Investigation of the Selectivity of Hydrogen Abstraction in the Nonenzymatic Formation of Hydroxyeicosatetraenoic Acids and Leukotrienes by Autoxidation*

The biosynthetic conversions of arachidonic acid to hydroperoxyeicosatetraenoic acids (HPETEs) and the further conversion of leukotriene epoxides are accom-panied by stereoselective hydrogen abstraction from the reaction substrate. Furthermore, this hydrogen removal has always been found to occur in fixed stereo- chemical relationship to carbon-oxygen chiral cen-ter(s) in the substrate or product. We have used ster- eospecifically labeled 10-’H-substrates with 14C internal standard to investigate whether the same relation- ships bear in HPETE and leukotriene formation during autoxidation. After autoxidation of labeled arachidonate, both the 8(R)- and 8Q-HPETE enantiomers (re- solved as diastereomer derivatives) and the 12(RS)- HPETE were observed to retain 41-47% ‘H relative to the starting material. In autoxidative formation of leukotrienes from labeled 15(S)-HPETE the four main leukotrienes, including two derived from 14,15-leu-kotriene A, hydrolysis, were observed to have retained an average of 46% ‘H. Primary and secondary isotope effects were found to accompany these reactions. The results prove that stereorandom hydrogen abstraction occurs in autoxidation and that the hydrogen loss bears no stereochemical relationship to chiral oxygen cen- ter(s) in the HPETE product, (8(R) or 8(S)), or the 15(S)-hydroperoxy The four 8J5-dihydroxy products with triene chromophores were isolated by RP-HPLC (system RP-2) and SP-HPLC (system SP-1). The menthoxy carbonyl derivatives were prepared, isolated by RP-HPLC, and subjected to oxidative ozonolysis. After re-esterification, analysis of the 2-hydroxyheptanoate MeMC fragments by GLC indicated that the original (2-15 hydroxyl group from all four compounds was of the S configuration (<5% R enantiomer). HPLC, UV Spectroscopy, and GC-MS-HPLC was performed with on-line UV detection at 270 or 235 nm. RP-HPLC separations were run on a Bio-Rad ODS 5s column, 250 X 4 mm. Dihydroxy acids were separated using methanol/water/acetic acid, 65:35:0.01 (v/v/v) (system RP-l), their methyl esters using the proportions 70:300.01 (system RP-2), and monohydroxy acids and methyl esters using 75:25:0.01 (system RP-3). SP-HPLC was performed on an Alltech 5 pm silica column, 250 X 4.6 mm, using hexane/isopropanol, 100:3 (v/ v) for dihydroxy methyl esters (system SP-l), the proportions 1000.5 for HETE methyl esters (system SP-2) and 1OO:O.l for the MeMC derivatives of HETEs (system SP-3). To enhance chromatographic resolution, a relatively low flow rate of 0.5 ml/min was used in all experiments. UV spectra were recorded using a Beckman DU-7 instrument using holmium oxide (279.4 nm) for calibration. Reference samples of all the products analyzed were prepared from unlabeled fatty acids and their identity confirmed by HPLC, UV spectroscopy, and GC-MS (9, 12). Mass spectra were obtained on methyl ester trimethylsilyl ether derivatives using a Ribermag R10-10B instrument operated in the electron impact mode. Steric

The biosynthetic conversions of arachidonic acid to hydroperoxyeicosatetraenoic acids (HPETEs) and the further conversion of leukotriene epoxides are accompanied by stereoselective hydrogen abstraction from the reaction substrate. Furthermore, this hydrogen removal has always been found to occur in fixed stereochemical relationship to carbon-oxygen chiral center(s) in the substrate or product. We have used stereospecifically labeled 10-'H-substrates with 14C internal standard to investigate whether the same relationships bear in HPETE and leukotriene formation during autoxidation. After autoxidation of labeled arachidonate, both the 8(R)-and 8Q-HPETE enantiomers (resolved as diastereomer derivatives) and the 12(RS)-HPETE were observed to retain 41-47% 'H relative to the starting material. In autoxidative formation of leukotrienes from labeled 15(S)-HPETE the four main leukotrienes, including two derived from 14,15-leukotriene A, hydrolysis, were observed to have retained an average of 46% 'H. Primary and secondary isotope effects were found to accompany these reactions. The results prove that stereorandom hydrogen abstraction occurs in autoxidation and that the hydrogen loss bears no stereochemical relationship to chiral oxygen center(s) in the HPETE product, (8(R) or 8(S)), or the 15(S)-hydroperoxy substrate. We conclude that the chiral features of the biosynthetic reactions are a reflection of enzymatic control of stereochemistry. Nonetheless, the findings of primary and secondary isotope effects in autoxidation which are similar to those observed in the analogous biosynthetic reactions suggests that, except for stereochemical control, the autoxidative and enzymatic reactions may be mechanistically similar.
