Identification of triacylglycerols containing two short-chain fatty acids at sn-2 and sn-3 positions from bovine udder by fast atom bombardment tandem mass spectrometry

Identification of triacylglycerols containing twoshort-chain fatty acids at sn-2 and sn-3 positionsfrom bovine udder by fast atom bombardmenttandem mass spectrometry

Young Hwan Kim1*, Keun-Young So2, Jin-Kyung Limb 2, Gil-Ja Jhon2* and So-Yeop Han2*1Complex Carbohydrate Research Center, University of Georgia, 220 River Bend Road, Athens, GA 30602-4712, USA2Department of Chemistry and Division of Molecular Life Sciences, Ewha Women’s University, Seoul 120-750, Korea

Several triacylglycerol (TAG) molecular species, that contain two short-chain fatty acids (C4 to C8) at thesn-2 and sn-3 positions of the glycerol backbone, were isolated from bovine udder by using solvent extrac-tion and silica gel column chromatography. Their structures were identified by fast atom bombardment(FAB) tandem mass spectrometry (MS/MS), based on the information obtained from collision-induceddissociation (CID) spectra of sodium-adducted molecules ([M� Na]�) of model TAG compounds which hadbeen synthesized from glycerol and appropriate fatty acids. For each species, the relative positions of thethree fatty acids on the glycerol backbone, as well as fatty acid composition and double-bond position in thefatty acyl group, were determined. A majority of sodium-adducted molecules observed in the FAB massspectrum were mixtures of at least two components that have different fatty acid composition but the samemolecular mass. In addition, all the components present in mixtures of all the species contain a long-chainfatty acid (C12 to C18) at thesn-1 position, a short-chain fatty acid (C4 to C8) at thesn-2 position, and abutyric acid uniquely at the sn-3 position. Copyright # 2000 John Wiley & Sons, Ltd.

Received 15 August 2000; Revised 19 September 2000; Accepted 2 October 2000

Triacylglycerols (TAGs) play an important role in nutritionand other biological processes. They are the primary meansof energy storage in animals and humans, and theirhormonally controlled hydrolysis and oxidation releaseenergy to meet the energy generation needs of organisms.1

In addition, they constitute the major lipid fraction in bovinemilk (98%)2 and thus have been extensively investigated interms of type and amounts of fatty acids present and in termsof their stereospecific distribution over the glycerol back-bone.3–6 A characteristic feature of the triacylglycerols inthe milk from ruminants is that they contain short-chainfatty acids such as butyric and hexanoic acid, which havebeen not found in triacylglycerols from other tissues ofruminants and normally not in the milk from non-ruminants.7–12 For example, short-chain fatty acids con-stitute about 15% of total fatty acids found in triacylglycer-ols of bovine milk.6,10,12 Probably the most characteristicfeature is the positioning of butyric and hexanoic acidsexclusively at thesn-3 position of the milk triacylglycerolmolecules.3,13 The physiological significance of the short-chain fatty acids at thesn-3 position of ruminant milktriacylglycerols should be emphasized because they areresponsible for melting points sufficiently low to be readilysecreted as liquid droplets at ambient temperature.14

Recently, we first isolated the monoacetyldiglycerides

(MADGs) containing an acetyl group at thesn-3 positionfrom a sample of animal tissue, bovine udder,15 and provedthat synthetic MADGs have remarkable biological functionssuch as hematopoietic stem cell stimulating activity (S.Kim, S.-Y. Han and G.-J. Jhon, unpublished data) and Ca2�

mobilizing activity in pancreatic acinar cells.16 In contrast,the importance of TAGs in biological functions andnutrition have been well-known for some time, and there-fore, many analytical methods involving mass spectrometry(MS) and tandem mass spectrometry (MS/MS), coupledwith a variety of ionization methods, have already been usedto determine their structures.17–28

In fact, collision-induced dissociation (CID) of sodium-adducted molecules of glycerolipids including glycogly-cerolipids,29 phospholipids30 and monoacetyldiglycerides(MADGs),31 have provided practical and essential informationon the regiospecificity of the fatty acyl groups on the glycerolbackbone from our previous work. Furthermore, it has offereddecisive evidence on the polar head group and fatty acidcomposition, as well as the double-bond position in the fattyacyl chains. On the basis of these results, the structuralanalyses of total lipids isolated from the cyanobacteriumSynechocystissp. PCC 6803 were performed at individualmolecular species level, using tandem mass spectrometry of[M � Na]� ions of each species.32,33 Thus, utilizing synthe-sized model TAGs in determining the structures of TAGsisolated from bovine udder became a great challenge.

