Identification of metabolites from Type III F2-isoprostane diastereoisomers by mass spectrometry

Journal of Lipid Research

Volume 43, 2002

495

Identification of metabolites from Type III F

2

-isoprostane diastereoisomers by mass spectrometry

Chiara Chiabrando,

1,

* Claudia Rivalta,* Renzo Bagnati,* Anna Valagussa,* Thierry Durand,

†

Alexandre Guy,

†

Pia Villa,

2,

* Jean-Claude Rossi,

†

and Roberto Fanelli*

Istituto di Ricerche Farmacologiche ‘Mario Negri,’* Department of Environmental Health Sciences,Via Eritrea 62, 20157 Milano, Italy; and UMR CNRS 5074,

†

Faculty of Pharmacy, UM I, 15 Av. Ch. Flahault,F-34060 Montpellier, France

Abstract F

2

-isoprostanes (F

2

-iPs) are prostaglandin (PG)-likeproducts of non-enzymatic free radical-catalyzed peroxida-tion of arachidonic acid that are now widely used as indicesof lipid peroxidation in vivo. Knowledge of the metabolicfate of F

2

-iPs in vivo is still scant, despite its importance fordefining their overall formation and biological effects invivo. Type III F

2

-iPs, which are diastereoisomers of cyclo-oxygenase-derived PGF

2

a

, may be metabolized through thepathways of PG metabolism. We therefore studied the invitro metabolism of eight synthetic Type III F

2

-iP diastereo-isomers in comparison with PGF

2

a

. We used gas chroma-tography-mass spectrometry and high performance liquidchromatography-electrospray-tandem mass spectrometryfor structural identification of metabolites formed after in-cubation of the various compounds with isolated rat hepato-cytes. PGF

2

a

was metabolized to several known products,resulting from a combination of

b

-oxidation, reduction of

D

5

and/or

D

13

double bonds, and 15-OH oxidation, plusother novel products deriving from conjugation with tau-rine of PGF

2

a

and its metabolites. Of the eight F

2

-iP diaste-reoisomers, some were processed similarly to PGF

2

a

, whereasothers showed peculiar metabolic profiles according to spe-cific stereochemical configurations. These data representthe first evidence of biodegradation of selected Type IIIF

2

-iP isomers other than 8-

epi-

PGF

2

a

, through knownand novel pathways of PGF

2

a

metabolism. The analyticalcharacterization of these products may serve as a basis foridentifying the most significant products formed in vivo.

—Chiabrando, C., C. Rivalta, R. Bagnati, A. Valagussa, T.Durand, A. Guy, P. Villa, J-C. Rossi, and R. Fanelli.

Identifi-cation of metabolites from Type III F

2

-isoprostane diastereo-isomers by mass spectrometry.

J. Lipid Res.

2002.

43:

495–509.

Supplementary key words

PGF

2

a

•

taurine conjugation

•

isolated rathepatocytes

•

gas chromatography-mass spectrometry

•

high perfor-mance liquid chromatography-electrospray-tandem mass spectrometry

F

2

-isoprostanes (F

2

-iPs), a complex family of 64 com-pounds isomeric to cyclooxygenase-derived PGF

2

a

, areproducts of non-enzymatic free radical-catalyzed peroxi-dation of arachidonic acid (1–3). Because these com-pounds are now widely used as indices of lipid peroxida-

tion in vivo (2–5), knowledge of their metabolic fatemay be useful, in general, to better define their overallformation. In fact, the identification of major metabo-lites of F

2

-iPs might help in finding new analytical targetsto monitor in addition to, or instead of, the parent com-pounds. Moreover, given that selected F

2

-iP isomers havepotent biological effects (2–5), it is important to estab-lish whether and how these isomers are specifically de-graded, to relate their levels in vivo to a putative biologi-cal effect.

Depending on the position of the free radical-initiatedhydrogen abstraction, four classes of F

2

-iP regioisomerscan theoretically be formed by peroxidation of arachi-donic acid. These families were first called Type I, II, III,and IV (1), but are now known as 5-, 12-, 8-, and 15-Series(2), or Type VI, V, IV, and III (6). Using the latter nomen-clature (6), removal of a hydrogen at C-10 gives rise to theformation of two regioisomer families (Type IV and TypeV), whereas at C-7 or C-13, it leads to the Type VI or TypeIII family, respectively. Regioisomer families of Type VIand Type III, theoretically predicted as the most abun-dant, did indeed predominate over Type V and IV in vivo(1, 6, 7).

F

2

-iPs of Type III are of particular interest becausethey are diastereoisomers of cyclooxygenase-derivedPGF

2

a

(

Fig. 1

), and may therefore mimic some of the bi-ological properties of PGF

2

a

or other prostanoids. Al-though the biological effects of most F

2

-iP isomers arelargely unknown, 8-

epi

-PGF

2

a

and 12-

epi

-PGF

2

a

do in-deed have biological effects that appear to be mediated

Abbreviations: GC, gas chromatography; iP, isoprostane; MO,methoxime; MS, mass spectrometry; MS/MS, tandem mass spectrome-try; NICI, negative ion chemical ionization; PFB, pentafluorobenzylester; PG, prostaglandin; SIR, selected ion recording; SPE, solid phaseextraction; TMS, trimethylsilyl ether.

1

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

2

Also affiliated with CNR, Cellular and Molecular PharmacologyCenter, Via Vanvitelli, 32, 20129 Milano, Italy.

methods

by guest, on January 12, 2019w

ww

.jlr.orgD

ownloaded from

http://www.jlr.org/

496 Journal of Lipid Research

Volume 43, 2002

by prostanoid receptors (TP and/or FP) (2 –5). In viewof their structural similarity with PGF

2

a

, Type III F

2

-iPsmay also interact with the enzymes that degrade prosta-glandins in vivo, but it is difficult to predict the influ-ence of the different steric configurations on the variousmetabolic pathways.

To date, the only F

2

-iP metabolites identified as prod-ucts of selected isomers are metabolites of 8-

epi-

PGF

2

a

, thebest-known and most-studied isomer (2–5). 2,3-Dinor-5,6-dihydro-8-

epi-

PGF

2

a

was reported as the only noteworthymetabolite of 8-

epi

-PGF

2

a

in humans (8). We have, how-ever, recently shown that at least two major metabolitesderiving from endogenous 8-

epi-

PGF

2

a

exist in humanand rat urine (2,3-dinor-5,6-dihydro-8-

epi-

PGF

2

a

and 2,3-dinor-8-

epi-

PGF

2

a

) (9). We also found that these samemetabolites, in addition to 2,3,4,5-tetranor-8-

epi-

PGF

2

a

,are formed when 8-

epi-

PGF

2

a

is incubated with isolatedrat hepatocytes (9). In the rabbit, 13,14-dihydro-15-keto-2,3,4,5-tetranor-8-

epi-

PGF

2

a

was identified as the most

abundant urinary metabolite of exogenous 8-

epi-

PGF

2

a

(10).As we showed recently (9), searching for unknown en-

dogenous metabolites of F

2

-iPs is very complex, because ofthe simultaneous presence in vivo of a myriad of isomericcompounds. In fact, to obtain stereo- and enantio-specificidentification of selected products, special analytical ap-proaches have to be devised ad hoc (9). To obtain a gen-eral indication, as a first step, about which metabolites canbe expected in vivo, we studied in vitro the metabolic fatesof PGF

2

a

and Type III F

2

-iPs. Among the 16 diastereoiso-mers of this family, we studied the eight with

cis

ring sub-stituents, which are believed to be the most abundant (6)(Fig. 1). The different diastereoisomers were incubatedwith isolated rat hepatocytes, and their metabolites identi-fied by gas chromatography-negative ion chemical ioniza-tion mass spectrometry (GC-NICIMS) and high-performanceliquid chromatography-electrospray ionization-tandem massspectrometry (HPLC-MS/MS).

Fig. 1. Structures of PGF2a and 8,12-cis-9,11-cisType III F2-iPs.


ww

.jlr.orgD

ownloaded from

http://www.jlr.org/

Chiabrando et al.

