Oxidation of 5,8,11,14,17-eicosapentaenoic acid by hepatic and renal microsomes

Biochimica et Biophysica Acta, 966 (1988) 133-149

133

Elsevier BBA 22938

Oxidation of 5,8,11,14,17-eicosapentaenoic acid by hepatic and renal microsomes Mike VanRollins

a, Peter D. Frade b and Oscar A. Carretero

a

a Hypertension Research and b Pharmacology Divisions, Henry Ford Hospital, Detroit, M I (U.S.A.)

(Received 17 November 1987) (Revised manuscript received 21 January 1988)

Key words: Eicosapentaenoicacid; Docosahexaenoicacid; (n - 3) Fatty acid; Fish oil; Cytochrome P-450 Liver and kidney microsomes were isolated from rats raised on high-fat diets. In terms of energy, the high-fat diets contained 4% vegetable and 40% fish, vegetable or coconut oils. Each microsomal preparation was fortified with 1 m M N A D P H and incubated with 5,8,11,14,17-eicosapentaenoic acid (20:5(n - 3)). The number of metabolites formed was assessed by reverse-phase high-performance liquid chromatography (HPLC). To identify the major metabolites, large-scale incubations were done with 2 0 : 5 ( n - 3) and microsomes from phenobarbital-treated rats. After extracts from the phenobarbital and dietary studies were combined, individual products were isolated by reverse- and normal-phase H P L C . The metabolites were identified by mass spectrometry, by chromatographic properties, and by comparing their retention times and mass spectra with those of chemically synthesized standards. For liver microsomes, the major metabolites were: 17,18-, 14,15-, 11,12- and 8,9-dihydroxyeicosatetraenoic acids, 20-hydroxyeicosapentaenoic acid, and 19-hydroxyeicosatetraenoic acid. For renal microsomes, the major metabolites were 20-hydroxyeicosapentaenoic and 19-hydroxypentaenoic acids. Because formation of these metabolites required N A D P H and was enhanced by phenobarbital pretreatment, 2 0 : 5 ( n - 3) appears to be oxidized by cytochrome P-450 monooxygenases. Based on reverse-phase high performance liquid chromatograms, all three high-fat diets may produce the same types of monooxygenase metabolites from 2 0 : 5 ( n - 3). It remains unknown whether fish-oil diets induce the synthesis of monooxygenases to oxidize n - 3 fatty acids, because these preliminary studies involved only two animals per dietary group.

Introduction Abbreviations: n : d ( n - a), 'n' and ' d ' represent the number of carbons and double bonds, respectively, the locant (upper) of the terminal double bond being (n - a) carbons from the carboxyl of the fatty acid; ODS, octadecasilyl; RP- and NPHPLC, reverse-phase and normal-phase high performance liquid chromatography; ECL, equivalent chain length; GC-MS, gas chromatography-massspectrometry. Correspondence: M. VanRollins, Hypertension Research Division, Henry Ford Hospital, 2799 W. Grand Boulevard, Detroit, MI 48202, U.S.A.

Diets rich in saltwater fish appear to reduce the incidence of heart attack and stroke. These diets are accompanied by hematologic changes: increases in bleeding time and reductions in blood viscosity, platelet numbers, platelet responsiveness to aggregating agents, and plasma-lipid levels [1,2]. Similar hematologic changes also occur with diets supplemented with fish oils rich in the n - 3 fatty

0304-4165/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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acids docosahexaenoic acid ( 2 2 : 6 ( n - 3)) and eicosapentaenoic acid (20:5(n - 3)). Thus, the n 3 fatty acids may be responsible for the antithrombotic effects of fish diets; however, the mechanisms by which 22:6(n - 3) and 20:5(n - 3) produce these effects remain largely unknown. It is likely that some of the anti-thrombotic effects of fish oil diets are due to oxidative metabolites of n - 3 fatty acids. Two established routes for the oxidation of polyunsaturated fatty acids involve cyclooxygenase and lipoxygenase enzymes. Like arachidonic acid (20:4(n - 6)), 20:5(n - 3) is oxidized by the cyclooxygenase system to prostaglandin, thromboxane, and prostacyclin products [1]. Also like 2 0 : 4 ( n - 6 ) , 2 0 : 5 ( n - 3) and 2 2 : 6 ( n - 3) are oxidized by lipoxygenases. However, the resulting oxidative products of n - 3 fatty acids are usually much less potent than the 20:4(n - 6) homologs [1,3]. Based primarily on 2 0 : 4 ( n - 6) oxidations, recent studies suggest an alternative pathway for the oxidation of polyunsaturated fatty acids [4,5]. Microsomal cytochrome P-450 monooxygenases oxidize 20:4(n - 6) to n-hydroxy, (n - 1)-hydroxy, and optically active epoxide derivatives. In turn, the epoxides are rapidly and stereospecifically hydrolyzed to vicinal diols by cytosolic and microsomal hydrolases [6,7]. Even though the hydrolases show high activity in vitro, high concentrations of epoxides occur in vivo, for instance, up to 2 0 / ~ g / g wet weight of rat liver or rabbit kidney [8]. The presence of significant epoxide concentrations in tissues may alter hemostasis because epoxides can dilate blood vessels [9] a n d / o r inhibit platelets from aggregating [10]. As a minor pathway, cytochrome P-450 monooxygenases also oxidize 2 0 : 4 ( n - 6) to 'lipoxygenase-like' metabolites, i.e., to regioisomeric monools, each containing a conjugated diene. The structures of lipoxygenase-like metabolites appear to differ from those of lipoxygenase products only in their chiral properties [11]. Nevertheless, lipoxygenase-like metabolites appear to have distinctly different biological actions when compared to those of the lipoxygenase enantiomers. In contrast to the detailed metabolic information available for 2 0 : 4 ( n - 6), relatively little is known about cytochrome P-450 metabolism of n - 3 long-chain fatty acids. We recently showed

that docosahexaenoic acid, one of the two major n - 3 fatty acids present in fish oils, is oxidized by hepatic cytochrome P-450 monooxygenases [12]. As with 2 0 : 4 ( n - 6 ) , 2 2 : 6 ( n - 3) is n-hydroxylated, ( n - 1)-hydroxylated or epoxygenated. Like 2 0 : 4 ( n - 6 ) , 2 2 : 6 ( n - 3 ) is also metabolized to small amounts of lipoxygenase-like metabolites. It is unknown whether 2 0 : 5 ( n - 3), the other major n - 3 fatty acid in fish oils, is similarly oxidized by cytochrome P - 450 monooxygenases. We now report that homologs of the 22:6(n - 3) metabolites are formed from 20:5(n - 3) and microsomes fortified with NADPH. The identities of the 20:5(n - 3) metabolites were determined from electron impact mass spectra, and by matching the resulting spectra with those generated from chemically synthesized standards. In these studies, microsomes were prepared from liver and kidney. Each organ, normally high in cytochrome P-450 activity [15], was isolated from rats on high-fat diets or treated with phenobarbital. High-fat diets, particularly those rich in fish oils, increase the synthesis of cytochrome P-450 monooxygenases [13,14]. Phenobarbital is even more effective than high-fat diets in inducing the synthesis of cytochrome P-450 monooxygenases [15]. In the present study, the same 2 0 : 5 ( n - 3) metabolites appeared to be formed by the different microsomal preparations, as judged from reverse-phase (RP)HPLC chromatograms. These results suggest that the chronic ingestion of high amounts of fish oil does not induce unique monooxygenases for the oxidation of 20:5(n - 3).

Experimental procedures Composition of high-fat diets Each diet contained the same amount of oil supplement, 18% by weight (40% by energy). However, they differed in the type of oil supplement: hydrogenated coconut oil (ICN), sunflower oil (70% 18:2(n - 6)) or MaxEPA (a fish oil concentrate containing 17% 2 0 : 5 ( n - 3 ) and 11% 2 2 : 6 ( n - 3), British Cod Liver Oils, Ltd., Hull, U.K.). To minimize autoxidation, the antioxidant 2,6-di-tert-butyl-p-cresol (Aldrich Chemical Co., Milwaukee, WI) was mixed with each of these oils (0.02%, w/w). All the diets also contained 26.26 (26)% vitamin-free casein (Research Organics,

135 Cleveland, OH), 30.78 (30)% dextrin powder (American Maize, Hammond, IN), 2 (4)% sunflower oil (Sunbrite, Hunt-Wesson Foods, Memphis, TN), and 15.80% non-nutritive bulk fiber (Alphacel, ICN). In addition, all the diets contained vitamin and mineral supplements: OLmethionine, 0.32%; choline chloride, 0.21%; AIN76A vitamin mixture (ICN Biochemicals, Cleveland, OH), 1.26%; AIN-76 mineral mixture (ICN), 5.26%; and DL-a-tocopherol acetate (250 IU/g), 0.10%. To minimize autooxidation during storage, diet batches were freshly prepared every 3 weeks. During this 3 week period, individual portions (25 g) were stored in air-tight containers (30 ml) at - 2 0 ° C . Each evening, individual portions were thawed and administered. HPLC materials and solvent preparation HPLC was done with one of two systems. For analytical and semi-preparative HPLC, the pump system consisted of a 50 or 200 ~1 loop injector (7125, Rheodyne, Cotati, CA), a column-temperature controller with solvent-heating module (CH1448, Systec, Inc., Minneapolis, MN), two dual-reciprocating-piston pumps (M6000A, Waters Associates, Milford, MA), a dynamic dual mixer (Beckman, San Ramon, CA), a gradient controller (720, Waters), and two in tandem variable-wavelength detectors (SF783G and SF770 fitted with 12 and 8 ~tl flow cells, respectively, Kratos Analytical Instruments, Ramsey, N J). For microbore analytical HPLC, the pump system consisted of a 60 nl loop injector (C214W, Valco Instruments Co., Houston, TX), a columntemperature controller with solvent-heating module, a dual-syringe pump with controller (MicroPump, Brownlee Labs, Santa Clara, CA), and a variable-wavelength detector (SF783G) fitted with a 2.4/xl flow cell. Organic solvents and H3PO 4 were of HPLC grade, and were purchased from Burdick and Jackson Laboratories, Inc. (Muskegon, MI) or from Fisher Scientific Co. (Pittsburgh, PA). House-deionized water was used after fresh treatment with resins which adsorbed carbonaceous or ionic materials (Nanopure II system fitted with an Organicfree cartridge, Barnstead, Boston, MA), and after a 30 min exposure to ultraviolet light

