the lipidome is a complex 
 mixture of fatty acid and sterol molecular 
 species , which include the fatty acid esters of sterols , triglycerides , 
 and glycerophospholipids , such as the ethanolamines , cholines , and 
 inositols . polyunsaturated fatty acids 
 ( pufas ) such as linoleic acid ( la ) ( 18:2 ) and arachidonic acid ( aa ) 
 ( 20:4 ) and their esters are particularly important molecular species . these essential fatty acids are involved in a number of consequential 
 metabolic transformations via their oxidation by lipoxygenase ( lox ) 
 and cyclooxygenase ( cox ) enzymes . oxidized lipids play a significant role 
 in a number of physiological and pathophysiological events , including 
 cardiovascular disease , cancer , and neurodegenerative diseases . the nonenzymatic peroxidation of both la and 
 aa by molecular oxygen generates intermediate peroxyl free radicals . products that result from this lipid peroxidation include peroxides 
 and hydroperoxides , as well as secondary electrophilic products capable 
 of covalently modifying biomolecules , potentially altering their function . tracking lipids , lipid metabolites , and lipid decomposition 
 products 
 in cells is a formidable task as the complexity of the mixture challenges 
 the most powerful analytical tools . stable isotope derivatives of 
 lipids have been used with some success to track the distribution 
 of molecular species into different lipid classes in organelles , but 
 detecting a minor metabolic byproduct from thousands of different 
 lipid species is particularly difficult since stable isotope - labeled 
 compounds are often isobaric with endogenous species . radiolabels have also been used successfully in many applications , 
 but associating a particular radioactive chromatography fraction with 
 the structure of a molecular species in a complex mixture is a challenge . recently , an affinity labeling technique that makes use of terminal 
 alkynyl lipid analogues was reported . in this approach , cobalt complexes 
 are formed on a phosphine - modified silica to isolate alkynyl lipids . this strategy has been used to monitor the distribution of alkynyl 
 fatty acids into various cellular phospholipid classes along with 
 the subsequent lipase - catalyzed metabolism of those lipids . in recent studies , terminal alkynyl analogues 
 of 4-hydroxy-2e - nonenal ( hne ) and 4-oxo-2e - nonenal ( one ) , lipid - derived electrophiles known to modify 
 proteins and nucleic acids , were used to globally profile electrophile 
 adduction of proteins . uv - cleavable biotin azide was used to isolate , 
 identify , and quantify cellular protein this affinity chemistry allows 
 protein  lipid adducts to be concentrated and identified by 
 standard proteomic protocols . one shortcoming of adding electrophiles 
 exogenously to cells is that it does not mimic endogenous lipid electrophile 
 diversity , concentration , time course of generation , and location 
 of formation . to address these issues , we have developed a series 
 of -alkynyl pufas to investigate endogenous lipid oxidation 
 and its cellular consequences . tracking lipid incorporation 
 and metabolism in cellular systems 
 has long been a goal for chemists , biochemists , and biologists alike . alkynyl analogues of lipid and cholesterol species have been utilized 
 to facilitate the tracking and retrieval of these species in cells . during these studies , it is assumed 
 that the -alkyne confers only a minimal structural change to 
 its lipid analogue , resulting in nearly identical chemical , biochemical , 
 and biological properties . here , we report the oxidation chemistry 
 for the alkynyl lipid analogues , aaa and ala , revealing that alkyne substitution has no effect on 
 fatty acid free radical autoxidation , and minimal effect on enzymatic 
 oxidation . this establishes that alkynyl pufas provide a method to 
 selectively study lipid distribution , chemistry , and lipid metabolite 
 interactions with cellular macromolecules . all reagents are from sigma , st . louis , mo , 
 unless otherwise noted . all native fatty acids and deuterated lipid 
 metabolites are from cayman chemical , ann arbor , mi , unless otherwise 
 noted . the alkynyl fatty acids , ala and aaa , were synthesized as previously described.n - methyl benzohydroxamic acid ( nmbha ) 
 has been prepared as previously described . benzene ( hplc grade ) was passed through a column of neutral alumina 
 and stored over molecular sieves ( benzene is a carcinogen and mutagen , 
 and should be used with extreme care ) . diazomethane was prepared by portion - wise 
 addition of nitrosomethylurea into heterogeneous mixture of 40% aqueous 
 koh and ethanol at 0 c . to a solution of ala in benzene was added 2,2-azobis(4-methoxy-2,4-dimethylvaleronitrile ) 
 ( meoamvn ) in benzene , and the mixture was incubated at 37 c . after 45 min , a solution of butylated hydroxytoluene ( bht ) and triphenyl 