As an extension of our work on the structural determina-tion of glycerolipids isolated from biological origins,15,32,33

we herein wish to report that TAGs containing two short-chain fatty acids have been isolated from bovine udder, andtheir structures completely determined by CID-MS/MS of

*Correspondence to: Y. H. Kim, Complex Carbohydrate ResearchCenter, University of Georgia, 220 River Bend Road, Athens, GA30602-4712, USA, or G.-J. Jhon and S.-Y. Han, Department ofChemistry and Division of Molecular Life Sciences, Ewha Women’sUniversity, Seoul 120-750, Korea.E-mail: [email protected], [email protected], [email protected]

Copyright# 2000 John Wiley & Sons, Ltd.

RAPID COMMUNICATIONS IN MASS SPECTROMETRYRapid Commun. Mass Spectrom.14, 2230–2237 (2000)

their sodium-adducted molecules desorbed by fast atombombardment(FAB).

EXPERIMENTA L

All solventsandreagents were the highest gradecommer-cially available.All aqueous solutionswerepreparedusingdeionizedanddistilled water. Purity of all compoundswasverified by 1H-NMR, 13C-NMR, 2D COSY NMR, IR, andhigh-resolution FAB massmeasurements.

Synthesesof rac-triacylglycerols

All three rac-triacylglycerols were synthesized fromcommerciallyavailableglycerolandappropriatefatty acids.Their structureswere confirmedby spectroscopicmethods.All reactions were conducted carefully, and monitoredclosely by thin-layer chromatography (TLC) in order toobtain rac-triacylglycerolswith high regioselectivity. Thestructuresof threetargetrac-triacylglycerolsareshown inFig. 1. The general synthetic procedureis asfollows.

1-Palmitoyl-2,3-dibutyryl-rac-glycerol (1). Palmitic acid(Aldrich, Milwaukee, WI, USA) was addedto glycerol inacetoneat 0°C. After stirring the reaction mixture withdicyclohexylcarbodiimide (DCC; Fluka, Switzerland) anddimethylaminopyridine (DMAP; Aldrich) at 0°C for 12h,pure1-palmitoyl glycerol wasobtained by silica gel flash-columnchromatography. Treatmentof 1-palmitoylglycerolwith butyric anhydride (Aldrich) in the presenceof DMAPat 0°C, followed by silica gel columnpurification,afforded1-palmitoyl-2,3-butyryl-rac-glycerol (1) in two stepsand35%overall yield from palmitic acid.

1-Oleoyl-2-caproyl-3-butyryl-rac-glycerol (2). Glycerolwas reactedwith oleic acid (Aldrich) in the presenceofDCC and DMAP at 0°C to form 1-oleoyl glycerol as themajor productby regioselective monoesterification. Treat-ment of 1-oleoyl glycerol with butyric anhydride in thepresence of DMAP at ÿ78°C for 30min, followed bycolumn purification, afforded the C3-butyrylated product.Theresulting1-oleoyl-3-butyryl glycerolwasthenacylatedwith n-caproic acid (Aldrich) in the presenceof DCC andDMAP in dichloromethane at 0°C. After silica gel columnpurification,thedesiredproductwasobtainedin threestepsand26%overall yield from the starting oleic acid.

1-Caproyl-2-palmitoyl-3-butyryl-rac-glycerol (3). Com-pound3 wassynthesizedby following the sameprocedure

usedin thesynthesisof compound 2. Af tersilicagelcolumnpurification, 1-caproyl-2-palmitoyl-3-butyryl-rac-glycerolwasobtained in 25%overall yield.

