ma at pH 8.0 at 37°C. Similar conversion was also found by incubating PGD2 .... via a Van den Berg type solventless injector. ..... Whittle, B. J. R., Moncada, S. & Vane, J. R. (1978) Prosta- ... Lowry, Q. H., Rosebrough, N. J., Farr, A. L. & Randall,.
Proc. Natl. Acad. Sci. USA Vol. 81, pp. 1317-1321, March 1984 Biochemistry
9-Deoxy-A9,A12-13,14-dihydroprostaglandin D2, a metabolite of prostaglandin D2 formed in human plasma (dehydration product of prostaglandin D2/serum albumin/cell growth inhibition)
YOSHIHARU KIKAWA*, SHUH NARUMIYA*, MASANORI FUKUSHIMAt, HIROHISA WAKATSUKAt, AND OSAMU HAYAISHI§ *Department of Medical Chemistry, Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto 606, Japan; tDepartment of Internal Medicine and Laboratory of Chemotherapy, Aichi Cancer Center, Chikusa-ku, Nagoya 464, Japan; tResearch Institute, Ono Pharmaceutical Co., Shimamoto, Mishima, Osaka 618, Japan; and §Osaka Medical College, Daigaku-cho, Takatsuki, Osaka 569, Japan
Contributed by Osamu Hayaishi, November 8, 1983
Incubation of prostaglandin D2 (PGD2) with ABSTRACT human plasma yielded a product that has been identified as 9-
deoxy-9,10-didehydro-12,13-cdidehydro-13,14-dihydro-PGD2 (9-deoxy-_9,9'2-13,14-dihydro-PGD2). The identification was based on mass spectrometry, UV spectrometry, mobilities and retention time on TLC and HPLC, and NMR. The conversion of PGD2 to this product was dependent on the incubation time and the amount of plasma added to a reaction mixture and was abolished by prior boiling. The conversion rate of PGD2 to this metabolite was 0.03 nmol/min per mg of protein of whole plasma at pH 8.0 at 37°C. Similar conversion was also found by incubating PGD2 with human serum albumin added at the concentration found in plasma. These results suggest that the conversion of PGD2 to this product is catalyzed by the enzymatic action of a plasma protein, probably serum albumin. The biological activities of this compound were examined in several systems. It showed negligible activity in inhibition of human platelet aggregation and relaxation of rabbit stomach strip. On the other hand, it exhibited a three times stronger inhibitory activity (IC50, 1.8 ,M) than PGD2 (IC50, 5 ,uM) on the growth of L-1210 cultured cells.
Prostaglandin (PG) D2 is formed in a variety of tissues and cells and modulates their functions under various physiological and pathological conditions. For example, it is produced during platelet aggregation and works as a negative feedback modulator of the aggregation process (1-3). It is produced by mast cells during IgE stimulation and modifies the anaphylaxis process (4, 5). It is also produced in the central nervous system of mammals and is involved in brain functions such as hypothermia, sleep, and luteinizing hormone secretion (610). A potential antineoplastic effect of PGD2 has been reported (11) and it has been found that 9-deoxy-A9-PGD2, a dehydration product of PGD2, has about three times stronger growth inhibitory effect than PGD2 on L-1210 leukemia cultured cells (12). The present study was undertaken to explore the possible formation of this PGD2 dehydration product in mammals. We report here the conversion in human plasma of PGD2 to a compound that has been identified as 9deoxy-A9, A12-13,14-dihydro-PGD2 and describe some properties of this metabolite.$
MATERIALS AND METHODS Materials. [5,6,8,9,12,14,15-3H]PGD2 (100 Ci/mmol; 1 Ci = 37 GBq) was purchased from New England Nuclear. PGB2, PGD2, and 9-deoxy-A9-PGD2 were synthetic products from Ono Pharmaceutical (Osaka, Japan). Human serum albumin (fatty acid free) was from Miles. ADP was obtained
