PROSTAGLANDINS

PGEl METAE!OLISMBYTHE PERFUSED RATLIVER D.J. Osborne, J.R. Boot, A.F. Cockerill, KG. Cranstone,W. Dawson, J. Harvey D.N.B.!&llenand C.W. Smith Lilly PesesrchCentre Ltd Erl WocdManor Windlesham, Surrey, GD20 6PH, England

The hepatic andbiliarymetabolites of PGEl have been isolated rat liver andidentifiedafter infusionsofPGE1 into isolatec! preparations. The results demonstratethat in gasral PGEl undergoes metabolism smlar to that of PGE2 in the rat and reveals the possibilityof a selective PG metabolite transport system across the biliary canalicularmembrane. IXlPODETION The in vivo metabolism of PGE2 in the rat is thought to proceed via three separate pathways. PGE2 is attacked by 15-h;rdroxy-PG dehydrcgenaseand 13,14-PGreductase, and then O-oxidisedto the ll-oxo-9,lCkdihydro-tetranor-PGE2 which is then i*roxidised to the hydroxy and csrboxy metabolites respectively. A second pathway leads to the formation of ll-oxo-9,lC+dihydro-tetranor-PGF 3. The third pathway is the @-oxidationof pc;E2to tetranor-PGE2$allowed o-oxidised to the terminal by dehydration to tetranor-PGB2,which is hydroxy and carboxy metabolites (1). Incubationof PGEl and PGFlc with ratlivermitcchondria revealed thatPGE1 and PGE'lcwere transformedinto their respectivedinor mlogues (2). A later study (3), using the perfused rat liver revealed that PGEl was rtatabolisedtopolarprcductswhichwere excreted into thebile and w effluent. This studywas undertaken inanattemptto isolate, identify and quantitate these metabolites of PGEl from the isolated perfused liver.

Livers from fasted Sprague-Dawleyrats (250 + 10 g bodyweight) were perfused via the hepatic portal vein with defibrinatedwhole blocd (KG ml) (3). The bile duct was cannulatedand bile collected. After 30 min perfusion, during which time the livers attained a steady state, PGEl (10 mg) and 5,6&-PGEl (20 Gi/160 ng) were infused at a rate of 200 jq/min via the portal vein and allowed to recirculatethrough the livers for at least an i-ourafter ccspletion

JUNE 1979 VOL. 17 NO. 6

863

PROSTAGLANDINS of the perfusion The liver, blcodandbile ccmponentswere collected separatelyand extracted. The liverms l-dnzgenised inO.9% saline (5 ml/g tissue) at 4oC. Ice cold ethanol was added to a concentration of 95% (v/v) and left overnight. After r-1 of the precipitated protein by filtration,the alchoholic filtrate was concentrated under reduced pressure to an approximateconcentrationof 65% (v/v) ethanol, and extracted three times with equal volumes of petroleum ether (4OWW. The alcoholic phasewas further concentrated under reduced pressure, acidified to pH 3 with 0.2 M citric acid and extracted three tines with ethyl acetate and then twice with equal volumes of n-butanol. The radioactive contents of the aqueous residue, petroleum ether, ethyl acetate ard n-butanol extracts were determined. Ethanol extracts of the blood and bile were similarly extracted and the distributionof the radioactivitydetermined. The PGs in the ethyl acetate and nbutanol extracts were separatedby silicic acid column chrcmatcgraphy, and identifiedby gas chrcaetcgraphy-massspectrcmetry (GC-_S) analysis. The conditions for the silicic acid chranatqraphy and chemical derivatisationhave been previously described (4). The PGs me eluted frcanthe silicic acid column in a stepwisemanner with increasingamounts of methanol in chloroform (3-129)and analysed as their methyl ester, rrethoxime,trimethylsilyl ether derivatives(ye. P".. (CH313SiO). GC was performed on a Pye 104 instrumentfitted with a flame ionisationdetector (FID) using a 9 ft x 4 rsni.d. glass column packed with 2% SE30 on X+120 GasChrcmQ. The column was kept at a teqerature of 24CW or programmed from 22CR to 25CW at 2°C,Mn with a nitrogen carrier gas flow of 42 ml/min. C-MS analysis on an IKB 9occ6 instrumentused similar chrcsmtcgraphicconditions but required a helium carrier gas flow of 30 ml-. An ionisirq voltage of 20 ev and an acceleratinguoltage of 3.5 Kv were used. The levels of prostaglandinsweremeasuredbothbygas chrcmatcgraphy and radioactivitymeasurements. The Gc of PG metabolites wasmeasuredbyapeakarea nonnalisationprocedure. The FID response of synthetic ll-oxc-9,X-dihydro-tetranor-PGEl and PGSl ~shownto be similar by peak area ratiomeasurements usingmethyl arachidateas an internal standard. Consequentlyit was assumed for the above calculationsthatallthe PG metabolites isolated gave a sjmj.1a.r FID response.

The measurement of radioactivitywas performed using a Packard Tri-Garb model 3320 liquid scintillatorspectrcmeterand corrected for quenchingwith respect to an internal standard. RESULTS Chrcn-atcgraphic properties of the PGs obserred are listed in Table 1. As expected $oxidation of the PGs necessitateduse of increasedpolarity solvents to elute them fran thesiliciczidcolumn.