Identification of metabolites from Type III F

2

-isoprostanes 497

MATERIALS AND METHODS

Materials

8-

Epi-

PGF

2

a

, 3,3

9

,4,4

9

-[

2

H

4

]-8-

epi-

PGF

2

a

, and 2,3-dinor-5,6-dihydro-8-

epi

-PGF

2

a

were purchased from Cayman Chemical Co.(Ann Arbor, MI). PGF

2

a

and 13,14-dihydro-15-keto-PGF

2

a

werefrom Upjohn Co. (Kalamazoo, MI). 15

R

-8-

epi

-PGF

2

a

,

ent

-8-

epi

-PGF

2

a

(15

R

and 15

S

), 12-

epi

-PGF

2

a

(15

S

and 15

R

),

ent

-12-

epi-PGF2a (15Rand 15S), 2,3-dinor-8-epi-PGF2a, 2,3-dinor-ent-8-epi-PGF2a, 2,3-dinor-5,6-dihydro-ent-8-epi-PGF2a, and 13,14-dihydro-15-keto-2, 3-dinor-5,6-dihydro-ent-8-epi-PGF2a were synthesized in our laboratories(11–13).

Isolated rat hepatocytesHepatocytes were isolated as described previously (9) from fed

male Crl:CD (SD) BR rats.3 Briefly, hepatocytes obtained after per-fusing the liver with a collagenase solution were washed and sus-pended in Leibovitz L-15 medium (Gibco, Scotland) supplementedwith HEPES 18 mM, insulin (1 mg/ml), gentamicin (50 mg/ml),and 5% FCS (Gibco) at a density of 1 3 106 cells/ml. After 3 h ad-hesion the medium was replaced, but without FCS and gentamicin.

Incubation of PGF2a and related F2-iPsPlates were prepared with 3 3 106 cells. The incubation me-

dium was enriched with 670 ng/ml of PGF2a, 8-epi-PGF2a, or theother F2-iPs under test. In a preliminary experiment to test theexperimental conditions, cells were incubated for 10, 20, and 40min with or without PGF2a and 8-epi-PGF2a. In the other experi-ments, PGF2a and the various F2-iPs were incubated separatelyfor 20 min. At the end of the incubation, the medium was col-lected and spun at 48C to eliminate non-adhering hepatocytes.Cell-free medium ad plates were stored at 2208C until analyzed.

ExtractionMetabolites were extracted from the incubation medium (2.5

ml) after addition of [2H4]-8-epi-PGF2a and [2H4]-PGF2a (50 ngeach) as internal standards. The medium was diluted to 10 mlwith 1 mM HCl, acidified to pH 3, and extracted on C18 solidphase extraction columns (SPE; Bakerbond) preconditionedwith methanol and 1 mM HCl. After two washing steps (waterand petroleum ether, 10 ml each), the columns were eluted with2 ml methyl formate (eluate A) and then 2 ml methanol (eluateB). After drying, eluates were assayed separately as follows. Elu-ate A (containing the unmetabolized parent compounds andunconjugated metabolites), was derivatized (see below) and ana-lyzed by GC-NICIMS, or reconstituted in the HPLC initial mo-bile phase (see below) and directly analyzed by HPLC-MS/MS.Eluate B (containing conjugated metabolites), was analyzed byHPLC-MS/MS. Metabolites were also extracted from plate-adhering hepatocytes by 1) adding ethanol and internal stan-dards to plates (48C overnight), 2) suspending the cell lysate, 3)vigorously vortexing, centrifuging and drying the ethanol sus-pension, and 4) resuspending the residue in 1 mM HCl and ex-tracting as above. In experiments to measure the unmetabolizedparent compounds, 0.025 ml medium and 5 ng internal stan-dards were used.

3 Procedures involving animals and their care were conducted inconformity with the institutional guidelines that are in compliancewith national (D.L. n. 116, G.U., suppl. 40, 18 Febbraio 1992, CircolareNo. 8, G.U., 14 Luglio 1994) and international laws and policies (EECCouncil Directives 86/609, OJ L 358, 1, Dec. 12, 1987; Guide for theCare and Use for Laboratory Animals, U.S. National Research Council,1996).

DerivatizationSamples were converted to pentafluorobenzyl ester (PFB),

methoxime (MO), and trimethylsilyl ethers (TMS), or PFB-TMSas follows: 1) add 30 ml pentaflourobenzyl bromide-acetonitrile(1:5, v/v) and 5 ml diisopropylethylamine to the dry residue(408C for 5 min); 2) repeat previous step; 3) add 50 ml meth-oxyamine HCl in pyridine (Pierce, Rockford, IL), (608C for 1 h);4) dry; and 5) add 50 ml N,O-bis-(trimethylsilyl)trifluoroaceta-mide (Fluka, Buchs, Switzerland) (608C for 1 h).

GC-NICIMSAnalyses were as described (9), using a Finnigan 4000 mass

spectrometer. Briefly, GC operating conditions were: NB-54 (5%phenyl, 95% dimethylpolysiloxane) fused silica capillary column(25 m length, 0.32 mm ID, 0.12 mm film thickness); solvent-splitinjection (508C to 3008C); oven temperature, isothermal at1608C for 1 min, then programmed to 3008C at 108/min; heliumas carrier gas. Negative ion chemical ionization (NICI) operat-ing conditions were: selected ion recording (SIR) of carboxylateanions (M-181, loss of PFB); methane as reagent gas; electronenergy, 100 eV.

HPLC-MS/MSAnalyses were done on an API 3000 triple quadrupole instru-

ment (PE Sciex, Thornhill, Ontario, Canada), inter faced withtwo Series 200 micro liquid chromatography (LC) pumps (Perkin -Elmer, Norwalk, CT). HPLC conditions were as follows: column,Superspher C18e, 3.5 mm, 2 3 125 mm (Merck, Darmstadt, Ger-many); flow, 0.2 ml/min; eluent A, 0.1% formic acid; eluent B,acetonitrile; gradient, from 10% to 52% B in 28 min; loop, 20 ml.The HPLC effluent was directed to the MS instrument through asplitting T-connection, reducing the flow into the ion source toabout 40 ml/min. MS conditions were as follows: source, Turbo-IonSpray in negative ion mode; heater gas, 2808C; ion sprayvoltage, 24,000 V; orifice voltage, 256 V; ring voltage, 2250 V;collision cell gas, nitrogen at a pressure of 2.6 3 1025 torr; col-lision energy, 26 to 56 eV (see Results). To optimize ionizationand Q2 decomposition, authentic standards were directly in-fused into the ion source, or MS/MS loop experiments with dif-ferent collision energies were run on the HPLC-eluted biologicalmaterial.

Metabolite quantitationTo compare the metabolic profiles of unconjugated metabo-

lites of the various diastereoisomers, we assayed the hepatocytemedium by GC-NICIMS in the SIR mode. In these experiments,2H4-8-epi-PGF2a (5 ng) was added to 0.25 ml of incubation me-dium before C18 solid phase extraction (SPE). Quantitation wasdone by comparing peak area ratios of the carboxylate anion ofeach metabolite to that of the internal standard, using for refer-ence the calibration curve of 8-epi-PGF2a/2H4-8-epi-PGF2a (9).This was done assuming that all metabolites gave an approxi-mately similar molar response on the NICIMS recording of theirrespective carboxylate anions (9). For compounds bearing the15-keto group, giving syn/anti isomers of the methoxime deriva-tive, the total peak area was used for calculation.

RESULTS

Identification strategy for PGF2a and F2-iPsThe hepatocyte incubation medium was first screened

for the presence of major metabolites of PGF2a and 8-epi-PGF2a by GC-NICIMS. We based ourselves on earlier de-scriptions of PGF2a metabolism in various species (14–19)


ww

.jlr.orgD

ownloaded from

http://www.jlr.org/

498 Journal of Lipid Research Volume 43, 2002

and our previous characterization of b-oxidation productsof PGF2a and 8-epi-PGF2a (9), and used full scan and SIRto screen for the carboxylate anion clusters of the PFB es-ters of metabolites possibly formed from the nine diastereo-isomers. Figure 2 shows a representative SIR tracing of theunconjugated metabolites of 8-epi-PGF2a.

The unconjugated products tentatively identified bythis screening method were then structurally identifiedafter HPLC separation by examining the mass spectra ofthe product ions formed after collision-activated decompo-

sition (CAD) of the carboxylate anions produced by electro-spray ionization (HPLC-MS/MS) (Table 1). Figure 3 showsa representative HPLC-MS/MS tracing of the unconju-gated metabolites of 8-epi-PGF2a.