from Sigma. Dimethylisopropylsilyl (Me2iPrSi) imidazole and methoxyamine hydrochloride were from Tokyo Kasei (Tokyo). Sep-pak silica and Sep-pak C18 cartridges were from Waters Associates. Precoated silica gel plates [G60(F254)] with concentration zones and silica gel 60 for column chromatography were from Merck. Sephadex LH-20 was a product of Pharmacia. Solvents used in the extraction of PGD2 metabolites for identification were distilled before use. All other chemicals were of reagent grade. Pregaration of 9-Deoxy-A9,Al2-13,14-dihydro-PGD2. 9-Deoxy-A -GD2 was synthesized as described (12), and conversion of 9-deoxy-A9-PGD2 to 9-deoxy-A9,A&12-13,14-dihydroPGD2 was carried out as described by Bundy et al. (13) for the conversion of PGD2 to A12-13,14-dihydro-PGD2. 9Deoxy-A9-PGD2 (48 mg) was dissolved in 3 ml of tetrahydrofuran and the solution was stirred at 4°C. 1,5-Diazabicyclo(4.3.0)non-5-ene (17.8 mg) in 0.18 ml of tetrahydrofuran was added to the solution and the mixture was stirred at room temperature overnight. The mixture was diluted with 30 ml of water, and the product was separated out by extraction with 50 ml of ethyl acetate. The ethyl acetate extract was washed successively with 1.2 M hydrochloric acid, three times with excess water, and finally with a saturated NaCl solution. After being dried over anhydrous MgSO4 and concentrated in vacuo, the crude product was purified in silica gel column chromatography (30 g of silica gel 60) with a solvent of ethyl acetate/n-hexane/methyl alcohol (50:50:2) to afford 9 mg of 9-deoxy-A9,Al2-13,14-dihydro-PGD2 as a colorless oil; Rf, 0.36 (silica gel TLC with ethyl acetate/benzene/acetic acid, 50:50:2); IR, 2930, 1700, 1640, 1580, 1232, and 1028 cm-1; NMR (C2HCl3), 6 7.5 (1H, dd C9 H), 6.56 (1H, t, C13 H), 6.35 (1H, dd, C10 H), 5.48 (2H, m, C5,C6 H), 3.88 (1H, m, C15 H), and 3.44 (1H, m, C8 H); mass spectrum (direct inlet), ions at m/z 334 (M), 316, 245, and 236; UV (EtOH) Xmax 244 nm (e 6100). Preparation of Human Plasma. Blood, 50 ml, was obtained from a healthy volunteer into a plastic tube containing 5 ml of 3.8% sodium citrate, and the mixture was centrifuged at 1000 x g for 15 min. Plasma was then dialyzed against two changes of 2 liters each of 50 mM Tris HCl buffer at pH 7.4 or pH 8.0. Enzyme Assay. Enzymatic activity was assayed as follows. The standard mixture contained 150 uM [3H]PGD2 (4000 cpm/nmol), dialyzed plasma, and 50 mM Tris HCl buffer (pH 7.4 or pH 8.0) in a final reaction volume of 0.4 ml. Reactions were carried out by incubating the mixture at 37°C with constant shaking and terminated by acidification with 1 M Abbreviations: PG, prostaglandin; Me2iPrSi, dimethylisopropylsilyl; GC/MS, gas chromatography/mass spectrometry. part of this work was presented at the Fifty-Sixth Annual Meeting of the Japanese Biochemical Society, Sept. 29-Oct. 2, 1983, Fukuoka, Japan.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1317
1318
Biochemistry: Kikawa et
Proc. NatL Acad Sci. USA 81
at
HCl to pH 3.0. The acidified mixture was directly applied on Sep-pak C18 cartridge, and products were eluted with 10 ml of ethyl acetate as described (14). After evaporation in vacuo, the residues were dissolved in 50 1.d of ethyl ether and applied 2 cm wide on a silica gel thin layer. Authentic PGB2, PGD2, 9-deoxy-A9-PGD2, and 9-deoxy-A9,A'2-13,14-dihydro-PGD2 were applied as markers at least 2 cm away from the samples. TLC was carried out with a solvent of benzene/ethyl acetate/acetic acid (50:50:2). After development, radioactive zones on the TLC plates were located by a radiochromatogram scanner Packard model 7201, and markers were visualized by exposure to iodine vapor. Silica gel zones
