Metabolism of Prostaglandin E, in Guinea Pig Liver

Vol.

THE JOURNAL OF Bm~oorca~ 245, No. 19, Issue of October Printed

Metabolism I. IDENTIFICATION

CHEMISTRY 10, pp. 5107-5114,

1970

in U.S.A.

of Prostaglandin OF SEVEN

E, in Guinea

Pig Liver

METABOLITES* (Received for publication,

May 19, 1970)

MATS HAMBERG AND ULF ISRAELSSON From the Department

of Medical Chemistry, Royal Veterinary

The present paper reports the isolation and identification seven metabolites formed from PGEz in a preparation guinea pig liver.

SUMMARY

In an earlier study liar-hydroxy-9,15-diketoprost-5-enoic acid and 1lcu ,15-dihydroxy-9-ketoprost-5-enoic acid were isolated after incubation of PGEgl with the soluble fraction of homogenates of guinea pig lung (1). PGE, and PGEI were metabolized by similar reactions, i.e. reduction of the Al3 double bond and dehydrogenation of the C-15 alcohol group (2, 3). These reactions seemed to have biological significance as judged from the structures of the urinary metabolites of PGEs in guinea pig (4), rat,* and man (5). * This work was supported by grants from the Swedish Medical Research Council (Projects 13X-2828 and 13X-217, the latter given to Professor Bengt Samuelsson). 1 The abbreviations used are: PGEI, prostaglandin El (lla, 15(S)-dihydroxy-9-ketoprost-13-enoic acid); PGE2, prostaglandin Ez (lla, 15(S)-dihydroxy-9-ketoprosta-5,13-dienoic acid) ; PGEI, prostaglandin Es (1101,15(S)-dihydroxy-9-ketoprosta-5,13,17-trienoic acid) ; PGF*,, prostaglandin F~*(9a, llcy, 15(S)-trihgdroxgprosta-5,13-dienoic a&d); PGFzp, prostaglandin F28 (9p, ll,, 15(i)trihvdroxvprosta-5.13.dienoic acid) : PGB2. m-ostaelandin BI (15(&-hy&Eoxy-9-kktoprosta-5,8(12j,’l3-trien& acid);TMSi, tri; methylsilyl; MO, 0-methyloxime. 2 K. G&en, data to be published.

EXPERIMENTAL

of of

PROCEDURE

Materials PGEz was generously provided by Dr. J. Pike, The Upjohn Company, Kalamazoo, Michigan. [17,18-3Ha]PGEa was prepared as previously described (1) and added to unlabeled PGE2 to make a specific activity of 2.8 PCi per pmole. PGFz, and PGFv were prepared by reduction of PGEz with sodium borohydride followed by reversed phase partition chroma@zrapb (6, 7). lla, 15.Dihydroxy-9-ketoprost-5-enoic acid and lla-hydroxy9,15-diketoprost-5-enoic acid were obtained by incubation of PGEz with the 100,000 X g supernatant of a homogenate of guinea pig lung as earlier described (1). methyl Methyl 9a!, 11 Q, 16(S)-Trihydroxyprostanoate-The ester of PGFk, 5 mg, was dissolved in 3 ml of ethanol and stirred with 10 mg of 5% palladium on carbon under hydrogen gas for 60 min. After purification by preparative thin layer chromatography 4 mg of methyl 9a, lla!, 15(S)-trihydroxyprostanoate were obtained (Rp = 0.64). The TMSi ether derivative was analyzed by gas-liquid chromatography (C-24.4 with 1.5 To SE-30, C-23.2 with 10% EGSS-X; see Table I) and by mass spectrometry. Ions of high intensity were present at m/e 517 (M - 71; loss of .(CHg)&Ha), 498 (M - 90; loss of TMSiOH), 467 (M - (90 + 31); loss of TMSiOH plus .0CH3), 427 (M (90 + 71) ; loss of TMSiOH plus +(CHt)&Ha), 408 (LW - 2 x 90; loss of 2 TMSiOH), 355 (M - (90 + 143) ; loss of TMSiOH plus .(CH,)&OOCH3), 337 (M - (2 x 90 + 71); loss of 2 TMSiOH plus .(CHp)&Ha), 297 (M - (90 + 201); loss of TMSiOH plus .(CH&CH(OTMSi)(CH2)&Ha), 217 (probably due to [TMSiO-CH=CH-CH=OTMSi]+ eliminated from the five-membered ring), 191 ([TMSiO=CH-OTMSi]+), 173 ([CH(=OTMSi)(CHf)&H3]+), and 129. Methyl S/3,11 a!, 16(S)-Trihydroxyprostanuatt+Hydrogenation of 5 mg of the methyl ester of PGFu as described above followed by preparative thin layer chromatography yielded 4 mg of methyl 9fl, 1la!, 15(S)-trihydroxyprostanoate (RF = 0.46). The retention times of the TMSi ether derivative corresponded to C-23.9 (SE-30) and C-22.4 (EGSS-X). The mass spectrum was similar to that of the TMSi ether derivative of the corresponding Sar-epimer.

5107

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Seven metabolites were isolated by reversed phase partition chromatography and thin layer chromatography after incubation of tritium-labeled prostaglandin Et with the soluble fraction of homogenates of guinea pig liver. The two major compounds (forming about 61% of the recovered radioactivity) were identified as 1ICC,15-dihydroxy-g-ketoprost-5-enoic acid and lla-hydroxy-9,15-diketoprost-5enoic acid. Three compounds (together about 26% of the recovered radioactivity) belonged to the F series of prostaglandins, and were identified as prostaglandin Fz,, Oor,Ilcr,lS-trihydroxyprost-5-enoic acid, and Pcr,lla-dihydroxy-15-keto-prost-5-enoic acid. The isolation of these compounds after incubation of prostaglandin Ez for the tist time showed that prostaglandin F, compounds can be formed from prostaglandin E compounds in animal tissue. Two minor compounds (together about 5% of the recovered radioactivity) were identified as S-isoprostaglandin Et and, tentatively, 84soprostaglandin Fzu.