Chromatographic and mass spectral features of parentcompounds, their metabolites, and authentic standardsare summarized in Table 2 (GC-NICIMS) and Table 3(HPLC-MS/MS). PGF2a and its two epimers at C-8 (8-epi-PGF2a) and C-12 (12-epi-PGF2a) gave excellent separationby both GC and HPLC, and their corresponding epimeric

Fig. 2. Gas chromatography-negative ion chemicalionization mass spectrometry (GC-NICIMS) selectedion recording tracing of carboxylate anions (M-PFB)2 of 8-epi-PGF2a and its unconjugated metabo-lites formed by isolated rat hepatocytes. The com-pounds were extracted from the incubation mediumand derivatized to pentafluorobenzyl ester/meth-oxime/trimethylsilyl ether (PFB/MO/TMS).13,14DH-15K, 13,14-dihydro-15-keto; 2,3-dinorDH,2,3-dinor-5,6-dihydro. The peak labeled with anasterisk is from the isotopic cluster of 2,3-dinormetabolite.

TABLE 1. Metabolites of PGF2a and F2-iPs identifieda in isolated rat hepatocyte incubation media

Modifications of PGF2 Structure PGF2a 8-epi ent-8-epi 15R-8-epi 15S-ent-8-epi 12-epi ent-12-epi 15R-12-epi 15S-ent-12-epi

C201-Tauro 1 1 1 1 1 1 1 1 113,14DH-15K 1 1 11-Tauro-13,14DH-15K 1 1 1

C182,3-Dinor 1 1 1 1 1 11-Tauro-2,3-dinor 1 1 1 113,14DH-15K-2,3-dinor 11-Tauro-13,14DH-15K-2,3-dinor 1

C182,3-dinorDH 1 1 1 1 11-Tauro-2,3-dinorDH 1 1 113,14DH-15K-2,3-dinorDH 1 11-Tauro-13,14DH-15K-2,3dinorDH 1

C162,3,4,5-Tetranor 1 1 1 1 1 1 1 1 113,14DH-15K-2,3,4,5-tetranor 1 1 1 1 1 1

1-Tauro, taurine conjugate at C-1; 13,14DH-15K, 13,14-dihydro-15-keto; 2,3-dinorDH, 2,3-dinor-5,6-dihydro.a Unconjugated metabolites were identified by both GC-NICIMS and HPLC-MS/MS, and taurine conjugates by HPLC-MS/MS.


ww

.jlr.orgD

ownloaded from

http://www.jlr.org/

Chiabrando et al. Identification of metabolites from Type III F2-isoprostanes 499

Fig. 3. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) tracing of total ion current (TIC) of prod-uct ions generated by collision-activated decomposition of carboxylate anions (M-H)2 of 8-epi-PGF2a and its unconjugated metabolitesformed by isolated rat hepatocytes. The compounds were extracted from the incubation medium. 13,14DH-15K, 13,14-dihydro-15-keto; 2,3-dinorDH, 2,3-dinor-5,6-dihydro.

Fig. 4. Tandem mass spectrometry (MS/MS) spectra obtained from collision-activated decomposition of the carboxylate anions (m/z 353;38 eV) of parent PGF2a and 8-epi-PGF2a, plus authentic 13,14-dihydro-15-keto-PGF2a and hepatocyte-derived 13,14-dihydro-15-keto-8-epi-PGF2a. HPLC retention time is indicated for each metabolite.


ww

.jlr.orgD

ownloaded from

http://www.jlr.org/


TA

BL

E3.

HPL

C-M

S/M

S be

hav

ior

of P

GF 2

a, C

lass

III

F2-

iPs

and

thei

r un

con

juga

ted

met

abol

ites

[M-H

]2

HPL

C R

eten

tion

Tim

e

Mod

ifica

tion

s of

PG

F 2 S

truc

ture

PGF 2

a8-

epi

ent-8

-epi

15R

-8-ep

i15

S-en

t-8-ep

i12

-epi

ent-1

2-ep

i15

R-1

2-ep

i15

S-en

t-12-

epi

m/z

min

Pare

nt P

GF 2

com

poun

ds (

PGF 2

a o

r F 2

-iP)

353

21.1

19.7

19.7

19.7

19.7

20.7

20.7

21.5

21.5

13,1

4DH

-15K

met

abol

ites

35

324

.221

.921

.9St

anda

rd: 1

3,14

DH

-15K

-PG

F 2a

353

24.2

2,3-

Din

or m

etab

olit

es

325

16.3

16.3

16.4

17.1

17.1

Stan

dard

: 2,3

-din

or-en

t-8-ep

i-PG

F 2a

325

16.3

13,1

4DH

-15K

-2,3

-din

or m

etab

olit

es32

518

.9

2,3-

Din

or-5

,6D

H m

etab

olit

es

327

17.5

16.2

16.2

16.5

16.5

Stan

dard

: 2,3

-din

or-5

,6-d

ihyd

ro-8

-epi-P

GF 2

a32

716

.2

13,1

4DH

-15K

-2,3

-din

or-5

,6D

H m

etab

olit

es

327

21.0

18.7

Stan

dard

: 2,3

-din

or-5

,6-d

ihyd

ro-1

3,14

DH

-15K

-ent

-8-ep

i-PG

F 2a

327

18.7

2,3,

4,5-

Tet

ran

or m

etab

olit

es29

914

.413

.213

.213

.913

.912

.912

.9(1

3.8)

(13.

8)

13,1

4DH

-15K

-2,3

,4,5

-tetr

anor

met

abol

ites

299

17.5

15.9

15.9

16.0

18.5

18.5

13,1

4DH

-15K

, 13,

14-d

ihyd

ro-1

5-ke

to; 5

,6D

H, 5

,6-d

ihyd

ro.

TA

BL

E2.

GC

-NIC

IMS

beh

avio

r of

PG

F 2a, C

lass

III

F2-

iPs

and

thei

r un

con

juga

ted

met

abol

ites

as

PFB

-TM

S or

PFB

-MO

-TM

Sa de

riva

tive

s

[M-P

FB]2

GC

Ret

enti

on T

ime

Mod

ifica

tion

s of

PG

F 2 S

truc

ture

PGF 2

a8-

epi

ent-8

-epi

15R

-8-ep

i15

S-en

t-8ep

i12

-epi

ent-1

2-ep

i15

R-1

2-ep

i15

S-en

t-12e

pi

m/z

min

Pare

nt P

GF 2

com

poun

ds (

PGF 2

a o

r F 2

-iP)

569

11.9

11.6

11.6

11.5

11.5

11.8

11.8

11.8

11.8

13,1

4DH

-15K

met

abol

ites

52

612

.011

.8/1

1.9

11.7

/11.

8St

anda

rd: 1

3,14

DH

-15K

- PG

F 2a

526

12.0

2,3-

Din

or m

etab

olit

es

541

10.4

10.4

10.4

10.6

10.6

Stan

dard

: ent

-2,3

-din

or-8

-epi-P

GF 2

a54

110

.4

13,1

4DH

-15K

-2,3

-din

or m

etab

olit

es49

810

.7/1

0.8

2,3-

Din

or-5

,6D

H m

etab

olit

es

543

10.8

10.5

10.5

10.8

10.8

Stan

dard

: 2,3

-din

or-5

,6-d

ihyd

ro-8

-epi-P

GF 2

a54

310

.5

13,1

4DH

-15K

-2,3

-din

or-5

,6D

H m

etab

olit

es

500

11.0

10.8

/10.

9St

anda

rd: 1

3,14

DH

-15K

-2,3

-din

or-5

,6D

H-e

nt-8

-epi-P

GF 2

a50

010

.8/1

0.9

2,3,

4,5-

Tet

ran

or m

etab

olit

es51

59.

59.

29.

29.

29.

29.

49.

49.

49.