 phosphine ( pph3 ) in benzene was added , and the mixture 
 was vortexed for 1 min . benzene was evaporated under a stream of argon , 
 and the residual material was reconstituted in 1.2% isopropanol in 
 hexanes with 0.1% acetic acid for hplc - uv analysis . for direct infusion 
 ms studies of ahode , eluted peaks from hplc - uv analysis 
 were collected , and solvents were evaporated under a stream of argon ; 
 then the residues were reconstituted in methanol . for nmr studies 
 of ahode , corresponding peaks collected from hplc - uv 
 analysis were combined , solvents were evaporated , and the residues 
 were dried under high vacuum for 2 h. these dried materials were reconstituted 
 in benzene - d6 and placed into 1.7 mm od 
 sample tubes for nmr analysis . to a mixture of aaa and nmbha in acetonitrile was added meoamvn in acetonitrile . after 35 h of incubation at 37 c , bht / pph3 in acetonitrile 
 was added , and the mixture was vortexed for 2 min . acetonitrile was 
 removed under a stream of argon , and the remaining material was reconstituted 
 in 1.2% isopropanol in hexanes with 0.1% acetic acid for hplc - uv analysis . ms studies of ahete and nmr studies of ahete were performed as described above for ahode . all hplc analyses of ( a)hete and ( a)hode were carried out on a single beckman 
 5 m ultrasphere silica column ( 250 mm  4.6 mm ) using 
 isocratic normal phase conditions ( 1.2% ipa in hexanes containing 
 0.1% acetic acid ) . chiral hplc analyses 
 of hete and hode methyl esters were performed on a chiralpak ad column 
 ( 250 mm  4.6 mm ) produced by chiral technologies inc . , exton , 
 pa . ahete products have been eluted with 2% ethanol 
 in hexanes , whereas ahode were eluted with 5% methanol 
 in hexanes . direct infusion ms experiments were performed on thermofinnigan 
 tsq quantum triple quadrupole mass spectrometer , whereas all hplc / ms 
 analyses were conducted on the same instrument coupled with a surveyor 
 ms pump and surveyor autosampler ( for rp - hplc ) or with waters alliance 
 2690 separation module ( np - hplc ) . detailed information about solvent 
 gradients and ms settings applied during these analyses is given in 
 the appropriate protocols presented below . unless stated otherwise , 
 all the hplc separations were conducted with a solvent flow rate of 
 1 ml / min . meoamvn was 
 added to a 165 mm solution of aaa in benzene , and the mixture was 
 incubated at 37 c for 24 h. solvent was then evaporated , and 
 the residue was treated with mixture of 1 mmol bht and 10 mmol p(ome)3 in 3:1 , acetonitrile / water and vortexed for 5 min . the following 
 solvent gradient was applied : 10% ( 95:5 , acetonitrile / methanol ) in 
 2 mm ammonium acetate was held for 10 min , then ramped to 25% ( 95:5 , 
 acetonitrile / methanol ) in 2 mm ammonium acetate over 45 min . lc / ms / ms 
 with negative ion mode electrospray ionization ( esi ) was used , and 
 the important mass spectrometer parameters were optimized for commercially 
 available pgf2. the m / z transitions monitored were for a5f2-isop ( 349 > 115 ) , a8f2-isop 
 ( 349 
 > 127 ) , a12f2-isop ( 349 > 
 151 ) , 
 and a15f2-isop ( 349 > 193 ) . control 
 oxidations of aa were performed using analogous reaction conditions ; 
 however , slightly different analytical conditions were applied to 
 analyze the aa oxidation products . the following solvent gradient 
 was applied for aa oxidation products : 20% ( 95:5 , acetonitrile / methanol ) 
 in 2 mm ammonium acetate was held for 10 min , then ramped to 40% ( 95:5 , 
 acetonitrile / methanol ) in 2 mm ammonium acetate over 40 min . the m / z transitions monitored were for 5f2-isop ( 353 > 115 ) , 8f2-isop ( 353 
 > 127 ) , 12f2-isop ( 353 > 151 ) , and 15f2-isop ( 353 > 193 ) . quantification 
 of cyclooxygenase activity was performed in a thermostated cuvette 
 at 37 c with stirring and monitored using a polarographic electrode 
 with an instech system 203 oxygen monitor ( instech laboratories inc . , 
 plymouth meeting , pa ) . activity assays were performed in 100 mm tris buffer containing 
 500 m phenol , with hematin - reconstituted cyclooxygenase protein . substrate concentration was varied ( 150 m ) , and maximal 
 reaction velocity data were obtained from the linear portion of the 
 oxygen uptake curves . the data were analyzed by nonlinear regression 
 with graphpad prism ( graphpad , san diego , ca ) . lox activity 
 was detected by monitoring the absorbance of the conjugated diene 
 product , hpete , at 235 nm . uv assays were monitored using a hewlett - packard 
 8453 diode array spectrophotometer equipped with a thermostated cuvette 
 at 25 c , with stirring at 180 rpm . the enzyme reactions included 
 reaction buffer 50 mm tris ph 7.4 with 0.03% tween-20 and substrate , 
 and were initiated by the addition of enzyme . compounds were dissolved 
 in acetonitrile containing 10% acetic acid before addition to the 
 reaction buffer ; acetonitrile was kept below 1% of the total reaction 