College, Stockholm, Sweden

5108

Metabolism

of Prostaglandin

Pig

Liver.

I

Vol.

245, No.

19

8iso-PGES (RF = 0.73; reference, methyl ester of PC&, RF = 0.66). Gas-liquid chromatographic analysis of the TMSi-MO derivative showed a single peak (cf. Reference 10) with retention times corresponding to C-23.9 (SE-30) and C-26.1 (EGSS-X). The mass spectrum was similar to that recorded on the major peak of the TMSi-MO derivative of the methyl ester of PG&. Ions of high intensity were present at m/e 539 (AI), 524 (I/ - 15; loss of .CH,), 508 (J/r - 31; loss of .OCH,), 468 (Jf - 71; loss of .(CH2)&H3), 418 (M - (90 + 31); loss of TMSiOH plus .0CH3), 366 (!11 - 173; loss of TMSiO-CH-CH,C= NOCH, (carbons C-9 to C-11) or its equivalent), 295 (JI (173 + 71); loss of TMSiO-CH-CH-C=NOCH, plus .(CHz)&HJ, 225 (JI - (173 + 141); loss of TMSiO-&I.CH,CH=CH-(CI~,),COOCI-I,), CH,C=NOCH, plus and 173 ([CH(=OTMSi) (Cl-IZ)4CI~3]+). That the compound obtained by treatment of PGE, with potassium acetate in ethanol differed from PGEs only with respect to the stereochemistry at C-8 was further proved by its conversion to PGB2 by treatment with sodium hydroxide in ethanol-water. The methyl ester of the PGB2 was identified by ultraviolet spectrometry (XgkYi = 278 mp), thin layer chromatography with the authentic compound as reference, and gas-liquid chromatographic analysis of the TMSi ether derivative (C-24.0 with SE-30). Reduction of Methyl Ester of 8-Iso-PGE-Treatment of the methyl ester of 8-iso-PGEB (1 mg) with sodium borohydride (5 mg) in 0.5 ml of methanol afforded two 8-iso-prostaglandin F compounds as judged by thin layer chromatographic analysis (Compound I, RF = 0.41; Compound II, RF = 0.47. References, methyl ester of PGF?,, RF = 0.45; methyl ester of PGF,b, RF = 0.37). The TMSi ether derivatives of the compounds formed from the methyl ester of 8-iso-PGEz appeared at C-22.8 (Compound I) and C-22.3 (Compound II) with EGSS-X, and The mass C-23.5 (Compound I and Compound II) with SE-30. spectra of the TMSi ether derivatives were similar to the mass spectrum of the TMSi ether derivative of the methyl ester of PGF,,. The behavior of Compounds I and II on thin layer chromatography and gas-liquid chromatography as well as the mass spectra showed that both compounds possessed three hydroxyl groups located at C-9, C-lloc, and C-15, and that the compounds only differed with respect to the stereochemistry at C-9. The absolute configurations of this carbon were not determined in the present investigation. In a paper describing the preparation of the 8-iso-PGF1 compounds from 8-iso-PGE1 (7) it was suggested on the basis of chromatographic mobilities that the less polar derivative had the 90 configuration (corresponding to Compound II), and therefore Compound I was tentatively identified as methyl 9a!, lla, 15(S)-trihydroxy-8-isoprosta-5,13dienoate. Methods Incubation Procedure and Isolation of Products-Guinea pig livers were minced and homogenized in 3 volumes of the followM KzHP04-0.028 M nicotining medium: 0.02 M KH2P04-0.072 amide-O.004 M MgCL.. The homogenate was centrifuged at 8,000 x g for 12 mm and the resulting supernatant was then centrifuged at 100,000 x g for 60 min. Tritium-labeled PGE, was dissolved in ethanol and added to the high speed supernatant (final concentration of PGE?, 30 pg per ml; final concentration of ethanol, 0.2%) and the mixture was shaken aerobically at 37” for 60 min. The prostaglandins were extracted from the incuba-