Isolation of triacyl glycerols containing two short-chainfatty acids from bovine udder

Freshbovine udder(2.7 kg), purchasedat a meatmarketinSeoul, Korea,wasminced,washedwith distilled water, andthen repeatedly rinsed with phosphate-buffered salinesolution (PBS, pH 7.4), while cooling in ice water. Thebovine udder was homogenized in 20g portions with amixer for 15min in CHCl3/MeOH 2:1 (v/v).34 Thehomogenized bovine udder was extracted overnight at4°C by stirring with CHCl3/MeOH 2:1 (v/v), andthentheextract wascentrifugedat 4°C and5000 rpm (JA-14R) for30min. The supernatant in chloroform was concentratedunder reduced pressureto afford 63.4g of a lipid mixture.The resultantresiduewas subjected to silica gel columnchromatography using hexane/diethyl ether 4:1 (v/v) aseluent, collecting a total of eight fractions.NMR analysisshowed that the fifth fraction contained TAGs, so thisfraction (285mg) wasthenchromatographedon a silica gelcolumn (35� 1.3cm i.d.) using hexane/ethyl acetate9:1(v/v) for further purification. The third (55.8mg) of fivefractionscollected wasfurtherpurified by repeatedchroma-tography on a silica gel column (16� 1.0cm i.d.) elutedwith hexane/ethylacetate 20:1 (v/v). The first fraction(11.1mg) was identified as a mixture of TAGs, whichcontained two short-chain fatty acids, and monoacetyldi-glycerides(MADGs)with anacetic acid atthesn-3 position,by NMR andmass spectrometric analysis.

Massspectrometry

All massspectrometric analyseswere performedusing aJMS-HX110/110A tandem mass spectrometer (JEOL,Tokyo, Japan), a four-sectorinstrument of E1B1E2B2 con-figuration described previously.35 Briefly, the ion sourcewasoperated at 10kV acceleratingvoltagein the positive-ion modewith amassresolution of 1000(10%valley). Ionswereproducedby FAB usingthecesiumion beamoperatedat 22keV. Approximately 10mg of each sample weredissolved in chloroform/methanol1:1 (v/v) andmixedwith1 mL of 3-nitrobenzyl alcohol (3-NBA; Sigma,St. Louis,MO, USA) with andwithoutNal (Sigma) on theFAB probetip. CID of thesodium-adductedmolecule selectedwith thefirst massspectrometer(MS-1)occurredin thecollisioncelllocated between B1 and E2 and floated at 3.0kV. Theresultant productionswereanalyzedby theB/E linked scanmethod in the second mass spectrometer (MS-2). Thecollision gas, helium, was introduced into the collisionchamberat a pressuresufficient to reduce theprecursor ionsignal by 70%. Signal averaging of two scansyielded thefinal spectra. MS-1 was operated with the resolutionadjustedso that only the 12C-species of the precursor ionswere transmitted. MS-2 was operated at a resolution of1000.

RESULTS

Tr iacylglycerols synthesizedfrom glycerol andappropriate fatty acids

The two rac-TAGs with two short-chain fatty acids at thesn-2 and sn-3 positions were synthesized from glycerol.

Figure 1. Structuresof synthetictriacylglycerolsthat containa long-chain fatty acid and two short-chainfatty acids,including a butyrylgroupat the sn-3 position.

Copyright# 2000JohnWiley & Sons,Ltd. Rapid Commun.MassSpectrom.14, 2230–2237(2000)

MS OF TAGS WITH TWO SHORT-CHAIN FAS FROM BOVINE UDDER 2231

Palmitic or oleic acid was treated with freshly distilledglycerol in thepresenceof DCCandDMAP to affordasn-1-monoacylated glycerol as the major product together withsn-1,2,3-triacylatedglycerol andsn-1,2-diacylatedglycerolasminor products. Monoacylation at the sn-1 position wasachieved at a low reaction temperature. The sn-2,3-diacylation could also be easily accomplished by carefuloperation of thereaction.Thus,thesynthesesof TAGswerecompleted in two or three steps.The massspectrometricbehavior of TAGs with a long-chain fatty acid (C16:0 orC18:1, whereC16:0 or C18:1 represent carbonnumber:de-greeof unsaturation) at the sn-1 position and two butyricacids(C4:0), or a caproicacid (C6:0) anda butyric acid,atthesn-2 andsn-3 positions,respectively,werefirst studied.Whentheyweredesorbedby fastatombombardment(FAB)using 3-nitrobenzyl alcohol (3-NBA) as matrix, theirpositive-ion mass spectraindicated a very low-intensitypeakcorresponding to the protonatedmolecule[M � H]�,and intensepeaksfor a variety of fragment ions includingacylium ions [Rn'CO]�, where n = 1, 2, 3 in the Rn'designationcorrespond to theacyl groupsat thesn-1, sn-2,and sn-3 positions, respectively, [Rn'CO� 74]� anddiglyceride ions [M � H-Rn'COOH]�, from which thecarbon number andthe degreeof unsaturation of eachacylgrouparedetermined,asshownin Fig.2(a). However, whenthey were FAB-produced using 3-NBA saturated withsodium iodide (NaI), the prominentpeakcorrespondingtothe sodium-adducted molecule,[M � Na]�, was observedasshownin Fig. 2(b).This ion is very importantin studyingthe structureof the correspondingmolecular species. In