(Organipure, Barnstead). Just before use, organic solvents and water were vacuum-filtered (0.22 #m GVW, Millipore Corp., Bedford, MA) and mixed in 2000 ml volumes to nearly fill the solvent reservoirs (Omnifit, Cambridge, U.K.). Each premixed solution was sonicated under vacuum for 1 min, and returned to ambient pressure with helium. Solutions were sparged for 5 min, and maintained thereafter under a helium blanket. Solutions prepared in this manner virtually eliminated the water-soluble contaminants seen at 192 nm during gradient steps, allowed reproducible retention times even with small (2.5 or 4.0%) changes in mobile-phase compositions, and minimized drift in absorption at 192 nm due to 02 influx. Substrate preparation Unlabeled 2 0 : 5 ( n - 3) was prepared in mg amounts with greater than 99% purity. In brief, ethyl 20:5(n - 3) (91% pure) was purchased from Nippon Chemical Feed Co. (Hokkaida, Japan). The oil (2-20 #1) was dissolved in 200 /zl CH 3 CN, injected onto a 1 (i.d.) × 25 cm column containing 5 /~m ODS particles (Ultrasphere ODS, Beckman), and developed at 4.7 ml/min and 1 300 psig in C H 3 C N / H 2 0 (70:30, v/v). Ethyl 20:5(n - 3 ) eluted at 48 min, and was visualized at 192 nm, collected, concentrated under vacuum, partitioned into CH2C12, and dried under a N 2 stream. To eliminate methylation artifacts [17], saponification-extraction was done in the following manner. About 30 mg of the RP-HPLC isolate was suspended in 5 ml N a O H / C H 3 O H / H 2 0 (4:90:10, w / v / v ) and stirred under N 2 for 16 h at 30 o C. The saponification mixture was placed on ice, 20 ml ice-cold H20 was added, and the pH was adjusted to 3 with HCOOH. After being neutralized, 2 0 : 5 ( n - 3) was rapidly partitioned into 25 ml hexane precooled to - 2 0 ° C , concentrated under N2, and suspended in 1-2 ml CH3OH. Purity of the product was assessed using microbore RP-HPLC. In this procedure, an aliquot (60 nl) of the 20:5(n - 3) fraction was injected onto a 0.1 (i.d.)× 25 cm column containing 5 /~m ODS particles (ASTEC, ANSPEC, Ann Arbor, MI) and developed (80 # l / m i n ) using C H 3 C N / H 2 0 (65:35, v/v; pH adjusted to 2.2 with H3PO4). A solitary peak at 8.8 min (non-esterified 2 0 : 5 ( n -

136

3)) and no peaks at 19.4 min (methyl 20:5(n - 3) or 26.2 rain (ethyl 2 0 : 5 ( n - 3)) confirmed that saponification was complete and was unaccompanied by methylation artifacts. The final product (over 99% pure) was dried under N2, dissolved in CH3OH/toluene (1:9), sealed under argon in glass ampoules, and stored at - 8 0 ° C . Under these conditions, 2 0 : 5 ( n - 3 ) was stable for over 6 months as judged by microbore RP-HPLC. The radiotracers [5,6,8,9,11,12,14,15,17,18(n)3H]20:5(n - 3) (79 Ci/mmol, > 99% radiopurity) and [ 1 J 4 C ] 2 0 : 5 ( n - 3 ) (56.9 Ci/mol, > 97% radiopurity) were purchased from New England Nuclear (Boston, MA). In all dietary studies, microsomal incubations were done with [3H]20:5(n - 3) diluted with 20:5(n - 3) to a specific activity of 0.4 Ci/mol. In the phenobarbital/vehicle studies, where metabolite structures were to be elucidated by gas chromatography-mass spectrometry (GC-MS), microsomal incubations were done with [14C]20:5(n-3) diluted with 2 0 : 5 ( n - 3 ) to a specific activity of 0.2 Ci/mol. Both fatty acid substrates were converted to sodium salts before being used in incubations [12]. Incubations

In one study, hepatic and renal microsomes were isolated from rats raised on high-fat diets. In brief, three groups (n = 8) of 6-week-old male rats (Wistar, Charles River Laboratories, Wilmington, MA) were fed a high-fat diet ad libitum for 9 weeks. Diet compositions (see above) differed only in their lipid supplements (40 energy %): hydrogenated-coconut, vegetable or fish oil. Profile analysis indicated that overall the three dietary groups had the same growth rates (parallelism) and that there were no detectable differences in the mean weights of the three groups. After being anesthetized with diethyl ether, these animals were killed by exsanguination. As before [12], microsomes were also isolated from livers of rats pretreated with phenobarbital. With such phenobarbital pretreatments, the cytochrome P-450 specific content increases to 4.3 n m o l / m g microsomal protein, about a 2-fold increase over vehicle values [12]. These preparations were used to generate enough products from 2 0 : 5 ( n - 3) for structural identifications. In brief, two groups (n = 2 or 3) of ll0-day-old Wistar rats

were injected intraperitoneally on 4 consecutive days with phenobarbital (20.2 mg dissolved in 2.5 ml trioctanoylglycerol) or vehicle (2.5 ml trioctanoylglycerol). After being deprived of food overnight, the rats were killed by decapitation. Fresh microsomes from the dietary and phenobarbital/vehicle studies were prepared and incubated as before [12]. In brief, livers (and kidneys in the dietary studies) were rapidly excised from two or three animals per group and rinsed in ice-cold saline (0.9 g/dl). All subsequent steps were performed at 4 ° C. The organs were blotted, weighed, minced with scissors, and added to 4 vol. of buffered salt solution (150 mM KC1, 50 mM Tris buffer (pH 7.5)). Tissues were homogenized with eight strokes from a loose-fitting Teflon pestle. Microsomal pellets were isolated at 4 ° C by differential centrifugations: 10 000 × g for 10 min, 1 0 0 0 0 0 x g for 60 min, and 100000Xg for 60 min. Each microsomal pellet was suspended so that 1.0 ml of buffered salt solution contained the equivalence of 0.5 g liver or 1.0 g whole kidney. N A D P H (1 mM) was added to the microsomal suspensions after they were equilibrated at 37 °C for 3 rain. In the dietary studies, 100 /~M [3H]20:5(n-3) (0.22 /~Ci) was added to each microsomal suspension (5 ml) and stirred for 15 rain. In the phenobarbital/vehicle studies, 130 ~tM [ 1 4 C ] 2 0 : 5 ( n - 3) (1.33 #Ci) was incubated with a 50 ml microsomal suspension (300 ml total volume) for 30 min. Reactions were stopped by the addition of 4 vol. of ice-cold ethanol. Metabolite H P L C and isolation

To assess the variety of microsomal metabolites formed after dietary and phenobarbital pretreatments, RP-HPLC was used. In brief, after the microsomal suspensions were centrifuged, the supernatants were concentrated under vacuum and acidified with HCOOH to pH 3. Metabolites were extracted into ethyl acetate as before [12] and resolved by RP-HPLC. A 0.46 (i.d.)× 1.5 cm cartridge guard column (5 /~m ODS particles; New Guard, Brownlee Labs, Santa Clara, CA) and a 0.46 (i.d.) x 25 cm analytical column (7/~m Zorbax-ODS particles, packed by Phenomenex HPLC Technology, Palos Verdes Estates, CA) were used. Metabolites dissolved in 50/~1 CH3OH were injected and developed in C H 3 C N / H z O (33:67,

137 pH 2.2) at 2 ml/min and 2200 psig. For liver metabolites, linear CH3CN/H20 gradients were initiated at 75 min (33-37% over 1.5 min), at 140 min (37-39.5% over 2 min), and at 188 min (39.5-100% over 34 min). For renal metabolites, run times were reduced by initiating gradients at 65 min (vs. 75), 130 min (vs. 140), and at 167 min (vs. 188). Both absorbance (at 192 and 237 nm) and radioactivity were monitored. Effluent fractions (0.8 ml) were collected every 0.4 min, mixed with 5 ml Instagel (Packard Instruments, Downers Grove, IL), and counted for 10 min in a liquid scintillation counter (TriCarb, Packard Instruments). Once individual radiochromatograms were obtained, microsomal extracts f~om phenobarbital and dietary studies were pooled and preparatively chromatographed for product identification by GC-MS. For these studies, pooled extracts equivalent to 300 ml hepatic microsomal suspensions were used - less than 10% of the pooled extracts came from the dietary studies. Extracts were concentrated at room temperature under a N 2 stream, suspended in 30-50 ~1 CH3OH, and chromatographed as described above. Following RP-HPLC, metabolites with absorbance at 192 nm were collected over ice, concentrated under vacuum, and extracted into CHzC12. Possible contaminations from closely eluting substances were minimized by not collecting during the leading and trailing edges of each peak of interest. After solvent evaporation under a N z stream, the RP-HPLC isolates were methylated with ethereal CH/N2, and subjected to normal-phase (NP)-HPLC as before [12]. Each NP-HPLC isolate was stored in CH3OH at - 8 0 °C until GC-MS could be performed.

