identiﬁ cation of novel oxidized levuglandin d in marine ... · ﬁ ed from this alga as follows....

This article is available online at http://www.jlr.org Journal of Lipid Research Volume 52, 2011 2245

Copyright © 2011 by the American Society for Biochemistry and Molecular Biology, Inc.

donic acid, to generate a large array of lipid peroxida-tion products, including a series of stereoisomers and regioisomers of PGH 2 (H 2 -isoprostanes) ( Fig. 1 ) ( 2, 3 ). These H 2 -isoprostanes undergo nonenzymatic rearrangement to form D 2 - and E 2 -isoprostanes, as well as isolevuglandins as shown in Fig. 1 . Isolevuglandins (isoLG) are sometimes referred to by the chemically inaccurate name of isoketals. LGs and isoLGs are the most reactive aldehydes in the mammalian biological system, reacting almost instanta-neously to form covalent adducts with free amines in pro-teins ( 4–6 ), aromatic amines in DNA ( 7, 8 ), and the ethanolamine headgroup of phosphatidylethanolamine ( 9, 10 ). The levels of LG and isoLG in these protein and DNA adducts are known to increase in several pathologi-cal conditions ( 11, 12 ).

Marine red algae of the genus Gracilaria are unique plants containing prostaglandins. PGE 2, PGA 2 , PGF 2 � , 15-keto PGE 2 , and PGE 1 were found in G. lichenoides ( 13 ), G. ver-rucosa ( vermiculophylla ), G. choda ( 14, 15 ), and G. asiatica ( 16 ). PGs in G. verrucosa and G. chorda were suspected as causative toxins for lethal food poisoning that occurred in Japan ( 14, 17 ). Although the biosynthetic and metabolic pathways and functions of PGs in red algae have not been fully clarifi ed, it has been suggested that, in G. vermiculo-phylla , arachidonic acid is released from the membrane lipids, such as monogalactosyldiacylglycerol and digalacto-syldiacylglycerol, by the action of glycerolipid acyl-hydro-lase ( Fig. 1 ) ( 18 ). LG, isoLG, and isoprostane esters have not yet been identifi ed in red algae.

Abstract In animals, the product of cyclooxygenase react-ing with arachidonic acid, prostaglandin(PG)H 2 , can un-dergo spontaneous rearrangement and nonenzymatic ring cleavage to form levuglandin(LG)E 2 and LGD 2 . These LGs and their isomers are highly reactive � -ketoaldehydes that form covalent adducts with proteins, DNA, and phosphati-dylethanolamine in cells. Here, we isolated a novel oxidized LGD 2 (ox-LGD 2 ) from the red alga Gracilaria edulis and de-termined its planar structure. Additionally, ox-LGD 2 was identifi ed in some tissues of mice and in the lysate of phor-bol-12-myristate-13-acetate (PMA)-treated THP-1 cells incu-bated with arachidonic acid using LC-MS/MS. These results suggest that ox-LGD 2 is a common oxidized metabolite of LGD 2 . In the planar structure of ox-LGD 2 , H8 and H12 of LGD 2 were dehydrogenated and the C9 aldehyde was oxi-dized to a carboxylic acid, which formed a lactone ring with the hydrated ketone at C11. These structural differences imply that ox-LGD 2 is less reactive with amines than LGs. Therefore, ox-LGD 2 might be considered a detoxifi cation metabolite of LGD 2 . —Kanai, Y., S. Hiroki, H. Koshino, K. Konoki, Y. Cho, M. Cayme, Y. Fukuyo, and M. Yotsu-Yamashita. Identifi cation of novel oxidized levuglandin D 2 in marine red alga and mouse tissue. J. Lipid Res. 2011. 52: 2245–2254.

Supplementary key words prostaglandins • Gracilaria • LC-MS/MS • NMR

In animals, the product of cyclooxygenase (COX) react-ing with arachidonic acid, prostaglandin(PG)H 2 , can un-dergo spontaneous rearrangement and nonenzymatic ring cleavage to form highly reactive levuglandin (LG)E 2 or LGD 2 ( 1 ), if PGH 2 is not utilized by cellular PG syn-thases. However, reactive oxygen species (ROS) can also interact with polyunsaturated fatty acids, such as arachi-

This work was supported by the Cabinet Offi ce of the Government of Japan through its funding program for the Next-Generation World-Leading Research-ers (LS012) and by the Japan Society for the Promotion of Science through Grant-in-Aid for Scientifi c Research No. 21380070.

Manuscript received 10 May 2011 and in revised form 30 August 2011.

Published, JLR Papers in Press, September 5, 2011 DOI 10.1194/jlr.M017053

Identifi cation of novel oxidized levuglandin D 2 in marine red alga and mouse tissue

Yoshikazu Kanai , * Sadahiko Hiroki , * Hiroyuki Koshino , † Keiichi Konoki , * Yuko Cho , * Mirriam Cayme , § Yasuo Fukuyo , ** and Mari Yotsu-Yamashita * ,1

Graduate School of Agricultural Science,* Tohoku University , Sendai, Miyagi 981-8555, Japan ; RIKEN Advanced Science Institute , † Wako, Saitama 351-0198, Japan ; National Fisheries Research and Development Institute , § Quezon City 1103, The Philippines ; and Asian Natural Environmental Science Center,** The University of Tokyo , Bunkyo-ku, Tokyo 113-8657, Japan

Abbreviations: COSY, correlation spectroscopy; HMBC, heteronu-clear multiple-bond correlation; HSQC, heteronuclear single-quantum coherence; LG, levuglandin; isoLG, isolevuglandin; ox-LGD 2 , oxidized levuglandin D 2 ; MRM, multiple reaction monitoring; PG, prostaglandin; PMA, phorbol-12-myristate-13-acetate; ROESY, rotating-frame Overhauser enhancement spectroscopy; TOCSY, total correlation spectroscopy.