previousstudies of glycoglycerolipids,29 glycerophospho-lipids30 andMADGs,15,31 it wasproved that CID of thesesodium-adducted molecules generateda wide variety ofproductionsinformativeontheregiospecificity of fatty acyllinkageson the glycerol backbone,aswell ason fatty acidcomposition andpolarheadgroup.In addition, thedouble-bondpositionsin thefatty acylgroupweredeterminedfromanalysisof the spectral pattern of the homologous ionsformed by charge-remotefragmentation (CRF) occurringalongthe long chainsof fatty acyl groups.36

Thus, to match the structural details of the syntheticTAGs with the product ions generated by CID of theirsodium-adducted molecules, tandem mass spectrometry(MS/MS)of the[M � Na]� ionswasinvestigated.Basedonthe resultsobtained, completestructural determination forseveralmolecularspecies of TAGs with short-chain fattyacids, which were isolated from bovine udder, will bediscussed below. The CID spectraof the [M � Na]� ions(m/z493 and521) of the syntheticTAGs, 1-palmitoyl-2,3-dibutyryl-rac-glycerol (1; C16:0/C4:0/C4:0-TAG) and 1-caproyl-2-palmitoyl-3-butyryl-rac-glycerol(3; C6:0/C16:0/C4:0-TAG), are shown in Figs. 3(a) and (b), respectively.Thelattercompoundwassynthesizedto obtaininformationaboutlocating threeacyl groupson the glycerol backbone,by comparingthespectral patternsof thesetwo compounds.Thesespectralpatternsareverysimilar to thoseobservedinthe CID spectraof [M � Na]� ions of MADGs studiedpreviously.31 The fragmentationpathways of the syntheticTAG compound1 are illustrated in Fig. 4. The nomen-clatureusedis takenfrom Refs.29 and37. Ionsof thesame

Figure 2. Positive-ionFAB massspectraof 1-palmitoyl-2,3-dibutyryl-rac-glycerol (C16:0/C4:0/C4:0-TAG) in a matrix of 3-nitrobenzylalcohol(a) without NaI and(b) with NaI.

Rapid Commun.MassSpectrom.14, 2230–2237(2000) Copyright# 2000JohnWiley & Sons,Ltd.

2232 MS OF TAGS WITH TWO SHORT-CHAIN FAS FROM BOVINE UDDER

type are differentiatedby the subscript in their notations,with eachdigit denoting thelocationof theacylgroupthatisremoved in the fragmentation. The superscript in thenotationsof some ionsrepresentsthecleavedbond positionrelative to the carbonyl carbon in the fatty acyl group.Forexample, thedesignation 3I1 in Fig. 3(a)meansthat the ionis formedby lossof thesn-1 acyl groupdueto thecleavageof the bond at the g-position to the carbonylcarbon. Also,these3I ions, except for the absence of that due to lossofbutyryl group, indicate distinctly intensepeaksamong a

series of the homologous ions generated by losses ofCnH2n � 2 along the fatty acyl chainsvia CRF eliminationreactions,becausetheyhaveextrastability dueto theira,b-unsaturated carbonyl structure. Therefore, these ionsprovide information on the composition of fatty acylgroups. However, in the caseof butyryl groups, the 3I ioncannotbeformedvia CRFelimination. In addition,thethreeG ions corresponding to sodium-adducted diglyceride ions([M � Na-R'COOH]�) also provide the sameinformation.In Fig. 3(b), themassdifferences betweentheprecursor ion

Figure 3. FAB tandemmassspectraof [M � Na]� ionsof (a) 1-palmitoyl-2,3-dibutyryl-rac-glycerol(C16:0/C4:0/C4:0-TAG) and (b) 1-caproyl-2-palmitoyl-3-butyryl-rac-glycerol (C6:0/C16:0/C4:0-TAG). Thesubscriptin thesymbolannotatingeachpeakrepresentstherelativeposition(sn-1,sn-2, orsn-3) of thefatty acyl group(or groups)removedfrom theglycerolbackbone,andthesuperscriptthecleavedbond position relative to the carbonyl carbonof the fatty acyl group. The fragmentationpathwaysfor the productionsobservedin (a) areillustratedin Fig. 4.

