. Heat was released and a precipitate formed when alcohols were present. Then 1 ml of water was added, followed by 2 ml of hexane, vortexing after each addition. The precipitate dissolved upon addition of hexane. After centrifugation, the upper hexane layer was removed and the aqueous layer was extracted twice more with 2 ml each of hexane. Hexane fractions were combined, washed with 2 ml of water, and dried over anhydrous Na2SO4. The FFAs generated were then methylated with 10% BF3 /methanol as described in Iverson et al. (13Iverson S.J. Frost K.J. Lowry L.F. Fatty acid signatures reveal fine scale structure of foraging distribution of harbor seals and their prey in Prince William Sound, Alaska.Mar. Ecol. Prog. Ser. 1997; 151: 255-271Crossref Scopus (225) Google Scholar). Peak areas from GC chromatograms were compared with known amounts of starting material to determine the extent of conversion of alcohols to FAMEs in relation to the original amount of FAME present. This entire procedure was repeated three times on the original standard mixtures. To assess absolute yields of the reaction, 16:0 and 18:0 alcohol standards (15 mg each) were accurately weighed and dissolved in acetone. The modified Jones' reagent was added and the FFAs generated were methylated as described above. Recovered FAMEs were then determined by GC to ensure no other reaction products were present, and were evaporated to dryness and weighed. Because commercial standards of DMAs were not available, plasmalogens were isolated from bivalves known to contain substantial amounts of this lipid. First, lipids were extracted from blue mussel (Mytelis edulis) tissue using a modified Folch et al. (14Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal Biol. Chem. Google Scholar) procedure as described in Iverson et al. S.J. Comparison of the and and Folch for total lipid in a broad of marine Scopus Google Scholar) and transesterified with 10% BF3 DMAs were then from FAMEs by on of developed in of FAMEs and DMAs were with The DMAs were from the extracted with and by GC. DMAs were then evaporated to dryness and oxidized with modified Jones' reagent as described above. The FFAs generated from DMAs were then methylated and again by GC for To demonstrate the of the oxidation technique on samples known to contain alcohols or plasmalogens, lipids were extracted and transesterified from and samples and as described above. this hexane containing FAMEs and alcohols or DMAs, was by GC to the first oxidizing alcohols and This mixture was then into FAME, alcohol, DMA by in In this the containing FAMEs fatty alcohols the and DMAs the After the FAME was removed from the extracted from the with and The alcohol and DMA bands were and also extracted with both alcohols and DMAs were present in the same they were The mixture was then evaporated to dryness and with the modified Jones' reagent. The FFAs were methylated and the FAME were with the FAME fraction that was after The fractions were then by GC and compared with the oxidizing alcohols and FAME samples were using a GC with a using a column with and was as the gas and the gas was with an The was for 2 min, for after and for after to FAMEs were to Iverson et al. (13Iverson S.J. Frost K.J. Lowry L.F. Fatty acid signatures reveal fine scale structure of foraging distribution of harbor seals and their prey in Prince William Sound, Alaska.Mar. Ecol. Prog. Ser. 1997; 151: 255-271Crossref Scopus (225) Google Scholar). for FAMEs W.W. and A Press, Scholar) were and each FAME was as of FAMEs were described using the of where A the number of carbon the number of and the of the to the methyl group. are as A variety of marine are known to contain WEs and plasmalogens (7Sargent J.R. The structure, metabolism and function of lipids in marine organisms.in: Malins D.C. Sargent J.R. Biochemical and Biophysical Perspectives in Marine Biology. Vol. 3. Academic Press, London1976: 149-212Google Scholar, R.G. Comparison of lipids in marine and freshwater organisms.in: Arts M.T. Wainman B.C. Lipids in Freshwater Ecosystems. Springer-Verlag, New York1999: 263-298Crossref Google Scholar, J.R. Marine Chemistry and of Scholar). In we have WEs in and in some and while plasmalogens have been found in some and Here we present a method to and quantitatively alcohols and DMAs into FAMEs, which is to predator and prey fatty acid We first this quantitative conversion using a standard mixture of FAMEs and This containing equivalent of five saturated FAME, was carried the reaction with the same carbon series of five monounsaturated fatty alcohols in an original of for each of the five This was then compared with the derived from areas determined by GC after oxidation of the alcohols to FAMEs. In each case, the of original from alcohols similar to the original of In after the oxidation reaction, the quantitative of were with the conversion of all alcohols to FAMEs with no as well as of the original of FAMEs of saturated fatty acid methyl alcohol and after conversion of the monounsaturated alcohols to fatty acid methyl in a standard from the original total of each saturated FAME and each corresponding alcohol contained in the standard mixture to from alcohol by from GC chromatogram areas after the oxidation reaction that the monounsaturated alcohols to fatty acid methyl gas from the original total of each saturated FAME and each corresponding alcohol contained in the standard mixture to from GC chromatogram areas after the oxidation reaction that the monounsaturated alcohols to FAMEs. in a new FAME, fatty acid methyl gas We absolute yields of the reaction using 16:0 and 18:0 alcohol standards and a reaction of this oxidation reaction to In the procedure we the acid was and of alcohol to mg 16:0 or DMA mg 16:0 can be the of alcohols to assess was less this we can be that the of was not to reagent. In GC any alcohols in the Thus, we can that the of FAME was to in the hexane of the FFAs or FAMEs To ensure the reaction to when this method to marine we mg of lipid. course, if amounts of alcohol or DMAs must be the reaction can be up as is that both alcohols and DMA are in the GC chromatogram to determine if the oxidation reaction is for a if amounts of reagent are in the the will be in the and a oxidation can be carried to the We then lipids containing plasmalogens, isolated from the blue to demonstrate the of DMAs and their quantitative conversion to FAMEs. DMAs form on polar GC which