1 To whom correspondence should be addressed. e-mail: [email protected]

The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of four fi gures.

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2246 Journal of Lipid Research Volume 52, 2011

then 200 ml). After the volatile substance in the ethyl acetate ex-tract was evaporated under low pressure, ox-LGD 2 was purifi ed by sequential reversed phase chromatography. First, a Cosmosil 5C18AR-II (i.d. 1.0 × 25 cm, Nacalai tesque, Tokyo, Japan) was used with aqueous 20 mM ammonium formate and methanol (1:1-3:7, v/v, gradient, fl ow rate of 1 ml/min), and then a Develo-sil ODS-SR-5 (i.d. 0.8 × 25 cm, Nomura Chemical, Seto, Japan) was used with aqueous 20 mM ammonium formate and methanol (4:6, v/v, fl ow rate of 1 ml/min). Lastly, a Mightysil RP-18 GPII (i.d. 0.46 × 25 cm, 5 � m, Kanto Chemical, Tokyo, Japan) with an acetonitrile-water-acetic acid mixture (40:60:0.1, v/v/v, fl ow rate of 0.5 ml/min) was used. After repeating the same purifi cation procedure four times, pure ox-LGD 2 ( � 0.1 mg, estimated by 1 H NMR) was obtained from a total of 800 g of wet G. edulis .

Structural determination of ox-LGD 2 NMR spectra were obtained on a Varian 600 MHz NMR spec-

trometer (Agilent Technologies, Santa Clara, CA) with CD 3 CN as the solvent. The signals of CHD 2 CN at 1.9 ppm in the 1 H NMR spectra and those of CD 3

13 CN at 118 ppm in the 13 C NMR spectra were used as the internal references. Signals were assigned based on the analyses of correlation spectroscopy (COSY), total correla-tion spectroscopy (TOCSY; mixing time 80 ms), heteronuclear single-quantum coherence (HSQC), heteronuclear multiple-bond correlation (HMBC), and rotating-frame Overhauser enhancement spectroscopy (ROESY; mixing time 400 ms) spec-tra. High-resolution fast atom bombardment (HRFAB)-MS (neg-ative, matrix: m -nitrobenzyl alcohol) was recorded by a JEOL JMS700 MS Station (Akishima, Japan). The UV spectrum of ox-LGD 2 was measured in methanol by a Shimadzu UV1800 spec-trometer (Kyoto, Japan). LC-MS/MS was performed by an API2000 mass spectrometer (AB SCIEX, Foster City, CA) with an ESI ion source in the negative ion mode.

Chemical derivation of ox-LGD 2 from PGH 2 Ox-LGD 2 was chemically derived from PGH 2 by two-step reac-

tion. First, LGs (as a mixture with PGE 2 and PGD 2 ) were de-rived from PGH 2 (5 � g) by incubation in 1.5 � l of DMSO at 37°C

In the present study, we found a novel oxidized deriva-tive of LGD 2 , named ox-LGD 2 , in the red alga Gracilaria edulis , and we determined its planar structure by spectro-scopic methods. Furthermore, we identifi ed ox-LGD 2 in some tissues of mice using LC-MS/MS and in a phorbol-12-myristate-13-acetate (PMA)-stimulated THP-1 cell lysate incubated with arachidonic acid.

MATERIALS AND METHODS

Materials Authentic PGs were purchased from Cayman Chemical (Ann

Arbor, MI). All solvents were HPLC grade and purchased from Wako Pure Chemical Industries (Osaka, Japan). 3,5-Di- tert -butyl-4-hydroxytoluene (BHT) was obtained from Sigma Chemical Co. (St. Louis, MO). Other reagents were special grade and pur-chased from Wako Pure Chemical Industries. Gracilaria edulis was collected in December 2004 at a beach in La Union, The Philip-pines. The frozen alga was transported to Tohoku University, Ja-pan, and kept at � 20°C until use. G. vermiculophylla was collected in May 2005 in Sendai Bay, Japan.

Purifi cation of ox-LGD 2 from the red alga G. edulis The presence of a novel PG-related compound in G. edulis was

suggested by an unknown ion detected at m/z 365 in an ESI-MS spectrum of the ethyl acetate extract from G. edulis, operating in the negative ion mode and scanning between m/z 100 and 500. The compound that gave this unknown ion at m/z 365 was puri-fi ed from this alga as follows. PGs and other polar lipids were extracted from G. edulis according to the method reported by Fusetani and Hashimoto ( 14 ), with slight modifi cations. Frozen G. edulis (200 g wet weight) was cut into approximately 1 cm pieces and soaked in water (600 ml) at room temperature overnight. The supernatant of the water extract, obtained by centrifugation at 1,000 g at 4°C for 10 min, was adjusted to pH 3.5 with 1 N HCl, and then the lipids were extracted with ethyl acetate (300 ml and

Fig. 1. Pathways leading to the formation of PGs, LGs, and isoLGs esters in the red algae genus Gracilaria and animals. PL, phospholipid; GL, galactolipid.