methyl Methyl 9a, 11 cy-Dihydroxy-1 &ketoprostarwate-The ester of PGF2,, 15 mg, was dissolved in 40 ml of chloroform and stirred with 150 mg of manganese dioxide for 72 hours at room temperature. After partial evaporation of the chloroform, ethanol was added and the mixture was centrifuged to remove the MnOz. The supernatant was extracted three times with diethyl ether. The residue obtained after evaporation of the ether was subjected to silicic acid chromatography yielding 10 mg of methyl 9a, 1 lot-dihydroxy-15-ketoprosta-5,13-dienoate (column packed with 1 g of silicic acid, elution with 30 ml of ethyl acetate-benzene (4 : 6, v/v)). The purity of the material Analysis obtained was checked by thin layer chromatography. by ultraviolet spectrometry showed an absorption band with Xethanol = 233 rnp, e = 10,000, due to the unsaturated C-15 max keto group. Infrared spectrometry showed absorption bands at 5.78 p (carbonyl of the carbomethoxy group), 6.00 p (a,&unsaturated keto group at C-15), 6.16 Jo (Ai3 double bond conjugated with the keto group at C-15), and 10.2 p (Ai3-trans double bond). Hydrogenation of methyl 9a, lla-dihydroxy-15-ketoprosta5,13-dienoate (10 mg) with rhodium on carbon (5%, 9 mg) in ethanol (3 ml) followed by preparative thin layer chromatography with Solvent System MI (8) afforded 6 mg of methyl The TMSi 9or, llcr-dihydroxy-15-ketoprostanoate (RF = 0.72). ether-MO derivative of this material gave a single peak on gasliquid chromatography (C-24.4 on SE-30, C-25.6 on EGSS-X). The mass spectrum of this derivative showed ions at m/e 543 (M), 528 (M - 15; loss of .CH3), 512 (f! - 31; loss of .OCHJ, 456 (M - (31 + 56) ; loss of .OCH, from the 0-methyloxime group plus CH*=CH-CH-CH3 (carbons C-17 to C-20)), 453 (M - 90; loss of TMSiOH), 422 (Al - (90 + 31); loss of TMSiOH plus .0CH3), 399 (M - 144; probably loss of .CHz-C(=NOCH,)(CH?)&H3 plus 2 H.), 366 (M - (90 + 31 + 56); loss of TMSiOH plus .0CH3 plus CHt=CH-CHz-CH3), 332 (M - (2 X 90 + 31); loss of 2 TMSiOH plus .OCH,), 309 (M - (90 + 144); loss of TlMSiOH plus =CH2-C(=NOCH3)(CHJ&H3 plus 2 H.), 297 (1Q’ - (90 + 156); loss of TMSiOH plus .(CH&--C(=NOCH,)(CHJ4CH3), 217 (probably due to [TMSiO-CH=CH-CH=OTMSi]+ eliminated from the five-membered ring), 191 ([TMSiO=CHOTMSi]+), and 156 ([a (CH,),C(=NOCH3)(CH2)&H3]+). Methyl 9p, 11 ol-Dihydroxy-15-ketoprostanoate-Oxidation of 15 mg of the methyl ester of PGF,p with MnOZ as described above followed by purification by silicic acid chromatography afforded 9 mg of methyl 9/3,llc~-dihydroxy-15-ketoprosta-5,13dienoate. Ultraviolet spectrometry showed X$~~nol = 233 showed absorption w, E = 10,000, and infrared spectrometry bands at 5.78 p, 6.00 p, 6.16 p, and 10.2 p (see above). Hydrogenation followed by preparative thin layer chromatography with Solvent System MI yielded 5 mg of methyl SD, lla-dihydroxy-15-ketoprostanoate (RF = 0.67). The retention times of the TMSi-MO derivative corresponded to C-23.9 (SE-30) and C-24.7 (EGSS-X) and the mass spectrum was similar to that of the TMSi-MO derivative of the Sar-compound. Methyl Ester of 8-Iso-PGE2---PGE2, 10 mg, was dissolved in 5 The ml of 96% ethanol containing 220 mg of potassium acetate. solution was kept under an atmosphere of argon for 110 hours at room temperature (these conditions have earlier been used for the preparation of 8iso-PGEi from PGEi, cj References 7 and 9). The material obtained by extraction with diethyl ether was treated with diazomethane and subjected to preparative thin layer chromatography yielding 0.5 mg of the methyl ester of

Eg in Guinea

Issue

of October

10, 1970

M.

Hamberg

and

RESULTS

Preliminary experiments with high speed supernatants of homogenates of guinea pig liver showed the formation of several compounds from PGE2. In order to obtain the single compounds in pure form consecutive separations by reversed phase partition chromatography and thin layer chromatography were necessary. The results of a large scale incubation with 42 g of guinea pig liver and 3 mg of tritium-labeled PGE2 are described. Three peaks of radioactivity appeared on reversed phase partition chromatography (Fig. 1). The materials present in Peaks A, B, and C were treated with diazomethane and sub-

IO

20

30 40 FRACTION

50 6.0 NUMBER

7’0

so

FIG. 1. Reversed phase partition chromatography of product isolated after incubation of 3 me: of 117.18~3HzlPGE2 with the high speed supernatant of a homog&ate’of’42 g ofguinea pig liver.

Column, 9 g of hydrophobic Hyflo Super-Cel; fractions, 5 ml. Aliquots of every other fraction were used for determination of the radioactivity. 7 18000 E d 2 14400

1 PGF2d -Me

18

16 14 DISTANCE

12

IO FROM

8 ORIGIN

6

4

2

0

(cm)

FIG. 2. Thin layer radiochromatogram of methyl esters of labeled compounds present in Peak A of the reversed phase partition chromatography shown in Fig. 1. References, methyl esters of PGEx and PGFz,.

jetted to thin layer chromatography (Figs. 2, 3, and 4). These separations yielded eight labeled compounds, viz. Al, A2, A3, A4, Bl, B2, Cl, and C2, which were identified as described below. Structure of Compound Al-This compound formed about 2% of the radioactivity recovered after incubation of tritium-labeled PGE*. On thin layer chromatography Al appeared as a band (RF = 0.41) just below the band of Compound A2 (RF = 0.45). The radioactivities of these bands were not resolved by the thin layer radiochromatographic scanner (Fig. 2). The behavior on thin layer chromatography indicated the presence of three hydroxyl groups in Compound Al (cj. Table II). This was also indicated by the C value of the TMSi ether derivative with EGSS-X (C-22.8) which lay within the group of C values found for a number of methyl prostanoates containing three trimethylsilyloxy groups (Table I, Fig. 5). The mass spectrum showed a molecular ion at m/e 584, indicating the presence of three hydroxyl groups and two double bonds in Compound Al. Thin layer chromatography with the methyl esters of PGF?, and