Autoxidation of polyunsaturated lipids was definitively characterized as a free radical reaction by kinetic and thermodynamic studies in the 1940s and early 1950s (reviewed in Ref. 1). When lipoxygenase enzymes were identified, their reaction mechanism was likened to the process of autoxidation, although important differences were also recognized (2). Typically, a lipoxygenase will oxygenate in only one position and in one stereospecific configuration on a fatty acid sub-strate, whereas a mixture of hydroperoxides are formed during autoxidation. Currently, there are some aspects of the lipoxygenase reaction which have been studied in more detail than the process of autoxidation. Using substrates labeled with one tritium atom on a critical methylene unit, it has been established that lipoxygenases effect a stereoselective hydrogen abstraction and add oxygen onto the opposite face of the substrate molecule (2, 3). This antarafacial relationship between hydrogen abstraction and oxygenation is now a well recognized feature of the lipoxygenase reaction (2-10). The first part of the studies described in this report was designed to elucidate whether the same relationship might also hold true in autoxidation.
During the propagation phase of autoxidation, the kinetics are such that the successful abstraction of hydrogen is almost inevitably followed by oxygenation (1). Because hydrogen abstraction and oxygenation always accompany one another, the possibility exists that to some extent the two reactions are interdependent. In other words, the propagation of autoxidation might have some of the elements of a concerted type of reaction mechanism in which oxygen participates in the reaction before hydrogen abstraction is complete. In this way oxygen might lower the activation energy of the rate-limiting step of hydrogen abstraction and thus facilitate the overall event. Clearly, autoxidation gives rise to racemic products. However, there could be an antarafacial relationship between hydrogen abstraction and oxygenation in the formation of each individual hydroperoxide enantiomer. Conceivably, the close proximity of oxygen on one side of the substrate in such a concerted reaction could influence and direct stereospecific hydrogen abstraction from the other face of the molecule. Random access of oxygen to the substrate would give the racemic mixtures always observed during autoxidation, but each individual enantiomer might demonstrate the same antarafacial relationship found in hydrogen removal in the lipoxygenase reaction. We have examined the stereochemical features of nonenzymatic oxygenation using arachidonic acid stereospecifically labeled with one tritium atom in a prochiral position on the methylene group at carbon 10 of arachidonic acid (Scheme 1). In autoxidation, hydrogen or tritium is lost from carbon 10 in the formation of four hydroperoxide products, namely 8(R)-, 8  products would retain tritium (Scheme 1, right side). If there was no fixed relationship then each of the four products would be radiolabeled, nominally at half the original activity, assuming a 50:50 chance of hydrogen or tritium abstraction. The latter alternative, in which there is no fixed relationship (Scheme 1, left side), would be compatible either with a concerted reaction without fixed chirality or with a distinct step of hydrogen abstraction followed by racemic addition of oxygen.
Recently, the stereochemical aspects of hydrogen abstraction have been extended to the study of leukotriene biosynthesis. Leukocytes affect dehydration of the lipoxygenase product 5(S)-HPETE to the unstable 5,6-epoxide leukotriene & (LT&) (11). Platelets and leukocytes can also transform 15(S)-HPETE to 14,15-LT& (9,12). These reactions are initiated by stereoselective hydrogen abstraction, and in both cases there is an identical and fixed relationship in the conformations of the hydroperoxy group of the substrate and the hydrogen removed during the reaction (Scheme 2). This fixed relationship has an important implication with regard to the biosynthesis of leukotrienes. Until it is established that different relationships bear in the biological and nonenzymatic syntheses of leukotriene epoxides, there remains the possibility that the biosynthetic reactions are themselves nonenzymatic. Because leukotriene biosynthesis begins with a chkal substrate (5(S)-or 15(S)-HPETE), the finding of a fixed stereochemical relationship of hydrogen abstraction and hydroperoxy group could merely reflect chirality predetermined in the structure of the substrate. This concern also has been stated recently by Hamberg (13). To resolve this latter question, we also have investigated the stereoselectivity of hydrogen abstraction in the nonenzymatic conversion of 15(S)-HPETE to leukotrienes.