Figure 4.Fragmentationpathwaysof theproductionsobservedin theCID spectrumof the[M � Na]� ion (m/z493)of 1-palmitoyl-2,3-dibutyryl-rac-glycerol (C16:0/C4:0/C4:0-TAG)shownin Fig. 3(a).



(m/z521) andthe threeG ions (m/z265,405,and433)are256, 116, and 88u, respectively, indicating that thereareone long-chain saturated (C16:0) and two short-chainsaturated (C6:0 and C4:0) acyl groups in the TAGcompound 3. Also, eachG ion accompanies eachcorre-sponding diglyceride ion ([M � Na-R'COONa]�) observedat m/z243,383, and411,respectively.

On the other hand, several types of product ionsinformativeon theregiospecificity of thefatty acyl linkageson theglycerolbackbonewereobservedin theCID spectraof the[M � Na]� ions.26,31Amongtheseions,threepairsof2D, 3D, andE ions, whichhavethestructurescorrespondingto five-, six-membered lactoneringsandpropenyl esterionswith anouter(sn-1 or sn-3) acylgroup,respectively, maybeformedfrom the [M � Na]� ion via the sameintermediatethat doesnot contain the sn-2 acyl group. Thus, one candetermine the location of the fatty acyl groupsat the sn-1andsn-2 positionsby ananalysisof theseions. In addition,theJ ionscorrespondingto sodiatedvinyl esterswith a sn-2acylgroupareobservedatm/z137and305in Figs.3(a)and(b), respectively. Thus, from thesemeasurements, it is astraightforward conclusionthatcompound1 hasaC4:0acylgroup and compound 3 a C16:0 acyl group at the sn-2position. Therefore,these2D, 3D, E, andJ ionsprovidetheinformationnecessaryto locate thethreeacyl groupson theglycerol backbone.

On the basis of these results, we conclude that thecompletestructureof theintactmoleculecanbedeterminedby theanalysisof theCID spectrum of the [M � Na]� ion,

exceptfor the chirality of the sn-2 carbon(in cases wherethe sn-1 andsn-3 substituents aredifferent).

Triacylglycerols isolated from bovine udder

SeveralTAG molecular speciescontaining two short-chainfatty acids were isolated from bovine udder by solventextraction and silica gel column chromatography.Figures5(a)and(b) showthepositive-ion mass spectraobtainedbyFAB of thesample in a matrix of 3-NBA without andwithNal, respectively. Thepeaksobservedat m/z633, 659,661,and687 in Fig. 5(b) correspond to monoacetyldiglyceridescontainingan acetylgroup at the sn-3 position.They werealso isolatedin pure form in other fractionsand, as theirstructures were completely identified in a previousstudy,15

only the resultsare summarizedin Table 1. On the otherhand,the peaksat m/z493,519,521,and547 in the samespectrumcorrespond to the TAG speciescontaining twoshort-chain fatty acids. Thesespecies differ in the chainlengthsof the fatty acidsandthe degree of unsaturation inthe fatty acyl group. These complete structures wereidentified, basedon comparisons with the resultsobtainedby the mass spectrometric analysesof the synthetic TAGcompoundsdiscussed above.

Figures6(a)and(b) showtheCID tandemmassspectraofthe[M � Na]� ionsof themolecularspeciesobservedatm/z547 in Fig. 5(b), and of synthetic 1-oleoyl-2-caproyl-3-butyryl-rac-glycerol (C18:1/C6:0/C4:0-TAG), respectively.Thepeakpatternsof thetwo spectraareverysimilar to each

Figure 5. Positive-ionFAB massspectraof triacylglycerolsisolatedfrom bovineudderthat containtwo short-chainfatty acids.A matrix of 3-nitrobenzylalcohol(a) without NaI and(b) with NaI wasused.



other. Fromthe threeG ionsobservedat m/z265,431,and459 accompanying the correspondingdiglyceride ions atm/z243,409,and437,respectively, this specieswasfoundto containC18:1,caproyl, andbutyryl groups.The fact thatthepeaksof thestable3I ionsformedby lossesof C18:1andcaproyl groups were observed at m/z 337 and 503 withgreater intensities than those of other neighboring ionsconfirms the aboveconclusion. Charge-remote fragmenta-tion along theC18:1 acylchainyieldedahomologousseriesof ions observedat m/z531,517,503,489,475,461,447,