4

13,1

4DH

-15K

-2,3

,4,5

-tetr

anor

met

abol

ites

472

9.6

9.5/

9.6

9.5/

9.6

9.5/

9.6

9.6

9.6

13,1

4DH

-15K

, 13,

14-d

ihyd

ro-1

5-ke

to; 5

,6D

H, 5

,6-d

ihyd

ro.

aPe

ak d

oubl

ets

for

MO

der

ivat

ives

of 1

5K c

ompo

unds

are

due

to s

yn/a

nti

isom

ers.


ww

.jlr.orgD

ownloaded from

http://www.jlr.org/


metabolites behaved similarly (Tables 2 and 3). For allcompounds, the steric configuration of the hydroxyl atC-15 had little or no influence on the retention time inboth techniques in the conditions used (Tables 2 and 3).The enantiomeric pairs (e.g., 8-epi-PGF2a and ent-8-epi-PGF2a)showed the expected identical behavior, since they obviouslyneed a chiral separation step to be resolved (Tables 2 and3). All diastereoisomers gave identical MS/MS spectra, asshown for example for PGF2a and 8-epi-PGF2a (Fig. 4), asreported by Waugh et al. (7).

Most unconjugated compounds were identified by di-rect comparison with the corresponding standard synthe-sized ad hoc in our laboratories (Tables 2, 3, and Table 4).Metabolites lacking the corresponding reference com-pounds were identified by comparison with an authentic di-astereoisomeric analog (Tables 2 and 3). Additional identi-fication criteria are described below in the specific sections.MS/MS spectra of selected diastereoisomeric unconjugatedmetabolites are shown in Fig. 4, Figs. 5 and 6.

For simplicity, the various diastereoisomeric metabo-lites identified for PGF2a and F2-iPs are described by refer-ring to their common chemical structure. Identificationcriteria for conjugated metabolites are reported below.

Metabolites of PGF2a and Type III F2-iPsMetabolism of PGF2a by isolated rat hepatocytes was

studied as a reference for the eight 8,12-cis-9,11-cis TypeIII F2-iP diastereoisomers (Fig. 1). Some of the com-pounds identified here as PGF2a metabolites are productsof known metabolic pathways, i.e., one or two steps ofb-oxidation, reduction of D5 and/or D13 double bonds,and 15-OH dehydrogenation (Fig. 7). We also identifiednovel taurine conjugates of PGF2a and its metabolites.Some, but not all, of these compounds were also formedfrom F2-iPs, which were in fact metabolized according tostereoselective patterns (Table 1).

The overall view of the metabolites identified for thenine PGF2 diastereoisomers (Table 1) shows that: 1) allmetabolites of PGF2a were also present as products ofsome, although not all, F2-iPs; 2) some metabolites wereexclusive products of F2-iPs (i.e., all 2,3-dinor metaboliteswith intact D5 double bond); 3) among F2-iPs, 8-epi-PGF2a

had the largest number of products in common withPGF2a; 4) the only products common to all nine diastereo-isomers were the 2,3,4,5-tetranor metabolites and the tau-rine conjugates of the parent compounds; 5) within the8-epi series, those with the 15R configuration were nottransformed into any of the seven possible metabolitesbearing the 13,14-dihydro-15-keto modification; and 6)within the 12-epi series, each enantiomeric pair showed acharacteristic profile.

Unconjugated metabolites. To help interpret the MS/MSspectra of the metabolites, the product ions formed fromthe precursor carboxylate anions of the metabolites arelisted in Table 4 in comparison with those of parent PGF2a

or F2-iPs, plus 3,39,4,49-2H4-8-epi-PGF2a, and 1,19-18O2-2,3-dinor-5,6-dihydro-8-epi- PGF2a. All mass spectral data referto authentic standards and corresponding metabolites, ex-cept for hepatocyte-derived 13,14-dihydro-15-keto-2,3-

dinor metabolite and the two tetranor compounds. Thestructural interpretation given in Table 4 is mainly basedon work by Murphy’s group (7, 20–21), or deducted fromthe comparison of the various metabolites and stable iso-tope-labeled analogs. Product ions of the carboxylateanion of 8-epi-PGF2a or PGF2a, that reportedly comprisethe a-chain, had the expected counterparts in the a-chainshortened metabolites, with shifts of 28 amu less (2,3-dinor), 26 amu less (2,3-dinor-5,6-dihydro), and 54 amuless (2,3,4,5-tetranor) (Table 4). This and other specificfeatures described below allowed identification of thethree compounds for which no authentic standard wasavailable.

2,3-dinor metabolite. No compound with a 2,3-dinormodification and intact D5 double bond was found forPGF2a, confirming previous observations in the rat in vitroand/or in vivo by ourselves (9) and others (16–18). The2,3-dinor metabolite was, however, identified as a promi-nent product of several F2-iP diastereoisomers, namely 8-epi-PGF2a, 15R-8-epi-PGF2a, ent-8-epi-PGF2a, 15S-ent-8-epi-PGF2a, 12-epi-PGF2a, and ent-12-epi-PGF2a (Tables 1, 2, 3).2,3-Dinor-8-epi-PGF2a, which we had characterized previ-ously as a prominent endogenous metabolite in humanand rat urine, as well as a major product of 8-epi-PGF2a me-tabolism by rat hepatocytes, was now compared with au-thentic 2,3-dinor-8-epi-PGF2a recently synthesized in ourlaboratories (Fig. 5). While the carboxylate anions of par-ent compounds and other metabolites produced severalfragment ions mainly due to single and combined lossesof H2O, CO2, and C2H4O from the ring, and various partsof the alkyl chains (7, 20–21), the carboxylate anion of the2,3-dinor metabolite (m/z 325) gave a peculiar fragmenta-tion (Table 4 and Fig. 5). Even at a low collision energy(22 eV), its MS/MS spectrum was dominated by a mainfragment ion at m/z 237, probably due to the loss ofC2H4O plus CO2 from the carboxyl. Other minor ionswere observed at m/z 263 (loss of H2O and C2H4O), m/z219 (loss of H2O, C2H4O and CO2), m/z 163 (loss of H2O,CO2 and hexanal), and m/z 137. Similar MS/MS spectra(not shown) were recorded for the corresponding metabo-lites derived from the other F2-iPs.

2,3-dinor-5,6-dihydro metabolite. This was a promi-nent product of PGF2a, 12-epi-PGF2a, and ent-12-epi-PGF2a,and a minor product of 8-epi-PGF2a and ent-8-epi-PGF2a (Ta-bles 1–3). These compounds were identified by directcomparison with commercial 2,3-dinor-5,6-dihydro-8-epi-PGF2a and synthetic 2,3-dinor-5,6-dihydro-ent-8-epi-PGF2a.Figure 6 shows the MS/MS spectra of authentic 2,3-dinor-5,6-dihydro-8-epi-PGF2a and hepatocyte-derived 2,3-dinor-5,6-dihydro-PGF2a, as an example. The precursor ion (m/z327) was fragmented according to the typical pattern ofthe PGF2 compounds (Table 4). The MS/MS spectrum of2,3-dinor-5,6-dihydro-8-epi-PGF2a labeled with 18O2 en-abled us to confirm the proposed fragmentation by ob-serving the expected shift of 4 amu in all fragment ionsconserving the carboxyl (Table 4). However, the ion at m/z167 did not shift to m/z 171, as expected, when the carboxylwas labeled with 18O2. The putatively corresponding ion of8-epi-PGF2a at m/z 193 shifts in fact to m/z 197 upon label-


ww

.jlr.orgD

ownloaded from

http://www.jlr.org/


Fig. 5. MS/MS spectra obtained from collision-activated decomposition of the carboxylate anions of authentic 2,3-dinor-8-epi-PGF2a andhepatocyte-derived 2,3-dinor-8-epi-PGF2a (m/z 325; 22 eV), and hepatocyte-derived 2,3,4,5-tetranor-8-epi-PGF2a and 13,14-dihydro-15-keto-2,3,4,5-tetranor-8-epi-PGF2a (m/z 299; 30 eV). HPLC retention time is indicated for each metabolite.

Fig. 6. MS/MS spectra obtained from collision-activated decomposition of the carboxylate anions (m/z 327; 40 eV) of authentic 2,3-dinor-5,6-dihydro-8-epi-PGF2a, hepatocyte-derived 2,3-dinor-5,6-dihydro-PGF2a, authentic 13,14-dihydro-15-keto-2,3-dinor-5,6-dihydro-ent-8-epi-PGF2a, and hepatocyte-derived 13,14-dihydro-15-keto-2,3-dinor-5,6-dihydro-PGF2a. HPLC retention time is indicated for each metabolite.


ww

.jlr.orgD

ownloaded from

http://www.jlr.org/


TA

BL

E4.