GC-MS The methylated metabolites, isolated following NP-HPLC, were either directly silylated or catalytically hydrogenated [18] and then silylated. Each derivative was subjected to capillary GC and characterized by electron impact (70 eV) mass spectrometry and by equivalent chain length (ECL) measurements. In brief, separations were achieved with a wall-coated (0.25/~m dimethyl-polysiloxane film; DB-1; J&W, Rancho Cordova, CA), fusedsilica column (0.25 mm (i.d.) × 15 or 60 m) and a gas chromatograph (5 890, Hewlett Packard). After

being dissolved in 50 ~1 CH3CN plus 50 /xl of N-m ethyl-N-trim ethylsilyltrifluoroacetamide (Pierce Chemical Co., Rockford, IL), samples were heated at 60°C for 15 min. After being concentrated under a nitrogen stream and dissolved in isooctane, the silylated derivatives (1 /~1) were injected while the column was positioned outside the chromatograph (on-column injector; J&W). The column, once charged with sample, was plunged into the chromatograph oven. Immediately thereafter, the oven temperature was increased stepwise from 115°C to 230°C. The linear velocity of the mobile phase (helium) was 40 c m / s at 230°C. For MS characterizations, a quadrupole instrument (5970B, Hewlett Packard, Palo Alto, CA) was used. For ECL characterizations, a plot of carbon-number vs. log (retention time) was generated using 22:0, 23:0, 24:0 and 26:0 methyl ester standards. The ECL of metabolites were determined from the interpolated log (retention times).

Synthesis and identification of oicinal diol standards A mixture of cis-5(6)-epoxy-8,11,14,17-20:4, cis-8(9)-epoxy-5,11,14,17-20:4, cis-11(12)-epoxy5,8,14,17-20:4, cis-14(15)-epoxy-5,8,11,17-20:4, and cis-17(18)-epoxy-5,8,11,14-20:4 was produced from methyl 2 0 : 5 ( n - 3) and m-chloroperoxybenzoic acid (VanRollins, M., Frade, P. and Carretero, O.A., unpublished data). In brief, methyl 20:5(n - 3) was dissolved in 1 ml CHzC12 and stirred with 0.1 Eq m-chloroperoxybenzoic acid at 30 °C for 15 rain. Upon removal of CHzCI 2 with a nitrogen stream, the residue was suspended in hexane/isopropanol (100:0.175) and analyzed by NP-HPLC. Most of the regioisomers were baseline-resolved using a 25 × 0.46 i.d. column containing 5 ~tm silica particles (Ultrasil, Beckman) and hexane/isopropanol (100:0.175) flowing at 1 ml/min. Regioisomers had the following NP-HPLC retention times (rain): 14,15- (32.8), 11,12- (35.6), 17,18- (44.6), 8,9- (44.6), and 5,6epoxy-20:4 (61.6). Each component, absorbing at 192 nm, was collected. The coeluting 17,18- and 8,9-epoxy-20:4 regioisomers were baseline-resolved using a 25 x 0.46 i.d. column containing 5 txm ODS particles (Ultremex, Phenomenex) and CH3CN/H20 (56:44) flowing at 2 ml/min. Again, absorption at 192 nm was monitored. Retention

138

times for the 17,18- and 8,9-epoxy-20:4 regioisomers were 49.4 and 58.4 min, respectively. The five isolates ( > 99% pure as judged by NPand RP-HPLC) were analyzed as methyl esters using electron impact GC-MS. All five compounds shared a common ECL (20.9). In contrast, the corresponding hydrogenated derivatives had the following ECL: 17,18- (21.8), 14,15- (21.6), 11,12- (21.6), 8,9- (21.5) and 5,6-epoxy-20:4 (21.5). By MS the five compounds were found to have a molecular weight of 332 and four double bonds; the latter was established from the spectra of hydrogenated derivatives in which there was an 8 Da increase in M + , ( M - H 2 0 ) and ( M OCH3) ions. In addition, the spectra and retention times of four of the hydrogenated compounds matched those of hydrogenated standards derived from 20:4(n - 6). Thus, the epoxide positions were directly established for four of the compounds, and indirectly (by exclusion) for 17,18-epoxy-20:4. Once identified, each epoxide regioisomer was converted to its corresponding vicinal (threo) diol as described [16]. Results and Discussion

RP-HPLC of microsomal metabolites from dietary and phenobarbital studies Rat microsomes from fiver or kidney were fortified with N A D P H , incubated with radiolabeled 2 0 : 5 ( n - 3), and precipitated with ethanol. Metabolites not precipitated in this manner were extracted into ethyl acetate. In the phenobarbital/ vehicle studies, the ethyl acetate extracts contained 86 and 87%, respectively, of the radioactivity added to the pooled hepatic microsomes. In the dietary studies, 78.6 _+ 4.8% (mean _+ S.E.; n = 6) and 85.8 ___3.1% (n = 6) of the added radioactivity were recovered from incubations with hepatic and renal microsomes, respectively. Thus, recoveries in the phenobarbital/vehicle studies are similar to those in the dietary studies. The similar recoveries are somewhat surprising due to the varying amounts of products formed (Tables I and II). Cytochrome P-450 metabolites of polyunsaturated fatty acids appear to be lost during processing because of irreversible binding to proteins or other macromolecules [20].

TABLE I DISTRIBUTION OF RADIOACTIVITY BETWEEN MAJOR GROUPS OF HEPATIC METABOLITES Liver microsomes were isolated from rats treated with phenobarbital or vehicle, or from rats raised on diets supplemented with oils (40 energy %). While fresh, the liver microsomes were fortified with 1 mM N A D P H and incubated for 30 rain (phenobarbital and vehicle studies) or 15 min (dietary studies) with radiolabeled (20: 5(n - 3 ) . After extractive isolation, products were applied to ODS columns for RP-HPLC. Compounds absorbing at 192 nm were collected, and their radioactive content was measured. Radioactivity is shown as percent of total collected counts which represented 92.4±6.7% of the applied radiolabel. Treatment

Metabofitegroup a A B C D 20:5(n - 3) (33-54) (83-94) (94-128) (144-168) (204-213)

Phenobarbital b Vehicle Coconut oil c Vegetable oil Fish oil

25.8 7.2

7.3 7.9

7.9 3.5

4.6 3.9

33.6 65.4

5.7

4.0

2.6

3.8

74.1

3.4 6.8

3.6 3.6

2.4 2.4

3.4 2.2

78.6 73.5

a Group retention times (min) refer to Fig. la. b Values from phenobarbital and vehicle studies represent results of microsomes pooled from two or three rats. ¢ In the dietary studies, each value represents the mean from two rats. Each group value ( A - D ) varied less than ± 19% with two exceptions: group D 3.4 (vegetable oil) and 2.2 (fish oil) varied by + 57% and ± 31%, respectively. Each 20:5(n 3) value varied less than +0.8%.

To assess how many 2 0 : 5 ( n - 3 ) metabolites were formed and how much 2 0 : 5 ( n - 3 ) was oxidized, RP-HPLC was used. In this procedure, both radioactivity and absorbance at 192 and 237 nm were monitored. From liver microsomes, at least 15 radiolabeled metabolites were formed from 2 0 : 5 ( n - 3) (Fig. 1A). Both the metabolites and the unreacted 2 0 : 5 ( n - 3) showed strong absorbance at 192 nm, indicating that the products contained multiple isolated double bonds [21,22]. The absorbance at 192 nm was used to quantitatively isolate products and substrate. By summing the radioactivity associated with the ultravioletabsorbing peaks, the percent oxidation of 20:5(n - 3) was assessed (Table I). Hepatic microsomes in the vehicle or dietary studies oxidized 20:5(n 3) by only 21-35% (Table I). In contrast, hepatic

139 T A B L E II DISTRIBUTION OF RADIOACTIVITY BETWEEN M A J O R G R O U P S OF R E N A L M E T A B O L I T E S Renal microsomes were isolated from rats raised on diets supplemented with oils (40 energy %). While fresh, the renal microsomes were fortified with 1 m M N A D P H and incubated for 15 min with radiolabeled 2 0 : 5 ( n - 3). The products were extractively isolated and subjected to RP-HPLC. Fractions were collected every 0.4 min and counted. Radioactivity is shown as percent of total counts recovered from ODS columns which represents over 77% of the applied radiolabel. Dietary/

Metabolite group a,b

oil supplement

A B C D (n - 3) ¢ (34-50) (82-91) (91-120) (139-165) (181-199)