433, 421,407,393,379,365, 351,and337 (correspondingto the 3I ion), in the high-mass region. From the alkyl-terminussideof the C18:1acyl chain,the massdifferencebetween adjacentpeaksis 14u dueto a seriesof CnH2n � 2

fragment losses via CRF elimination reactions.31,36 How-ever, if adoublebondis encountered,themassdifferenceisreducedto 12u andthenagain becomes14u dueto aseriesof CnH2n fragment losses,to an eventualabundant 3I ion.Identification of theabundant even-massproduct ion (of m/z448) formedby allylic cleavagealsoenablestheposition ofthe double bondto be located. Therefore, the doublebondon the C18:1 acyl chain is at the 9th position from thecarbonyl carbon.TheC18:1acylgroupcorrespondsto oleicacid (i.e., 9-octadecenoicacid).

Informationon theregiospecificity of threeacyl linkagesontheglycerolbackbonewasobtainedfrom themassvaluesof the product ions designated as 2D, 3D, E, and J in Fig.6(a). TheJion observedatm/z165containsacaproylgroup.From threepairsof 2D, 3D, andE ions, the sn-1 andsn-3positions are occupiedby an oleoyl and a butyryl group,respectively. Thus, this speciesof m/z 547 is 1-oleoyl-2-caproyl-3-butyryl-rac-glycerol. Similar structural analyseswerealsoappliedto othermolecularspecies. Theresultsaresummarizedin Table 1. According to these results, otherionic species, exceptfor thatof m/z547,weremixturesof atleast two components,which have different fatty acidcompositionsbut the samemolecularmass.For example,the speciesof m/z 493 indicatesa CID spectral patternsimilar to that of the syntheticcompound1 (C16:0/C4:0/C4:0-TAG), asshownin Fig. 7. However, additional peaks

Table 1. Structural identification of triacylglycerol (TAG) mol-ecular species containing two short-chain fatty acylgroups from bovine udder

MW

[M � Na]�, m/zStructureof fatty acyl groups

(sn-1/sn-2/sn-3)aObserved Calculated

470.4 493.3513 493.3505 C16:0/C4:0/C4:0� C14:0/C6:0/C4:0�C12:0/C8:0/C4:0

496.4 519.3670 519.3662 C18:1/C4:0/C4:0� C16:1/C6:0/C4:0498.4 521.3807 521.3818 C18:0/C4:0/C4:0� C16:0/C6:0/C4:0524.4 547.3972 547.3975 C18:1/C6:0/C4:0610.4 633.5 C16:0/C16:0/C2:0636.5 659.5 C18:1/C16:0/C2:0638.5 661.5 C18:0/C16:0/C2:0664.5 687.5 C18:0/C18:1/C2:0

a Thefatty acylgroupsof individualmolecularspeciesof TAGsaresymbolizedby the convention,carbonnumber:degreeof unsaturation[i.e., C2:0 (acetyl),C4:0 (butyryl), C6:0 (caproyl), C8:0 (capryloyl), C12:0 (lauroyl), C14:0(myristoyl), C16:0(palmitoyl), C16:1(palmitoleoyl),C18:1(oleoyl)].

Figure 6. FAB tandemmassspectraof [M � Na]� ions (m/z547) of (a) the speciesisolatedfrombovineudderand(b) synthetic1-oleoyl-2-caproyl-3-butyryl-rac-glycerol (C18:1/C6:0/C4:0-TAG).



not observedin Fig. 3(a),anddifferentabundancesof somepeaksobservedin the high-massregion, suggestthat thisionic speciescontains two other minor components inaddition to the major component,1-palmitoyl-2,3-dibuty-ryl-rac-glycerol. In particular, in additionto theJ ion of m/z137 containing a butyryl group, two other J ions areobserved at m/z 165 and 193, implying that the minorcomponentscontainacaproyl (C6:0) andacapryloyl (C8:0)group at the sn-2 position, respectively. The presenceofthese fatty acyl groupsis further confirmedby the G2 ionsobserved at m/z 377 and 349 due to losses of the corre-spondingacyl groups,accompanying thediglyceride ionsatm/z 355 and 327. In the caseof a long-chain acyl grouplocatedat thesn-1 position, in addition to theG1 ion of m/z237 formed by loss of a palmitoyl group from the majorcomponent,theproduct ionsat m/z265and293 correspondto theG1 ionsformedby lossesof amyristoyl (C14:0)andalauroyl (C12:0) group from the two minor components,respectively. Thus, the two minor componentswith totalfatty acid composition of C24:0are1-myristoyl-2-caproyl-3-butyryl-rac-glyceroland1-lauroyl-2-capryloyl-3-butyryl-rac-glycerol. The m/z values of the characteristicproductions for the threecomponentsarelisted in Table 2.