a
corresponding to PGD2, 9-deoxy-A9,A12-13,14-dihydroPGD2, and 9-deoxy-A9-PGD2 were scraped, respectively, and the rest of the silica gel was scraped altogether. Radioactivity in the silica gel was measured with a Packard liquid scintillation spectrometer (model 460C) in a toluene scintillator. Since 3H at position 12 was lost during conversion of PGD2 to 9-deoxy-A9,A12-13,14-dihydro-PGD2, quantification of the latter compound was carried out on the basis of the specific activity corrected for the loss of this tritium. Isolation of 9-Deoxy-A,9 A2-13,14-dihydro-PGD2. The reaction product, 9-deoxy-A ,A 2-13,14-dihydro-PGD2, was isolated on a large scale. The reaction mixture was 50 mM Tris HCl, pH 8.0/1 mM [3H]PGD2 (140 cpm/nmol) and a 4570% ethanol fraction of plasma dialyzed at pH 8.0 (800 mg of protein) in a total vol of 16 ml. The reaction was carried out at 370C for 180 min and terminated by acidification with 1 M HCl to pH 3.0. The acidified reaction mixture was divided into 16 tubes and partially purified by the use of Sep-pak C18 cartridges. After evaporation in vacuo, the residues of ethyl acetate extract from the Sep-pak cartridges were dissolved in 100 Al of ethyl ether and applied 15 cm wide on a silica gel thin layer with a concentration zone. Chromatography was carried out with a solvent of benzene/ethyl acetate/acetic acid (50:50:2). The major product with an Rf value of 0.36 was extracted from the silica gel with ethyl acetate. After evaporation to dryness, the residues were dissolved in 100 ,l of acetonitrile and the product was further purified by reversed-phase HPLC. HPLC was carried out using a system from Waters Associates (a 6000A solvent-delivering systems with a model U6K injector) and a semi-preparative Waters Bondapak C18 column (7.8 mm x 30 cm). Elution was accomplished under isocratic conditions with a solvent of 0.017 M KH2PO4/CH3CN/isopropanol (30:17:5) at 1 ml/min. Fractions were collected every 2 ml, and the radioactivity in an aliquot of each fraction was measured to establish the chromatographic profile. 9-Deoxy-A9,A'&2-13,14-dihydroPGD2 typically eluted at 18 min. Gas Chromatography/Mass Spectrometry (GC/MS) of the Product. Derivatizations were carried out essentially as reported (15) except that methylation with diazomethane was done at -20°C to avoid the formation of by-products. GC/MS was carried out using a Hitachi model M80A instrument. Data were analyzed with a Hitachi model M-003 data analyzer computer system. The column used was a crosslinked OV-1 fused silica capillary column (25 m x 0.31 mm i.d., Hewlett-Packard). Helium was used as a carrier gas with a flow rate of 10 ml/min. The column temperature was maintained isothermal at 240°C, and samples were applied via a Van den Berg type solventless injector. Mass spectrometry was done at an ionization potential of 20 eV and an ionization current of 120 uA. Bioassays. Relaxation of a rabbit transverse stomach strip was examined as follows. The strip was prepared as described (16). The tissue was suspended vertically and superfused with a Krebs solution containing a mixture of antagonists at 5 ml/min at 370C (12). Relaxation of the tissue was recorded via an isometric transducer T7-8 (Toyo Boldwin, Tokyo) coupled to an amplifier type 7236 (San-ei Instrument,
(1984)