Coconut 0.2 Vegetable 0.1 Fish 0.3

2.1 1.6 2.8

0.2 0.1 0.2

20:5

0.8 0.4 0.5

95.1 96.0 94.7

a G r o u p retention times (min) refer to Fig. lb. b Each group value ( A - D ) varied less than + 18% in duplicate rats with one exception: the 0.4 value (group D metabolites, vegetable oil study) varied by + 37%. c Each 2 0 : 5 ( n - 3 ) value varied less than +0.5% in duplicate rats.

microsomes from phenobarbital-treated animals oxidized 20:5(n - 3) by 66% (Table I). Thus, more 2 0 : 5 ( n - 3) was oxidized when microsomes were isolated from rats pretreated with phenobarbital, a potent inducer of cytochrome P-450 monooxygenases. Significantly, 20:5(n- 3) was minimally oxidized (< 2%) by hepatic microsomes not fortified with NADPH. Liver microsomes from rats on high-fat diets produced over 15 metabolites which eluted in four clusters: group A (peaks I-IV), group B (peaks V and VI), group C (five peaks) and group D (four peaks) (Figs. la and 2). The same group components appeared to be present in all dietary studies (Fig. 2). Based on the number of components per group and the RP-HPLC properties of homologous groups from 2 0 : 4 ( n - 6) [6,8,20] and 22:6(n - 3 ) [12], the metabolites in groups A - D were assigned the following structures: A, vicinal diols (see below); B, 20- and 19-hydroxy-20:5(n-3) (see below); C, lipoxygenase-like metabolites; and D, epoxides. Concerning the last two group assignments, group C metabolites had high A 2 3 7 / A I 9 2 ratios (Fig. la) and co-migrated with

standards prepared by autoxidation of 20:5(n - 3) (VanRollins, M., unpublished data). In contrast, group D metabolites showed little absorbance at 237 nm (Fig. la), and the RP-HPLC elution pattern was similar to that of epoxide standards. Aside from groups A-D, there were no other major groups of metabolites. The absence of major products eluting before the group A metabolites may mean that the microsomes used in the present studies were uncontaminated with cytosolic epoxide hydrolases [23]. In summary, over 15 metabolites of 20:5(n - 3) were formed by hepatic microsomes from rats fed high-fat diets. The 15 plus products were readily apparent by their absorbance at 192 nm. In all three dietary studies, the major metabolites were epoxygenase (groups A + D), n-hydroxylase (group B), and (n-2)-hydroxylase (group B) products. In all three dietary studies, the minor metabolites were products of the lipoxygenase-like pathway (group C). Most importantly, no metabolites appeared to be unique to one of the diets. Thus, even prolonged exposure to a diet high in n - 3 fatty acids may not result in novel oxidations of these fatty acids. Hepatic microsomes from vehicle-treated rats produced essentially the same metabolite profiles as seen in the dietary studies (Figs. la and 2). In contrast, microsomes from phenobarbital-treated rats produced higher proportions of group A (vicinal diols) and group C (lipoxygenase-like) metabolites (Table I). Likewise, with 2 2 : 6 ( n - 3) and 2 0 : 4 ( n - 6 ) as substrates, microsomes from phenobarbital-treated rats produced high proportions of vicinal diols and lipoxygenase-like metabolites [12,23]. In the present phenobarbital study, compounds I-IV (group A) represented 7.3, 4.7, 2.5 and 2.8% of the recovered radioactivity, respectively. Compared to vehicle and dietary values (Fig. la), phenobarbital treatments had selectively increased the microsomal capacity to form compounds I and II (I > II). Compounds I and II are 17(18)- and 14(15)-dihydroxy-20:4 (see below). Likewise, in studies on 2 0 : 4 ( n - 6) [6,23] and 22:6(n - 3) [12] oxidations, microsomes and purified cytochrome P-450 monooxygenases from phenobarbital-treated animals preferentially epoxygenate toward the 0~ ends of fatty acids. A similar response to phenobarbital in the present study

140

A

B

C

,..i~ p.___.~_----~

D•

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, 0075

002

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300

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'v'hv~'.

.... ........ . ....... ...:

i

A237

L ii!i!i!i

,l[,.,v

iiiiiiii

~iiii! 1(30

CH3CN

(%)

A

150

3,.5 1

3zoN I

,

33.0~11

i

I 75

0

i

I00

J

•

/

I 150

c.,c,

tt3r.o

~

(.~

~33.0 225

MINUTES

A

B

C

D

20 5w3 ~olo~l

oo38 ~00050

=I i

ii!i! 250'

'%,

•

k~l%

xO.5

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ill

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,ton, CH3CN

(%)

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Fig. 1. Reverse-phase chromatogram of metabolites formed by incubating [3H]20:5(n - 3 ) with hepatic (a) or renal (b) microsomes from a rat on a high-fat diet. Microsomes were isolated from the liver or kidneys of a rat raised on a diet supplemented with 40 energy % hydrogenated coconut oil. Upon fortification with 1 mM NADPH, the hepatic and renal microsomes were incubated with [3H]20:5(n- 3) for 15 min at 37° C. After being extractively isolated, the products (86000 cpm aliquots) were resolved on ODS columns using acetonitrile gradients (shown beneath figures); details on HPLC chromatographic conditions are provided in the Experimental procedures section. Column effluents were monitored at 237 (upper tracing) and 192 nm (middle tracing), and were collected in 0.4 min fractions for radioactivity determinations (lower tracing).

141 KIDNEY

LIVER

=-..3-~ A

tSO

125 15¢

6

tu I-

13

25C

z a: UJ a.

t,~ I-Z :~

oO

m

I

o "l

mo

250

~,:~3

C

12 ~.

13

MINUTES

Fig. 2. Radiochromatogramsof 20:5(n- 3) metabolites from hepatic (left) and renal (right) microsomesof rats raised on high-fat diets containing (A) hydrogenatedcoconut, (B) vegetableor (C) fish oils. Experimentaland chromatographicconditions are described in Fig. 1.

provides further evidence that the 2 0 : 5 ( n - 3 ) metabolites were produced by the cytochrome P450 system. The oxidation of 2 0 : 5 ( n - 3) by renal microsomes from rats on high-fat diets was also examined. In these experiments, 20:5(n - 3) was incubated with microsomes equivalent to twice the amount of tissue used in the liver studies. Even so, renal microsomes oxidized only 4.7 + 0.3% (mean _+ S.E.; n = 6) of the added 20:5(n - 3) (Table II). Over the same time period, liver microsomes oxidized 2 0 : 5 ( n - 3 ) by about 25% (Table I). It therefore appears that renal microsomes may be one-tenth as active as liver microsomes in oxidizing 20:5(n - 3). Similar differences in hepatic and renal capacities have been observed when xenobiotics are used as substrates [15]. In all three dietary studies, the principal products of renal microsomes were group B metabolites (2-3% of the added 20:5(n - 3), Table II). In addition, RP-HPLC chromatograms (Fig. 2) sug-

gested that no metabolites were unique to any of the diets. In contrast to liver, group B compounds V and VI were produced in almost equal amounts (Fig. lb). Also in contrast to liver, only small amounts (0.4-0.8%) of group D metabolites and trace amounts (0.1-0.3%) of group A metabolites were formed (Table II). Thus, microsomes from rat kidney formed primarily 20-hydroxy-20:5(n 3) and 19-hydroxy-20:5(n - 3) (see below for compound identifications). To our knowledge, this is the first report on polyunsaturated fatty acid oxidations by NADPH-fortified microsomes from rat kidney. However, in comparable studies with rabbit kidney [19,24,25], 2 0 : 4 ( n - 6) is oxidized primarily to 2 0 - h y d r o x y - 2 0 : 4 ( n - 6) and 19-hyd r o x y - 2 0 : 4 ( n - 6). Thus in both rat and rabbit kidney, the primary microsomal metabolites of polyunsaturated fatty acids seem to be 20-hydroxy and 19-hydroxy derivatives. In summary, the R P - H P L C chromatograms suggested that various microsomal preparations

142 m a y differ q u a n t i t a t i v e l y b u t n o t qualitatively in the way they oxidize 20:5(n - 3).

99% pure. After silylation, c o m p o u n d s I - V I were also f o u n d to be over 99% pure b y capillary GC.