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Identifi cation of novel oxidized levuglandin D 2 2247

4 weeks if age, 20-21 g body weight) purchased from Japan SLC (Hamamatsu, Japan) were euthanized with diethyl ether. The tis-sues (testis, liver, kidney, lung, heart, brain, and muscle) from fi ve mice (labeled mouse A–E) and the liver from one mouse (mouse F) were dissected on ice and immediately frozen in liquid nitrogen. Eicosanoids were extracted according to the method utilized by Yang et al. ( 23 ) with slight modifi cations. Briefl y, the frozen tissues were ground to fi ne powders using a mortar in liq-uid nitrogen. Parts of the ground tissues (20-25 mg) were trans-ferred into sealed microcentrifuge tubes, and three volumes of ice-cold PBS buffer containing 0.1% (w/v) BHT and 1 mM EDTA were added to the tissue samples. The samples were then homog-enized by an ultrasonic processor (Vibra cell, Sonics and Materi-als Inc., Danbury, CT) on ice for 2 min. The homogenate was transferred into a glass tube (i.d. 1.0 × 10 cm), and 20 � l of 1 N citric acid was added. The eicosanoids were then extracted with 1 ml of hexane-ethyl acetate (1:1, v/v) and mixed by vortex for 2 min. Samples were centrifuged at 1,800 g at 4°C for 10 min, and then the upper organic layer was collected. Next, the solvent was evap-orated to dryness under a stream of nitrogen gas at room tem-perature, and then the residue was dissolved in 200 � l of methanol. An aliquot (10 � l) was injected into an LC-MS/MS in MRM mode for quantitation of PGs and ox-LGD 2 (see below). For the samples in which ox-LGD 2 was not detected, the remain-ing sample solutions were concentrated to 70 � l by a stream of nitrogen gas, and an aliquot (20 � l) was applied to LC-MS/MS (MRM) again. The fi nal limit of detection for PGE 2 , PGD 2 , and ox-LGD 2 was 0.004, 0.008, and 0.004 � g/g tissue, respectively. Af-ter MRM analysis, the remaining sample solution (19/20, v/v) prepared from the liver of mouse B was concentrated to 40 � l, and 20 � l of this solution was injected on column for product ion scan analysis for ox-LGD 2 ([M-H] � m/z 365).

To examine the effect of BHT on the level of ox-LGD 2 , the frozen and ground liver from mouse F was divided into two por-tions, and a part (20 mg) of each was homogenized in the same buffer as described above with or without BHT. The sample solu-tions for LC-MS/MS (MRM) analysis were prepared as described above.

Formation of ox-LGD 2 in the lysate of PMA-treated THP-1 cells

The human monocytic leukemia cell line THP-1 was obtained from the Cell Resource Center for Biomedical Research, Tohoku University, and RIKEN BioResource Center (Tsukuba, Japan). The cells were maintained in a RPMI 1640 culture medium contain-ing 10% (v/v) fetal bovine serum and penicillin/streptomycin

for 1 h ( 19 ). Second, we applied Pinnick oxidation ( 20 ) to oxi-dize aldehyde in LGs to carboxylic acid. To the reaction mixture of the fi rst reaction, 1 � M KH 2 PO 4 (10 � l, aqueous), water (10 � l), t -butanol (30 � l), 2-methyl-2-butene (10 � l), and 56 mM NaClO 2 (5 � l, aqueous) were added, and the mixture was kept at 10°C for 1 h. After the reactions, the mixture was acidifi ed with 1 N HCl, and lipophilic compounds were extracted with ethyl acetate (500 � l). The ethyl acetate layer was washed with 500 � l of water and dried by a stream of nitrogen gas. The resultant residue was dissolved in 200 � l of methanol, and an aliquot (10 � l) of the methanolic solution was applied to LC-MS/MS in the multiple reaction mode (MRM) as described below.

Examination of reactivity of ox-LGD 2 with the thiol in cysteine

As ox-LGD 2 has conjugated double bonds with carbonyl group, purifi ed ox-LGD 2 was examined for its reactivity with the thiol in cysteine as a nucleophile under the similar reaction condition reported for the Michael addition of glutathione to � 7 -PGA 1 methyl ester ( 21 ). Purifi ed ox-LGD 2 (12 ng, 0.033 nmol) dis-solved in 0.05 ml of methanol was mixed with a solution of L -cysteine (4 ng, 0.033 nmol) in 0.1 M phosphate buffer (pH 7.4, 0.05 ml) and incubated at 30°C for 4 h. For reference, purifi ed ox-LGD 2 was similarly incubated without L -cysteine. Next, metha-nol was removed from the reaction mixture by a stream of nitro-gen gas, and the remaining ox-LGD 2 was extracted with ethyl acetate from the aqueous layer acidifi ed with 1 N HCl. After re-moval of ethyl acetate by stream of nitrogen gas, the remaining residue was dissolved in methanol (0.1 ml), and an aliquot (10 � l) of this solution was applied to LC-MS/MS (MRM) to quantify unreacted ox-LGD 2 . This experiment was performed three times.