Prod

uct i

onsa

dete

cted

by

MS/

MS

afte

r fr

agm

enta

tion

of c

arbo

xyla

te a

nio

ns

[M-H

]2 o

f PG

F 2a, C

lass

III

F2-

iPs

and

thei

r un

con

juga

ted

met

abol

ites

Mod

ifica

tion

s of

PGF 2

Str

uctu

reC

ompo

undb

[M-H

]2-H

2O-2

(H2O

)-C

2H4O

-3(H

2O)

-H2O

-C2H

4O

-2(H

2O)

-(C

O2

or

C2H

4O)

-C2H

4O-C

O2

-H2O

-(C

16-C

20)

-3(H

2O)

-CO

2

-H2O

-C

2H4O

-CO

2

-C2H

4O-h

exan

al

-C2H

4O-(

C14

-C20

)

-C2H

4O-C

O2

-(C

16-C

20)

-H2O

-hex

anal

-CO

2

m/z

PGF 2

a o

r C

lass

III

F2-iP

St35

333

531

730

929

929

127

326

325

524

720

919

319

13,

39,4

,49-2 H

4-PG

F 2a

St35

733

932

131

330

329

527

726

725

925

121

319

719

513

,14D

H-1

5KSt

; M35

333

531

729

127

320

919

12,

3-di

nor

St; M

325

263

245

237

219

163

13,1

4DH

-15K

-2,3

-din

orM

325

263

245

163

2,3-

din

or-5

,6D

HSt

; M32

730

929

128

327

326

524

723

722

922

118

316

716

51,

19-18

O2-

2,3-

din

or-5

,6D

HSt

331

313

295

287

269

247

241

229

221

187

167

165

13,1

4DH

-15K

-2,3

-din

or-

5,6D

HSt

; M32

730

929

126

524

716

52,

3,4,

5-te

tran

orM

299

281

255

237

219

211

201

193

137

13,1

4DH

-15K

-2,3

,4,5

-te

tran

orM

299

281

237

219

137

aFo

r ot

her

pro

duct

ion

s la

ckin

g ob

viou

s in

terp

reta

tion

, see

Fig

s. 4

–6.

bD

ata

from

: Sta

nda

rd (

St)

and/

or h

epat

ocyt

e-de

rive

d m

etab

olit

e (M

).

TA

BL

E5.

HPL

C-M

S/M

S be

hav

ior

of ta

urin

e co

nju

gate

s of

PG

F 2a, C

lass

III

F2-

iPs

and

thei

r m

etab

olit

es

Mod

ifica

tion

s of

PG

F 2 S

truc

ture

[M-H

]2

HPL

C R

eten

tion

Tim

e

PGF 2

a8-

epi

ent-8

-epi

15R

8-ep

ien

t-8-ep

i-15S

12-ep

ien

t-12-

epi

15R

-12-

epi

15S-

ent-1

2-ep

i

m/z

min

1-T

auro

460

13.3

12.4

12.1

12.4

12.1

12.5

12.5

12.7

12.7

1-T

auro

13,

14D

H-1

5K46

014

.613

.3—

—13

.3—

——

—1-

Tau

ro 2

,3-d

inor

432

—9.

1—

——

——

——

1-T

auro

-13,

14D

H-1

5K-2

,3-d

inor

432

—10

.5—

——

——

——

1-T

auro

-2,3

-din

or-5

,6D

H43

410

.4—

——

——

——

—1-

Tau

ro-1

3,14

DH

-15K

-2,3

-din

or-5

,6D

H43

412

.3—

——

——

——

—

1-Ta

uro,

taur

ine

con

juga

te a

t C-1

; 13,

14D

H-1

5K, 1

3,14

-dih

ydro

-15-

keto

; 5,6

DH

, 5,6

-dih

ydro

.

TA

BL

E6.

Prod

uct i

ons

dete

cted

by

MS/

MS

afte

r fr

agm

enta

tion

of m

olec

ular

an

ion

s [M

-H]2

of t

auri

ne

con

juga

tes

of P

GF 2

a, C

lass

III

F2-

iPs

and

thei

r m

etab

olit

es

Mod

ifica

tion

of

PGF 2

Str

uctu

re[M

-H]2

-H2O

-C2H

4O-H

2O-C

2H4O

-(C

16-C

20)

-H

-H2O

-(C

16–C

20)

1H

-C2H

4O-

hex

anal

CH

2 (C

H) 2

(CH

2)2

CO

NH

(CH

2)2

SO32

(CH

2)2

CO

NH

(CH

2)2

SO32

CH

2C

ON

H(C

H2)

2SO

32

CO

NH

(CH

2)2

SO32

NH

(CH

2)2

SO32

CH

2)2

SO32

SO32

m/z

1-T

auro

460

442

416

388

372

316

233

178

165

151

124

107

801-

Tau

ro-1

3,14

DH

-15K

460

442

233

178

151

124

107

801-

Tau

ro-2

,3-d

inor

432

388

370

151

124

1-T

auro

-13,

14D

H-1

5K-2

,3-d

inor

432

414

151

124

107

801-

Tau

ro-2

,3-d

inor

-5,6

-DH

434

390

372

362

346

165

124

107

801-

Tau

ro-1

3,14

DH

-15K

-2,3

-din

orD

H43

441

616

512

410

780

1-Ta

uro,

taur

ine

con

juga

te a

t C1;

13,

14D

H-1

5K, 1

3,14

-dih

ydro

-15-

keto

; 5,6

DH

, 5,6

-dih

ydro

. by guest, on January 12, 2019w

ww

.jlr.orgD

ownloaded from

http://www.jlr.org/


ing with 18O2, indicating retention of the carboxyl (22).Our data indicate that for 2,3-dinor-5,6-dihydro-8-epi-PGF2a,this fragment is instead due to the loss of CO2, and notC2H4O, in addition to part of the v-chain.

2,3,4,5-tetranor metabolite. A metabolite with a2,3,4,5-tetranor structure, resulting from two b-oxidationsteps, was common to PGF2a and all F2-iPs (Table 1), indi-cating that this pathway [in addition to conjugation withtaurine (see below)] is the least affected by the stere-ochemical structure of the parent compound. This was aprominent product for all diastereoisomers, except 15S-ent-8-epi-PGF2a, which had a much more abundant 13,14-dihydro-15-keto-2,3,4,5-tetranor metabolite (see below).Our previous characterization of the 2,3,4,5-tetranor me-tabolites of PGF2a and 8-epi-PGF2a (9) helped prove theidentity of the other diastereoisomeric metabolites. Thechromatographic and mass spectral characteristics of thenine diastereoisomeric 2,3,4,5-tetranor metabolites (Tables2 and 3) clearly support their identity. The MS/MS spectraof the carboxylate anion (m/z 299) showed a fragmenta-tion consistent with the proposed structure (Table 4 andFig. 5). All ions conserving the a-chain were found, as ex-pected, at a m/z value 54 amu less than the correspondingions of the parent compound.

13,14-dihydro-15-keto metabolite. A metabolite withthis structure was found for PGF2a and for two of the F2-iPs, namely 8-epi-PGF2a and 15S-ent-8-epi-PGF2a (Table 1).

The product deriving from PGF2a had GC-NICIMS andHPLC-MS/MS behavior identical to authentic 13,14-dihy-dro-15-keto-PGF2a (Tables 2 and 3, and Fig. 4). On GC,the two syn/anti isomers due to derivatization of the ketogroup to methoxime eluted shortly after the parent com-pounds, as a single broad peak for 13,14-dihydro-15-keto-PGF2a but as separate peaks for the metabolites from 8-epi-PGF2a and 15S-ent-8-epi-PGF2a (Table 2 and Fig. 2). OnHPLC, the 13,14-dihydro-15-keto diastereoisomeric metab-olites eluted about 3 min later than their parent com-pounds (Table 3 and Fig. 3). The MS/MS spectra of hepato-cyte-derived and authentic 13,14-dihydro-15-keto-PGF2a

are shown in Fig. 4. On account of their identical molecu-lar weight, the 13,14-dihydro-15-keto metabolite and theparent compound have the same mass-to-charge ratio forthe carboxylate anion and several product ions (Table 4).The MS/MS spectrum of the 13,14-dihydro-15-keto metab-olite can, however, be clearly distinguished by the promi-nent ions at m/z 113, 183, and 195, and the very low abun-dance of m/z 193 (base peak in the parent compound).The corresponding metabolites from 8-epi-PGF2a and 15S-ent-8-epi-PGF2a had identical MS/MS spectra (not shown).

