Formation of 14,15-Hepoxilins of the A3 and B3 Series through a 15-Lipoxygenase and Hydroperoxide Isomerase Present in Garlic Roots*

We report herein for the first time the formation by freshly grown garlic roots and the structural characterization of 14,15-epoxide positional analogs of the hepoxilins formed via the 15-lipoxygenase-induced oxygenation of arachidonic acid. These compounds are formed through the combined actions of a 15(S)-lipoxygenase and a hydroperoxyeicosatetraenoic acid (HPETE) isomerase. The compounds were formed when either arachidonic acid or 15-HPETE were used as substrates. Both the “A”-type and the “B”-type products are formed although the B-type compounds are formed in greater relative quantities. Chiral phase high performance liquid chromatography analysis confirmed the formation of hepoxilins from 15(S)- but not 15(R)-HPETE, indicating high stereoselectivity of the isomerase. Additionally, the lipoxygenase was of the 15(S)-type as only 15(S)-hydroxyeicosatetraenoic acid was formed when arachidonic acid was used as substrate. The structures of the products were confirmed by gas chromatography-mass spectrometry of the methyl ester trimethylsilyl ether derivatives as well as after characteristic epoxide ring opening catalytically with hydrogen leading to dihydroxy products. That 15(S)-lipoxygenase activity is of functional importance in garlic was shown by the inhibition of root growth by BW 755C, a dual cyclooxygenase/lipoxygenase inhibitor and nordihydroguaiaretic acid, a lipoxygenase inhibitor. Additional biological studies were carried out with the purified intact 14(S),15(S)-hepoxilins, which were investigated for hepoxilin-like actions in causing the release of intracellular calcium in human neutrophils. The 14,15-hepoxilins dose-dependently caused a rise in cytosolic calcium, but their actions were 5–10-fold less active than 11(S),12(S)-hepoxilins derived from 12(S)-HPETE. These studies provide evidence that 15(S)-lipoxygenase is functionally important to normal root growth and that HPETE isomerization into the hepoxilin-like structure may be ubiquitous; the hepoxilin-evoked release of calcium in human neutrophils, which is receptor-mediated, is sensitive to the location within the molecule of the hydroxyepoxide functionality.

In an attempt to learn more about the enzymatic formation of the hepoxilins, we investigated other sources for the "hepoxilin synthase" which may provide an abundant supply for the purification of the enzyme. Herein, we report the formation of hepoxilins via the 15-lipoxygenase pathway which is abundant in freshly grown roots of garlic. The isolated compounds (14,15hepoxilins) constitute structural analogs of the parent 11,12hepoxilins and provide further information on the specificity of the hepoxilin receptor in human neutrophils toward hepoxilins derived from 12-lipoxygenase.

EXPERIMENTAL PROCEDURES
Materials-Garlic bulbs were purchased from a local grocery store. They were placed individually in glass beakers in contact with tap water for 2-3 weeks until sufficient roots emerged, water being changed every day. The roots were cut off, rinsed with tap water and stored at Ϫ70 o until enough tissue was collected for the experiments. Ionomycin, Dextran T-500, and Ficoll-Paque were purchased from Amersham Pharmacia Biotech, Sweden, and Indo-1-AM from Calbiochem. ADAM reagent was purchased from Research Organics Inc., Cleveland, Ohio. Racemic 15(S/R)-HPETE was prepared by photo-oxidation as described previously for 12(S/R)-HPETE (3). RPMI 1640 medium, hemin (bovine), and all reagent grade chemicals for buffers and NDGA were purchased from Sigma. BW 755C was a gift of Dr. Salvador Moncada (Burroughs-Wellcome, UK).