 volume . substrate concentration was varied ( 150 m ) , 
 and maximal reaction velocity data were obtained from the linear portion 
 of the absorbance curves . rates were converted from absorbance units / s 
 to m ahpete / s using the molar absorptivity 
 constant of 0.027 m cm . s15lox1 , la , aa , ala , and aaa were all diluted to 2 final concentration 
 in 100 mm borate ph 9 at 25 c . one ml fatty acid was added to 
 a 1 cm cuvette in a beckman - coulter du-800 spectrophotometer as a 
 blank control . one ml enzyme was added , and the reaction was monitored 
 at 235 nm sampling every 1.5 s in triplicate . the slope was averaged 
 over 10 points in the linear portion of the curve to get the absorbance 
 per second , which was converted to vo using 
 the molar extinction coefficients of 0.023 m cm for hpode and 0.027 m cm for hpete . soybean 15 lipoxygenase 1 ( s15lox1 ) ( cayman chemical ) 
 was diluted to have a final concentration ratio of 100:1 , fatty acid / enzyme 
 in 100 mm borate ph 9 . potato 5 lipoxygenase ( 5lox ) ( cayman chemical ) 
 was diluted to have a final concentration ratio of 100:1 , fatty acid / enzyme , 
 in 100 mm phosphate ph 6.3 . la and ala were added 
 from 100 stocks in dmso , and incubated 15 min at 25 c . the reactions were quenched and fatty acid metabolites extracted with 
 ethyl acetate containing 0.5% acetic acid , pph3 ,  
 9-hode - d4 , and 13-hode - d4 . organic layer was dried under inert gas and dissolved 
 in methanol for lc / ms / ms analysis . metabolites were analyzed on a 
 thermo finnigan tsq quantum with esi source interfaced to surveyor 
 ms pumps and surveyor autosampler in both full scan and srm modes . metabolites were separated by reverse - phase gradient hplc on a c18 ( 50 mm  2.1 mm , 3 m ) column ( supelco , st . louis , 
 mo ) using 0.1% formic acid in water and 0.1% formic acid in acetonitrile 
 as the a and b mobile phases , respectively . full scan samples were 
 separated by holding 20% b for 2 min , then ramping to 98% b over 6 
 min , holding 98% b for 3 min , then equilibrating to 20% b for 3 min . m / z to 380 m / z in 1 s. srm 
 samples were separated by holding 40% b for 0.5 min , then ramping 
 to 98% b over 2 min , holding at 98% b for 2 min , then equilibrating 
 to 40% b for 2.5 min . metabolites were detected by srm in negative 
 ion mode observing the m / z transitions 
 for a9-hode ( 291.2 > 171.2 ) , a13-hode 
 ( 291.2 > 195.2 ) , 9-hode ( 295.2 > 171.2 ) , 13-hode ( 295.2 > 
 195.2 ) , 
 9-hode - d4 ( 299.2 > 172.2 ) , and 13-hode - d4 ( 299.2 > 198.2 ) for 100 ms each . mouse 
 cox2 ( mcox2 ) was diluted to have a final concentration ratio of 100:1 , 
 fatty acid / enzyme in 100 mm tris , 500 m phenol , 2 [ mcox2 ] 
 hematin ph 8 . la and ala were added from 100 stocks in dmso , and incubated 
 15 min at 37 c . the reactions were quenched and fatty acid metabolites 
 extracted and analyzed as detailed for the lipoxygenase enzymes . s15lox1 was diluted to a final concentration ratio 
 of 100:1 , fatty acid / enzyme , in 100 mm borate ph 9 . fatty acids 
 were separately added to enzyme solutions , and incubated 15 min at 
 25 c . the reactions were quenched and fatty acid metabolites 
 extracted with ethyl acetate containing 0.5% acetic acid , pph3 , and 15-hete - d8 . the organic 
 layer was dried under inert gas and dissolved in methanol for lc / ms / ms 
 analysis . metabolites were analyzed on a thermo finnigan tsq quantum 
 with esi source interfaced to surveyor ms pumps and surveyor autosampler 
 in both full scan and srm modes . metabolites were separated by gradient 
 hplc on a c18 ( 50 mm  2.1 mm , 3 m ) column 
 using 0.1% formic acid in water and 0.1% formic acid in acetonitrile 
 as the a and b mobile phases , respectively . full scan samples were 
 separated by holding 20% b for 2 min , then ramping to 98% b over 6 
 min , holding 98% b for 3 min , then equilibrating to 20% b for 3 min . q1 was scanned in negative ion mode from 250 m / z to 380 m / z in 1 s. srm 
 samples were separated by holding 40% b for 0.5 min , then ramping 
 to 98% b over 2 min , holding at 98% b for 2 min , then equilibrating 
 to 40% b for 2.5 min . metabolites were detected by srm in negative 
 ion mode observing the m / z transitions 
 for a15-hete ( 315.2 > 253.2 ) , 15-hete ( 319.2 > 
 257.2 ) , 
 and 15-hete - d8 ( 327.2 > 264.2 ) for 
 100 
 ms each . mcox2 was diluted to a final concentration ratio of 
 100:1 , fatty acid / enzyme in 100 mm tris , 500 m phenol , 2 
 [ mcox2 ] hematin ph 8 . aa and aaa were added from 100 stocks in dmso , 
 and incubated 15 min at 37 c . the reactions were quenched and 
 fatty acid metabolites extracted and analyzed as detailed for the 
 lipoxygenase enzyme full scan mode experiment . s15lox1 , la , aa , ala , 
 and aaa were diluted to 2 final concentration 
 in 100 mm borate ph 9 at 25 c . one ml fatty acid was added to 
 a 1 cm cuvette in a beckman - coulter du-800 spectrophotometer and blanked . 