13,14-dihydro-15-keto-2,3-dinor metabolite. A com-pound with this structure was found only as a minor productof 8-epi-PGF2a (Table 1). This metabolite was identified byobserving its GC-NICIMS and HPLC-MS/MS characteris-tics, which are consistent with its putative structure. In

Fig. 7. Structures of unconjugated metabolites identified in this study. Formation of taurine conjugates at C1 (taurine-conj) is also indi-cated (structures shown in Fig. 8).


ww

.jlr.orgD

ownloaded from

http://www.jlr.org/


particular, GC-NICIMS analysis showed the peak doubletof the syn/anti isomers (RT 5 10.7 and 10.8 min) at m/z498 when derivatized to methoxime, but a single peak(RT 5 10.9 min) at m/z 469 (29 amu less, correspondingto the different groups at C-15, i.e., C5NOCH3 vs. C5O),when the keto group was not derivatized (not shown).The MS/MS spectrum, although poorly informative, wasvery similar to that of the corresponding metabolite with-out the 13,14-dihydro-15-keto modification (Table 4), ex-cept for a difference in the most abundant ion (base peakat m/z 263).

13,14-dihydro-15-keto-2,3-dinor-5,6-dihydro metabo-lite. A compound with this structure was found only as aminor product of PGF2a and 8-epi-PGF2a (Table 1). This me-tabolite was identified by direct comparison with authentic13,14-dihydro-15-keto-2,3-dinor-5,6-dihydro-ent-8-epi-PGF2a

using both GC-NICIMS and HPLC-MS/MS (Tables 2 and3). The MS/MS spectra of the standard and the metabolitefrom PGF2a are shown in Fig. 6. The mass spectrum wassimilar for the corresponding metabolite derived from 8-epi-PGF2a (not shown). As observed for the C20 analogs,although the MS/MS spectra of the 13,14-dihydro-15-keto-2,3-dinor-5,6-dihydro metabolite and its 2,3-dinor-5,6-dihydro analog have the same mass-to-charge ratio for theprecursor (m/z 327) and some product ions, the former canbe clearly distinguished by the prominent ion at m/z 113,typical of all metabolites bearing the 13,14-dihydro-15-ketomodification, and the absence of an ion at m/z 111 (Fig. 6).

13,14-dihydro-15-keto-2,3,4,5-tetranor metabolite. Aprominent product with this structure was found forPGF2a, and among F2-iPs, for 8-epi-PGF2a, 15S-ent-8-epi-PGF2a, 12-epi-PGF2a, and ent-12-epi-PGF2a (Table 1). For15S-ent-8-epi-PGF2a, this was by far the most abundant me-tabolite. These compounds were identified by comparingtheir MS/MS spectra (Fig. 5) with two authentic C20 andC18 homologs, 13,14-dihydro-15-keto-PGF2a and 13,14-dihydro-15-keto-2,3-dinor-5,6-dihydro-ent-8-epi-PGF2a (Figs.4 and 6). As expected, the 13,14-dihydro-15-keto-2,3,4,5-tetranor metabolite had the carboxylate anion (m/z 299)and most of the product ions at the same m/z value as the2,3,4,5-tetranor metabolite, except for the expected majorion at m/z 113 (Fig. 5). HPLC and GC behavior, as well asthe NICIMS features of all isomers, were consistent withthis putative structure (Tables 2 and 3). The presence ofthe keto group was also confirmed, e.g., for 13,14-dihydro-15-keto-2,3,4,5-tetranor-8-epi-PGF2a, by observing that thetwo peaks (syn/anti isomers) eluting after 9.5 and 9.6 minat m/z 472 disappeared when the keto group was not de-rivatized to methoxime, whereas a peak appeared at m/z443 after 9.7 min, as expected (not shown).

Taurine conjugates. Taurine conjugates of parentPGF2a, F2-iPs, and their prominent C20 and C18 metabo-lites were identified on the basis of their HPLC behavior(Table 5 and Fig. 8) and MS/MS spectra (Table 6, Fig. 8,and Fig. 9). Some of these spectra showed striking similar-ities with those described by Hankin et al. (20) for the cor-

Fig. 8. HPLC-MS/MS tracing of TIC and MS/MS spectra obtained from collision-activated decomposition of the molecular anions (m/z 460;56 eV) of hepatocyte-derived 1-tauro-PGF2a, and 1-tauro-13,14-dihydro-15-keto-PGF2a. HPLC retention time is indicated for each metabolite.


ww

.jlr.orgD

ownloaded from

http://www.jlr.org/


responding taurine conjugates of PGE2 and its metabo-lites. Because no reference compounds were available, wewere not able to quantify these metabolites. However, tak-ing the intensity of the total ion current generated byeach of these compounds on HPLC-MS/MS as an approx-imate indicator of its relative abundance, for each diaste-reoisomer the total amount of taurine conjugates and un-conjugated products was of about the same order ofmagnitude. This is in accordance with Hankin’s study(20), showing comparable overall amounts of radioactivityassociated with taurine-conjugated or unconjugated me-tabolites of 3H-PGE2 formed by isolated rat hepatocytes.

Excluding the taurine conjugates of 2,3-dinor and13,14-dihydro-15-keto-2,3-dinor metabolites which gave apeculiar fragmentation, like their unconjugated counter-parts, the MS/MS spectra of taurine conjugates showed:1) prominent ions derived from detachment of the entireor fragmented taurine group (m/z 124, H2N-CH2-CH2-

; m/z 107, CH2 5 CH- ; m/z 80, ), or 2) ionsderived from retention of the charge on the PGF back-bone with loss of H2O and/or C2H4O (Table 6). Otherprominent ions correspond to fragments of the a-chainconjugated with taurine (for the parent compounds, m/z151, 178, and 233, corresponding to taurine attached toC1, C1–C3, and C1–C7). Of these, the smallest fragmentat m/z 151 (CONH(CH2)2 ) was common to all C18

SO32 SO3

2 SO32

SO32

and C20 conjugates. As shown by Hankin et al. (20) fortaurine conjugates of PGE2 and its metabolites, theseproducts tend to be scarcely fragmented under standardMS/MS conditions, except for the taurine-dinor metabo-lite, which shows easy detachment of the taurine group,but no other significant fragment except at m/z 124. Highcollision energy (56 eV) was therefore used to obtainstructurally informative MS/MS spectra of the taurineconjugates, except for 1-tauro-2,3-dinor and 1-tauro-13,14-dihydro-15-keto-2,3-dinor metabolites (42 eV).

As observed for unconjugated metabolites, the 13,14-dihydro-15-keto modification does not alter the molecularweight, so that MS/MS spectra have the molecular anionsand many fragment ions at the same m/z values. However,a common feature of the MS/MS spectra of all tauro-conjugates with the 13,14-dihydro-15-keto modificationwas the absence of prominent ions in the central part ofthe mass spectrum (Table 6, and Figs. 8 and 9).

Taurine conjugates of c20 compounds. A taurine con-jugate of the parent compound was observed for PGF2a

and all F2-iP diastereoisomers (Table 1). This was by far themost abundant of all conjugates for all diastereoisomers,except for 15S-ent-8-epi-PGF2a, which, instead, had a moreprominent 1-tauro-13,14-dihydro-15-keto metabolite, in ac-cordance with 15-OH dehydrogenation being a preferen-tial pathway for this diastereoisomer. The least abundant

Fig. 9. MS/MS spectra obtained from collision-activated decomposition of the molecular anions of hepatocyte-derived 1-tauro-2,3-dinor-8-epi-PGF2a and 1-tauro-13,14-dihydro-15-keto-2,3-dinor-8-epi-PGF2a (m/z 432; 42 eV) plus 1-tauro-2,3-dinor-5,6-dihydro-PGF2a and 1-tauro-13,14-dihydro-15-keto-2,3-dinor-5,6-dihydro-PGF2a (m/z 434; 56 eV). HPLC retention time is indicated for each metabolite.


ww

.jlr.orgD

ownloaded from

http://www.jlr.org/


epi-PGF2a > ent-8-epi-PGF2a > 15S-ent-8-epi- PGF2a @ 15S-ent-12-epi- PGF2a).