Incubations-Separate incubations were carried out with arachidonic acid (Cayman Chemical Co., Ann Arbor, MI) or with 15(S/R)-HPETE as substrates. Typically, 1 g of garlic roots was homogenized in 2 ml of phosphate-buffered saline of the following composition: NaCl (140 mM), NaH 2 PO 4 (10 mM), and Na 2 HPO 4 (10 mM), pH 7.2. The * This work was supported by a grant from the Medical Research Council of Canada (MT-4181) (to C. R. P.-A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ʈ homogenate was centrifuged at 2000 rpm for 5 min, and 400-l aliquots were added to siliconized tubes containing either arachidonic acid (1 g) or 15(S/R)-HPETE (1 g) and made up with buffer to a total volume of 1 ml. The samples were incubated at 37°C for 60 min. The incubation was terminated by cooling on ice and addition of 1 ml of methanol and 2 ml of ethyl acetate containing ADAM reagent (140 g/sample). The samples were stirred at 23°C in the dark for 60 min to form the ADAM esters, and the ethyl acetate phase was separated and evaporated to complete dryness with a stream of N 2 gas. The residue containing the ADAM esters of the products was analyzed directly by HPLC. To generate standards of 14,15-hepoxilins, 15(S/R)-HPETE (1 g) was treated in phosphate-buffered saline containing hemin (10 g) for 60 min and worked up as with the garlic root incubations.
GCMS-Samples were analyzed as the MeTMSi derivatives in the EI mode. Because the ADAM esters of the metabolites are unsuitable for hydrolysis and conversion into the methyl ester form for GCMS analysis, separate large scale experiments using 100 g of 15-HPETE were performed in which the crude ethyl acetate extracts were converted directly into the methyl ester form (instead of the ADAM esters) with a solution of ethereal diazomethane. The methyl esters were then purified by SP-HPLC on a Porasil column (hexane/isopropanol 99.7/0.3, v/v, detection 210 nm). The purified fractions were converted into the TMSi ether derivatives by reaction with TriSilZ (Chromatographic Specialties, Brockville, Ontario, Canada, 20 l), 5 min at 60°C. Aliquots were injected into the GCMS directly. Samples were also analyzed after hydrogenation (platinum oxide/methanol, 30 s) of the methyl esters and subsequent conversion into the TMSi ether derivatives. A DB-1 methyl silicone capillary column (Chromatographic Specialties, Brockville, Ontario, Canada, 30 m length ϫ 0.25 mm ID, 0.25 m film thickness) was used; the column temperature was programmed from 200°C initially to 300°C at 10°C/min.
Measurement of Intracellular Free Calcium-Neutrophils were prepared according to procedures previously published by our group (7). Intracellular free calcium concentrations were monitored continuously in a Perkin-Elmer fluorescence spectrophotometer (Model 650 -40) using Indo-1-AM-loaded neutrophils. Excitation wavelength was set at 331 nm and emission wavelength at 410 nm, with slits of excitation and emission set at 3 and 15 nm, respectively. Neutrophil suspensions (10 7 cells) were loaded with 3 l of 1 mM (final concentration 3 M) of the acetoxymethyl ester precursor of Indo-1 for 30 min at 37°C. Unloaded dye was removed by centrifugation, and the cells were resuspended in fresh RPMI 1640 (1 ml). Dye-loaded cells were kept at room temperature on a rotator (Roto-Torque, Cole-Palmer model 7637, USA) turning at about 10 -15 rotations/min. Typical measurements involved 2 ϫ 10 6 cells in 1 ml of cell medium in a temperature controlled plastic cuvette (Diamed Labs., Canada) at 37°C with constant stirring. Compounds were added in 1 l of Me 2 SO. Each measurement was followed by a calibration for maximum and minimum calcium release with ionomycin (1 M final concentration) and MnCl 2 (3 mM final concentration), respectively, according to Grinstein and Furuya (23). Responses were recorded on a chart recorder (LKB model 2210, Amersham Pharmacia Biotech, Sweden) at 1 cm/min chart speed.
Measurement of Garlic Root Growth-Garlic bulbs were purchased from a local grocery store, care being taken to select bulbs of similar weight and size. They were cleaned and placed on glass beakers (one/ beaker) filled with tap water (100 ml) with the bottom of the bulb touching the water. Four groups of 6 bulbs/group were set up. Two groups contained the two inhibitors which were dissolved in Me 2 SO and added to selected containers at a final concentration of 4 mg/100 ml. Two other groups contained only Me 2 SO and served as control. The water was replaced every third day for two weeks. At the end of the study period, the roots were cut off with scissors, dried on paper, and weighed.