Unlabeled arachidonic and dihomo-y-linolenic acids were supplied by Nu-Check Prep Inc. Soybean lipoxygenase Type IV (Sigma) was used to prepare 15(S)-HPETE from arachidonic acid; the product was purified by RP-HPLC (system RP-3) prior to use. a-Tocopherol (Sigma) was quantified in ethanol (Azo" = 292 nm, = 75.8 (15)) and used without further purification. Sodium borohydride was supplied by Fisher and triphenylphosphine by Eastman. (-)-Menthyl chloroformate was obtained from Regis Chemical Co. (Morton Grove, IL). Solvents were distilled-in-glass grade from Burdick and Jackson.

Autoxidation Experiments
Methyl Arachidonute-Autoxidation was conducted by mixing 200 pg of [3-"C, lO-~s-~HIarachidonate methyl ester with 10% (w/w) atocopherol in ethanol, followed by evaporation to dryness in a 5-ml Reactivial (Pierce) and incubation in the dark under an oxygen atmosphere at 37 "C for 18 h. Under these conditions, about 15-20% conversion to HPETEs was found to occur, as judged by the appearance of a prominent diene chromophore on UV analysis. A small amount of triene was evident, indicative of secondary oxygenations. The presence of mixed chromophores (a-tocopherol, HPETEs, and diHPETE trienes) precluded a precise determination of the extent of reaction. Samples were reduced with triphenylphosphine in methanol (100 pl of a 1 mg/ml solution, 15 min at room temperature), and the resulting HETE methyl esters plus unreacted arachidonate were then separated by SP-HPLC (system SP-2). The partially resolved peaks of 8-and 9-HETE methyl esters were collected together, converted to the menthoxy carbonyl (MC) derivatives, repurified by RP-HPLC, and then resolved by SP-HPLC (system SP-3) and counted. The other HETE methyl esters were either analyzed by SP-HPLC as the by SP-HPLC (system SP-2) and RP-HPLC (system RP-3) and MeMC derivatives in essentially the same way, or they were repurified counted. The unreacted methyl arachidonate was repurified by RP-HPLC, and an aliquot was counted. Arachidonic Acid-When batches of stereospecifically labeled fatty acids were isolated after biosynthesis by the fungus S. parasitica, the back shoulders of the arachidonic acid peaks on RP-HPLC were contaminated with linoleic acid. One such mixture of linoleic acid and [3-"C, lO-~s-~HIarachidonic acid (1.1 mg of arachidonic acid by GLC analysis) was kept at -20 "C under air for 4 months in the RP-HPLC solvent. At this stage, UV analysis in methanol indicated a conjugated diene chromophore was present, corresponding to about 45 pg of hydroperoxy fatty acids. The sample was reduced with sodium borohydride, and the resulting hydroxy fatty acids were separated by RP-HPLC (system RP-3). Approximately 300 pg of unchanged arachidonic acid (27% of original) was recovered in later fractions. The RP-HPLC peaks corresponding to the 11-HETE and the 8-plus 12-HETEs were pooled and resolved from each other by SP-HPLC of the methyl ester derivatives (system SP-2). The MC derivatives of the 8(RS)and 11(RS)-HETEs were prepared, and the R and S diastereomers were subsequently resolved by SP-HPLC (system SP-3).
lS(S)-HPETE Methyl Ester-Autoxidation of stereospecifically labeled (15(S)-HPETE methyl ester prepared from [3-"C, ~O-LS-~H] arachidonic acid was conducted with hemoglobin as catalyst (16). A solution of hemoglobin (3 mg) in 10 ml of pH 7 phosphate buffer was warmed to 37 "C and then 300 pg of the 15(S)-HPETE methyl ester was added in 6 pl of ethanol and the incubation continued for 5 min. The sample was then acidified and extracted with diethyl ether. The ethereal extract was subsequently chromatographed on RP-HPLC (system RP-Z), and the four major 8J5-dihydroxy compounds containing triene chromophores were isolated. After recording the UV On the Autoxidative Formation of HPETEs and Leukotrienes spectrum of each product, they were individually re-purified by SP-HPLC (see "Results" and Fig. 2) and counted.