Identification of microsomal metabolites

Mass spectral properties of group A metabolites

Peaks I - I V a n d VI (Fig. l a ) were f o u n d b y N P - H P L C to c o n t a i n only one c o m p o n e n t . I n contrast, peak V c o n t a i n e d a second c o m p o n e n t with a n N P - H P L C r e t e n t i o n time of 30.5 min. I n the microsomal sample used for m e t a b o l i t e identifications, peak area a n d radioactivity of the seco n d c o m p o n e n t were f o u n d to be 20% of that for c o m p o u n d V (retention time 63.4 min, T a b l e III). Because the microsomes used i n this study were pooled, it is u n k n o w n whether the m i n o r c o m p o n e n t was present in all t r e a t m e n t groups. Based o n similar N P - a n d R P - H P L C properties of a 20:4(n - 6 ) h o m o l o g [19,24], the m i n o r c o m p o n e n t was p r o b a b l y 1 9 - o x o - 2 0 : 5 ( n - 3 ) . U n f o r t u n a t e l y , the sample was lost before its structure could be confirmed by MS. R e c h r o m a t o g r a p h i n g c o m p o u n d s I - V I b y N P - H P L C a n d m o n i t o r i n g a b s o r b a n c e at 192 n m indicated that the final isolates were over

Electron i m p a c t mass spectra were generated from trimethylsilyl ether, methyl ester derivatives of group A metabolites. The spectra of the four metabolites (Table IV) shared some high-mass ions: 494 ( M + ) , 479 ( M - C H 3 ) , 463 ( M OCH3), 404 ( M - M e 3 S i O H ) , 373 ( M - ( M e 3 S i O H + O C H 3 ) ) a n d 314 ( M - 2 × Me3SiOH ). The shared high-mass ions suggested a c o m m o n molec-

100-

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TABLE llI

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460

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m/z

CHROMATOGRAPHIC CHARACTERIZATION OF MICROSOMAL PRODUCTS OF 20:5(n - 3 ) Peak "

g ,5.0

o

HPLC retention times (min) RP b

NP ~

36.3 41.7 44.5 50.2 90.3 94.9

67.6 43.5 47.2 62.1 63.4 44.2

131T363

Me3Si010SiMe

GC retention times (ECL) d 100

23.5 (24.5) 22.8 (23.5) 22.6 (23.3) 22.6 (23.2) 22.5 (23.5) 21.7 (22.8)

" Peak assignments are portrayed in Fig. la. b Retention times represent values for underivatized metabolites. Chromatography was done using an ODS column with a CHaCN/H20 (pH 2.2) mobile phase. Gradient conditions are detailed in the Experimental procedures section. c Retention times for methyl ester derivatives were measured. Chromatography was done isocraticaily on a silicic acid column with hexane/isopropanol mixtures: 99:1 (v/v) for peaks I-IV, or 99.5:0.5 (v/v) for peaks V and VI. d Retention times were determined for trimethylsilyl ether, methyl ester derivatives. Numbers in parentheses are values of corresponding hydrogenated derivatives. Chromatography was done isothermally (230 o C) using a non-polar (dimethylpolysiloxane) stationary phase and helium carrier gas.

~ b

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< 50.

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=

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OOCH3

Me~SiOI

Fig. 3. Electron impact (70 eV) mass spectrum of non-hydrogenated compound I (a) and hydrogenated compound 1 (b) a., trimethylsilyl ether, methyl ester derivatives.

143 TABLE IV CHARACTERISTIC F R A G M E N T S (Da) IN MASS SPECTRA OF M I C R O S O M A L METABOLITES OF 20:5(n - 3 ) a Peak No. b

a-Cleavages to -OSi(CH 3) 3 groups

Other high-mass ions

I

363(4.3) c, 334(9.1)T d, 233(32.0), 131(51.1)

494(4.3), 479(0.1), 463(0.6), 404(0.4), 375(0.6), 314(0.5), 282(1.0), 273(4.0) e, 260(1.1), 241(2.2), 143(21.6) e

II

425(1.5), 323(3.0), 294(10.1)T, 273(20.1), 171(24.8)

494(1.0), 479(0.2), 463(0.7), 404(0.8), 373(0.8), 335(12.5) e, 314(0.9), 245(5.1) e, 243(2.5), 233(3.0) e

III

385(1.6), 313(5.0), 283(6.0), 254(8.8)T, 211(3.2), 181(2.6)

494(1.4), 479(0.4), 463(0.4), 404(1.5), 373(0.8), 335(0.4), 295(46.4) e, 263(1.4), 234(1.4), 223(12.8) e, 205(5.6) e

IV

353(2.9), 345(4.1), 251(0.7), 243(25.6), 214(4.8)T, 149(2.2), 141(0.7)

494(1.6), 479(0.3), 463(0.4), 404(0.8), 373(0.7), 335(0.7), 317(0.8), 314(0.8), 263(6.3) e 255(51.2) e, 211(6.2)

V

301(0.4), 103(58.5)

389(4.5), 314(0.9), 273(0.9), 260(1.0), 247(1.4), 234(2.2), 227(1.5), 220(7.2), 213(3.6)

VI

360(0.2)T, 117(28.9)

404(0.2), 389(2.2), 373(0.1), 355(0.2), 314(0.5), 285(0.5), 259(1.8), 246(0.9), 234(1.9), 227(1.0), 220(6.3)

a Electron impact (70 eV) mass spectra were generated from trimethylsilyl ether, methyl ester derivatives. b Compounds were isolated from the peaks labeled in Fig. la. c The base (largest) ion peak for all compounds was rn/z 73. Values in parentheses represent intensities as percentage of the base peak. d ' T ' rearrangement ions reflect a-cleavages to an oxygen heteroatom and follow transfer of the adjacent -Me3Si to the COOCH 3 group [29,30]. Formation of this ion also reflects a-cleavages in that it occurs by a loss of one or two Me3SiOH groups from an a-cleavage fragment listed in the second column.

ular weight of 494. This value is 178 Da greater than 316, the molecular weight of methyl 20:5(n - 3), and suggested that each metabolite possesses four double bonds and two Me3SiO groups. The occurrence of m/z 73 (Me3Si), 75 (MeESiOH), 103 (MeaSiO=-CH2), 129 (CH2=CHCH=OSiMe3) and 147 (MeaSiO=-SiMe2) (Fig. 3a) confirmed the presence of MeaSiO groups in group A metabolites. Thus, the four group A metabolites are dihydroxy 20:4 isomers. Mass spectra were also generated from trimethylsilyl ether, methyl ester derivatives of hydrogenated group A metabolites (Table V). High-mass ions 487 ( M - C H 3 ) , 471 ( M - O C H 3 ) , 397 (M - (CH 3 + Me3SiOH)), 381 (M - - ( O C H 3 + Me3SiOH)), and 291 (M - (OCH 3 + 2 x MeaSiOH)) were present in all four spectra, and indicated a common molecular weight of 502.

Thus, hydrogenation increased the molecular mass by 8 Da (494 ~ 502; Tables IV and V) and demonstrated that four double bonds were present in the original group A metabolites. A molecular weight of 502 is 176 Da greater than 326, the molecular weight of methyl 20:0, and indicated the presence of two Me3SiO moieties. The occurrence of m/z 73, 75, 103, and 147 (Fig. 3b) also confirmed the presence of Me3SiO groups in group A metabolites. In summary, hydrogenation studies reaffirmed that group A metabolites are dihydroxy-20:4 isomers. The electron impact mass spectra of compound I (Fig. 3a) contained ions at 375 ( M - (CH3CH 2 + Me3SiOH)), 363 ( M - CH3CH2CH(OSiMe3) ), 334 ( M - CHaCHECH(OSiMe3)CHOx) *, 273 * ' T ' means that formation of this ion involved prior transfer of a Me3Si moiety to the COOCH 3 group [29,30].

144 TABLE V CHARACTERISTIC FRAGMENTS (Da) IN MASS SPECTRA OF HYDROGENATED METABOLITES OF 20:5(n -3) a Peak No. b

a-Cleavages to

-OSi(CH3) 3

groups

Other high-mass ions

444(5.0) CTd, 371(100.0), 233(1.4),

487(0.4), 471(6.0), 429(1.4), 397(0.4), 383(2.0), 381(0.4), 355(2.9), 339(2.7), 323(1.5), 297(0.5), 291(0.9), 281(1.2) e, 267(1.4), 249(4.9), 231(2.6)

II

431(0.2), 402(6.8)T, 329(100.0), 275(0.8), 173(52.7)

487(0.4), 471(3.4), 413(0.2), 411(0.2), 397(0.4), 387(1.7), 381(0.4), 341(1.4) e, 313(3.1), 291(0.8), 281(1.1), 247(0.7), 238(1.1), 225(1.1)

III

389(0.4), 360(12.7)T, 317(1.6), 287(100.0), 215(52.7), 185(1.4)

487(0.7), 471(3.4), 411(0.3), 397(0.7), 381(0.8), 345(1.7), 317(1.6), 299(0.8) e, 291(0.8), 271(6.6), 227(0.8) e

IV

359(1.5), 318(13.1)T, 257(72.1), 245(100.0), 155(1.4), 143(2.7)

487(0.5), 471(3.3), 411(0.4), 397(0.7), 381(0.8), 331(0.4), 303(0.4), 291(1.1), 241(2.0), 229(6.5), 215(6.1), 213(3.8)

V

103(11.5), 311(0.1)

414(0.2), 399(50.1), 383(3.3), 367(100.0), 341(0.9), 324(1.4), 297(0.7), 292(2.9), 117(0.6)

VI

370(24.1)T, 117(100.0)

399(6.8), 383(3.1), 367(24.2), 341(0.3), 327(1.4), 324(0.4), 313(0.3), 292(2.3), 215(1.5)

a Electron impact (70 eV) mass spectra were generated from microsomal metabolites after the metabolites were methylated, catalytically hydrogenated, and trimethylsilylated. b Compounds were isolated from the peaks labeled in Fig. la. c Values in parenthesis represent intensities as percentages of the base peak. a 'T' rearrangement ions reflect a-cleavages to an oxygen heteroatom and follow transfer of the adjacent Me3Si- to the C O O C H 3 group [29,30]. e Formation of this ion also reflects a-cleavages in that it occurs by a loss of one or two Me3SiOH groups from an a-cleavage fragment listed in the second column.