RESULTS AND DISCUSSION
Incubation of arachidonic acid with a cell-free homogenate of garlic roots caused the formation of products that migrated on reverse phase-HPLC after conversion into fluorescent ADAM derivatives as characteristic doublets (19.4, 20.4, and 21.5, 22.1 min, Fig. 1E) in a region where authentic 11,12-hepoxilins A 3 and B 3 migrate (doublets at 23.0, 23.9 min for the two epimers of HxA 3 and 25.5, 26.3 for HxB 3 , Fig. 1A). Hence, the pattern and relative elution of these products indicated that they were hepoxilin-like products (Fig. 1). Further confirmation of the hepoxilin-like nature of these compounds was obtained by their disappearance from the chromatograms after treatment of the samples with acid (data not shown). Arachidonic acid comprises approximately 2% of the fatty acids in garlic roots and approximately 5% in garlic bulbs. 2 Previous studies had shown that an active 15-lipoxygenase was present in garlic roots (24) and suggested therefore that the new products may be derived from this pathway. In fact, when 15-HPETE was incubated with a garlic root cell-free homogenate, products similar to those formed from arachidonic acid were observed (compare Fig. 1, D and E). Identical products were also formed when 15-HPETE was incubated with hemin (in the absence of tissue,  Despite the nonenzymatic (hemin-catalyzed) conversion of 12-HPETE into the hepoxilins, we have previously observed that an enzyme system exists in the pineal gland which forms exclusively the 11(S),12(S)-hepoxilins (3). This was demonstrated by the selective utilization of 12(S)-but not 12(R)-HPETE during hepoxilin formation. In the present experiments, we employed a similar approach with 15(S/R)-HPETE to investigate whether a selective consumption of 15(S)-HPETE could be observed during incubation with garlic root homogenates to indicate the presence of an HPETE isomerase. Hence HPLC fractions corresponding to 15-HETE and the 14,15-hepoxilins were isolated and subjected to chiral phase analysis. Fig. 2 shows that garlic roots convert arachidonic acid exclusively into the 15(S)-HETE which accumulates in the incubation (Fig. 2D); conversely, when 15(S/R)-HPETE is used as substrate, the 15(S)-enantiomer is selectively consumed for further conversion into hepoxilin products while the 15(R)enantiomer remains unmetabolized (Fig. 2C). When a nonselective reaction of 15-HPETE is carried out, as with hemin, both 15(S)-and 15(R)-HETE are detected at the end of the incubation (Fig. 2B). These experiments indicate the stereospecificity of 15-lipoxygenase in garlic roots as well as the selective utilization of the 15(S)-enantiomer by HPETE isomerase to form the 14,15-hepoxilins. Confirmation of these findings was obtained when 14,15-hepoxilins were analyzed by chiral-phase HPLC. Fig. 3a shows that while 14,15-hepoxilin B 3 (the major hepoxilin formed by garlic roots) affords chiral specificity (compare panels C and D) when an enzyme system is used, note that the corresponding hepoxilin A 3 does not appear to, as its pattern is similar to that obtained during hemin catalysis (Fig.  3b).
Characterization of the products was achieved through In contrast to the highly stereoselective formation from arachidonic acid (AA) and 15(S)-HPETE of the 14(S),15(S)-hepoxilin B 3 (Fig. 3), chiral phase analysis of the A-series appears to indicate that there is no stereo-selection in its formation by the garlic root system. GCMS analysis both as the intact metabolites and after catalytic hydrogenation. Hydrogenation serves to stabilize the hepoxilin structure and leads to characteristic reductive opening of the epoxide group depending on whether the hydroxyl group is adjacent to it (B-like) or allylic to it (A-like) (25). Fig. 4 compares the mass spectrum of the two types of metabolites isolated from garlic roots; the top panel shows the 14,15-hepoxilin B 3 (Fig. 4A, Rt 16.5 min), and the lower panel shows the spectrum of 14,15-hepoxilin A 3 (Fig. 4C, Rt 17.3 min). Characteristic fragment ions in the spectra are: m/z 332 (M-TMSiOH), 309 (C1-C13) (Fig. 4A); and m/z 422 (M ϩ ), 407 (M-15), 241 (C11-C20) (Fig. 4C). Selected ion chromatograms for these spectra are shown in the accompanying Figs. 4, B and D. These spectra were identical to those of products obtained during hemin catalysis of 15-HPETE (not shown). Catalytic hydrogenation of these products gave a mixture of 13,14-and 13,15dihydroxy-arachidic acid (Rt 12.1 min) derived from 14,15hepoxilin B 3 (Fig. 5A)  FIG. 5. EI mass spectra of the isolated products after catalytic hydrogenation (platinum oxide/methanol). The mass spectra of hydrogenated products derived from 14,15-hepoxilin B 3 are shown in panels A (mass spectrum) and B (reconstructed ion chromatogram) and of the hydrogenated 14,15-hepoxilin A 3 in panels C (mass spectrum) and D (reconstructed ion chromatogram). between two vicinal OH groups) and 173 (C15-C20) (Fig. 5A for the former product); and m/z 173 (C15-C20), 317 (C11-C20), 287 (C1-C11) (Fig. 5C for the latter product). Selected ion chromatograms for these spectra are shown in the accompanying Fig. 5, B and D.