Controls for Hydroperoxide Epimerization during Autoxidation-The degree of epimerization of the 15(S)-hydroperoxy group of 15(S)-HPETE methyl ester (50 pg) was studied in mixtures containing methyl dihomo-y-linolenate (300 pg) as the major component undergoing autoxidation. This allowed subsequent isolation of the remaining 15(S)-HPETE methyl ester free from 20.3~6 hydroperoxide products. Autoxidation was allowed to proceed in a dry film under oxygen (37 "C, *a-tocopherol, 10% (w/w)) until UV analysis indicated 10-20% oxygenation of 20.3~6. The remaining 15-HPETE methyl ester was isolated by RP-HPLC, reduced with triphenylphosphine, and purified as 15-HETE methyl ester by SP-HPLC (system SP-2). Following conversion to the MC derivative, oxidative ozonolysis, and re-esterification, the configuration of the original C-15 hydroxyl group was determined by GLC analysis of the MC derivative of 2-hydroxyheptanoate methyl ester (17). When autoxidation was conducted in the presence of a-tocopherol, there was no detectable epimerization (<3% R enantiomer). In the absence of a-tocopherol, a peak corresponding to 5% R enantiomer was detected. This apparent, small degree of epimerization of 15(S)-HPETE was not investigated further.
To check for possible epimerization in the autoxidation of 15(S)-HPETE methyl ester to 8,15-diH(P)ETE products, 1 mg of unlabeled 15(S)-HPETE methyl ester was autoxidized with hemoglobin under the same conditions used for the stereospecifically labeled material. The four 8J5-dihydroxy products with triene chromophores were isolated by RP-HPLC (system RP-2) and SP-HPLC (system SP-1). The menthoxy carbonyl derivatives were prepared, isolated by RP-HPLC, and subjected to oxidative ozonolysis. After re-esterification, analysis of the 2-hydroxyheptanoate MeMC fragments by GLC indicated that the original (2-15 hydroxyl group from all four compounds was of the S configuration (<5% R enantiomer).
Reference samples of all the products analyzed were prepared from unlabeled fatty acids and their identity confirmed by HPLC, UV spectroscopy, and GC-MS (9,12). Mass spectra were obtained on methyl ester trimethylsilyl ether derivatives using a Ribermag R10-10B instrument operated in the electron impact mode.
Steric Analysis of Alcohols-The GLC method of Hamberg (17) was conducted as described previously (12). For HPLC analysis of Rand S-HETE enantiomers, the MC derivative of the HETE methyl ester was prepared (17) and purified by RP-HPLC (Bio-Rad ODSdS column, solvent system methanol/water, 1003 (v/v), retention volume -15 ml). In this system the MeMC diastereomers were not measurably resolved. Care was taken to collect the whole chromatographic peak in order not to alter the proportions of the diastereomers. Samples were then analyzed by SP-HPLC (system SP-3, see Results and Fig. 1). Liquid Scintillation Counting-A Searle Mark I11 instrument was used with a 3H/14C ratio program for cross-channel correction. All samples were evaporated to dryness in new glass scintillation vials (Kimble, Toledo, OH), prior to counting in ACS scintillation fluid (Amersham Corp). Results are expressed as counts/min or ratios of counts/min. For high level samples (e.g. Fig. 1 and Table I, A and B) counting times were at least 100 min/vial. This corresponds to background values of 2,000 counts 3H and 600 counts "C and values of at least 70,000 counts of 3H and "C for a single HETE MeMC diastereomer. For low level samples (Table IC and Fig. 2) counting times were at least 500 min/vial. This gave background values of about 10,000 counts 3H and 3000 counts 14C, with vials from chromatographic peaks having 2-5 times background counts.