Tokyo). Compounds were dissolved in ethanol at a concentration of 1 mg/ml and diluted with 50 mM Tris HCl (pH 8.0) before application. Platelet aggregometry was carried out using an aggregometer Hematracer 1 model PAT 2M (Niko Bioscience, Tokyo) as described (17). Inhibition of L-1210 cell growth was examined as described (11). Miscellaneous. UV absorption spectra were recorded on a Shimadzu UV300 spectrophotometer. NMR was recorded in C2HCl3 with a Varian NMR spectrometer XL-200. Protein concentration was determined according to the method of Lowry et al. (18) with bovine serum albumin as standard. RESULTS Formation and Identification of 9-Deoxy-&9, A1213914dihydro-PGD2. When PGD2 was incubated with dialyzed human plasma, it was converted to two products (Fig. 1). The major product (compound 2) accounted for more than 80% of the total amount of products, while the minor product (compound 1) accounted for only 10%. Boiling of plasma markedly decreased the formation of compound 2 but did not affect the formation of compound 1 significantly. These results suggest that compound 2 is formed by the enzymatic action of human plasma while compound 1 is a nonenzymatically degraded product. To identify the structure of compound 2, we isolated this product on a large scale and examined it by UV spectrometry, GC/MS, and NMR. The UV absorption spectrum of compound 2 is shown in Fig. 2; the compound has absorption maxima at 244 nm with an (EtOH) of 6100 and a small shoulder around 300 nm, suggesting an enone type structure. The mass spectrum of the methyl ester methoxime-Me2iPrSi ether derivative of compound 2 is shown in Fig. 3; ions are found at m/z 477 (M), 462 (M - 15, loss of *CH3), 446 (M 31, loss of -OCH3), 434 [M 43, loss of *CH(CH3)2], 406 (M 71, loss of -C5H11), 359 (M 118, loss of -HOMe2iPrSi), and 201 (M - 276, base peak, Me2iPrSiO+=CH-C5H11). The molecular ion at m/z 477 suggeste
-
-
I
II
I
+ Plasma
B0 l E u ._
._
Boiled ._
0
200'0
a
niIA15 0
5
Distance
10
(cm)
FIG. 1. TLC of reaction products. Reaction mixtures containing 50 jM [3H]PGD2 (200,000 cpm/nmol) in 100 of plasma that had been dialyzed at pH 8.0 were incubated at 370C for 2 hr. Aliquots were analyzed with a solvent of benzene/ethyl acetate/acetic acid (50:50:2). (Upper) Dialyzed human plasma (7 mg of protein) at pH 8.0. (Lower) Dialyzed human plasma was boiled (100'C) for 5 min prior to incubation. Markers and products were localized by exposure to iodine vapor. D2, PGD2; B2, PGB2; 1, compound 1; 2, compound 2. Incubation of PGD2 with undialyzed plasma yielded essen-
tially
same
results.
Biochemistry: Kikawa et aL
Proc. Natd Acad Sci USA 81 (1984)
0
.0
01 200
250
300
350
Wavelength (nm) FIG. 2. Comparison of UV spectra of compound 2 (upper curve) and authentic 9-deoxy-A9,A'2-13,14-dihydro-PGD2 (lower curve) in ethanol. Concentrations were 0.05 and 0.1 mM for the authentic compound and compound 2, respectively.
ed that compound 2 had lost one hydroxyl group from the precursor molecule, PGD2, because it is 118 mass units (= HOMe2iPrSi) less than that of the methyl ester methoxime Me2iPrSi ether of the latter, m/z 595. Compound 2, however, showed the base peak at m/z 201, which was also the base peak of PGD2, suggesting that the 15-hydroxyl group was retained in the molecule. These results suggested that compound 2 had lost a hydroxyl group at position 9 of PGD2 and undergone a double bond shift to form an enone structure. On the basis of these analyses, we synthesized 9-deoxy-A9,A12-13,14-dihydro-PGD2 and compared its properties with those of compound 2. As shown in Figs. 2 and 3, the UV and mass spectra of the two compounds were identical. Identification was further confirmed by NMR and mobilities on TLC and HPLC. Although NMR of purified compound 2 revealed the presence of contaminating materials in the aliphatic regions, it also showed signals at 8 (ppm) 7.57 (dd, C9 H), 6.56 (t, C13 H), 6.35 (dd, C10 H), 5.48 (m, C5,C6 H), 3.88