Preparation of crude PG mixtures from red algae PGs were extracted from G. edulis and G. vermiculophylla by two

different methods for comparison: water extraction and ethanol extraction. The frozen algae (10 g) were fi nely (2-3 mm) and quickly cut on ice and mixed completely. Next, 1.0 g was used for each extraction method. The water extraction was prepared ac-cording to Fusetani and Hashimoto ( 14 ) as follows. The algae (1.0 g) were homogenized with 8 ml of water containing 0.002% (w/v) BHT at 20°C for 60 s. The homogenate was centrifuged at 2,000 g for 15 min. The pH of the supernatant was adjusted to 3.0 with 1N HCl, and the PGs were extracted with 8 ml of ethyl ace-tate. After washing the ethyl acetate layer with 0.8 ml of water, the solvent was evaporated under nitrogen gas, and the residue was dissolved in 2 ml of methanol. The ethanol extraction was pre-pared according to a method by Powell ( 22 ). Briefl y, the algae (1.0 g) were homogenized in 10 ml of ethanol-water 4:1 (v/v) containing 0.002% (w/v) BHT at 20°C for 60 s. The homogenate was shaken at 5°C for 1 h and then centrifuged at 1,000 g for 10 min. A 3 ml volume of the ethanol soluble fat was diluted with the same volume of water, and the pH was adjusted to 3.0 with 1N HCl. A 2 ml volume of the acidic solution was applied to an ODS-silica Sep-Pak cartridge (Waters, Milford, MA), and polar and nonpolar lipids were removed from the column with 10 ml of ethanol-water 15:85 (v/v) and 10 ml of petroleum ether. Next, the PGs were eluted with 6 ml of ethyl acetate. The solvent was evaporated under a stream of nitrogen gas, and the residue was dissolved in 2 ml of methanol. Aliquots of the water (1/200) and ethanol (1/1000) extracts, each from 1.0 g (wet mass) of the al-gae, were injected into an LC-MS/MS in multiple reaction mode (MRM) as described below.

Animal experiments Animal experiments were approved by the Animal Ethical

Committee of Tohoku University. Six normal mice (ddY, male, Fig. 2. COSY spectrum of ox-LGD 2 (0.1 mg, CD 3 CN, 600 MHz). The signal corresponding to C H D 2 CN was set at 1.9 ppm.


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RESULTS

Planar structure of ox-LGD 2 Pure ox-LGD 2 , obtained from G. edulis, showed a UV max

at 267 nm ( � 25,000) in methanol, suggesting the presence of a conjugated triene- or dienone-like structure. Ox-LGD 2 has a molecular formula C 20 H 30 O 6 , as determined by HR-FAB-MS (found at m/z 365.1964 [M-H] � ; calculated for C 20 H 29 O 6 at 365.1964), which is indicative of six degrees of unsaturation. The 1 H NMR (supplementary Fig. I), COSY ( Fig. 2 ), and TOCSY (supplementary Fig. II) spectra were measured to elucidate the structure of ox-LGD 2 . Two car-bon chains of C2-C7 and C13-C20, which contain double bonds at C5-C6 and C13-C14, respectively, were deduced by analyses of the COSY and TOCSY. The stereochemistry of the latter double bond was determined to have an E geom-etry due to its large vicinal 3 J HH value of 16.2 Hz between the olefi nic proton signals of H13 and H14. Additionally, H14 had a correlation with an oxymethine proton signal at 4.19 ppm (H15) in the COSY. These partial structures suggest that ox-LGD 2 is an analog of PG, although a singlet methyl proton signal (Me10) at 1.58 ppm was also observed. In the HMBC spectrum ( Fig. 3 ), obvious long-range correlations

(100 U/ml, 100 � g/ml). The cells were differentiated into mac-rophage-like phenotypes by incubation with 40 nM of phorbol-12-myristate-13-acetate (PMA) at 37°C for 72 h in 5% CO 2 ( 24 ). After washing with PBS, the cells were harvested and frozen at � 80°C. Next, the cells (7.5 × 10 5 cells) were thawed in 1 ml of 40 mM Tris-HCl (pH 7.8) buffer containing 2 mM EGTA, 5 mM trypto-phan, and 2 � M hematin ( 24 ). An ethanolic solution of arachi-donic acid (2 � g/2 � l) was added to this cell suspension, and the suspension was vigorously vortex-mixed. After incubation at 25°C for 5 min, the cell suspension was acidifi ed by addition of 1 N HCl (50 � l), and the lipids were extracted by 4 ml of ethyl acetate. Ethyl acetate was evaporated by a stream of nitrogen gas, and the obtained residue was dissolved in 200 � l of metha-nol. An aliquot (10 � l) of this methanolic solution was injected into an LC-MS/MS in MRM mode for PGE 2 and ox-LGD 2 (see, below).

LC-MS/MS analysis of ox-LGD 2 and PGs The ion transitions m/z 365/177 and 365/195 were monitored

for quantitation of ox-LGD 2 by LC-MS/MS analysis in MRM mode. For further identifi cation of ox-LGD 2 , ox-LGD 2 in the liver of mouse B was analyzed in product ion scan mode, setting [M-H] � m/z 365 as the precursor ion and scanning between m/z 100 and 400. The collision energy was set at � 20 eV.

PGs were quantifi ed by LC-MS/MS in MRM mode by follow-ing the method of Cao ( 25 ) with slight modifi cation. The PGs and ox-LGD 2 were separated using a Cosmosil 5C18AR-II (i.d. 0.46 × 15 cm) column with acetonitrile-water-acetic acid (35:65:0.1, v/v/v) with a fl ow rate of 0.3 ml/min at 25°C for the algal samples to separate PGA 2 and PGB 2 ( Fig. 8 ). For the analy-sis of the chemical reaction products derived from PGH 2 ( Fig. 6 ), mouse tissues ( Fig. 10 ) and the lysate of the THP-1 cells ( Fig. 11 ), an alternative LC solvent, acetonitrile-water-acetic acid (40:60:0.1, v/v/v), was used for faster elution. The ion transi-tions monitored for PGE 2 and PGD 2 were m/z 351/333 and 351/271, and those for PGA 2 , PGB 2 , and PGJ 2 were m/z 333/315 and 333/271. The collision energy was set at � 15 eV for the PGs and at � 20 eV for ox-LGD 2 . The calibration curves were con-structed from the different amounts of each authentic PG and purifi ed ox-LGD 2 injected into the LC-MS/MS instrument and their peak areas in the MRM chromatograms. The calibration curve obtained for each PG and ox-LGD 2 showed good linearity (r 2 = 0.99).