tion mixture as earlier described (11) (95% recovery of radioactivity) and were subjected to reversed phase partition chromatography with Solvent System C-50 supplemented with acetic acid (11). The labeled material was extracted from the fractions with diethyl ether (after an initial extraction with petroleum ether to remove the high boiling 2-ethylhexanol) and was then treated with diazomethane. The product was subjected to thin layer chromatography. Thin layer chromatography was carried out with plates coated with Silica Gel G. When not otherwise indicated the solvent system used was the organic layer of ethyl acetate-methanol-water (16 : 1: 10, v/v/v). A Berthold Diinnschicht-scanner II was used for the assay of radioactivity of the plates. The compounds were also detected by spraying with 2’,7’-dichlorofluoresceine and viewing under ultraviolet light. The zones were scraped off and eluted with ethyl acetate. Gas-Liquid Chromatography-Gas-liquid chromatography was performed with an F and M Biomedical Gas chromatograph model 400. The stationary phases used were 1.5% SE-30 Ultraphase (purchased from Pierce Chemical Company, Illinois) on Gaschrom Q, and 15% silion Gaschrom Q, 1O7o EGSS-X cone grease (Dow Corning High Vacuum Grease) on Gaschrom Q. The last mentioned phase was used for the analysis of short chain length compounds formed on oxidative ozonolysis performed on derivatives of the isolated compounds. Methyl esters of fatty acids were used as standards. Diagrams were constructed by plotting the retention times of the standards on a logarithmic scale against the number of carbon atoms of the acids on a linear scale. These diagrams were used to convert the retention times of the isolated compounds into C values (cf. Reference 11). The metabolites formed from PGEz were analyzed as the methyl ester-TMSi ether derivatives or as the methyl esterTM&MO derivatives (cf. Reference 12). The use of 10% EGSS-X allowed a group separation of derivatives of prostaglandins containing zero, one, or two 0-methyloxime groups (Fig. 5). The number of keto groups of the metabolites could therefore provisionally be assessed by gas-liquid chromatography with this stationary phase. Gas-liquid chromatography-mass spectrometry was performed with the LKB 9000 instrument and a column packed with 1% SE-30. The energy of the electron beam was maintained at 22.5 ev. In order to facilitate the interpretation of the mass spectra, derivatives containing deuterium-labeled MO and TMSi groups were prepared (cf. References 5 and 12). Measurement of Radioactivity-Radioactivity was determined with a Packard Tri-Carb model 3375 liquid scintillation counter. The scintillation fluid consisted of 4.0 g of 2,5-diphenyloxazole and 50 mg of 1,4-bis-[2’(5’-phenyloxazolyl)]benzene in 1000 ml of toluene-99.5% ethanol (4:1, v/v).

U. Israelson

5110

Metabolism

of Prostaglandin

Ez in Guinea Pig Liver.

I

Vol.

TABLE

C values found

on gas-liquid and

The compounds derivatives.

were

analyzed

19

I

chromatography 1.5%

245, No.

with

10%

EGSS-X

SE-30 as the

TMSi

T

or TMSi-MO

c value

Compound 10%

Methyl 14

12 10 DISTANCE

8

6 FROM

4 ORIGIN

2

Methyl

(cm)

layer radiochromatogram of methyl esters of Iabeled compounds present in Peak B of the reversed phase partition chromatography shown in Fig. 1. References, methyl esters of PGE2 and PGFQ.. 2

s ;I E 2 2

c2

t

10800

ii E z0

3600 7200

i

14 12 DISTANCE

10 8 FROM

6 ORIGIN

i

i (cm 1

0

FIG. 4. Thin layer radiochromatogram of methyl esters of labeled compounds present in Peak C of the reversed phase partition chromatography shown in Fig. 1. References, methyl esters of PGEz and PGFz,.

PGF,o as well as Compounds I and II (formed on reduction of the methyl ester of 8-iso-PGEJ indicated that Al was identical with Compound I. Furthermore, the gas-liquid chromatographic behaviors of the TMSi ether derivatives of the latter two compounds were identical on EGSS-X and SE-30 (Table I). Finally, t,he finding that the TMSi ether derivatives of Al and of Compound I yielded indistinguishable mass spectra conclusively showed that Al was identical with Compound I. As described above, Compound I was tentatively identified as methyl 9cu, 110~) 15(X)-trihydroxy-&isoprostad, 13-dienoate and thus the parent acid of Al would be identical with 8-iso-PGFt,. Structure of Compound A$?-Compound A2, which formed about 9% of the recovered radioactivity, had a mobility on thin layer chromatography identical with that of the methyl ester of PGF,, (RF = 0.45, see Fig. 2 and Table II). Gas-liquid chromatography of the TMSi ether derivative showed a single peak with a retention time identical with that of the methyl ester and TMSi ether derivative of PGF2, (see Table I and Fig. 5). Conclusive evidence for the identity of Compound A2 with the methyl ester of PGFb was furnished by the finding that the mass spectrum of the TMSi ether derivative of Compound A2 was identical with that of the methyl ester-TMSi ether derivative of PGFZ,. Ions of high intensity were present at m/e 584 (M), 569 (n/r - 15; loss of .CH,), 513 (M - 71; loss of . (CHJ&H3), 494 (1M - 90; loss of TMSiOH), 423 (M - (90 + 71); loss of TMSiOH plus . (CHz)&Hs), 404 (M - 2 X 90; 10~s of 2

.................

Methyl 9a,lla,15(S)-trihydroxyprosta-5,13-dienoate. ............ Methyl 9@,lla,l5(S)-trihydroxyprosta-5,13-dienoate. ........... Met*hyl Sol,llol-dihydroxy-l5-ketoprostanoate ...................... Methyl SP,lla-dihydroxy-15-ketoprostanoate. .................. Methyl ll~l, 15-dihydroxy-9-ketopros&5-enoate. .................. Methyl llor,l5(S)-dihydroxy-S-ketoprosta-5,13-dienoate. ........... Methyl lla,l5(S)-dihydroxy-9-keto8-iso-prosta-5,Wdienoate ........