(363 - Me 3SiOH), 233 ( C H 3CH 2 (CH(OSiMe3)) 2 ) and 131 (CH3CH2CH(OSiMe3)). These ions, reflecting a-cleavages to Me3SiO groups, establish that Me3SiO groups were located at C-17 and C-18. Thus the structure of underivatized comp o u n d I is 17,18-dihydroxy-20:4. The mass spectrum of h y d r o g e n a t e d c o m p o u n d I (Fig. 3b) revealed ions at 444 ( M C H 3 C H 2 C H O T ) , 429 (444-CH3), 383 ( M ( C H 3 C H 2 + Me3SiOH)), 371 ( M - C H 3 C H 2 C H (OSiMe3)), 281 ( 3 7 1 - M e 3 S i O H ) , 233 ( M (CHz)asCOOCH3) a n d 131 ( C H 3 C H 2 C H (OSiMe3)). These ions confirmed the presence of a Me3SiO group at C-17 and C-18. H y d r o g e n a t i o n did not shift the a-cleavage fragments 131 and 233 to higher mass (Figs. 3a and b). Therefore, there were no double b o n d s between the methyl terminus (C-20) and the vicinal Me3SiO groups

(C-17 and C-18). In contrast, hydrogenation resulted in an 8 D a shift for the a-cleavage fragment 363 (Figs. 3a and b). Therefore, there were four double b o n d s between the vicinal Me3SiO and the C O O C H 3 groups. The mass spectral data suggested that the d o u b l e - b o n d positions remained intact, and c o m p o u n d I is identified as 17,18-dihydroxy-5,8,11,14-eicosatetraenoic acid. The mass spectrum of c o m p o u n d II (Table IV) showed ions at m/z 425 ( M - C H 3 C H 2 C H = C H C H 2 ) , 335 ( 4 2 5 - Me3SiOH), 323 ( M - C H 3 C H 2 C H = C H C H 2 C H ( O S i M e 3 ) ) , 294 ( M - C H 3 C H 2 C H = C H C H 2 C H ( O S i M e 3 ) C H O T ) , 273 ( C H 3 C H z C H = C H C H 2 ( C H ( O S i M e 3 ) ) 2 ) , 245 ( 4 2 5 - 2 × Me3SiOH ), 233 ( 3 2 3 - M e 3 S i O H ) , and 171 ( C H 3 C H z C H = C H C H a C H ( O S i M e 3)). These ions established the presence of Me3SiO groups at C-14 and C-15. The structure of underivatized com-

145 pound II is thus 14,15-dihydroxy-20:4. The spectrum of hydrogenated compound II (Table V) revealed ions at 431 ( M - CH3(CH2)4), 402 ( M - C H 3 ( C H 2 ) 4 C H O T ) , 387 ( 4 0 2 - C H 3 ) , 341 (431 - Me3SiOH), 329 ( M - CH3(CH2)aCH (OSiMe3)), 275 ( M - (CH2)12COOCH3), and 173 (CH3(CH2)4CH(OSiMe3)). Together, these ions confirmed the location of hydroxy groups at C-14 and C-15. Hydrogenation shifted by 2 Da the a-cleavage fragments 171 and 273 (Tables IV and V). The 2 Da shift indicates that there is a single double bond between the methyl terminus and the vicinal Me3SiO groups. Hydrogenation also increased by 6 Da the a-cleavage fragments 425 and 323 (Tables IV and V). This 6 Da shift indicated that there are three double bonds between the Me3SiO and COOCH 3 groups. Thus, hydrogenation experiments suggested that the double bonds retained their original positions, and compound II is identified as 14,15-dihydroxy-5,8,11,17-eicosatetraenoic acid. The spectrum of compound III (Table IV) demonstrated ions at 385 ( M - C H 3 ( C H 2 C H = C H ) 2 C H 2 ) , 313 ( C H 3 ( C H 2 C H = C H ) z C H 2 ( C H (OSiMe3))2), 295 (385-Me3SiOH), 283 ( M CH3(CH2CH=CH)zCH2CH(OSiMe3)), 263 (385 - (CH3OH + Me3SiOH)), 254 ( M - CH3(CH 2 CH=CH)zCH(OSiMe3)CHOT), 223 ( 3 1 3 - Me 3 SiOH), 211 ( C H 3 ( C H 2 C H = C H ) 2 C H 2 C H (OSiMe3)), 205 ( 3 8 5 - 2 × M e 3 S i O H ) and 181 ((CH2CH=CH)2(CH2)3COOCH3). These ions indicate that one Me3SiO group occurred at C-11 and another at C-12. Thus, the structure of underivatized compound III is ll,12-dihydroxy-20:4. The spectrum of hydrogenated compound III (Table V) contained ions at 389 ( M - C H 3 (CH2)7) , 360(M-CH3(CH2)TCHOT), 345(360 - CH3), 317 ( M - (CH2)9COOCH3), 299 (389 Me3SiOH), 287 (M-CH3(CH2)7CH(OSiMe3)), 227(317 - Me3SiOH), 215 (CH3(CH2)vCH (OSiMe3)) and 185 ((CH2)9COOCH3). These ions indicate the presence of Me3SiO moieties at C-11 and C-12. Hydrogenation increased by 4 Da the a-cleavage fragments 211 and 313 (Tables IV and V). Therefore, there are two double bonds between C-12 and C-20. Hydrogenation also increased by 4 Da the a-cleavage fragments 385, 283 and 181 (Tables IV and V), Thus, there are two double bonds between C-1 and C-11. In summary,

hydrogenation experiments suggested that the double bonds had not shifted from their original positions, and compound III is identified as 11,12-dihydroxy-5,8,14,17-eicosatetraenoic acid. The spectrum of compound IV (Table IV) demonstrated ions at 353 ( M - C H 2 C H = C H ( C H 2 ) 3 COOCH3), 345 ((CH(OSiMe3))2CH2CH--CH (CH2)3COOCH3) , 263 ( 3 5 3 - Me3SiOH), 255 (345 - Me3SiOH), 251 ( M - CH(OSiMe3)CH 2 CH=CH(CH2)3COOCH3), 243 (CH(OSiMe3) CH2CH=CH(CHa)3COOCH3), 214 (CH2CH= CH(CH2)3C(=OSiMe3)OCH3X), 211 ( 2 4 3 - C H 3 OH), 149 (CH3(CH2CH=CH)3CH2) and 141 (CH2CH=CH(CH2)3COOCH3). These ions demonstrated that Me3SiO groups were present at C-8 and C-9. Thus the structure of underivatized compound IV is 8,9-dihydroxy-20:4. The spectrum of hydrogenated compound IV (Table V) revealed the presence of ions at 359 ( M - (CH2)6COOCH3) , 318 ( M - CH3(CH2)10 CHOT), 303 ( 3 1 8 - CH3), 257 (CH3(CH2)10CH (OSiMe3)), 245 (CH(OSiMe3)(CH2)6COOCH3), 155 (CH3(CH2)10) and 143((CH2)6COOCH3). These ions reaffirmed the presence of Me3SiO groups at C-8 and C-9. In addition, hydrogenation increased by 6 Da the a-cleavage fragments 353, 251 and 149 (Tables IV and V). The 6 Da shift demonstrated that there were three double bonds between C-9 and C-20. Hydrogenation also increased by 2 Da the a-cleavage fragments 243 and 141 (Tables IV and V). The 2 Da shift indicates that there was a single double bond between C-1 and C-8. Thus, hydrogenation results suggested that the double-bond positions remained intact, and compound IV is identified as 8,9-dihydroxy5,11,14,17-eicosatetraenoic acid.

Chromatographic properties of group A metabolites The RP-HPLC properties of group A metabolites were compared to those of vicinal diols arising from cytochr0me P-450 oxidations of 20:4(n - 6 ) and 2 2 : 6 ( n - 3). For group A metabolites, the elution order was 17,18- (I), 14,15- (II), 11,12(III), and 8,9-dihydroxy-20:4 (IV) (Table III). For 2 0 : 4 ( n - 6)-derived isomers, the elution order is 14,15-, 11~12-, 8,9- and 5,6-dihydroxy-20:3 [6,23]. Similarly, the elution order for 22:6(n - 3)-derived diols is 19,20-, 16,17-, 13,14-, 10,11- and 7,8-dihydroxy-22:5 [12]. In all three series, retention time

146

increases as the distance between the vicinal hydroxyls and the carboxyl groups decreases. In addition, the regular spacing of the eluting metabolites suggests that the diol regioisomers did not differ in other properties as well - for instance, double-bond positions and geometries [21]. Thus the RP-HPLC properties of group A metabolites support the hydroxyl and double-bond assignments made by MS. The NP-HPLC properties of group A metabolites were compared to those of vicinal diols derived from other polyunsaturated fatty acids. Elution orders for methyl ester derivatives were examined. The elution order for group A metabolites was: 14,15- (II), 11,12- (III), 8,9- (IV), and 17,18-dihydroxy-20:4 (I) (Table III). The only reported elution sequence for 2 0 : 4 ( n - 6)-derived regioisomers is 14,15- and 11,12-dihydroxy-20:3 [19]. In both 20-carbon series, the retention time increases as the distance between the vicinal hydroxyl and the carbomethoxy groups decreases. Homologous series derived from 1 8 : 2 ( n - 6) [26], 1 8 : 3 ( n - 3 ) [26] and 2 2 : 6 ( n - 3 ) [12] show the same general relationship. In addition, comparable migration patterns occur during thin layer chromatography [27]. It thus appears that the closer the vicinal hydroxyls are to the carbomethoxy moiety, the greater the likelihood that both groups can simultaneously hydrogen bond with surface silanols. However, a single anomaly occurs in all n - 3 fatty acid series - an unusually long retention time is seen for the (n 3)-diol. Concerning this regioisomer, the vicinal hydroxy groups may actually be very close to the carbomethoxy moiety due to intramolecular arching [28] and hydrogen bonding. To summarize, the NP-HPLC ehition order for group A metabolites is similar, both in general and anomalous properties, to that reported for vicinal diol regioisomers derived from other fatty acids. Thus, the NP-HPLC elution order of group A metabolites supports the hydroxyl locations assigned by MS. The GC properties of group A metabolites were compared to those of vicinal diols derived from other polyunsaturated fatty acids. Elution orders for the trimethylsilyl ether, methyl ester derivatives on non-polar stationary phases were examined. For group A metabolites, the elution order was 8,9- (IV) or 11,12- (III), then 14,15- (II) and