The products isolated in this study are derived from activation of 15(S)-lipoxygenase (see Scheme 1). That this enzyme activity is of importance to the rooting system of garlic was demonstrated by the addition of two inhibitors of lipoxygenases, BW 755C and NDGA, prior to initiation of rooting. Both inhibitors blocked the appearance of roots which was abundant in the control groups (Fig. 6). 11(S),12(S)-Hepoxilin A 3 has been shown to evoke a dose-dependent rise in intracellular calcium in human neutrophils (7,9). We therefore examined the isolated 14,15-hepoxilins in this bioassay. Fig. 7 shows typical calcium profiles for the A-type and the B-type 14(S),15(S)-hepoxilins in comparison with the profiles seen with the 11,12-type hepoxilins A 3 and B 3 (top panels). It is clear that the 14,15-hepoxilins are able to dosedependently cause a rise in intracellular calcium although they are less active than 11,12-hepoxilin A 3 .
Garlic is widely used as a health supplement for a variety of conditions. It has been reported to have anti-platelet (26,27), anti-cancer (28 -30), and anti-atherogenic (31) properties although active ingredients additional to the selenium-containing compounds, allicin (32) and ajoene (32,33), have not been systematically investigated. A lipoxygenase pathway has recently been identified from garlic with the isolation of some unique divinyl ether metabolites of linoleic acid (34). The presence of a 15(S)-lipoxygenase in garlic may afford relevance to atherosclerosis. Reported evidence suggests that 15-lipoxygenase oxidizes LDL to a pro-atherogenic form (35). However, evidence with transgenic rabbits that overexpress 15-lipoxygenase also indicates that these animals are resistant to the development of atherosclerosis when fed a cholesterol-rich diet (36,37). Hence 15-lipoxygenase may act in both a pro-and anti-atherogenic manner depending on the time course of atherosclerosis (38). In the early stages, 15-lipoxygenase may serve an anti-atherogenic role, whereas in the later stages, it may act in a pro-atherogenic fashion (39). The products described herein are major products derived from 15-lipoxygenase present in garlic. Their actions on neutrophils in terms of intracellular calcium release may provide an insight into the antiinflammatory actions of these compounds as hepoxilins derived from the 12-lipoxygenase have been shown to inhibit the actions of inflammatory mediators (9). Whether the antiatherogenic actions of garlic are related to the formation of the 14,15-hepoxilins remains to be established.
In conclusion, we have demonstrated the presence in garlic roots of 15(S)-lipoxygenase activity, and this is coupled with an HPETE isomerase (see Scheme 1). The products are analogs of the 11,12-hepoxilins that we described previously from the pancreas, pineal gland, and brain formed through the enzymatic isomerization of 12(S)-HPETE. The present results show that 15(S)-lipoxygenase is functionally important to the rooting system of garlic although we do not know whether the isolated 14,15-hepoxilins play a role. The present study also provides further information into the structural specificity of the hepoxilin receptor for 11,12-hepoxilin A 3 in human neutrophils SCHEME 1. Pathway describing the formation by garlic roots of the 14,15-hepoxilins isolated in this study. and the potential ubiquity of the HPETE isomerase (or hepoxilin synthase).