Methyl 16

23.2

24.4

22.4

23.9

23.3

24.0

22.7

23.5

25.6

24.4

24.7

23.9

15(S)-trihydroxy-

26.9

(2G.3)

24.5

(24.1)

27.2

(26.4)

24.5

(23.9)

(30.3)

24.3

(24.0)

26.1

llor-hydroxy-9,15-diketo-

prost&enoate.

..................

Al ........................... A2 ................................ A3 ................................ A4 ......... ...................... Bl ................................

Hydrogenated Bl. ............... B2 ................................ Cl. ............................... Hydrogenated Cl .................. c2 .............

..................

30.8 22.8 23.3 27.2 26.1 23.0 23.2 26.9 25.6 25.6 30.8

(26.4)

(26.3)

(30.3)

23.5 24.0 24.5 23.9 24.1 24.4 24.5 24.0 24.4 24.3

(23.9)

(24.1)

(24.0)

TMSiOH), 397, 333 (iM - (2 x 90 + 71); loss of 2 TMSiOH 217 ([TMSiO=CH-CH=CHplus . (CH2)&Hz), 307, OTMSi]+), 191 ([TMSiO=CH-OTMSi]+), and 173 ([CH(= OTMSi)(CH2)&H#). Xtructure of Compound AS-Compound A3 (8% of the recovered radioactivity) behaved identically with the methyl ester of PGEz on thin layer chromatography (Fig. 2 and Table 11). Analysis of the TMSi-MO derivative by gas-liquid chromatography and mass spectrometry (cj. Reference 12) showed that A3 was identical with the methyl ester of PGE2 and was thus due to PGEz remaining after the incubation period. Structure of Compound A&-This compound (3% of the recovered radioactivity) formed a band (RF = 0.73) just above that of Compound A3 (Rr = 0.66). This chromatographic behavior as well as the finding that the TMSi-MO derivative on gas-liquid chromatography with EGSS-X appeared in the group of derivatives containing one MO group and two TMSiO groups (Fig. 5) suggested that Compound A4 contained one keto group and two hydroxyl groups. The mass spectrum of the TMSGMO


t

18000

9p, lh,

prostanoate.

FIG. 3. Thin

1.5% SE-30 (215”)

Sol,llol,l5(S)-trihydroxy-

prostanoate ...................

0

EGSS-X (175.y

Issue

of October

10, 1970

M. Hamberg and U. Israelsson II

TABLE

RF values found

The solvent methanol-water

on thin

layer chromatography

system used was the organic layer of ethyl (16: 1: 10, v/v/v).

acetate-

Position of Rp value Hydroxyl group(s)

Keto group(s) Double bond(s)

lla! 1101,15 9ol,lla! llor, 15” lla,15 901,1101,15 9~,llcz,15 9p,1101,15

a Derivative

9,15 9 15 9” 9

5 5 5 5,135 5,13 5 5,13 5,13

of 8-isoprostanoic

acid.

T

32,_

0.89 0.7g 0.79 0.73 0.66 0.64 0.45 0.37

c2a*-

POSITION

1‘M Sic

MO

OF ouble ondk

-11 M

9,15

5

1

3cI-

2Ei5 m >

A3-+.-----82+.-

1IId,

lld,15

9 9

5,13 5

CJ 2t j-

At,+.Cl-s..

lld,15d 9d, 114

9 * 15

5,13" -

,go,\1

15

-

-

.-

d

21

22 I

0 Number

I

I

-9 d,bi,15 -.9 d,lld,lE

-

-9 0, ILL,15 -9 0, lld,l!

-

5,13 5,13 -

L

12 of MO groups

FIG. 5. C values found on gas-liquid chromatography with 10% EGSS-S. The positions in the derivatives analyzed of trimethylsilyloxy (TMSiO) groups, MO groups, and double bonds are indicated in the righl panel. Asterisks indicate derivative of 8.isoprostanoic acid. In the cases in which two peaks appeared because of syn-anti isomerism of the MO group, the C value for the major peak is given.

anti isomerism

of the 0-methyloxime group (cf. Reference 12). The retention times were identical with those found for the TMSi-MO derivative of methyl lla!, 15-dihydroxy-9-ketoprost5-enoate (Table I and Fig. 5). The mass spectrum of the TMSiMO derivative of B2 is given in Fig. 7. Ions were present at m/e 541 (Al), 510 (IV - 31; loss of .OCHa), 470 (Al - 71; loss of .(CH,)&Hz), 440 (114 - 101; loss of .(CH~BCOOCHI), 420 (M - (90 + 31); loss of TMSiOH plus .OCHJ, 350 (IV (90 + 101) ; loss of TMSiOH plus . (CHr)KOOCHJ, 340 (Jf -