17,18-(Me3SiO)220:4 methyl esters (I) (Table III). In other regioisomer series of vicinal diols derived from 18:2(n - 6), 1 8 : 3 ( n - 3), 20:4(n - 6) or 22:6(n - 3), comparable elution orders are found [27,12]. In each of these series, increasing the distance between (CH(OSiMe3))2 and COOCH3 groups affects the ECL minimally until the n - 6 and n - 3 carbons are reached. Then there is a small ( n - 6 carbon) or large increase ( n - 3 carbon) in the ECL. Significantly, the published [27] ECL values (in parenthesis) for 8,9- (22.6), 11,12- (22.7), and 14,15-(OSiMe3)2-20:3 (22.8) methyl esters are close to the ECL of homologous metabolites from 20:5(n - 3) (Table III). Such GC data provide supportive evidence for the hydroxyl and double-bond positions indicated by MS. GC properties of hydrogenated group A metabolites were compared to those of hydrogenated vicinal diols derived from 22:6(n - 3); we were unable to find a published elution order for the 20:4(n - 6) homologs. The elution order of trimethylsilyl ether, methyl ester derivatives on non-polar stationary phases was compared. Group A metabolites eluted in the following order: 8,9(IV), 11,12- (III), 14,15- (II), and 17,18-dihydroxy20:0 (I) (Table III). After hydrogenation, regioisomers of vicinal diols from 22:6(n - 3) also show an analogous elution order [12]. Moreover, in both fatty acid series, increasing the distance between (CH(OSiMe3))2 and COOCH 3 groups produces little change in ECL values until the n - 6 or n - 3 carbon is reached. At these positions there is a small ( n - 6) or large ( n - 3) increase in the ECL value. Concerning how individual retention times were affected by hydrogenation, the ECL of group A metabolites were increased by 0.6-1.0 units. This increase was similar to that seen for 22:6(n - 3) metabolites [12]. If the relative contributions of 20- and 22-carbon skeletons to ECL are considered, such increases are consistent with compounds I - I V having four double bonds. In summary, the RP-HPLC, NP-HPLC and GC properties of group A metabolites further substantiate the hydroxyl and double-bond positions assigned by MS.

GC-MS properties of vicinal diol standards To establish more conclusively the fine structure of group A metabolites, vicinal diol standards

147 were chemically synthesized from 20:5(n-3). These standards have the same structure as 20:5(n - 3 ) except for the presence of (threo) vicinal hydroxyl groups at one of the original double-bond sites. Upon capillary GC-MS, these standards possessed the same retention times and mass spectra as the corresponding group A metabolites. The results suggest that the standards and metabolites have the same functional group positions and geometries. Thus, like the vicinal diols derived from 20:4(n - 6) [6], group A diols may also have the threo configurations.

Mass spectral properties of group B metabolites Electron impact mass spectra of the group B metabolites (Figs. 3 and 4) were also generated. High-mass ions at 404 (M+), 389 (M - CH3) , 314 ( M - Me3SiOH ) and 299 ( M - (CH 3 + Me3SiOH)) indicated a common molecular weight of 404. This value is 88 Da greater than 316, the molecular weight of methyl 20:5, and suggested the presence of a Me3SiO group and five double bonds. The occurrence of m / z 73 (Me3Si), 75 (Me2SiOH), 146 (CH2=C(OSiMe3)OCH3), and 159 (CH2=CHC(=OSiMe3)OCH3) (see Ref. 30 for origin) confirmed that there was a Me3SiO moiety in each group B metabolite. Thus, the underivatized group B metabolites are hydroxy20:5 isomers.

100

,5.0

#

9

,2.5 •

==

0400~

5O

z

JLt_L-.t= , . , . .

_=

300

N

O.C

li; ~6o

400

°

'tlL, l ~ t . . . . . . . . . . .

2rio

0.0

L,

360

400

m/7

1 0 3 ~ 0 0 C H

s 404

Mwt

Fig. 4. Mass spectrum of compoundV as trimethylsilylether, methylester derivative.

Mass spectra of hydrogenated group B metabolites (Table V) revealed high-mass ions at 414 (M÷), 399 (M - CH3), 383 ( M - OCH3), 367 ( M - (CH 3 -t- CH3OH)) , 341 ( M - Me3Si), 324 ( M - Me3SiOH), and 292 (M - (CH3OH + Me3SiOH)). These ions indicated a common molecular weight of 414. Thus, hydrogenation shifted the molecular mass by 10 Da (404--, 414; Tables IV and V) and demonstrated that five double bonds were originally present in group B metabofites. A molecular weight of 414 is 88 Da greater than 326, the molecular weight of methyl 20:0, and indicated the presence of a Me3SiO group. The occurrence of m / z 73, 75,146 and 159 reaffirmed the presence of a Me3SiO moiety in group B metabolites. Thus, hydrogenation studies confirmed that group B metabolites are hydroxy20:5 isomers. The mass spectrum of compound V (Fig. 4) showed ions at m / z 301 (M-CH2(OSiMe3)) and at 103 (CHz(OSiMe3)). These ions suggested the presence of a Me3SiO moiety at C-20. Therefore, the structure of underivatized compound V is 20-hydroxy-20:5. The mass spectrum of hydrogenated compound V (Table V) demonstrated ions at 311 ( M CH2(OSiMe3) ) and 103 (CHz(OSiMe3)). These ions confirmed the presence of a Me3SiO moiety at C-20. Hydrogenation did not shift the a-cleavage fragment 103 but hydrogenation did increase the a-cleavage fragment 301 by 10 Da (301 ---, 311; Tables IV and V). The 10 Da shift confirmed that five double bonds were originally present. If it is assumed that the isolated double bonds retained their original positions, compound V is identified as 20-hydroxy-5,8,11,14,17-20: 5. The mass spectrum of compound VI (Fig. 5) revealed ions at rn/z 360 ( M - C H 3 C H O T) and 117 (CH3CH(OSiMe3)). These ions suggested the presence of a Me3SiO group at C-19. Thus, underivatized compound VI is identified as 19-hydroxy-20: 5. The mass spectrum of hydrogenated compound VI (Table V) demonstrated ions at 370 ( M CH3CHO T) and 117 (CH3CH(OSiMe3)). These ions indicated a Me3SiO group occurring at C-19. Hydrogenation did not increase the mass of the a-cleavage fragment 117 (Tables IV and V). However, hydrogenation increased by 10 Da the a-

148

100' 3.0 ,¢

¢o

.= z~

i./i i

1.5>

~5o o)

.-¢"0 ~

•

~/3.0m

o 0.0

•

300

400

300

400

0

100

200 m/z 117-~

OSiMe3

404 Mwt

Fig. 5. Mass spectrum of compound VI as trimethylsilyl ether, methyl ester derivative.

cleavage fragment 360 (Tables IV and V). The 10 Da shift indicated that there were five double bonds between C-1 and C-19. Thus, hydrogenation results suggested that the double-bond positions had remained intact, and compound VI is identified as 19-hydroxy-5,8,11,14,17-20:5.