 one ml enzyme was added , and the reaction was monitored at 235 nm 
 sampling every 1.5 s , until the absorbance reached 0 . the data 
 was fit to a one - phase exponential association in graphpad prism to 
 get r values and maximum absorbances . mcox2 was incubated 
 5 min in 100 mm tris , 500 m phenol , and 2 [ mcox2 ] hematin 
 ph 8 at 37 c . aaa was added , and the reaction 
 was allowed to proceed for 15 h at 37 c . the reaction was extracted 
 with two volumes ethyl acetate , and the organic layer was removed 
 and evaporated under inert gas . the residue was dissolved in ethanol 
 and separated by reverse - phase hplc on a supelcosil c18 ( 150 mm  3.0 mm , 5 m ) column , eluted with a linear 
 gradient with a and b buffers consisting of 0.1% acetic acid in water 
 and 0.1% acetic acid in acetonitrile . the gradient was held at 10% 
 b for 10 min , then ramped to 100% b over the next 20 min , then held 
 at 100% b for 5 min , all at the flow rate of 1.0 ml / min . peaks were collected , 
 dried under inert gas , and dissolved in cdcl3 for nmr analysis . h and h  h cosy spectra were 
 recorded on bruker av - ii 600 mhz spectrometer equipped with a cryoprobe . chemical shifts are reported in parts per million relative to the 
 signal of residual nondeuterated solvent . metabolites of mcox2 
 do not have an absorbance that can be used to perform kinetic measurements . therefore , lc / ms / ms was used to measure kinetic parameters based on 
 specific metabolites . mcox2 was diluted to 25 nm in 100 mm tris , 500 
 m phenol , 50 nm hematin ph 8 , and incubated at 37 c . aa and aaa were added from 100 stocks in dmso , 
 and incubated at 37 c . the reactions were quenched 
 and fatty acid metabolites extracted with ethyl acetate containing 
 0.5% acetic acid , pge2-d4 , 
 and 13-hode - d4 . metabolites were separated 
 by reverse - phase gradient hplc on a c18 ( 50 mm  2.1 
 mm , 3 m ) column using 0.1% formic acid in water and 0.1% formic 
 acid in acetonitrile as the a and b mobile phases , respectively . metabolites 
 were separated by holding 25% b for 0.5 min , then ramping to 99% b 
 over 2.5 min , holding at 99% b for 3 min , then equilibrating to 25% 
 b for 3 min . metabolites were analyzed in negative ion mode by srm , 
 monitoring the transitions for a11-hete ( 315.2 > 
 167.2 ) , apg ( 347.2 > 267.2 ) , pg ( 351.2 > 271.2 ) , 
 13-hode - d4 ( 299.2 > 198.2 ) , pge2-d4 ( 355.2 > 275.2 ) on an abi / sciex 
 3200 
 qtrap interfaced to a shimadzu lc system . no deuterated standard exists 
 for 11-hete . the signal intensity ratio between a11-hete and 13-hode - d4 was observed to 
 be similar in both full scan ms and srm modes , indicating that 13-hode - d4 can be used to quantify a11-hete . the amount of each product formed was converted into a concentration , 
 and then into initial reaction rates ( vo ) using the incubation time . raw264.7 macrophages 
 ( atcc , manassas , va ) were plated at 10% confluence in dmem+glutamax 
 ( life technologies , grand island , ny ) supplemented with 10% fetal 
 bovine serum ( atlas biologicals , fort collins , co ) . fatty acid free 
 bsa / aaa ( 6:1 ) was prepared as previously described . after 24 h , media was replaced with serum free 
 dmem+glutamax containing aaa / bsa added to 15 m 
 final concentration of aaa . after 24 h , the cells 
 were washed with one volume dmem+glutamax to remove any unincorporated aaa , and treated with dmem+glutamax 100 ng / ml kdo2-lipid a ( kla ) ( avanti polar lipids , alabaster , al ) , prepared 
 as previously described . after 24 h , 
 cells were scraped into media and extracted with two volumes ethyl 
 acetate containing 0.5% acetic acid , pge2-d4 , and 13-hode - d4 . the organic 
 layer was dried under an inert gas stream and dissolved in methanol 
 for lc / ms / ms analysis . metabolites were separated by reverse - phase 
 gradient hplc on a c18 ( 50 mm  2.1 mm , 3 m ) 
 column using 0.1% formic acid in water and 0.1% formic acid in acetonitrile 
 as the a and b mobile phases , respectively . metabolites were separated 
 by holding 25% b for 0.5 min , then ramping to 99% b over 2.5 min , 
 holding at 99% b for 3 min , then equilibrating to 25% b for 3 min . metabolites were analyzed in negative ion mode by srm , monitoring 
 the transitions for a11-hete ( 315.2 > 167.2 ) , a11 - 8,9-heet ( 331.2 > 165.2 ) , apg ( 347.2 
 > 267.2 ) , pg ( 351.2 > 271.2 ) , 13-hode - d4 ( 299.2 > 198.2 ) , and pge2-d4 ( 355.2 > 275.2 ) on an abi / sciex 3200 qtrap interfaced 
 to a shimadzu 
 controller , autosampler , and hplc pumps . la and ala were oxidized under conditions that 
 would allow us to compare autoxidation rates and products . as shown 
 in figure 1 , four -alkynyl conjugated 
 diene hydroperoxides ( alkynyl hydroperoxyoctadecadienoic acids , ahpodes ) were produced as primary products from ala autoxidation , a result directly analogous to the chemistry 
 observed with la . reduction of the hydroperoxides 
 to the corresponding alcohols , ahodes , was carried 
 out immediately after peroxidation since the hodes are more stable 
 and better suited for hplc analysis than hpodes . hplc - uv analysis 
 revealed that parallel oxidation of equimolar amounts of ala and la generated equivalent amounts of ahodes 
 and hodes , with elution orders identical to those of the natural compounds . the isolated products were then analyzed by 
 esi - ms and nmr to confirm hydroxyl position and conjugated diene geometry 
 ( supporting information [ si ] figure 1 ) . 