1319
(m, C15 H), and 3.44 (m, C8 H), which were identical with those of the authentic compound. On TLC, compound 2 showed exactly the same mobilities as authentic 9-deoxyA9,A12-13,14-dihydro-PGD2 with three solvent systems. Solvent systems used in TLC analyses were A [ethyl acetate/ benzene/acetic acid (50:50:2)], B [benzene/dioxane/acetic acid (66:33:1)], and C [chloroform/tetrahydrofuran/acetic acid (10:2:1)]. The Rf values were 0.36, 0.45, and 0.83 with solvents A, B, and C, respectively. On HPLC, compound 2 showed a single peak that had a retention time identical with authentic 9-deoxy-A9,A'2-13,14-dihydro-PGD2. On the basis of these analyses, we have identified compound 2 as 9-deoxy-A19,A12-13,14-dihydro-PGD2. Compound 1 was not isolated on as large a scale as compound 2 and, therefore, the detailed structural analyses were not carried out for this compound. However, it showed the same mobilities as authentic 9-deoxy-A9-PGD2 on TLC with the three solvent systems; the Rf values were 0.30, 0.43, and 0.81 with solvents A, B, and C, respectively. As shown in Fig. 4A, the formation of 9-deoxy-A9,4a213,14-dihydro-PGD2 from PGD2 was proportional to the volume of dialyzed plasma added to the reaction mixture and was completely abolished by prior boiling. It was also proportional to the time of incubation up to 120 min (Fig. 4B). The reaction rate at pH 8.0 was about 1.5 times that at pH 7.4. These results suggested the enzymatic nature of this reaction. On the contrary, the formation of 9-deoxy-A9-PGD2 was not proportional to either plasma volume in the reaction volume or incubation time (data not shown). Fitzpatrick and Wynalda (19) have reported that, in the presence of serum albumin, PGD2 undergoes time-dependent degradation to an unidentified compound. We examined the possibility that serum albumin catalyzed the above reaction by adding equivalent amounts of human serum albumin instead of dialyzed plasma. As shown in Fig. 4A, serum albumin catalyzed the conversion of PGD2 to 9-deoxy-A9,412-13,14-dihydro-PGD2 as efficiently as whole plasma, suggesting that serum albumin in dialyzed plasma worked as a catalyst in the reaction. A
100
B C
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_i O
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-
c
>0 0
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0 c
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>%
._1
._
12 0 6 Plasma Protein (mg) 0 4 8 Serum Albumin(mg)
0 100
5 30 60 Time (min)
0
50
o
ILl
100
m/z
FIG. 3. Mass spectra of the methyl ester methoxime Me2iPrSi ether derivatives of compound 2 (A) and authentic 9-deoxy-A94,12-
13,14-dihydro-PGD2 (B).
FIG. 4. Effects of amounts of dialyzed human plasma and human serum albumin on rate of conversion of PGD2 to 9-deoxy-A9,A'213,14-dihydro-PGD2 (A) and time course of conversion of PGD2 to 9-deoxy-A9,'A2-13,14-dihydro-PGD2 (B). (A) Various amounts of human plasma that had been dialyzed at pH 7.4 (O) or pH 8.0 (e) were added to the reaction mixture (total vol, 0.4 ml) and incubation was carried out at 37°C for 60 min. In control experiments, dialyzed plasma at pH 7.4 (O) and pH 8.0 (-) was boiled (100°C) for 5 min before addition to the reaction mixture. Human serum albumin was dissolved in 50 mM Tris-HCl at pH 7.4 (A) or at pH 8.0 (A) and then added to the reaction mixture at concentrations equal to its plasma concentration. (B) PGD2 (60 nmol) was incubated with dialyzed human plasma (5.6 mg of protein) at pH 8.0 (-) or pH 7.4 (o) in a total
vol of 0.4 ml for the indicated times. In control experiments, boiled dialyzed plasma was used in incubations at pH 8.0 (n) and pH 7.4 (o).