Chromatographic properties of group B metabofites The RP-HPLC properties of group B metabolites were compared to those of 22- and 21-hydroxy-22:6(n- 3). Compound V (20-hydroxy2 0 : 5 ( n - 3)) eluted before compound VI (19-hydroxy-20:5(n - 3)) during the RP-HPLC of group B metabolites (Table III). Under almost identical RP-HPLC conditions, 22-hydroxy-22:6(n- 3) elutes before 21-hydroxy-22:6(n-3) [12]. Thus, the RP-HPLC properties of group B metabolites support the hydroxyl positions assigned by MS. The NP-HPLC properties of group B metabolites were compared to those of the ( n - 1)- and n-hydroxy derivatives of 22:6(n - 3) and 20:4(n 6). In the latter studies, 2 2 : 6 ( n - 3) metabolites were chromatographed as methyl esters [12] whereas 2 0 : 4 ( n - 6) metabolites were chromatographed as free acids [19,24]. During the NPHPLC of 2 0 : 5 ( n - 3) metabolites, compound VI (19-hydroxy-20:5(n - 3)) eluted before compound V (20-hydroxy-20:5(n- 3)) (Table III). Likewise, the (n - 1)-hydroxy derivatives of 20:4(n - 6) and 22:6(n- 3) elute before the corresponding n-hy-

droxy derivatives during NP-HPLC [12,19,24]. Thus, the hydroxyl positions determined by MS are confirmed by the NP-HPLC properties of group B metabolites. The GC properties of group B metabolites were compared to those of n-hydroxy and ( n - 1)hydroxy derivatives of 22:6(n - 3) or 20:4(n - 6). Comparisons were done on the trimethylsilyl ether, methyl ester derivatives. Compound V (20-hydroxy-20:5(n - 3)) eluted after compound VI (19hydroxy-20:5(n- 3)) during GC on a non-polar stationary phase (Table III). For analogous metabolites derived from 2 0 : 4 ( n - 6) and 22:6(n - 3 ) , the n-hydroxy isomer elutes later than the ( n - 1)-hydroxy isomer [19,12]. Significantly, the ECL of compound V (22.5) was only 0.1 units less than that reported for 20-hydroxy-20:4(n - 6) [19]. The ECL of metabolite VI (21.7) was also only 0.1 units less than that reported for 19-hydroxy2 0 : 4 ( n - 6) [19]. Such GC data indicate that the group B metabolites are very similar in structure to 2 0 : 4 ( n - 6)-derived metabolites - that is, the corresponding regioisomers are probably identical except for the additional n - 3 double bond in the group B metabolites. The GC properties of hydrogenated group B metabolites were compared to those of 22- and 21-hydroxy-22:0. Hydrogenation increased the ECL of group B metabolites by 1.0 (compound V) and 1.1 (compound VI) ECL units (Table III). A 1.1 shift in ECL is also observed after 22-hydroxyor 21-hydroxy-22:6(n - 3) are hydrogenated [12]. If the contributions from different chain lengths are considered, the GC data are consistent with group B metabolites having five double bonds. In summary, the RP-HPLC, NP-HPLC and GC properties of group B metabolites confirmed that hydroxyl groups were present at C-20 or C-19. In addition, the GC studies indicated that group B metabolites have double-bond properties similar to those of 20-hydroxy-20:4(n- 6) and 19-hydroxy-20:4(n - 6).

Conclusions After fortification with 1 mM NADPH, microsomes from rats on high-fat diets or from rats pretreated with phenobarbital oxidized 20: 5(n - 3) to over 15 products. The major products were

149 identified b y MS, b y c h r o m a t o g r a p h i c p r o p e r t i e s , a n d b y m a t c h i n g their mass s p e c t r a a n d c h r o m a t o g r a p h i c p r o p e r t i e s with those of s t a n d a r d s . M a j o r m e t a b o l i t e s of liver m i c r o s o m e s were: 17(18)-, 14(15)-, 11(12)-, a n d 8 ( 9 ) - d i h y d r o x y e i c o s a t e t r a e n o i c acids, 2 0 - h y d r o x y e i c o s a p e n t a enoic acid, a n d 1 9 - h y d r o x y e i c o s a p e n t a e n o i c acid. M a j o r m e t a b o l i t e s of r e n a l m i c r o s o m e s were 20h y d r o x y e i c o s a p e n t a e n o i c acid a n d 1 9 - h y d r o x y e i c o s a p e n t a e n o i c acid. Based on the types of p r o d ucts formed, the o r g a n differences in m e t a b o l i t e f o r m a t i o n , the altered m e t a b o l i t e profiles seen w h e n m i c r o s o m e s f r o m p h e n o b a r b i t a l - t r e a t e d rats were used, a n d the r e q u i r e m e n t for N A D P H in m e t a b o l i t e synthesis, it a p p e a r s that 20:5(n - 3) is o x i d i z e d b y c y t o c h r o m e P-450 m o n o o x y g e n a s e s . It also appears, b a s e d on R P - H P L C c h r o m a t o grams, that diets enriched in fish oil m a y n o t result in novel o x i d a t i o n s of the n - 3 f a t t y a c i d s b y c y t o c h r o m e P-450 m o n o o x y g e n a s e s . H o w e v e r , m o r e d e t a i l e d s t r u c t u r a l analyses of the e p o x i d e s a n d l i p o x y g e n a s e - l i k e m e t a b o l i t e s are n e e d e d to c o r r o b o r a t e these findings.

Acknowledgment This w o r k was s u p p o r t e d in p a r t b y N I H Research G r a n t n u m b e r H L 28982-05 of the N a t i o n a l H e a r t , L u n g a n d B l o o d Institute, a n d b y the A m e r i c a n H e a r t A s s o c i a t i o n . W e are d e e p l y grateful to Dr. J.R. F a l c k ( U n i v e r s i t y of T e x a s H e a l t h Science Center, Dallas, T X ) for p r o v i d i n g us with e p o x i d e s t a n d a r d s d e r i v e d f r o m 2 0 : 4 ( n - 6) (cis5(6)-epoxy-8,11,14-20:3, cis-8(9)-5,11,14-20:3, cisl l ( 1 2 ) - e p o x y - 5 , 8 , 1 4 - 2 0 : 3 , a n d cis-14(15)-epoxy5,8,11-20:3).

References 1 Lands, W.E.M. (1986) Fish and Human Health, Academic Press, Orlando. 2 Simopoulus, A.P. (1986) in Health Effects of Polyunsaturated Fatty Acids in Seafoods (Simopoulos, A.P., Kifer, R.R. and Martin, R.E., eds.), pp. 3-33, Academic Press, Orlando. 3 Lewis, R.A., Lee, T.H. and Austen, K.F. (1986) in Health Effects of Polyunsaturated Fatty Acids in Seafoods (Simopoulos, A.P., Kifer, R.R. and Martin, R.E., eds.), pp. 227-238, Academic Press, Orlando.

40liw, E., Granstrom, E. and Angaard, E. (1983) in New Comprehensive Biochemistry (Pace-Asciak, C. and Granstrom, E., eds.), Vol. 5, pp. 1-44, Elsevier, New York. 5 Capdevila, J., Saeki, Y. and Falck, J.R. (1984) Xenobiotica 14, 105-118. 60liw, E.H., Guengerich, F.P. and Oates, J.A. (1982) J. Biol. Chem. 257, 3771-3781. 7 Falck, J.R., Manna, S., Jacobson, H.R., Estabrook, R.W., Chacos, N. and Capdevila, J. (1984) J. Am. Chem. Soc. 106, 3334-3336. 8 Capdevila, J., Pramanik, B., Napoli, J.L., Manna, S. and Falck, J.R. (1984) Arch. Biochem. Biophys. 231, 511-517. 9 Proctor, K.G., Falck, J.R. and Capdevila, J. (1987) Circ. Res. 60, 50-59. 10 Fitzpatrick, F.A., Ennis, M.D., Baze, M.E., Wynalda, M.A., McGee, J.E. and Liggett, W.F. (1987) J. Biol. Chem. 261, 15334-15338. 11 Capdevila, J., Yadagiri, P., Manna, S. and Falck, J.R. (1986) Biochem. Biophys. Res. Commun. 141, 1007-1011. 12 VanRollins, M., Baker, R., Sprecher, H. and Murphy, R.C. (1984) J. Biol. Chem. 259, 5776-5783. 13 Century, B. (1973) J. Pharmacol. Exp. Ther. 185, 185-194. 14 Rowe, L. and Wills, E.D. (1976) Biochem. Pharmacol. 25, 175-179. 15 Burke, M.D. and Orrenius, S. (1978) Pharmacol. Ther. 7, 549-599. 16 Chacos, N., Falck, J.R., Wixtrom, C. and Capdevila, J. (1982) Biochem. Biophys. Res. Commun. 104, 916-922. 17 Aveldano, M.I. and Horrocks, L.A. (1983) J. Lipid Res. 24, 1101-1105. 18 VanRolfins, M. and Murphy, R.C. (1984) J. Lipid Res. 25, 507-517. 19 Oliw, E.H., Lawson, J.A., Brash, A.R. and Oates, J.A. (1981) J. Biol. Chem. 256, 9924-9931. 20 Oliw, E.H. and Oates, J.A. (1981) Biochim. Biophys. Acta 666, 327-340. 21 VanRollins, M., Aveldano, M.I., Sprecher, H. and Horrocks, L.A. (1982) Methods Enzymol. 86, 518-530. 22 Aveldano, M.I., VanRollins, M. and Horrocks, L.A. (1983) J. Lipid Res. 24, 83-93. 23 Chacos, N., Capdevila, J., Falck, J.R., Manna, S., MartinWixtrom, C., Gill, S.S., Hammond, B.D. and Estabrook, R.W. (1983) Arch. Biochem. Biophys. 223, 639-648. 24 Morrison, A.R. and Pascoe, N. (1981) Proc. Natl. Acad. Sci. USA 78, 7375-7378. 25 Jacobson, H.R., Corona, S., Capdevila, J., Chacos, N., Manna, S., Womack, A. and Falck, J.R. (1985) in Advances in Ion Transport Regulation, Vol. 1, pp. 311-318, Raven Press, New York. 26 Oliw, E.H. (1983) Biochem. Biophys. Res. Commun. 111, 644-651. 27 Oliw, E.H. (1983) J. Chromatogr. 275, 245-259. 28 Corey, E.J., Iguchi, S., Albright, J.O. and De, B. (1983) Tetrahedron Lett. 24, 37-40. 29 Eglinton, G., Hunneman, D.H. and McCormick, A. (1968) Org. Mass Spectrom. 1, 593-611. 30 Capella, P. and Zorzut, C.M. (1968) Anal. Chem. 40, 1458-1463.