 utilizing chiral chromatography , it was determined that the autoxidation 
 of ala produces a mixture of r- 
 and s - hode enantiomers , similar to that of la ( si figure 2 ) . autoxidation products of of la ( a ) , ala ( b ) , aa 
 ( c ) , aaa ( d ) . la / ala peaks are labeled 
 with the hydroxyl position on either the 9 or 13 carbon , and the double 
 bond configuration which resulted in either cis / trans ( c , t ) or trans / trans ( t , t ) . aa / aaa peaks are labeled with the position 
 of the oxygen on either the 5 , 8 , 9 , 11 , 12 , or 15 carbon . both fatty 
 acid pairs oxidize to similar product profiles with similar elution 
 orders . contrary to the simplicity of 
 products generated from la autoxidation , 
 peroxidation of aa yielded a much more complex mixture of products . in addition to acyclic hydroxy and hydroperoxy products ( hetes and 
 hpetes ) analogous to hodes and hpodes , a mixture of diastereomeric 
 isoprostanes ( isops ) was produced from aa peroxidation . oxidation of aaa under conditions that gave hpetes 
 as major products was promoted by nmbha . as shown in figure 1 , characterization of 
 peroxidation products uv 
 elution profile for ahetes was similar to the profile 
 obtained for their natural analogues . ms analyses established the 
 position of oxygen substitution on the carbon chain ( si table 1 ) , and nmr analysis provided information about stereoisomeric 
 geometry ( si figure 1 ) . the major hete 
 stereoisomers have z , e - conjugated 
 diene geometry , analogous to the structure of aa - derived hetes . these 
 experiments show that the elution order of ahetes 
 is identical to the elution order observed for hetes . additionally , 
 oxidation of equimolar mixtures of aa and aaa generated 
 nearly equimolar mixtures of hete and ahete . hetes are not the only autoxidation products formed from aa , so aaa was exposed to the radical initiator meoamvn in the 
 absence of nmbha under conditions of oxidation and workup that were 
 expected to yield significant quantities of the af2-isops . lc / ms / ms analysis of the major ( a)f2-isops formed in this sequence are 
 shown in si figure 3 . chromatograms of 
 only the 5- and 15-series ( a)f2-isops are presented because these compounds are 
 formed in a large excess compared to the 8- and 12-regioisomers . the 
 preference for formation of the 5- and 15-regioisomers and the elution 
 profiles observed for both the natural and -alkynyl analogues 
 are consistent with a previous report . aa kinetic values taken from the literature are in parentheses.vmax/km values are reported for crude enzyme preparations , while kcat / km values are 
 reported for purified enzymes . using alkynyl fatty acids to further probe the biochemistry of 
 cellular systems requires detailed knowledge of the chemistry of enzymatic 
 oxidation . kinetic parameters were determined for the transformations 
 of aaa in the presence of several lox and cox enzymes 
 by measuring alkene formation or o2 consumption , respectively . data presented in table 1 demonstrate small 
 differences in kcat / km for the alkynyl and natural fatty acids , suggesting 
 that aaa is an efficient substrate for both cox-1 
 and cox-2 . the catalytic efficiency of human platelet - type 12-lox 
 in the presence of aaa was also found to be similar 
 to the efficiency observed for aa as a substrate . on the other hand , 
 porcine leukocyte - type 12-lox , rabbit reticulocyte 15-lox1 , and s15-lox1 
 did not oxidize aaa as efficiently as aa , illustrated 
 by the relatively large differences in vmax / km and kcat / km values between these substrates . 
 despite these differences in kinetic parameters , aaa is completely converted by a15lox1 when reacted for long enough 
 times ( figure 2 ) . la , ala , aa , and aaa were all 
 incubated with s15lox1 and observed at 235 nm until abs = 0 . 
 despite having different kinetic parameters , fatty acid pairs ala / la ( a ) and aaa / aa ( b ) , are eventually 
 oxidized completely . the kinetic parameters for the transformations of ala and la by s15lox1 were also measured , and kcat / km values were determined 
 to 
 be 0.51  0.07 m s and 5.2  0.6 m s , respectively . these values are similar to those observed for aaa and aa ( table 1 ) , and like aaa , ala is also eventually completely 
 reacted ( figure 2 ) . ala and 
 la give a similar product profile of primarily ( a)9-hode or ( a)13-hode for the enzymatic transformation 
 by 5-lox or s15-lox1 respectively ( si figures 
 4 and 5 ) . a13-hode produced from s15lox1 was assessed 
 for optical purity , and determined to be entirely the s isomer , as anticipated from the stereochemistry of la oxidation 
 ( si figure 3 ) . ovine cox1 generated a product 
 profile similar to that of mcox2 when reacted with la , and neither 
 enzyme oxygenated ala ( si figure 5 ) . xic analysis of the metabolite profiles of aaa 
 ( a ) and aa ( g ) catalyzed by mcox2 show very different products . the 
 metabolism of aaa by mcox2 shows some aaa remaining ( b ) and four products corresponding to the addition 
 of one ( c ) , two ( d ) , three ( e ) , and four ( f ) atoms of oxygen . the 
 profiles in b . the metabolism of 
 aa by mcox2 shows a single major product showing the addition of three 
 atoms of oxygen and having the same m / z as pge2/pgd2 ( k ) . in addition to the major 
 metabolite and remaining aa ( h ) , metabolism of aa by mcox2 shows three 
 other products corresponding to the addition of one ( i ) , two ( j ) , 
 and four ( l ) oxygens . these aa products correspond to the nonalkynylated 
 versions of the products seen when aaa is metabolized 
 by mcox2 . we compared the kinetics of mcox2 