DISCUSSION

F2-isoprostanes, a family of 64 compounds isomeric toPGF2a, are products of free radical-mediated non-enzymaticperoxidation of arachidonic acid (1–3). These com-pounds can be used to evaluate local or systemic lipid per-oxidation in vivo, since they are detected esterified to tis-sue and plasma lipids and in the free form in body fluids(2–5). Increased F2-iP levels were found in several experi-mental and clinical conditions associated with free radical-mediated oxidant damage (2–5). Given that F2-iPs are be-coming increasingly popular as oxidative stress markers, itis important to expand our knowledge about their forma-tion and fate in vivo. Identification of the major metabo-lites of the various F2-iP isomers, with the developmentof specific assays, may help evaluate the in vivo formation ofselected compounds. This is of particular interest for theType III regioisomer family, since the only compoundswith proven biological activity so far are these diastereo-isomers of PGF2a.

Isolated hepatocytes represent a well-characterized, rathersimple system to screen for potential pathways of hepaticmetabolism of endogenous and exogenous compounds(23). This in vitro system has the advantage of employingfreshly isolated cells, with their intact enzymatic machinery,that reproducibly yield enough material for mass spectrom-etry identification of metabolites after rather simple puri-fication procedures. This technique has been successfullyused in the field of eicosanoids and isoprostanes (9, 17, 20).

We compared the metabolic fate of PGF2a versus TypeIII F2-iPs. Among the 16 possible diastereoisomers of theType III family, we considered only the eight cis ring-substituted diastereoisomers that are believed to be themost abundant (6). We identified several products result-ing from a combination of b-oxidation, reduction of D5

and/or D13 double bonds, or 15-OH oxidation, plus tau-rine conjugates of parent compounds and their promi-nent C20 and C18 metabolites.

This study showed slight-to-marked stereo- and enantio-selectivity in the different enzymatic steps involved in themetabolic transformations of PGF2a and Type III F2-iPs.Some compounds were extensively metabolized to a vari-ety of products, while others were poorly metabolized,with specific stereochemical configurations favoring orimpeding selected reactions. For example, the steric con-figuration of the hydroxyl at C-15 must be S, as in the cy-clooxygenase-derived PG, for 15-OH dehydrogenation tooccur easily. In fact, no metabolites with the 15-keto modi-fication (unconjugated or conjugated) were formed fromdiastereoisomers with the 15R configuration, except forthe 13,14-dihydro-15-keto-2,3,4,5-tetranor metabolites ofent-8-epi-PGF2a (minor product) and ent-12-epi-PGF2a. TheD13 double bond of prostaglandin (PG) cannot be re-duced without prior 15-OH dehydrogenation (17). Thisappears to be true for F2-iPs too, as all metabolites with the

conjugate was found for 15S-ent-12-epi-PGF2a, in agree-ment with its scarce overall metabolism. In addition tothe prominent 1-tauro-13,14-dihydro-15-keto metaboliteof 15S-ent-8-epi-PGF2a, other 1-tauro-13,14-dihydro-15-keto-metabolites were found as minor products of the isomersforming the corresponding unconjugated metabolite, i.e.,PGF2a and 8-epi-PGF2a, (Table 1). This was taken in addi-tion to the chromatographic and mass spectral behavior(Tables 5 and 6, and Fig. 8), as further proof of their iden-tity in the absence of reference material.

Taurine conjugates of c18 metabolites. Taurine con-jugates were identified for all major diastereoisomeric C18metabolites (Table 1). In particular, we identified: 1) 1-tauro-2,3-dinor metabolites of 8-epi-PGF2a, 15R-8-epi-PGF2a,12-epi-PGF2a, and ent-12-epi-PGF2a; 2) 1-tauro-2,3-dinor-5,6-dihydro metabolites of PGF2a, 12-epi-PGF2a and ent-12-epi-PGF2a; 3) 1-tauro-13,14-dihydro-15-keto-2,3-dinor metabo-lites of 8-epi-PGF2a (minor metabolite); and 4) 1-tauro-13,14-dihydro-15-keto-2,3-dinor-5,6-dihydro metabolites ofPGF2a. HPLC-MS/MS features of these metabolites areshown in Table 5, and representative MS/MS spectra areshown in Fig. 9 and interpreted in Table 6.

Taurine conjugates of c16 metabolites. AlthoughMS/MS spectra of sufficient intensity could not be re-corded for taurine conjugates of C16 metabolites, prelimi-nary evidence obtained by multiple reaction monitoringof the precursor-to-product ion transition from m/z 406 tom/z 124 suggests that very small amounts of these conju-gates were present in the hepatocyte medium. The mostprominent unconjugated C16 metabolites with and with-out the 13,14-dihydro-15-keto modification had their cor-responding taurine conjugate as a small product (data notshown). Hankin et al. (20) also found that the taurineconjugate of tetranor-PGE2 was a minor component of themetabolic profile.

Metabolic profiles of PGF2a and F2-iPsIn a first experiment to establish the best experimental

conditions for the identification study, PGF2a and 8-epi-PGF2a were incubated separately for 10, 20, and 40 min.Quantitation by GC-NICIMS of unconjugated metabolitesin the incubation medium and cell lysate indicated that:1) all unconjugated metabolites accumulated with time inthe medium (sum of metabolites formed per plate: ,5–10 ng/min), while their abundance remained constant intime in the cell lysate; and 2) the amount of the parentcompounds decreased with time in the medium, reaching,150 ng/ml at 40 min, but was low at any time in thehepatocyte lysate (,20 ng).

Other GC-NICIMS measurements showed that, after 20min of incubation, the unmetabolized compound in themedium was reduced to 50–60% of the initial amount forPGF2a, 8-epi-PGF2a, and 12-epi-PGF2a, ,70% for 15R-8-epi-PGF2a, ent-12-epi-PGF2a, and 15R-12-epi-PGF2a, ,80% forent-8-epi-PGF2a and 15S-ent-8-epi-PGF2a, and 95% for 15S-ent-12-epi-PGF2a. This is in agreement with the abundance pro-file of total metabolites (unconjugated plus conjugated)from the various diastereoisomers (PGF2a > 8-epi-PGF2a >12-epi-PGF2a .15R-8-epi-PGF2a > ent-12-epi-PGF2a > 15R-12-


ww

.jlr.orgD

ownloaded from

http://www.jlr.org/


13,14-dihydro modification had the concomitant pres-ence of the 15-keto group.

Another reaction that was markedly influenced by thesteric structure was the reduction of D5 double bond,which proceeded easily for PGF2a, but was more or less im-peded for the various enantiomeric pairs of F2-iPs. Thepathways that were the least affected by the stereochemi-cal configuration were b-oxidation and taurine conjuga-tion, which gave rise to at least one product each, even forthe scantly metabolized 15S-ent-12-epi-PGF2a.

The finding of significant amounts of taurine conju-gates of PGF2a, F2-iPs, and their prominent C20 and C18metabolites drew our attention to the fact that, apart fromthe 13,14-dihydro-15-keto metabolites, all the other prod-ucts identified here are formed through activation of thecarboxyl group to a CoA-thioester. It is generally acceptedthat both b-oxidation and taurine conjugation of carboxylicacids occur after the formation of a reactive CoA thioesterintermediate (24). For PGF2a and F2-iPs, this commonprecursor appears to be easily formed for C20 and C18products, because there is subsequent formation of 1) un-conjugated C18 and C16 products through b-oxidation,and 2) taurine conjugates of C20 and C18 productsthrough taurine-N-acyltranferase activity. The fact thatb-oxidation does not proceed further to C14 products andthat the C16 metabolites, although abundant, are poorlyconjugated with taurine, suggests, among other possibili-ties, a scant formation of the precursor CoA-thioester ofthe C16 metabolites. Due to the presence of this commonactivation step for most of the prominent metabolites ofPGF2a and F2-iPs, xenobiotic carboxylic acids that are me-tabolized through the corresponding CoA-thioesters mightcause inhibition and/or diversion of PGF2a and F2-iPs me-tabolism in vivo. An example of such drugs are the widelyused 2-arylpropionic non-steroidal anti-inflammatory drugs(24). This should be taken into consideration when mea-surements of F2-iPs or their selected metabolites in bodyfluids are used for assessing their formation in vivo.