derivative showed a molecular ion at m/e 539, indicating the presence in A4 of one keto group, two hydroxyl groups, and two double bonds. Compound A4 was thus an isomer of the methyl ester of PGE*. This contention was supported by the formation of the methyl ester of PGB2 on treatment of A4 with sodium hydroxide in ethanol-water. Comparison of A4 with the methyl ester of S-iso-PGEz by thin layer chromatography (cf. Table II), gas-liquid chromatography (Table I, Fig. 5)) and mass spectrometry established that A4 was identical with the methyl ester of 8-iso-PGE2 and thus differed from the methyl ester of PGEZ only with respect to the stereochemistry at C-8. Structure 0s Compound Bi-Analysis by thin layer chromatography showed that two labeled compounds were present in Peak B of the reversed phase partition chromatography shown in Fig. 1. Compound Bl (10% of the recovered radioactivity) formed a band (RF = 0.64, see Fig. 3 and Table II) below that of Compound B2 (RF = 0.79). Gas-liquid chromatographic analysis of the TMSi ether derivative indicated the presence of three hydroxyl groups in Bl (Table I, Fig. 5). The mass spectrum of the TMSi ether derivative is shown in Fig. 6. As seen, ions are present at m/e 496 (JJ - 90; loss of TMSiOH), 425 (iv - (90 + 71); loss of TMSiOH plus (CH2)&H3), 406 (1M 2 x 90; loss of 2 TMSiOH), 355 @I - (90 + 141); loss of TMSiOH plus . CH-CH=CH-(CH1)pCOOCH$, 316 (M 3 X 90; loss of 3 TMSiOH), 295 (dl - (90 + 201); loss of TMSiOH plus .(CHJ2CH(OTMSi)(CHa)&H3), 255 (M - 331; formed by unknown mechanism. Labeling with 2Hs-TMSi groups showed that this ion contained one TMSi group (shift to m/e 264), 220 (Al - (2 x 90 + 186); loss of 2 TMSiOH plus CHa=C(OTMSi)(CH2)&H3 or its equivalent), and 173 ([CH(= OTMSi)(CHp)4CH3]+). From this mass spectrum it could be deduced that Compound Bl had no double bond in the side chain attached to C-12 (elimination of the saturated side chain in the formation of the ion at m/e 295) but had a double bond in the carboxyl side chain (elimination of a monounsaturated carboxyl side chain in the formation of the ion at m/e 355). Catalytic hydrogenation of Bl with 5% palladium on carbon resulted in the formation of methyl 9ar, 1 la, 15-trihydroxyprostanoate as judged from gas-liquid chromatographic (Table I, Fig. 5) and mass spectrometric analysis of the TMSi ether derivative and comparison with the authentic material. The position of the double bond in Bl was established by oxidative ozonolysis performed on the triacetate derivative (cf. Reference 13). The product was treated with diazomethane and analyzed by gas-liquid chromatography with 15yc silicone grease on Gaschrom Q. A major peak with a retention time identical with that of dimethyl glutarate appeared (C-8.0, cf. Reference 13), thus showing that the double bond of the degraded derivative was located at As, i.e. in the same position as that of the double bond of the carboxyl side chain of PGE2. The structural work described thus showed that Compound Bl was identical with methyl 9o(, 1 lar ,15-trihydroxyprost-5enoate. Structure of Compound B&-Compound B2, which formed 37% of the recovered product, had a mobility on thin layer chromatography identical with that of the methyl ester of lloc, 15-dihydroxy-9-ketoprost-5-enoic acid (RF = 0.79, see Fig. 3 and Table II). The latter compound was prepared by incubation of PGEz with the 100,000 X g supernatant of a homogenate of guinea pig lung (1). Gas-liquid chromatographic analysis of the TMSGMO derivative of B2 showed two peaks due to syn-

5111

Metabolism of Prostaglandin Ez in Guinea Pig Liver.

5112

T

I

Vol.

245, No.

19

2

316

255

425 406

20 IO 0 m/e

FIG. 6. Mass spectrum

of TMSi

ether derivative

of Compound

Bl

100 265 340

.T B 2

510 420

40

470

I

30 20

0 20

60

100

IL0

FIG. 7. Mass spectrum

160

recorded

220

260

on the major

300

360

360

peak of the TMSi-MO

201; loss of . (CH&CH(OTMSi)(CH2)&HJ, 330 (M - (2 x 90 + 31); loss of 2 TMSiOH plus .OCHs), 265 (M - (90 + 186) ; loss of TMSiOH plus CH2 = C(OTMSi)(CH2)&H3) or its equivalent (this cleavage was supported by the mass spectra of the following derivatives: (a) bis(2Hg-TMSi) derivative, m/e 265, (b) 2H3-M0 derivative, m/e 268, (c) ethyl ester of the parent acid of B2, m/e 279, and (d) TMSi-MO derivative of hydrogenated B2, m/e 267)), 199 (formed by unknown fragmentation. The ion contained one TMSi group as judged frorll the mass spectrum of the bis(2Hg-TMSi) derivative (shift to 208)) and 173 ([CH(==OTMSi)(CHr)&H$). This mass spectrum was identical with that of the TMSi-MO derivative of methyl llar,15dihydroxy-9-ketoprost-5-enoate. The ions at m/e 440 and 350 (formed by elimination of C-l to C-4 by cleavage (II to the A6 double bond) confirmed that the A5 double bond of PGE, remained in its position in the formation of B2. This compound was thus methyl lla, 15-dihydroxy-9-ketoprost-5-enoate. Structure of Compound Cl-Peak C of the reversed phase partition chromatography contained two labeled compounds as Compound Cl (7% of judged by thin layer chromatography. the recovered radioactivity) formed a band with Rp = 0.79 (Fig. 4 and Table II). Gas-liquid chromatographic analysis of the TMSi-MO derivative indicated the presence in Cl of two hydroxyl groups and one keto group (Table I, Fig. 5). The mass spectrum is given in Fig. 8. Ions of high intensity were present at m/e 541 (M), 510 (M - 31; loss of .0CH3), 451 420 (M - (90 + 31); loss (M - 90; loss of TMSiOH),