 oxidation of aaa determined by o2 uptake 
 ( table 1 ) to values determined by lc / ms / ms 
 and noticed that the product profile 
 from aaa was different from that of aa . as demonstrated 
 in figure 3 , four aaa - derived 
 oxygenation products were identified by ms , which correspond to the 
 addition of one ( m / z = 315.2 ) , two 
 ( m / z = 331.2 ) , three ( m / z = 347.2 ) , and four atoms of oxygen ( m / z = 365.2 ) . the product at m / z = 315.2 corresponds to a11-hete , and 
 the product at m / z = 347.2 corresponds 
 to apge2/d2 . one possibility 
 for the identity of the product at m / z = 331.2 is ahpete ; however , attempted reduction 
 of the hydroperoxide with either tcep or pph3 did not alter 
 the peak elution time ( data not shown ) , indicating a chemically distinct 
 species from the hydroperoxide . although the metabolite profile of 
 aa by mcox2 resulted in peaks with m / z values corresponding to the addition of one , two , three , and four 
 atoms of oxygen , similar to what was seen with aaa , 
 the intensity of the peaks displayed major differences . aa oxygenation 
 by mcox2 results in a major peak at m / z = 351.2 , corresponding to pge2/d2 , and a minor 
 peak at m / z = 319.2 , corresponding 
 to a single oxygen atom incorporation . the remaining oxygen addition 
 peaks were very minor by comparison , but have similar retention time 
 and molecular weight shifts relative to pg as was seen with aaa . ovine cox1 generated a product profile similar to that 
 of mcox2 for aaa and aa ( data not shown ) . h  h cosy spectrum of the collected 
 lc / ms peak with m / z = 315.2 with 
 peaks assigned , which was identified to be that of a11-hete . to identify the metabolites depicted 
 in figure 3 , product peaks at m / z = 
 315.2 and 331.2 were isolated and analyzed via 1d and 2d nmr . the 
 compound present at m / z = 365.2 
 was not stable through the isolation process and thus was not analyzed . figure 4 shows the structure and h  h cosy for the peak at m / z = 315.2 . it was determined that the identity of this peak 
 is ( 5z , 8z , 12e,14z)-11-hydroxyeicosa-5,8,12,14-tetraen-19-ynoic 
 acid ( alkynyl 11-hydroxyeicosatetraenoic acid , a11-hete ) . si table 2 shows the chemical shifts relative 
 to cdcl3 and coupling constants as determined from the h nmr ( si figure 6 ) . the coupling 
 constants for the alkene between c12 and c13 , j12,13 = 15.2 and 15.1 hz respectively , identify the bond as trans . figure 5 shows the structure 
 and h  h cosy for the peak at m / z = 331.2 . it was determined that the 
 identity of this peak is ( z)-7-(3-((3e , 5z)-2-hydroxyundeca-3,5-dien-10-yn-1-yl)oxiran-2-yl)hept-5-enoic 
 acid ( alkynyl 11-hydroxy-8,9-epoxy - eicosatrienoic acid , a11 - 8,9-heet ) . si table 3 shows the chemical 
 shifts relative to cdcl3 and coupling constants as determined 
 from the h nmr ( si figure 7 ) . the coupling constants j12,13 = 15.2 and 
 15.1 hz , assign the alkene between c12 and c13 as trans . the coupling constants for the epoxide were measured as j8,9 = 4.2 and 4.3 hz , identifying the epoxide 
 as cis . h  h cosy spectrum 
 of the collected 
 lc / ms peak with m / z = 331.2 with 
 peaks assigned , which was identified to be that of a11 - 8,9-heet . due to the distinct product 
 profile of aaa metabolism , aa kinetic parameters were determined using the product 
 pge2 , whereas the kinetic parameters for aaa were determined using apge2 and a11-hete . the catalytic efficiency for the formation of pge2 by mcox2 , 
 1.6  0.2 m s , 
 was similar to the oxygen uptake value for hcox2 seen in table 1 . the small difference between the two can be explained 
 by the formation of the nonenzymatic pg degradation product , 12-hydroxyheptadecatrienoic 
 acid ( hht ) , which accounts for approximately 20% of the total pg signal 
 ( data not shown ) . the catalytic efficiencies for aaa products were very different , however , at 0.019  0.005 m s for apg formation 
 and 0.4  0.1 m s for a11-hete formation . when these values are compared 
 to the hcox2 oxygen uptake during aaa metabolism 
 value , 2  1 m s , it can be seen that most of the oxygen consumption is due to the 
 formation of a11-hete . menten 
 plots and relevant kinetic parameters for 
 mcox2 metabolism of aa and aaa measuring the formation 
 of prostaglandins ( a ) , alkynyl prostaglandins ( b ) , and alkynyl 11-hete 
 ( c ) . to evaluate the potential of aaa as a tool in 
 cellular settings , bsa / aaa complexes 
 were formed as previously described and then were added to serum - free 
 cell culture medium for 24 h. cells were 
 then washed with medium to remove any unincorporated aaa and activated with 100 ng / ml kla for 24 h. fatty acid metabolites 
 were extracted from the combined media and cells . figure 7 shows the quantification of a11-hete , a11 - 8,9-heet , apg , and pg in cells enriched 
 with aaa and activated with kla . the alkynylated 
 products were only seen at high levels in the aaa - enriched 
 and kla - activated cells , which corresponds to the conditions where 
 levels of fatty acid release and cox2 expression are highest . further 
 correlating to the kinetic and in vitro experiments , a11-hete was more abundant than apg and a11 - 8,9-heet in cells . the ratio of a11-hete 
 to apg and a11 - 8,9-heet is increased 
 from the purified protein analyses indicating that cellular and purified 
 enzyme metabolite profiles may have slight differences . raw264.7 macrophages 
 were enriched aaa , 
 then activated with 100 ng / ml kla for 24 h. metabolite levels 
 were measured by lc / ms / ms - srm for the media and cells combined . understanding both the 
 enzymatic and nonenzymatic metabolism of 