Although a detailed quantitative description of PGF2a

and F2-iP metabolism by isolated rat hepatocytes was be-yond the scope of this study, for each diastereoisomer thetotal amounts of conjugated and unconjugated productswere in general of the same order of magnitude. Al-though this cannot be taken as proof that these productsare of comparable abundance in vivo, it certainly showsthat these cells have an efficient enzymatic machinery forboth pathways, and that these metabolites may thereforebe formed in vivo, at least in the rat.

Many in vivo studies in different mammalian speciesfound that exogenous PGF2a is metabolized through com-mon enzymatic pathways, including those discussed above(14–16, 18, 19). Nevertheless, not only are the prominentcirculating and urinary metabolites different in the vari-ous species, but even in the same species the mode of ad-ministration may alter the metabolic profile of PGF2a (16,18, 19). Searching for the major metabolites of the endog-enously formed, rather than exogenously administered,compounds in each species would hence be the best ap-proach, but this can be extremely difficult in the absence

of any preliminary analytical characterization of poten-tial in vivo metabolites. A simplified approach such asthat described here, despite suffering from the obviousintrinsic limitations of an in vitro study, allows, in prin-ciple, the obtaining of useful information for subse-quent in vivo studies aimed at identifying the main en-dogenous products.

If their presence is proved in vivo, some of the metabo-lites identified here might become useful targets of newassays for determining overall F2-iP formation, or for eval-uating the in vivo formation and fate of a specific bio-active diastereoisomer. Selected metabolites might also bechosen for their particular steric configurations or theirgreater abundance in vivo, for developing immunoassayswith minimal cross-reactivity against endogenous cyclooxy-genase-derived metabolites.

In conclusion, we showed how the different Type IIIF2-iP diastereoisomers are degraded through pathways ofPGF2a metabolism, with metabolic profiles depending ontheir steric configuration. These data, together with ourstructural and analytical characterization of the metabo-lites, may be useful as a basis for further investigating F2-iPdisposition in vivo.

Claudia Rivalta is a fellow of the Fondazione A. e A. Valenti.

Manuscript received 2 October 2001 and in revised form 19 November 2001.

REFERENCES

1. Morrow, J. D., K. E. Hill, R. F. Burk, T. M. Nammour, K. F. Badr,and L. J. Roberts, II. 1990. A series of prostaglandin F2-like com-pounds are produced in vivo in humans by a non-cyclooxygenase,free radical-catalyzed mechanism. Proc. Natl. Acad. Sci. USA. 87:9383–9387.

2. Roberts, II, L. J., and J. D. Morrow. 2000. Measurement of F2-isoprostanes as an index of oxidative stress in vivo. Free Radical Biol.Med. 28: 505–513.

3. Lawson, J. A., J. Rokach, and G. A. FitzGerald. 1999. Isoprostanes:formation, analysis and use as indices of lipid peroxidation in vivo.J. Biol. Chem. 274: 24441–24444.

4. Delanty, N., M. Reilly, D. Praticò, D. J. Fitzgerald, J. A. Lawson, andG. A. FitzGerald. 1996. 8-epi-PGF2a: specific analysis of an iso-eicosanoid as an index of oxidant stress in vivo. Br. J. Clin. Pharma-col. 42: 15–19.

5. Praticò, D. 1999. F2-isoprostanes: sensitive and specific non-invasive indices of lipid peroxidation in vivo. Atherosclerosis. 147:1–10.

6. Rokach, J., S. P. Khanapure, S-W. Hwang, M. Adiyaman, J. A. Law-son, and G. A. FitzGerald. 1997. The isoprostanes: a perspective.Prostaglandins. 54: 823–851.

7. Waugh, R. J., J. D. Morrow, L. J. Roberts, II, and R. C. Murphy.1997. Identification and relative quantitation of F2-isoprostaneregioisomers formed in vivo in the rat. Free Radical Biol. Med. 23:943–954.

8. Roberts II, L. J., K. P. Moore, W. E. Zackert, J. A. Oates, and J. D.Morrow. 1996. Identification of the major urinary metabolite ofthe F2-isoprostane 8-iso-prostaglandin F2a in humans. J. Biol. Chem.271: 20617–20620.

9. Chiabrando, C., A. Valagussa, C. Rivalta, T. Durand, A. Guy, E. Zuc-cato, P. Villa, J-C. Rossi, and R. Fanelli. 1999. Identification andmeasurement of endogenous b-oxidation metabolites of 8-epi-prostaglandin F2a. J. Biol. Chem. 274: 1313–1319.

10. Basu, S. 1998. Metabolism of 8-iso-prostaglandin F2a. FEBS Letters.428: 32–36.

11. Roland, A., T. Durand, T. Rondot, J-P. Vidal, and J-C. Rossi. 1996. Apractical asymmetric synthesis of ent-12-epi-PGF2a methyl ester.Bull. Soc. Chim. Fr. 133: 1149–1154.


ww

.jlr.orgD

ownloaded from

http://www.jlr.org/


12. Guy, A., T. Durand, J-P. Vidal, and J-C. Rossi. 1997. Total synthesisof 15(RS)-5,6-dehydro-8-epi-PGF2a methyl ester by a biomimeticprocess. Tetrahedron Lett. 38: 1543–1546.

13. Durand, T., A. Guy, O. Henry, J-P. Vidal, J-C. Rossi, C. Rivalta, A.Valagussa, and C. Chiabrando. 2001. Total syntheses of four me-tabolites of 15-F2t-isoprostane. Eur. J. Org. Chem. 4: 809–819.

14. Granström, E., and B. Samuelsson. 1971. On the metabolism ofprostaglandin F2a in female subjects. J. Biol. Chem. 246: 5254–5263.

15. Granström, E., and B. Samuelsson. 1971. On the metabolism ofprostaglandin F2a in female subjects. II. Structures of six metabo-lites. J. Biol. Chem. 246: 7470–7485.

16. Green, K. 1971. The metabolism of prostaglandin F2a in the rat.Biochim. Biophys. Acta. 231: 419–444.

17. Sago, T., R. Nakayama, T. Okumura, and K. Saito. 1986. Metabo-lism of prostaglandins D2 and F2a in primary cultures of rat hepa-tocytes. Biochim. Biophys. Acta. 879: 330–338.

18. Sun, F. F. 1974. Metabolism of prostaglandin F2a in the rat. Bio-chim. Biophys. Acta. 348: 249–262.

19. Granström, E., H. Kindahl, and M. L. Swahn. 1982. Profiles of pros-

taglandin metabolites in the human circulation. Identification of late-appearing, long-lived products. Biochim. Biophys. Acta. 713: 46–60.

20. Hankin, J. A., P. Wheelan, and R. C. Murphy. 1997. Identificationof novel metabolites of prostaglandin E2 formed by isolated rathepatocytes. Arch. Biochem. Biophys. 340: 317–330.

21. Zirrolli, J. A., E. Davoli, L. Bettazzoli, M. Gross, and R. C. Murphy.1990. Fast atom bombardment and collision-induced dissociation ofprostaglandins and thromboxanes: some examples of charge re-mote fragmentation. J. Am. Soc. Mass Spectrom. 1: 325–335.

22. Li, H., J. A. Lawson, M. Reilly, M. Adiyaman, S-W. Hwang, J.Rokach, and G. A. FitzGerald. 1999. Quantitative high perfor-mance liquid chromatography/tandem mass spectrometric analy-sis of the four classes of F2-isoprostanes in human urine. Proc. Natl.Acad. Sci. USA. 96: 13381–13386.

23. Berry, M. N., H. J. Halls, and M. B. Grivell. 1992. Techniques forpharmacological and toxicological studies with isolated hepato-cyte suspensions. Life Sci. 51: 1–16.

24. Hutt, A. J., and J. Caldwell. 1990. Amino acid conjugation. In Con-jugation Reactions in Drug Metabolism. G. J. Mulder, editor. Tay-lor & Francis, London. 273–305.


ww

.jlr.orgD

ownloaded from

http://www.jlr.org/

Documents

Identification of metabolites from Type III F2-isoprostane diastereoisomers by mass spectrometry