Fig. 3. HMBC spectrum of ox-LGD 2 (0.1 mg, CD 3 CN, 600 MHz, 2,3 J CH =8 Hz, 55 h). The cross peak of H13/C8 was shown more clearly in the spectrum of the crude ox-LGD 2 (data not shown), supporting the assignment of C8. The signal of C H D 2 CN was set at 1.9 ppm and that of CD 3

13 CN was set at 118 ppm as references.

Fig. 4. Planar structure of ox-LGD 2 . A: Ox-LGD 2 with key HMBC and ROESY correlations. B: PGB 2 . C: Predicted structure for ox-LGE 2 .


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Based on these data ( Table 1 , see also HSQC spectrum in supplementary Fig. III), the planar structure of ox-LGD 2 was elucidated as shown in Fig. 4A . From the molecular ion of ox-LGD 2 ([M-H] m/z 365), the major product ions were shown at m/z 177 and 195 ( Fig. 5A ), which were inter-preted as shown in Fig. 5B .

The structure of ox-LGD 2 was also confi rmed by chemi-cal derivation of ox-LGD 2 from PGH 2 as shown in Fig. 6A . The produced ox-LGD 2 was shown as the peak at the same retention time (24.5 min) as that of purifi ed ox-LGD 2 ( Fig. 6B ) by LC-MS/MS (MSM) monitoring the ion transi-tion of m/z 365/195 and 365/177. The yield of ox-LGD 2 from PGH 2 was roughly estimated as 1% (mol/mol) with-out optimization.

to four quaternary carbons at � 105.6 (C11), 127.2 (C8), 155.6 (C12), and 174.9 (C1) ppm were observed. The chemical shifts of C13 and C14 (117.5 and 145.5 ppm) with H13 and H14 (6.46 and 6.39 ppm) and C8 and C12 (127.2 and 155.6) suggests that C13, C14, C8, and C12 are conju-gated olefi n carbons. HMBC correlations clarifi ed these connectivities around the quaternary and carbonyl carbons by giving cross peaks between C8/H13, C11/Me10, C11/H13, C12/Me10, C12/H13, and C12/H14 ( Fig. 4A ). These HMBC correlations suggested that ox-LGD 2 has a fully sub-stituted double bond between C8 and C12, and it is conju-gated with another double bond between C13 and C14 and a methyl-substituted hemiacetal carbon at C11 ( Fig. 4A ). The remaining substitution group at C8 was speculated to be a carbonyl group on the basis of the chemical shift values of C8 (127.2 ppm), C12 (155.6 ppm), C13 (117.5 ppm), and C14 (145.5 ppm), in which the � and � position car-bons (C12 and C14) were shifted to low fi eld. Although the presence of a carbonyl group at C9 was not directly indi-cated by the C9/CH 2 -7 HMBC correlation, the molecular formula of ox-LGD 2 (C 20 H 30 O 6 ) and the resemblance of the UV and NMR spectral data of ox-LGD 2 with that of PGB 2 ( Fig. 4B ; PGB 2 : UV max at 269 nm, � 28500 in methanol; NMR data of PGB 2 , see Table 1 ) strongly suggested the presence of a conjugated carbonyl group at C9. Chemical shift differences for C8 and C12 between ox-LGD 2 and PGB 2 are due to the structural differences between the � , � -unsaturated- -lactone and the cyclopentenone. The LGE 2 -type structure (ox-LGE 2 , Fig. 4C ) was excluded as a possible structure for ox-LGD 2 because the HMBC correlation be-tween the hemiacetal carbon at 105.6 ppm and H13 should be explained by unlike 4 J CH correlation in this structure. The Z geometry of the C5-C6 double bond was indicated by ROE observed between H4/CH 2 -7 ( Fig. 4A ) in the ROESY.

Fig. 5. MS/MS analysis of ox-LGD 2 . A: Product ion spectrum of the [M-H] � ion at m/z 365 of ox-LGD 2 . Purifi ed ox-LGD 2 (18 ng) was injected directly into an MS/MS by an LC pump with methanol (0.2 ml/min). Collision energy: � 20 eV. B: Interpretation of the major product ions.

TABLE 1. NMR spectral data of ox-LGD 2 and PGB 2 in CD 3 CN

Position

ox-LGD 2 PGB 2

� H Multiplicity (coupling

constant J ) � C � H Multiplicity (coupling

constant J ) � C

1 — ND — 175.02 2.25 m 33.0 2.25 m 33.43 1.60 m 24.7 1.58 m 25.24 2.14 m 26.8 2.17 m 26.95 5.40 m 131.1 5.31 m 130.36 5.39 m 125.6 5.26 m 127.87 3.04, 2.95 m 21.5 2.94 m 21.98 — 127.2 — 139.79 — ND — 209.110 1.58 s 25.2 2.60 m 26.211 — 105.6 2.28 dd (10.9, 6.8) 34.112 — 155.6 — 164.313 6.46 d (16.2) 117.5 6.79 d (15.8) 123.414 6.39 dd (16.2, 5.4) 145.5 6.29 dd (15.8, 5.9) 143.915 4.19 brt 71.7 4.20 q (5.9) 72.116 1.48 m 37.5 1.48 m 37.717 1.36 m 25.5 1.37 m 26.218 1.27 m 32.2 1.27 m 32.419 1.27 m 23.1 1.27 m 23.120 0.85 t (7.0) 14.0 0.85 t (7.0) 14.3