RESULTS
Autoxidative Formation of HPETEs-Tritium loss from the 10-carbon of methyl [ lO-L~-~H]arachidonate (admixed with methyl [3-14C]arachidonate as internal standard) was examined after autoxidative conversion to 8(R,S)-HPETE and other HPETE products. a-Tocopherol, included in the autoxidation mixture in relatively large amounts, had little effect on the course of the initial oxygenation, but had a marked salutatory effect on minimizing the decomposition of the resulting hydroperoxides (18). The racemic HPETE products were reduced to HETEs and purified. This was followed in some cases by preparation of the MC derivative, to allow chromatographic resolution of the Rand S-HETE enantiomers. The MC diastereomers of 8(R)and 8(S)-HETE, following resolution by SP-HPLC, Fig. 1, were each found to contain 42% of the original tritium content of the methyl arachidonate. This result is compatible with stereorandom tritium loss from the 10-carbon in the formation of the 8(R)-and 8(S)hydroperoxides. The tritium retention is less than 50% due to a secondary isotope effect which slightly slows removal of 10-DR-protium when 3H occupies the geminal 10-Ls position on carbon 10. Results from the corresponding experiment performed with [ lO-~~-~H]arachidonate support this conclusion (see below).
The 9(R,S)-HETE MeMC diastereomers contained 105.9% of the original tritium content of the methyl arachidonate. Virtually the same tritium enrichment was found in 11-HETE, analyzed as the MeMC diastereomers (Table IA). Notably, the methyl arachidonate recovered from the reaction was even further tritium enriched (Table IA). These results are compatible with a primary isotope effect which retards removal of the 10-Ls-tritium atom from the arachidonate in the formation of 8-and 12-HETEs. Consequently, the pool of unreacted arachidonate becomes tritium-enriched. The enrichments found in the 9-and 11-HETEs reflect the average 3H content of the arachidonate (-104.2%). Of additional interest was the finding that the 5-HETE MeMC diastereomers were very highly enriched in tritium (Table IA). This is due to a primary isotope effect involving secondary oxygenation reactions centered on the 10-carbon, given 5,12-dihydroperoxide products.
An autoxidation of methyl [3-l4C, lo-~~-~H]arachidonate was conducted in similar fashion to verify the above results. The results (Table IB) completely support the findings from the pro-S 3H experiment. The 8and 12-HETEs contained slightly less than 50% of the original tritium, owing to the secondary isotope effect centered on the 10-carbon. The 9and 11-HETEs were enriched to an extent which reflects the average composition of the arachidonate during the reaction. The final arachidonate was correspondingly further enriched. Both the 5and 15-HETEs were highly tritium-enriched due to the primary isotope effect on secondary oxygenations involving the tritium/hydrogen at the 10-carbon.
We also analyzed the tritium retentions from an additional autoxidation experiment conducted with [lO-~s-~HIarachidonic acid in the absence of a-tocopherol. The yields of the mono-H(P)ETEs were considerably lower in this case because the hydroperoxides were free to undergo side reactions. In spite of this constraint on precise 3H/14C determinations, the results were in good agreement with the a-tocopherol autoxidations. As expected, the 11-HETE MeMC diastereomers retained the 1 0 -~, -~H label while there was stereorandom 3H loss in the formation of the 8-HETE McMC diastereomers (Table IC).
Hemoglobin-catalyzed Autoxidation of 15(S)-HPETE to Leukotrienes-Four 8,15-dihydroxy products were analyzed    from this reaction, each of which must lose a hydrogen from the 10-carbon of 15(S)-HPETE. Two largely arise from conversion of 15(S)-HPETE to 14,15-LTA4, followed by hydrolysis of the epoxide to two 8,15-dihydroxy diastereomers racemic at carbon 8. These products contain an all-trans-conjugated triene. The other two are 8,15-dihydroxy diastereomers which contain a trans,cis,trans-conjugated triene. This double bond configuration is that expected from an oxygenation reaction at carbon 8. Although this is the mechanism of formation of these compounds in platelets and leukocytes (9), analysis of incubations of 15(S)-HPETE with hemoglobin has indicated incorporation of "0 from HZ"0 at the 8-carbon as an additional route of formation of these trans,cis,trans-conjugated triene products (16). We have previously prepared these compounds in milligram amounts and characterized their structures by HPLC, UV, GC-MS, NMR, and steric analysis of alcohols (9,12). Several trial experiments were conducted with unlabeled 15(S)-HPETE autoxidized with On the Autoxidative Formation of HPETEs and Leulzotrienes hemoglobin, and the structures reported (16) for these for major 8,15-dihydroxy products were confirmed by RP-HPLC, SP-HPLC, UV, GC-MS, and steric analysis comparisons with our standards.