Biochemistry: Kikawa et aL
1320
Proc. NatL Acad Sd USA 81
Biological Properties of 9-Deoxy-A9,A92-13,14-dihydroPGD2. We examined the biological activity of 9-deoxyA9,A"2-13,14-dihydro-PGD2 in several systems by using the synthetic compounds and compared them with those of PGD2. The antiaggregatory activity of PGD2 is compared with that of 9-deoxy-A9,A4 -13,14-dihydro-PGD2 in Fig. 5A. PGD2 inhibited platelet aggregation induced by 10 ,uM ADP in a dose-dependent manner; the IC50 value for PGD2 was 40 nM and at 200 nM PGD2 platelet aggregation was completely inhibited. On the contrary, 9-deoxy-A9,A'2-13,14-dihydroPGD2 (200 nM) inhibited platelet aggregation by only 10% and, even at higher concentrations, the extent of inhibition never exceeded 10%. Similar results were also obtained on a c
A
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Concentrationf(/4M )
0
100
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Dose ( ng ) -100
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smooth muscle relaxing activity (Fig. SB). PGD2 relaxed the rabbit transverse stomach strip in a dose-dependent manner between 5 and 200 ng with an ED50 value of 30 ng. 9-DeoxyA&9,A12-13,14-dihydro-PGD2, on the other hand, caused only 20% relaxation at 1000 ng. Thus, in both inhibition of platelet aggregation and relaxation of stomach strip, 9-deoxy-A9,A1213,14-dihydro-PGD2 showed negligible activity. On the contrary, this compound elicited much stronger inhibitory activity on growth of L-1210 cultured cells than did the precursor, PGD2 (Fig. 5C). 9-Deoxy-A9,A'2-13,14-dihydro-PGD2 inhibited cell growth in a dose-dependent manner from 0.5 uM with an IC50 value of 1.8 gM. Under the same conditions, the IC50 value of PGD2 was 5 ,tM.
DISCUSSION
10
a'
(1984)
50
0
L)
1
2
5
10
Concentration ( uM )
FIG. 5. Dose-response curves of effects of 9-deoxy-A&9,A213,14-dihydro-PGD2 and PGD2 on the inhibition of human platelet aggregation (A), on the relaxation of isolated rabbit transverse stomach strip (B), and on the growth of L-1210 leukemia cells (C). (A) Human platelet-rich plasma (250 ;A) was incubated for 1 min at 370C and then for 1 min with various concentrations of 9-deoxy-A9,A1213,14-dihydro-PGD2 and PGD2, after which aggregation was initiated by addition of 10 ;LM ADP. (B) The tissue was suspended vertically and superfused with a Krebs solution and various doses of compounds were applied directly to the tissue. (C) L-1210 leukemia cells were cultured in the presence of various concentrations of 9deoxy-A9,Al12-13,14-dihydro-PGD2 and PGD2. After 4 days, the number of cells that excluded trypan blue was counted. In this experiment, 100%1o represents a cell number of 281 x 104 cells per ml. O, 9-Deoxy-A9,A12-13,14-dihydro-PGD2; *, PGD2.
A strong growth inhibitory activity has been found for 9deoxy-A9-PGD2, a dehydration product of PGD2, and the possible formation of this compound in biological systems has been suggested (12). This study has revealed that 9deoxy-A9,A12-13,14-dihydro-PGD2 instead of 9-deoxy-A9PGD2 is enzymatically formed from PGD2 in human plasma. Dehydration of PGE to PGA has been reported by several investigators (20, 21). Recently, Fitzpatrick and Wynalda (19) reported that serum albumin catalyzes such dehydration and that similar dehydration also occurs with PGD2 but they did not identify the structure of the PGD2 product. When we added, instead of dialyzed plasma, human serum albumin to the incubation mixture at concentrations found in plasma, we found that serum albumin catalyzes the conversion of PGD2 to 9-deoxy-A9,A12-13,14-dihydro-PGD2 with efficiency equal to that of dialyzed plasma (Fig. 4). These results suggest that the formation of 9-deoxy-A9,A12-13,14-dihydroPGD2 in dialyzed plasma is catalyzed by serum albumin itself. Because 9-deoxy-A&9-PGD2 was also present in the