Chemical shifts are in � values. Multiplicities and coupling constant J (in parentheses) are in Hertz. CD 3 13 CN

at 118.0 ppm and CHD 2 CN at 1.90 ppm. 13 C at 150 MHz and 1 H at 600 MHz. 13 C chemical shift was roughly determined by HSQC and HMBC correlations. ND, not determined; m, multiple; d, doublet; dd, double doublet; t, triplet; brt, broad triplet; s, singlet; q, quartet.


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chromatograms for the PGs and ox-LGD 2 in the ethanol extracts from the two species of red algae are shown in Fig. 8 . Additionally, the contents of the PGs and ox-LGD 2 in their respective water and ethanol extracts are summarized in Table 2 . Although PGE 2 and PGA 2 were detected in both of these algae, ox-LGD 2 and PGB 2 were identifi ed only in G. edulis , not in G. vermiculophylla , even though the level of PGE 2 was much higher in G. vermiculophylla than in G. edu-lis . PGD 2 and PGJ 2 were less than their limits of detection, 0.056 and 0.13 � g/g, respectively, in both G. edulis and G. vermiculophylla . Except for PGA 2 , PGs and ox-LGD 2 were higher in the ethanol extract than in the water extract.

Identifi cation and quantitation of ox-LGD 2 in mouse tissues

We examined the presence of ox-LGD 2 , PGE 2 , and PGD 2 in seven tissues (testis, liver, kidney, lung, heart, brain, and muscle) of fi ve normal ddY strain male mice (labeled

Examination of reactivity of ox-LGD 2 with the thiol in cysteine

Typical MRM mass chromatograms of purifi ed ox-LGD 2 incubated with or without cysteine are shown in Fig. 7 . The mean and SD values of recovered ox-LGD 2 after incuba-tion with or without cysteine in three repeated experi-ments were 8.5 ± 0.5 and 9.2 ± 0.7 ng, respectively, which were not signifi cantly different. These data suggest that ox-LGD 2 is not reactive with the thiol in cysteine under this condition, even though ox-LGD 2 possesses an � , � -unsaturated carbonyl group. Probably the presence of two constituents at C8 and C12, composed of a long carbon chain, decreases its reactivity with nucleophiles.

Quantitation of PGs and ox-LGD 2 in red algae PGs were extracted by two different methods from two

species of red algae, Gracilaria edulis and G. vermiculophylla , and quantifi ed using LC-MS/MS in MRM mode. MRM ion

Fig. 6. Chemical derivation of ox-LGD 2 from PGH 2. A: Scheme of chemical derivation of ox-LGD 2 from PGH 2 . B: LC-MS/MS (MRM) chromatograms of purifi ed ox-LGD 2 (2 ng) and chemically derived ox-LGD 2 obtained by monitoring the ion transition of m/z 365/195. LC was performed using a Cosmosil 5C18ARII (i.d. 0.46 × 15 cm) column with acetonitrile-water-acetic acid (40:60:0.1, v/v/v) with a fl ow rate of 0.3 ml/min at 25°C.

Fig. 7. Reactivity of ox-LGD 2 with the thiol in cysteine. Purifi ed ox-LGD 2 (12 ng, 0.033 nmol) in 0.05 ml of methanol was mixed with the solution of L -cysteine (4 ng, 0.033 nmol) in 0.1 M phosphate buffer (pH 7.4, 0.05 ml) and incubated at 30°C for 4 h. For reference, purifi ed ox-LGD 2 was similarly incubated without L -cysteine. After incubation, remaining ox-LGD 2 was quantifi ed using LC-MS/MS (MRM). The mean and SD values (n = 3) of recovered ox-LGD 2 after incubation with or without cysteine were 8.5 ± 0.5 and 9.2 ± 0.7 ng, respectively, which were not signifi cantly different between them. Typical MRM mass chromatograms of ox-LGD 2 incubated with (B) and without (A) cysteine are shown. LC was performed using a Cosmosil 5C18ARII (i.d. 0.46 × 15 cm) column with acetonitrile-water-acetic acid (40:60:0.1, v/v/v) with a fl ow rate of 0.3 ml/min at 25°C. MA, measured peak area; MH, measured peak height; RT, retention time.


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� g/g, respectively. When the same sample was homoge-nized in the buffer without BHT, the values were 0.017 � g/g (PGE 2 ) and 0.13 � g/g (ox-LGD 2 ), almost same as those with BHT.