Three hundred micrograms of stereospecifically labeled 15(S)-HPETE methyl ester prepared from [lO-~s-'H]arachidonic acid mixed with [3-14C]arachidonic acid were autoxidized in the presence of hemoglobin. Following ether extraction and purification of the 8,15-dihydroxy compounds by RP-HPLC and SP-HPLC, the 3H/14C ratios indicated in Fig.  2 were obtained. The results from the four 8,15-dihydroxy compounds are in general agreement and in distinct contrast to the corresponding values previously determined for these compounds in platelets and leukocytes. The present results indicate that between 38-55% of the tritium in the 15(S)-HPETE substrate is retained in the products formed by autoxidation, compared with only 1-4% retention of the pro-S-'H label in the biosynthetic reactions (9). Thus, the biosyn- thesis is stereoselective whereas autoxidation is stereorandom.
The yield of these products was low (-0.2% each), and therefore, the determination of precise 'H/14C ratios was adversely affected by counting background. Nevertheless, there are strong grounds in support of the validity of the results. Close inspection of the 3H and I4C profiles in Fig. 2 reveals that, in all but the third peak, the "C counts elute very slightly ahead of the 3H peaks. This is caused by the very slightly greater polarity of the 'H-labeled molecules. The same effect is apparent when larger amounts are chromatographed at high resolution on HPLC. As an example, the early elution of the 14C-labeled 8(R)and 8(S)-HETE MeMC diastereomers is evident in Fig. 1. It can also be seen in Fig. 1 that the 3H and 14C profiles of the 9(R,S)-HETE MeMC diastereomers co-chromatograph. Close scrutiny of the chromatographic behavior of the ll-HETE MeMC diastereomers on SP-HPLC reveals that the 11(R)-diastereomer shows the separation of 'H and "C whereas the ll(S)-diastereomer does not (not shown). These details provide a powerful argument in support of the validity of the 'H and 14C quantitations obtained with the 8,154ihydroxy compounds. They refute the possibility that the 3H and 14C peaks are artifacts, despite the very low levels of counts over background. The fact that background readings (chromatographic base-lines) differed on the four runs in Fig. 2 adds an unpredictable element of "noise" which may wholly or partially account for the apparent differences in 'H retention determined for each compound.
The 15(S)-HETE recovered from the hemoglobin autoxidation experiment showed a 12.5% increase in 'H/14C ratio compared with the 15(S)-HPETE starting material. This is the result expected from the primary isotope effect retarding abstraction of 3H from the substrate, as noted above in the autoxidations which formed 8-and 12-HPETEs from arachidonate. DISCUSSION This study was conducted to determine whether the stereoselective hydrogen abstractions which accompany the biosynthesis of HETEs and leukotrienes are also a part of the autoxidative formation of these compounds. It is evident from our results that the hydrogen abstractions in autoxidation are stereorandom and, furthermore, that there is no chiral relationship between hydrogen removal and newly formed or preexisting centers of chirality in the products. It is hardly surprising that hydrogen abstraction in autoxidation is stereorandom. The important new result is that the removal of one particular hydrogen does not influence (and/or is not influenced by) other chiral features of the reaction.
Substrates labeled with one tritium atom on the 10-Ls-(pros) or lO-DR-(prO-R) positions were used to study the stereoselectivity of HPETE and leukotriene formation during autoxidation. In the first set of experiments, we analyzed derivatives of the 8(R)-and 8(S)-HPETE enantiomers formed from [3-14C,10-~S-3H]-and [3-'4C,10-~R-3H]arachidonate. In principle, analysis of the 12-HPETE enantiomers could also be used to investigate the hydrogen abstractions from the 10carbon; however, the 12(R)-and 12(S)-HETE enantiomers could not be resolved by HPLC of the MeMC derivatives, thus precluding this alternative. We should also point out that control experiments were conducted with unlabeled substrates to check for possible racemization of hydroxy or hydroperoxy groups after their initial formation. Racemization was undetectable (<5%, see "Experimental Procedures"). The hemoglobin-catalyzed autoxidation of 15(S)-HPETE was used to study the formation of 14,15-LTA, and 8(R),15(S)-

TABLE I1
Tritium retention at the 10-carban in tramformutwns to lipoxygenuse and leukotriene products ND, not determined. D and L assignments refer to the configuration in arachidonic acid.