420

derivative

L60

500

of Compound

5LO

580

B2

of TMSiOH plus .OCH& 330 (M - (2 x 90 + 31); loss of 2 TMSiOH plus .OCHs), 310 (M - (90 + 141); loss of TMSiOH plus .CHt-CH=CH-(CH&COOCH,), 307 (M - (90 + 144); probably by loss of .CHt--C(=NOCHJ(CHZ)&H~ plus 2H. plus TMSiOH. This fragmentation was indicated by the mass spectra of the bis(2Hg-TMSi) derivative (shift to m/e 316) and of the 2H3-MO derivative (m/e 307), although the mechanism is unknown at present), 255 (M - 286; formed by unknown fragmentation. Labeling with 2Hs-TMSi groups and with 2H3-MO group showed that the ion at, m/e 255 contained one TMSi group and no MO group), and 217 (M - (2 X 90 + 144); loss of 2 TMSiOH plus .CH-C(=NOCHJ(CH2)&H3 plus 2 H.). The ions at m/e 307 and 217 indicated that, Compound Cl had a keto group located at C-15 and no double bond between C-13 and C-14. This was also shown by the ion at m/e 156 which is due to the side chain attached to C-12 (. (CH&the C(=NOCHJ(CHZ)&H~). The ion at m/e 310 indicated presence in Cl of a carboxyl side chain containing one double bond. Catalytic hydrogenation of Cl afforded methyl 9ar, lla-dihydroxy-15-ketoprostanoate as judged by gas-liquid chromatography (Table I, Fig. 5) and mass spectrometry. The C values and the mass spectrum were identical with those of the authentic methyl 9ar , liar-dihydroxy-15-ketoprostanoate prepared from PGF2, as described above. The double bond of the carboxyl side chain of Cl was located by oxidative ozonolysis performed on the diacetate derivative. Analysis of the ozonolysis product


.$ 60 5 .E 50

Issue

of October

M. Hamberg

10, 1970

and U. Israelsson

5113

100 307 90 2

20 330

80

I 70

a 5 5 c 2 4 d

60 50 40

310

217

I

30

?55

m/e

FIG.

8. Mass spectrum

of TMSi-MO

derivative

of Compound

Cl

9. Mass spectrum

recorded

on the major

peak of the TMSi-MO

by gas-liquid chromatography revealed a major peak with a retention time identical with that of dimethyl glutarate (C-8.0, with 15% silicone grease) showing that the double bond was located at As, i.e. in the same position as that of the double bond of the carboxyl side chain of PGE2. Compound Cl was thus identical with methyl Sa!, lla-dihydroxy-15-ketoprost-5-enoate. Structure of Compound Q&Compound C2, which formed 24% of the recovered radioactivity, behaved identically with the methyl ester of llcu-hydroxy-9,15-diketoprost-5-enoic acid on thin layer chromatography (Rr = 0.89, cf. Fig. 4 and Table II). On gas-liquid chromatographic analysis of the TMSi-MO derivative of C2 two peaks appeared due to the syn-anti isomers of the 0-methyloxime group at C-9. The retention times were identical with those of the TMSi-MO derivative of methyl llor-hydroxy9,15-diketoprostd-enoate (Table I, Fig. 5). The mass spectrum of the derivative of C2, given in Fig. 9, showed ions at m/e 465 (M - 31; loss of .OCHJ, 395 (2M - 101; loss of .(CH&COOCH8), 375 (iv - (90 + 31); loss of TMSiOH plus .OCHa), 340 (M - 156; loss of .(CHJzC(=NOCH3)(CH&CH& 325 (J4 - (31 + 140); loss of .OCH, plus carboxyl side chain minus The mechanism for this fragmentation is not known at He. present. That the carboxyl side chain was indeed lost in the formation of the ion at m/e 325 was shown by the mass spectra of the TMSi-MO derivative of the ethyl ester of liar-hydroxy-9,15diketoprostd-enoic acid (m/e 325) and of the TMSi-MO derivatives of methyl lla-hydroxy-9,15-diketoprostanoate and its CB and Cl6 homologues (m/e 325, cf. Reference ll)), 265 (M - (90 +

derivative

of Compound

C2

kOH ‘,. OH

H6 Prostaglandin

E2

(A3

1

.,.-COOH

FIG.

bation

10. Structures of compounds formed from PGEs on incuwith the soluble fraction of guinea pig liver homogenates.

141); loss of TMSiOH plus . CH?--CH=CH-(CHJ&OOCHJ, and 232 (M - (90 + 31 + 143) ; loss of TMSiOH plus .OCHa plus .CH-C(=NOCHJ(CH2)&H3 plus He). This mass spectrum was identical with that of the TMSi-MO derivative of authentic methyl lla-hydroxy-9,15-diketoprost-5-enoate. The ion at m/e 395 confirmed that the double bond of the carboxyl


m/e

FIG.

Metabolism

of Prostaglandin

side chain was located at As, i.e. in the same position as in the incubated PGE2. Compound C2 was thus methyl I lar-hydroxy-9,15-diketoprost5-enoate. DISCUSSION

3 M. Hamberg,

data to be published.

I

Vol.

245, Xo.

19

hydroxy-15-ketoprost-5-enoic acid identified in the present investigation. 8-Iso-PGE2 and its reduction product, tentatively identified as 8-iso-PGFp,, formed 3 and 2oj,, respectively, of the radioactivity recovered after incubation of PGE*. 8-Iso-PGE1 was earlier isolated in low yield after incubation of 8,11, lCeicosatrienoic acid with homogenates of sheep vesicular gland tissue (9). In this case it was suggested that at least part of the 8-isoPGEl isolated was formed by nonenzymatic enolization of the C-9 keto group of PGEr. Furthermore, treatment of PGEr with potassium acetate in ethanol afforded an equilibrium mixture consisting of 15% of 8-iso-PGEr and 85% of PGEr (9). In the present investigation incubation of PGEz with boiled 100,000 x g supernatant of guinea pig liver yielded 1 to 2% of This indicated that, in the case of fresh 100,000 X 8-iso-PGEt. g supernatant, at least part of the 8-iso-PGEn isolated was formed by a nonenzymatic reaction. However, even if the 8-iso-PGE compounds may be formed nonenzymatically it is conceivable that the equilibrium may be approached under physiological conditions, resulting in excretion of metabolites of 8-isoprostaglandins. wish to thank assistance.