DISCUSSION

This study wasundertakenin orderto completely determinethe structures of themolecular speciesin theTAG fractionisolated from bovine udder, which have two short-chainfatty acids. The study usedtandem mass spectrometry ofsodium-adducted molecules of eachspecies.CID-MS/MSof each [M � Na]� ion provided the information on the

structureof each acyl group (carbon number, degreeofunsaturation, position of double bond, etc.) and on thelocationof the threeacyl groupson the glycerol backbone(sn-1, sn-2, or sn-3). As shown in theresults summarizedinTables1 and2, eachspecieswasa mixture of at least twocomponents that havedifferent fatty acid composition butthesamemolecular mass.Furthermore,themost remarkablefeatureis thatall thecomponentspresentin mixturesof allthe speciescontaina long-chain fatty acid (C12 to C18) atthe sn-1 position,a short-chain fatty acid (C4 to C8) at thesn-2 position, and a butyric acid uniquely at the sn-3position. To our knowledge,therehavebeenno previousreportson finding these speciesin natural TAGs isolatedfrom animaltissues.

Theseresults areinconsistentwith thoseobtained from apreviousstudy38–40 of the biosynthesisof triacylglycerolscontaining butyric and hexanoic acids in lactating cowmammary gland. A major pathway of short-chain triacyl-glycerol biosynthesisin mammalian tissuesis the glycer-olphosphate pathway.40–42 The first step in this pathwayinvolves successiveacylations of glycerol-3-phosphatebylong-chain acyl-CoA at the sn-1 and sn-2 positions tophosphatidic acid.This is followedby dephosphorylation tolong-chain 1,2-diacylglycerol, which is finally acylatedbybutyryl-CoA or hexanoyl-CoA at the sn-3 position. It hasalso beenreportedthat the 1-acylglycerolphosphate acyl-transferasefrom cow mammary gland is not activetowardsbutyryl-CoA or hexanoyl-CoA but only towardslong-chainacyl-CoA,38,39whichis consistentwith theabsenceof short-chain fatty acids at the sn-2 position of cow milk triacyl-glycerols.3 However, according to our results, a non-trivialquantityof thespeciescontaining theshort-chain fatty acid

Figure 7. FAB tandemmassspectrumof the [M � Na]� ion observedat m/z493 in Fig. 5(b).

Table 2. Assignmentsof characteristic product ions observedin the CID spectrum of thespeciesof m/z 493 for the three components with the same total fatty acidcomposition

Components(sn-1/sn-2/sn-3)

Characteristicproductions,m/z2D1,2/2D2,3

3D1,2/ 3D2,3 E1,2/E2,3 G1/G2/G33I1/3I2 J

C16:0/C4:0/C4:0 209/377 223/391 151/319 237/405/405 309/N.D.a 137C14:0/C6:0/C4:0 209/349 223/363 151/291 265/377/405 337/449 165C12:0/C8:0/C4:0 209/321 223/335 151/263 293/349/405 365/421 193

a Becausethe formation of the 3I ion dueto lossof a butyryl groupis impossible,the ion of m/z477arosefrom lossof CH4 from otherfatty acyl groupsvia CRFeliminationreactions.



at the sn-2 position aswell asa butyryl group at the sn-3position wasisolatedfrom bovineudder.Ourresults suggestthat thesemight be biosynthesizedby a separate 1-acyl-glycerolphosphate acyltransferase,which is specific forshort-chain acyl-CoAs, or throughother pathways.

Acknowledgements

Financialsupportfrom theKoreaScienceandEngineeringFoundation(KOSEF1999-2-209-006-3for G.-J. JhonandS.-Y. Han), and fromthe Korean Ministry of Education (Brain Korea 21 program) forgraduatestudentfellowships(K.-Y. SoandJ.-K. Limb), aregratefullyacknowledged.

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Identification of triacylglycerols containing two short-chain fatty acids at sn-2 and sn-3 positions from bovine udder by fast atom bombardment tandem mass spectrometry