JUNE 1979 VOL. 17 NO.6

PROSTAGLANDINS

3

a s v

JUNE 1979 VOL. 17 NO. 6

865

PROSTAGLANDINS The Q- and $isaners of PGFl, dinor-PGFk 13-oxo-11,12_dihydrodtirPGFl and ll-oxo-9,UMihydrotetranor-PGFlwere resolved by CC, the B-isomer always showing a slightly lower C value on an SE30 column. The mass spectra of PGF2c, PGE2, PGEl, PGPlc, PGE'l6,dinor-PGEl, tetranor--1, tetranor-PGBl,ll-oxo-9,1O-dihydrotetranor-PCEl and 7a-hyc?roq+,ll-dioxo-tetraiior-prosta-1,X-dioic acid were identical to previously repotied spectra (1,2) and when available to the respective reference standards. Spectra of the c1-and B-iscmersof dinor-PCPlwere qualitatively similar except for the abundance of them/'e281[N-277]+and m/e 282 [M-276]+ions. Theseicnsarise from consecutivelosses of ~1+90+116] amu and [71+90+115]amurescectively. The 115 and 116 amu fragments arise fran less of [CECESi(CH3)J and [CH~CKX~(CH~)~] respectively frcm the ring of these F-t:!- PCs. The loss of the 115 amu fragment was more prevalent for t;lex-is-r ..&ereasfor Y:e 3-iscmer 'Lhe loss of the 116 amu fragment was ,moreabundant. The requisite icns and PGF13 shaJ a similar correlation,as (m/e 309, 310) frcm ?Q& do those frcm tetranor-PGF1,ard tetraror-PGF&, (m/e 253, 254) the latter being obtained by reduction of tetranor-PGElwith sodium borohydride. Significantmass spectral characteristicsof the appropriatemethyl ester, methoxtie and trimethylsilylether derivativesofdinor-PGFl(,/g),13-oxo-ll,12-dihydrod~or-P~(,/Q), tetranor-PGFl, and ll-oxo-9,lCMihydrotetranor-PGFl(3/3) are listed below. The notable absence of tetranor-PGFl3may be due to the difficulty of reccgnising its presence because of interferencefrcm sample background. All these molecules sb peaks of m/e 191 and 217 characteristicof a F-type prostaglandin.

: m/e 281 [~X-C5H~1-(CH3)3SiOH-l16]+ : The ions of m(e 173 [CjH11CH&i(m3)3] and m/e 199 [C5Hl1CHCH=CH~i(CH3)3]confirm the presence of an unchanged O-Cbiil.

13-Ox~ll,12-dihydrodinor-PGFl(Y/a) - &m/e 515 (CO.:%) : m/e 484 + : m/e 428 IN-871 : m/e 425 5M-(CH3)3S1OH]+':m/e 395 : m/e 394 [M-0X3-(CH3)3SiOH] : m/e 371 [M-144]+ : m/e 335 [W2(CH3) SiOH]+. : m/e 304 [M-~(CH~)JS~OH-EHJ]~: m/e 281 [fi(CH3)3&OH-144]+ : The absence of ions of m/e 173 and 199 indicatesmodificationofthe B-chain. Tetranor-PGE'lc- &-m/e 530 : m/e 515 [WCH3]+ : m/e 459 [K-C5Hll]+: m/e 440 [L+(CX~)SiOH]+': m/e 369 [WCgHll-(CH3)3SiOH]+: : m/e 350 [N-2(CH3 ?3SiOH]+' : ;n/e279 $?-2(CX3)3SiOH-C5Hl1]+ m/e 254 [M5Hll-(CH3)3SiCWllS]+ : m/e 253 [.~5Hli-(oI3)3SiOH116]+ : The m/e 173 and 199ionsagain confirm the presence of

866

JUNE 1979 VOL. 17 NO. 6

N.D. co.1 N.D.

Trace amounts of MI;E2and I?C;F2u, believed to be endogenous,were also observed.

N.D. Not detected

0.3

ll-Oxo-9,1+dihydro-tetranor-PGEl

2.6

0.5 1.3

N.D.

Tetranor-PGBl

N.D.

N.D.

N.D.

11-&o-9,10-dihydro-tetranor-PGFle ll-Oxo-9,10-dihydro-tetranor-PGFlB 1 7~hydroxy-5,11-dioxo-tetranor-prosta-1,16dioic acid

0.5 2.1 0.4

N.D.

Tetranor-PGFl'l,

Tetranor-DGEl

0.5

2.7

13-tko-11,12-dihydro-dinor-PGl?lu 13-Oxo-3.1,12-dihydro-dinor-PCE'l6 0.7

N.D.

Dinor-PGQ

0.5

3.7

N.D.

N.D.

1.7

N.D.

Dinor-FGFl,

5.0

0.3

0.5

5.7

N.D.

CO.1 33.2

N.D.

N.D.

0.4

N.D.

1.9

26.4

N.D.

Liver

Blood

Bile

Distributionof PGs following silicic acid chromatographyof ethyl acetate extracts measured by gas chranatography.

P%3 Dinor-PGEl

*1,

=1

Table 2

E

%

E

%

PROSTAGLANDINS

The spe&mm~ was also identical to a a unchanged B-chain. reference sample of tetranor-PGPl,obtained by reduction of tetranor-PGElwith sodium borohydride. ll-Oxo-9,lMihydrotetranor PQlca 6) - M+'m/e 487 (;G_;kG

-1

OH

OGLE

Fig-la

3

1

PROSTAGLANDINS

l-l

1’ \

872

t

JUNE 1979 VOL. 17 NO. 6

PGE1 metabolism by the perfused rat liver.

PROSTAGLANDINS PGEl METAE!OLISMBYTHE PERFUSED RATLIVER D.J. Osborne, J.R. Boot, A.F. Cockerill, KG. Cranstone,W. Dawson, J. Harvey D.N.B.!&llenand C...
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