Acknowledgments-We

skillful

technical

Mrs. 8. Regardt

for her

KEFEHENCES 1. ~NGG%RD, E., GREEN, K., AND SAMCELSSON, B., I. Biol. Chem., 240, 1932 (1965). 2. ~NGG~RD, E., AND SAMUELSSON, B., J. Biol. Ch,em., 289, -1097 (1964). 3. ANGGARD. E.. AND SAMUELSSON. B., Biochemistry, 4, 1864 (1965). 4. HaMBERG, &I., AND S.\MUELSSON, B., Biochem. Biophys. Res. Commun.. 34, 22 (1969). 5. HAMBERG,~., .WD ~AMUELSSON, B., J. Amer. Chem. Sot., 91, 2177 (19G9). 0. BERGRTR~M, S., KR.~IUSCA, I>., SAMUELSSON, B., AND ~.IGv.~~L, J., Ada Gem. Stand., 16, 969 (1962). 7. PIICE, J. E., LINCOLN, F. H., AND SCHNEIDER, W. P., J. Org. Chem., 34, 3552 (1969). 8. GREEN, K., AND SAMUELSSON, B., J. Lipid. Res., 6, 117 (1964). 9. DINIELS, E. G., KRTEGER, W. C., KUPIECIU, F. l’., PILE, J. E:., .\ND SCHNEIDER. W. P.. J. Amer. Chem. Sot.. 90, 5894 (1968). 10. VrQ~~, k., AND HORNING, M. G., Anal. I,ett., 2, 357 (1969) 11. HAMBERG. Nil.. Eur. J. Biochem.. 6, 135 (19681. 12. GROEN, Ii'., &em. Phys. Lipids,‘3, 254 (1969j. 13. HAMHERG, M., AND SAMCELSSON, B., J. Biol. Chem., 241, 257 (1966). 14. ~NGG~RD. E.. AND S.WCELSSON. B., J. Biol. Chem., 240, 3518 (1965). ’ 15. HAMTIERG,~LI., AND SAMVELSSOX, B., 1. Biol. Chem., 242, 5336 (1967). It. K., AND VAN I)ORP, 1). A., 16. NUGTEREN, L>. H., BEERTHUS, Rec. Trau. Chim., Pavs-Bas, 86, 405 (1966).


The present work shows the formation of seven metabolites from PGEz by enzymes present in the high speed supernatant of homogenates of guinea pig liver (Fig. 10). The two major compounds, viz. 1 lcll ,15-dihydroxy-9-ketoprost-5-enoic acid and lla-hydroxy-9,15-diketoprost&enoic acid, forming 37% and 24% of the recovered radioactivity, respectively, have earlier been obtained by incubation of PGEz with the soluble fraction of homogenates of guinea pig lung (1). In this case, lla, 15dihydroxy-9-ketoprost-5-enoic acid was isolated only in a small yield, the major metabolite being I la-hydroxy-9,15-diketoprost5-enoic acid. The pathways by which these metabolites are formed in guinea pig liver have recently been deduced with PGE, labeled with deuterium at, C-15.3 The presence in guinea pig liver of an enzyme catalyzing the reduction of the C-9 keto group of PGE2 was shown by the isolation of PGF,,, 9a, lla, 15-trihydroxyprost-5-enoic acid, and Scr, llcr-dihydroxy-15-ketoprost-5-enoic acid after incubation of PGE2. The formation of PGF2, from PGE2 is noteworthy since it is the first example of conversion of a PGE compound into a PGF, compound. Both PGE and PGF, compounds have earlier been isolated after incubation of unsaturated fatty acids with preparations of guinea pig lung (14) and sheep vesicular gland (15, 16). However, in these cases it was shown that the PGF, compounds were formed directly from the precursor acids, and that the PGE and PGF, compounds were not interconvertible. The pronounced differences in the spectra of biological activities of the PGF, and PGE compounds also make the demonstrated conversion of PGEz into PGFZ, of great interest. 9a!, 110~) 15-Trihydroxyprost-5-enoic acid and 9o(, 1 loc-dihydroxy-15-ketoprost-5-enoic acid bear the same relationship to PGFra as lla!, 15-dihydroxy-9-ketoprost-5-enoic acid and llac-hydroxy-9,15-diketoprost-5-enoic acid to PGE2. It is not known at present whether these compounds are products of further metabolism of PGFzo, or whether they arise by reduction of the C-9 keto group of the corresponding PGE compounds. Experiments designed to solve this problem are being carried out presently.3 Recently, the major urinary metabolite of PGEz in the guinea pig was identified as 5p, icu-dihydroxy-ll-keto-tetranorprostanoic acid (4). Interestingly, this compound has the fl configuration at the carbon atom corresponding to C-9 of the &prostaglandins, and therefore does not arise by /3 oxidation of the 9a, lla-di-

E2 in Guinea Pig Liver.

Metabolism of Prostaglandin E2 in Guinea Pig Liver : I. IDENTIFICATION OF SEVEN METABOLITES Mats Hamberg and Ulf Israelsson J. Biol. Chem. 1970, 245:5107-5114.

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