Formation of ox-LGD 2 in the lysate of PMA-treated THP-1 cells

In the lipophilic extract from the lysate of PMA-treated THP-1 cells (7.5 × 10 5 cells), after incubation with arachi-donic acid (2.0 � g) at 25°C for 5 min, the peaks corre-sponding to PGE 2 and ox-LGD 2 were found at 15.3 and 24.7 min, respectively, in the MRM ion chromatograms, as detected by the ion transitions m/z 351/271 and m/z 365/195 ( Fig. 11 ). The amounts of PGE 2 and ox-LGD 2 were quantifi ed as 13.6 ng and 3.8 ng, respectively. PGE 2 and ox-LGD 2 were less than the limits of detection (PGE 2 , 0.1 ng; ox-LGD 2 , 0.3 ng) when arachidonic acid was not

mouse A–E) using LC-MS/MS in MRM mode. As shown in Fig. 9 , ox-LGD 2 was detected ( 0.004 � g/g) in all tissues of three mice (A, B, C) but only in liver and lung of mouse E (0.004 � g/g), whereas ox-LGD 2 was less than limit of detection (<0.004 � g/g) in the tissues of mouse D and the tissues of mouse E (except liver and lung). The content of ox-LGD 2 in the mice tissues was not related to the content of PGE 2 and PGD 2 , and it was different in individual mice. By comparison among the tissues, ox-LGD 2 tended to be high in liver and lung, whereas PGE 2 and PGD 2 were sig-nifi cantly higher in lung (and brain for PGD 2 ) than other tissues. Representative MRM ion chromatograms of PGE 2 and ox-LGD 2 in the liver of mouse B are shown in Fig. 10C , displaying the peak of ox-LGD 2 at 24.6 min, almost same retention time (24.5 min) of purifi ed ox-LGD 2 from the red alga ( Fig. 10A ). Ox-LGD 2 in the liver of mouse B was further identifi ed by the product ion scan spectrum for the peak of ox-LGD 2 (24.0-25.0 min) ( Fig. 10D ), showing the same fragmentation pattern as that of purifi ed ox-LGD 2 ( Fig. 10B ).

Effect of BHT in the homogenization buffer on the level of ox-LGD 2

The level of ox-LGD 2 in mouse liver extracted with the buffer in the presence or absence of BHT was determined using LC-MS/MS in MRM mode to examine whether ox-LGD 2 is produced by autoxidation. In the lipophilic ex-tract from the liver of mouse F homogenized in PBS buffer containing 1 mM EDTA and 0.1% (w/v) BHT, the con-tents of PGE 2 and ox-LGD 2 were 0.017 � g/g and 0.12

Fig. 8. LC-MS/MS (MRM) analysis of PGs and ox-LGD 2 in red algae. MRM chromatograms obtained by monitoring the ion transition of m/z 351/271 for PGE 2 and PGD 2 ; m/z 333/271 for PGA 2 , PGJ 2 , and PGB 2 ; and m/z 365/195 for ox-LGD 2 . A: Authentic mixture of PGs (each 10 ng) and ox-LGD 2 (10 ng). B: Ethanol extract of G. edulis . C: Ethanol extract of G. vermiculophylla. LC was performed using a Cosmosil 5C18ARII (i.d. 0.46 × 15 cm) column with acetonitrile-water-acetic acid (35:65:0.1, v/v/v) with a fl ow rate of 0.3 ml/min at 25°C.

TABLE 2. Contents of PGs and ox-LGD 2 in Gracilaria edulis and G. vermiculophylla

Prostaglandin

Concentrations ( � g/g wet mass)

G. edulis G. vermiculophylla

Water extract Ethanol extract Water extract Ethanol extract

PGE 2 7.7 29 140 260PGD 2 <0.056 <0.056 <0.056 <0.056PGA 2 16 11 42 11PGB 2 3.0 9.4 <0.10 <0.10PGJ 2 <0.13 <0.13 <0.13 <0.13ox-LGD 2 0.72 1.9 <0.15 <0.15


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under the similar condition reported for the Michael addi-tion of glutathione to � 7 -PGA 1 methyl ester ( 21 ). There-fore, we predict ox-LGD 2 to be a detoxifi cation metabolite of LGD 2 .

The reactions that produce ox-LGD 2 from LGD 2 (a two-step oxidation, dehydrogenation of H8 and H12, and oxi-dation of the C9 aldehyde to a carboxylic acid) can be predicted. We presume that these oxidations are enzy-matic oxidations, because the possibility of autoxidation from LGD 2 to ox-LGD 2 in mice and red algae can be ex-cluded by the following observations: i ) ox-LGD 2 was de-tected in the extracts from mouse tissues, which were immediately frozen in liquid nitrogen just after dissection and homogenized in buffer containing BHT; ii ) the level of ox-LGD 2 in the liver of mouse did not increase even when the sample was homogenized in buffer without BHT; and iii ) ox-LGD 2 was found only in G. edulis , not in G. vermiculophylla , even though G. vermiculophylla contained higher levels of PGE 2 than did G. edulis ( Table 2 ).

The stereochemistry of C15 in ox-LGD 2 has not been fully determined by some chemical methods (e.g., Mosher’s method ( 26 )) due to the limited sample amount (0.1 mg). Ox-LGD 2 isolated from G. edulis was shown as a single isomer in the 1 H NMR spectrum (supplementary Fig. I)

added to the lysate (data not shown). We also confi rmed that the yield of ox-LGD 2 was not signifi cantly changed by exclusion of 5 mM tryptophan from the incubation buffer (40 mM Tris-HCl buffer, pH 7.8, containing 2 mM EGTA, 5 mM tryptophan, and 2 � M hematin) ( 24 ) used for this experiment (see supplementary Fig. IV).