 alkynyl fatty acids is important because lipid oxygenation products 
 and lipid electrophile formation has been reported to result from 
 both enzymatic and nonenzymatic mechanisms . our data indicate 
 that ala and la are kinetically equivalent substrates 
 for free radical chain oxidation ; ( a)hodes are formed 
 as two positional isomers with oxidation at the 9 and 13 carbons . additionally , the conjugated dienes are in two different conformations , 
 the ze kinetic product , and the ee thermodynamic product . analyzing the ze / ee product 
 ratios can be used as a  peroxyl radical clock  to measure 
 peroxidation propagation rate constants , further confirming that these two substrates are equivalently oxidized . the mechanism of hpete and isop formation has been 
 studied in great detail and it has been established that six major z , e - hpete products form with hydroperoxide substitution 
 at carbons 5 , 8 , 9 , 11 , 12 , and 15 of the 20 carbon eicosanoate chain . the isops are formed as a mixture of stereoisomers , 
 the four sets of regioisomers identified by the position of the allylic 
 alcohol in the chain , 5 , 8 , 12 , and 15 . quantification 
 of the isop isomeric mixture has been used in recent years as a measure 
 of oxidative stress in vivo . this is an important 
 finding for setting up future lipid oxidation studies since many disease 
 states , including models for cardiovascular disease and neurodegenerative 
 diseases , are characterized by a high level of oxidative stress . the kinetic values measured here indicate 
 that with some notable 
 exceptions , aaa is a reasonable enzymatic substrate 
 for both cox and lox classes of enzymes . enzymatically , both alkynyl 
 pufas are metabolized by various lox enzymes to product profiles similar 
 to those of the native pufas . despite the observed differences in 
 catalytic efficiency , aaa , aa , ala , and la are completely oxygenated by s15lox1 when allowed to react 
 to completion ( figure 2 ) . we hypothesize that 
 the reduced efficiency observed is the result of the alkyne altering 
 the conformation of the lipid within the lox active site . model systems 
 designed to study cellular processes all have limitations , and the 
 reduced enzymatic efficiency seen here may restrict the use of this 
 model to understand short - term enzymatic lipid metabolism . however , 
 in many biological settings , this reduced enzymatic efficiency remains 
 negligible in understanding and tracking lipid metabolism because 
 many studies will be looking at changes over long time periods . all products result from 
 the same first steps , abstraction of the 13-(s)-hydrogen 
 and addition of molecular oxygen to the 11-carbon . pathway a shows 
 the formation of a11-hete when the reaction is terminated 
 before 9,11-endoperoxide is formation . after endoperoxide formation , 
 closure of the 5-membered prostaglandin ring results in pathway b 
 and the formation of apgg2 , the precursor 
 for all apgs . however , if ring closure does not occur 
 ( pathway c ) , the endoperoxde can cleave , resulting in an epoxide and 
 an 11-alkoxyl radical . these data demonstrate 
 the potential usefulness of apufas for the study 
 of lipids in a biological setting ; however , one 
 major finding is the differential metabolism of aa and aaa by mcox2 and ocox1 . all of the products in our proposed mechanism 
 ( scheme 1 ) result from the same first two steps , 
 13-(s)-hydrogen abstraction and oxygen addition to 
 c11 forming the alkynyl 11-hydroperoxyl radical . the remaining reactions 
 proceed through two critical junctions , endoperoxide formation and 
 prostaglandin ring closure . the a11-hydroperoxyl 
 radical can be reduced by h atom transfer to form a11-hete ( scheme 1a ) , which was identified 
 as one of the major products by lc / ms / ms and 2d - nmr ( figure 4 ) . when endoperoxide formation is followed by prostaglandin 
 ring closure , and a final oxygenation at c15 , apgg2 , the precursor to all prostaglandins is formed ( scheme 1b ) . however , when endoperoxide formation is not 
 followed by prostaglandin ring closure , endoperoxide homolytic cleavage 
 results in 8,9-epoxide and 11-alkoxyl radical formation . the alkoxyl 
 radical can then be terminated to give a11 - 8,9-heet 
 ( scheme 1c ) , which was identified by 2d - nmr 
 ( figure 5 ) . the epoxide was identified as cis due to the coupling constants for h8 and h9 , which were 
 measured at 4.2 and 4.3 hz , respectively . this is an interesting observation 
 indicating that the epoxide is formed in the enzyme active site , before 
 the bond between c7 and c8 can rotate . nonenzymatic epoxidation from 
 endoperoxide scission would be expected to give a 3:1 trans : cis geometry due to the free rotation of the c7c8 bond . 11 - 8,9-heet was first identified when 8,9 epoxy - eicosatrienoic 
 acid was incubated with cyclooxygenase enzymes . the cyclooxygenase 
 enzymes were only able to add oxygen to c11 because the epoxide prevented 
 endoperoxide formation . the major mcox2 products 
 of aaa oxygenation we have identified are structurally 
 similar to previously reported cox2 variant aa metabolites . demonstrated that mutations 
 at gly526 and leu384 to larger amino acids restrict endoperoxide formation 
 and prostaglandin ring closure , resulting in the generation of multiple 
 products including 11-hpete and pgs . their proposed mechanism proceeds 
 through several intermediates that , when terminated , will give the 
 aa - derived products similar to those we have identified . while 
 we have been unable to solve a crystal structure of aaa in a productive conformation in the mcox2 active site , 