be for FAMEs. Thus, it was possible to simply areas and after oxidation In this particular after FAME preparation the two DMA were 18:0 DMA and which to the of and an a have been quantified a of of total fatty acids when it in all marine prey in the Atlantic Iverson S.J. R.G. and in fatty acid signatures of marine and on the and of J. Scopus Google Scholar) and in the blue Thus, this and that of an that where no other fatty acid is known to may as of the presence of DMAs on this polar less polar DMAs coelute with acids of the same carbon number (9Ackman R.G. Comparison of lipids in marine and freshwater organisms.in: Arts M.T. Wainman B.C. Lipids in Freshwater Ecosystems. Springer-Verlag, New York1999: 263-298Crossref Google Scholar) that amounts of chain acids may the presence of DMAs in that the polar column employed the in of 18:0 and FAME after oxidation are However, between the of all DMAs and the FAMEs after oxidation was also with of 16:0 the A that was carried the reaction did not any that the in 16:0 in FAMEs was not a result of To ensure of DMAs from the when those all material the of the was Thus, it is possible that of 16:0 alcohol to be present in blue Fatty acid biomarkers in a water marine of Scholar) were removed with the DMAs from the of 16:0 alcohol be oxidized during the reaction, a 16:0 FAME it be that this oxidation allowed to the DMA and To this is the first report of these DMAs in the marine A more procedure that we was to simply apply the oxidation reaction to the entire FAME sample first FAMEs from alcohols and The Jones' reagent is for the oxidation of alcohols J. 2nd edition. Scholar), that However, when FAME samples containing were carried the we found of the while of saturated and monounsaturated FAMEs were We a number of to this the use of an Jones' and of these the of that these are to oxidation to be to such a oxidizing any alcohols and DMAs present in marine samples contain one or no (7Sargent J.R. The structure, metabolism and function of lipids in marine organisms.in: Malins D.C. Sargent J.R. Biochemical and Biophysical Perspectives in Marine Biology. Vol. 3. Academic Press, London1976: 149-212Google Scholar), allowing this reagent to be in the oxidation of alcohols and DMAs, but their separation from the FAMEs. was this potential of of material that made the of the method with monounsaturated standards that an to the problem of with exposure to Jones' reagent is to simply use oxidizing In are a number of potential reagents that be employed to oxidize alcohols to and of compounds. Scholar), but we an that was to the oxidation to the of FFAs with the of Jones' of acidic is the of those oxidation of in it is the of oxidizing the available Its use with fatty alcohols also been S. of Lipid 1966; Google Scholar), it a We have that DMAs are also effectively oxidized to FFAs with this reagent. The with the use of Jones' reagent is the of compounds and the with also of the importance of the of the in which the alcohols and DMAs are this is hexane and can be easily accomplished using a of in with a water that also effectively of Jones' reagent is an aqueous the alcohols and DMAs must be dissolved in a that is with water, such as acetone. any of hexane alcohols and DMAs will in solution in hexane and will not with the Jones' and oxidation to FFAs will not This of however, effectively the application of this procedure to any sample that may contain alcohols or compounds be and cannot be quantified with this Although DMAs are this is not the for WE alcohols and FAMEs D.C. wax esters from the of the Scopus Google Scholar). we have the of fatty acids and after oxidation in three sample and These are prey that are typical of the samples processed in In addition to acyl lipids, and contained WEs, while contained WEs and of plasmalogens. in all three is a change after with Jones' reagent. In the in and are after oxidation and these are in with the that those structures are the alcohol of R.G. S. M. esters of a potential for whale Chem. Soc. Scopus Google Scholar). In several the fatty acids and are and are particularly in prey and for diet (13Iverson S.J. Frost K.J. Lowry L.F. Fatty acid signatures reveal fine scale structure of foraging distribution of harbor seals and their prey in Prince William Sound, Alaska.Mar. Ecol. Prog. Ser. 1997; 151: 255-271Crossref Scopus (225) Google Scholar, S.J. Frost K.J. and fatty acid composition of and in Prince William Sound, to and Ecol. Prog. Ser. Scopus Google Scholar), of these compounds and their to fatty acid signatures is is known about the DMA composition of but the in after oxidation that is an of this alkyl structure in this however, the of alcohols to these the of the fatty acid With these it is not possible to determine a alcohol or or is for this In the myctophid the in of fatty acids after oxidation in and which also with that those structures are the alcohols in this The fatty acid and fatty alcohol composition of the myctophid of wax esters and 1997; Google Scholar). to all three in are in several fatty such as and With such as that in of particular fatty such as will with in other fatty acids and a in one fatty acid may a in it appear as if is no change in the fatty Thus, the addition of an internal standard is to absolute of some However, since we are in the fatty acid signature available to the predator for lipid deposition (S. J. Iverson, C. Field, W. D. Bowen, and W. Blanchard, unpublished the change in signature after oxidation is most for of and myctophid samples not also similar in of fatty acids and these of the prey fatty acid signature available to the predator be if the FAME were for alcohols and In the method we have described a and effective means to fatty to their corresponding fatty acid which will appropriate of the fatty acids available for deposition in a predator's fat stores from that of prey. While we have concentrated on to marine this technique is not to those sample it can as easily be to the of found in and We present this technique as an alternative to the procedures that are to determine alcohols and DMAs This method in the of trophic and diets using fatty acid The to R. G. for to the of DMA in samples and of The also for on the digestion and deposition of wax esters and M. S. C. and two for on an of this This was by from the and and the North Marine fatty acid methyl gas chromatography wax

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