DISCUSSION

LGs, reactive byproducts of PG synthesis, have been re-ported to form covalent adducts with proteins ( 4–6 ), DNA ( 7, 8 ), and phosphatidylethanolamine ( 9, 10 ). However, the oxidized forms of LGs, isoLGs, and isoprostanes have not been reported. In the present study, we purifi ed an oxidized LGD 2 (ox-LGD 2 ) from the red alga Gracilaria edu-lis , and we determined its planar structure by spectroscopic methods and by chemical derivation from PGH 2 . Further-more, ox-LGD 2 was identifi ed in mouse tissues and the lysate of PMA-treated THP-1 cells incubated with arachi-donic acid. These results suggest that ox-LGD 2 is a com-mon oxidized metabolite of LGD 2 . Because ox-LGD 2 does not possess a reactive aldehyde in its molecule, it must be less reactive with amines than are LGs. We also confi rmed that the ox-LGD 2 was not reactive to the thiol in cysteine

Fig. 9. Contents of PGE 2 , PGD 2 , and ox-LGD 2 in the tissues of fi ve normal ddY strain male mice (Mouse A–E), 4 weeks of age, 20-21 g body weight. Eicosanoids were extracted from frozen and powdered tissues of mice in liquid nitrogen immediately after dissection. PGE 2 , PGD 2 (A), and ox-LGD 2 (B) were quantifi ed using LC-MS/MS (MRM) as described in the text.


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bearing a -hydroxybutenolide, like ox-LGD 2 , has been re-ported ( 27 ), although we have not tested such activity for ox-LGD 2 due to a limited amount of purifi ed ox-LGD 2 . The level of ox-LGD 2 should be examined in the tissues of several animals, including humans, under various physio-logical conditions.

In summary, our fi rst identifi cation of ox-LGD 2 as a probable detoxifi cation metabolite of LGD 2 provides methods to examine its formation in animal tissues and cells.

and the MRM ion chromatograms ( Fig. 8 ). Similarly, the peaks corresponding to ox-LGD 2 detected by LC-MS/MS (MRM) were single peaks in both the lipophilic extracts from mouse tissues ( Fig. 10 for liver) and the PMA-treated THP-1 cell lysate ( Fig. 11 ). We also confi rmed that the op-tical rotation and 1 H NMR of PGE 2 isolated from red algae were identical to those of authentic PGE 2 , which was de-rived from PGH 2 ( Fig. 1 ). Therefore, the stereochemistry at C15 of ox-LGD 2 is presumed to be in an S confi guration like PGE 2 ( Fig. 1 ). However, the stereochemistry at C11 in ox-LGD 2 could be racemic due to the nature of the he-miketal, which could mean that the two diastereomers of ox-LGD 2 were not separated by 1 H NMR and MRM. We admit that this point should be further confi rmed.

Although it is reported that approximately 20% of PGH 2 nonenzymatically converts to both of LGE 2 and LGD 2 ( 1 ), ox-LGE 2 ( Fig. 3C ), the regioisomer of ox-LGD 2 , was not found in this study in the red alga G. edulis by screening using LC-diode array detection and LC-MS in single ion monitoring (SIM) mode. The presence of ox-LGE 2 in mouse tissues and the THP-1 cell lysate has not been fully examined by LC-MS/MS due to lack of authentic ox-LGE 2 .

In this study, we measured the contents of PGE 2 , PGD 2 , and ox-LGD 2 in liver of six mice (mouse A–F). The con-tent of ox-LGD 2 in the mice was 15, 60, 14, <4, 5, and 130 ng/g, respectively, and the content of PGE 2 was 300, 10, 14, 6, 8, and 17 ng/g, respectively, suggesting that the con-tent of ox-LGD 2 in liver differed in individual mice and was unrelated to the content of PGE 2 .

Further study will be needed to examine the physiologi-cal functions of ox-LGD 2 and to identify the predicted en-zymes that catalyze the oxidation of LGD 2 to ox-LGD 2 . Interestingly, a synthetic inhibitor of prostanoid production

Fig. 10. Representative quantitation and identifi cation of ox-LGD 2 in the liver of mouse B. A: MRM ion chromatograms of the mixture of authentic PGE 2 (0.7 ng) and purifi ed ox-LGD 2 (2.5 ng). B: Product ion spectrum of the [M-H] � ion at m/z 365 of ox-LGD 2 (purifi ed, 1.0 ng) eluted at 24.0-25.0 min. C: MRM ion chromatograms of PGE 2 (0.01 ng) and ox-LGD 2 (0.06 ng) in an aliquot (1/20) of the lipophilic extract from the liver of mouse B (20 mg). D: Product ion spectrum of ox-LGD 2 (0.6 ng) eluted at 24.0-25.0 min in the liver of mouse B. LC was performed using a Cosmosil 5C18ARII (i.d. 0.46 × 15 cm) column with ace-tonitrile-water-acetic acid (40:60:0.1, v/v/v) with a fl ow rate of 0.3 ml/min at 25°C. Collision energy for MRM and the product ion scan: � 15 eV for PGE 2 , � 20 eV for ox-LGD 2 .

Fig. 11. Formation of PGE 2 and ox-LGD 2 by PMA-treated THP-1 cell lysate incubated with arachidonic acid. The lysate of PMA-treated THP-1 cells (7.5 × 10 5 cells) was incubated with arachidonic acid (2.0 � g) at 25°C for 5 min. The lipophilic extract from this lysate was subjected to LC-MS/MS analysis for PGE 2 and ox-LGD 2 in MRM mode. The amounts of formed PGE 2 and ox-LGD 2 were quantifi ed as 13.6 ng and 3.8 ng, respectively. LC was performed using a Cosmosil 5C18ARII (i.d. 0.46 × 15 cm) column with acetonitrile-water-acetic acid (40:60:0.1, v/v/v) with a fl ow rate of 0.3 ml/min at 25°C.


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identiﬁ cation of novel oxidized levuglandin d in marine ... · ﬁ ed from this alga as follows....

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