reaction mixture as a plausible nonenzymatic product, one conceivable reaction sequence is that first PGD2 is dehydrated to 9-deoxy-A9-PGD2 and then the latter compound undergoes the shift of the 13,14 double bond to the 12,13 position. In our preliminary work, we found that when 9-deoxy-A9[3H]PGD2 is added to dialyzed plasma, it is quickly converted to 9-deoxy-A9,A12-13,14-[3H]dihydro-PGD2. However, isotope trapping experiments using [ H]PGD2 and unlabeled 9-deoxy-A -PGD2 failed to trap the radioactivity, because excess amounts of 9-deoxy-A9-PGD2 inhibit the conversion of [3H]PGD2 (unpublished observation). Thus, an intermediate role of 9-deoxy-A9-PGD2 remains unsettled. The name of PGJ2 has been proposed for 9-deoxy-A9-PGD2 (12) and, in that context, 9-deoxy-A9,A'12-13,14-dihydro-PGD2 should be named A12-13,14-dihydro-PGJ2. The natural occurrence of 9-deoxy-A9,A12-13,14-dihydroPGD2 as well as 9-deoxy-A9-PGD2 has not yet been reported. In 1979, Ellis et al. (22) reported detailed analyses of urinary metabolites of PGD2 in monkey. According to their results, PGD2 is either metabolized on its side chain with its ring structure intact or first converted to PGF2a and then further metabolized. Their findings have led to discovery of the two PGD2-catabolizing enzymes-that is, NADP-linked PGD2 dehydrogenase (17, 23) and PGD2 11-keto reductase (24-26). Ellis et al. did not refer to any compound with a PGJ2-type ring structure. However, they left about 50% of the urinary products, containing most of the nonpolar metabolites, unanalyzed (22). Thus a possibility remains that 9-deoxyA9,A12-13,14-dihydro-PGD2 occurs as a natural metabolite of PGD2. We have found that 9-deoxy-A9,Al2-13,14-dihydro-PGD2 shows a unique spectrum of biological activities. It lacks antiaggregatory activity for human blood platelet and also is inactive for relaxation of some smooth muscle, in both of which PGD2 is quite active. On the contrary, as 9-deoxy-A9PGD2 (12), it showed three times stronger growth inhibitory
Biochemistry: Kikawa et aL activity on L-1210 cultured cells than PGD2 itself. Because PGD2 and 9-deoxy-A9-PGD2 are converted to 9-deoxyA9,A12-13,14-dihydro-PGD2 in the presence of plasma, most growth inhibitory activity of the former compounds would be exerted by their conversion to the latter. Thus, it is likely that 9-deoxy-A9,A12-13,14-dihydro-PGD2 is the ultimate compound that exerts antineoplastic action of PGD2 and, because of the lack of other PGD2 activities, it might serve as a prototype for further development of antineoplastic PG analogues. Note Added in Proof. Since completion of this manuscript, the catabolism of PGD2 by serum albumin and identification of reaction products have also been reported by Fitzpatrick and Wynalda (27).
We are grateful to Drs. T. Miyamoto and Y. Arai of Ono Central Research Institute for their kind assistance and discussion and to Prof. M. Sudo of Fukui Medical College for his encouragement to Y.K. This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science And Culture of Japan and by grants from the Japanese Foundation of Metabolism and Diseases and the Japan Brain Foundation. 1. Oelz, O., Oelz, R., Knapp, H. R., Sweetman, B. J. & Oates, J. A. (1977) Prostaglandins 13, 225-234. 2. Whittle, B. J. R., Moncada, S. & Vane, J. R. (1978) Prostaglandins 16, 373-388. 3. Watanabe, T.; Narumiya, S., Shimizu, T. & Hayaishi, 0. (1982) J. Biol. Chem. 257, 14847-14853. 4. Steinhoff, M. M., Lee, L. H. & Jakschik, B. A. (1980) Biochim. Biophys. Acta 618, 28-34. 5. Lewis, R. A. & Austen, K. F. (1981) Nature (London) 293, 103-108. 6. Abdel-Halim, M. S., Hamberg, M., Sjoquist, B. & Anggard, E. (1977) Prostaglandins 14, 633-643. 7. Narumiya, S., Ogorochi, T., Nakao, K. & Hayaishi, 0. (1982) Life Sci. 31, 2093-2103.
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