 we can look at other substrates to corroborate the idea that aaa may not be binding properly in the active site , resulting 
 in an altered product profile . the crystal structure 
 of its isomer , 1-ag , has been solved for mcox2 , and it was revealed 
 that it sits in the active site in two different conformations . the 
 structural difference in these confirmations is a slight change in 
 the position of the -tail in the active site . oxygenation may 
 occur in both conformations because abstractable hydrogens on c13 
 are in line with the catalytic tyr385 , but different distances in 
 each conformation . 2-ag has two major 
 products , pg - glycerol and 11-hete - glycerol , which further corroborates that there are multiple modes of binding . these data are potentially relevant to aaa binding 
 in the mcox2 active site , because they indicate that small changes 
 in the binding of the -tail has an impact on the oxygenation 
 and cyclization events at the center of the fatty acid . therefore , 
 we hypothesize that the alkynyl tail changes the way aaa sits in the cox2 active site , resulting in similar o2 consumption despite its altered product profile , as defined in these 
 studies . on the basis of all of the in vitro oxidation , 
 we investigated the viability of alkynyl probes for the analysis of 
 lipid metabolite detection and tracking in a biological setting . raw264.7 
 macrophages are the prototypical cell line used to study lipid metabolism 
 because their lipid chemistry has been extensively cataloged by the 
 lipid maps consortium ( www.lipidmaps.org ) . therefore , we 
 investigated if the in vitro mcox2 metabolites of aaa could be measured in cultured cells and observed a11-hete as the major aaa metabolite in 
 cells , with apg and a11 - 8,9-heet 
 also detected , but at a much lower level . this product ratio matches 
 the kinetic efficiencies measured in vitro for a11-hete and apg formation . many molecules 
 have been reported to potentiate cox2 activity in vitro , including free fatty acids . it is 
 not unreasonable to think that many of these species are present in 
 cells , and could potentiate the formation of a11-hete 
 as was seen in our data . 11-hete has been reported in many animal 
 and cell models as a cox2-derived metabolite . additionally , it has been reported 
 that hydroxy fatty acid metabolites of cox2 , including 11-hete , can be further oxidized by cellular dehydrogenases to oxo fatty 
 acids . these oxo fatty acids are electrophilic , 
 reacting with nucleophilic amino acids of proteins potentially changing 
 cellular functions . prostaglandins are not readily converted to electrophilic 
 species ; thus , hydroxy fatty acids are the most viable method to study 
 this chemistry in cells . 11-oxoeicosatetraenoic acid , the oxidized 
 product of 11-hete , has been detected in cells , and shown to be antiproliferative . this avenue of exploration is potentially viable using aaa as a part of a cox2-mediated metabolite study , in the appropriate 
 context . collectively , these studies demonstrate 
 that apufas are metabolized similarly to native pufas 
 and represent a viable 
 tool for studying lipid distribution , metabolism , and reactions between 
 lipid metabolites and cellular macromolecules in many physiological 
 and pathophysiological models . while there are some caveats regarding 
 the enzymatic metabolism of aaa , specifically the 
 metabolism of these surrogates by the cyclooxygenase enzymes , the 
 nonenzymatic metabolism is indistinguishable from that of the native 
 lipid species . therefore , apufas can be used as analogues 
 for pufas , especially in cellular disease models involving high amounts 
 of oxidative stress resulting in high levels of lipid oxidation .
lipid and lipid metabolite profiling 
 are important parameters in 
 understanding the pathogenesis of many diseases . 
 alkynylated polyunsaturated 
 fatty acids are potentially useful probes for tracking the fate of 
 fatty acid metabolites . 
 the nonenzymatic and enzymatic oxidations 
 of -alkynyl linoleic acid and -alkynyl arachidonic acid 
 were compared to that of linoleic and arachidonic acid . 
 there was 
 no detectable difference in the primary products of nonenzymatic oxidation , 
 which comprised cis , trans - hydroxy 
 fatty acids . 
 similar hydroxy fatty acid products were formed when 
 -alkynyl linoleic acid and -alkynyl arachidonic acid 
 were reacted with lipoxygenase enzymes that introduce oxygen at different 
 positions in the carbon chains . 
 the rates of oxidation of -alkynylated 
 fatty acids were reduced compared to those of the natural fatty acids . 
 cyclooxygenase-1 and -2 did not oxidize alkynyl linoleic but efficiently 
 oxidized alkynyl arachidonic acid . 
 the products were identified as 
 alkynyl 11-hydroxy - eicosatetraenoic acid , alkynyl 11-hydroxy-8,9-epoxy - eicosatrienoic 
 acid , and alkynyl prostaglandins . 
 this deviation from the metabolic 
 profile of arachidonic acid may limit the utility of alkynyl arachidonic 
 acid in the tracking of cyclooxygenase - based lipid oxidation . 
 the 
 formation of alkynyl 11-hydroxy-8,9-epoxy - eicosatrienoic acid compared 
 to alkynyl prostaglandins suggests that the -alkyne group causes 
 a conformational change in the fatty acid bound to the enzyme , which 
 reduces the efficiency of cyclization of dioxalanyl intermediates 
 to endoperoxide intermediates . 
 overall , -alkynyl linoleic acid 
 and -alkynyl arachidonic acid appear to be metabolically competent 
 surrogates for tracking the fate of polyunsaturated fatty acids when 
 looking at models involving autoxidation and oxidation by lipoxygenases .