PROSTAGLANDINS

- V0R.K IN PROGRESS -

HUhIAN COLONIC !dUCOSA FCDABOLISES PROSTAGLUDIN E2

P.R. Smith, D.J. Dawson and C.H.J. Swan,

Department of Gastroenterology

North Staffordshire Hospital Cenixe, Stoke-on-Trent, England.

exogenous prosta@,nZ.ns (X’s) exert effects on the whole of the gastrointestinal tract and production of PG's In the gut has previoT;sly been demonstrated in vitro, (Bennett, 1976). X’s :have rn-oi’ou& effects on colcnic motilizy (Uunt et al, 1975) ad. have been &Li.cated in the pathophysiology of ulcerative colitis (UC) (Gould, 1375). A-I increase in PS levels hrs been denonstrated in the mucosa of patients with active UC ax-d in vitro studies suggest an increase in PG synthesis (&rris et al, 1978). Although FG metabolism has been extensively investigated in a variety of tissues, including hunan gastric mucosa (Feskar and Peskar, 1976), the authors are not awzre of any reports of FG metabolism in hum= colonic mucosao This p&per presents preliminary resdts to suggest t!-at high speed supernatants of hum colonic mucosa significantly metabolise FGE2.

Inflamed color,ic tissue from ulcerative colitics with active disease, or macroscopically norm1 colon from patients with carcinoma

coli vrz.5 obtiinea at surgery. X&in five minutes of resection, tissue v;as placed in buffer (0.02M Tris-?iCl pH 7.6) ar.d dxansportea at LOC. Excess fatty tissue and nesentery were removed, t!!e colon 0per.d and Ylashed three tines in cold buffer. Kucosal ma sub- mucosal tissue ('musosa') were remove2 by scraping with an ice-cold tizroscooe slide ana homogenise3 for 15 seconds in four volumes of buf"er &in A 3 e Silverson Laboratory Zomogeriser, followed by three passes in a Camlab TX-R glass/teflon pestle honogeniser. The resulttig honsgenate was centrifcged a t 12OOOg for 30 minutes at 4OC znd the pellet disc&rded. Tne supernatant was further centrifuge& at 1170,OOOg for 60 minutes at 4 C. The fFne1 supernatant (Cytosol) was dividd into alio_nots 3fia stared zt -20':.

bssnys for FG netabolising activity were csrried out by incub- ating aliqucts cf high speed supernatacts with radioiabelled PGE . The reaction mixture was as foll071s: superr.stsnt (2-6mg protein , e ?J) 2.5%, PGE2 5@, bg3H-FGE2 (Amershaz, lSOCi/m.mol) in a final voilwe of 400~1 buffer, Incubaticns were for 30 min at 37'C; the reaction being terminated by acidification to pH3 by the addition of forirLc acia. Prosto,$ar.dins were then extracted tv&ce vzith 3ml ethyl acetate, the combined extracts evaporated to dryness and the residue dissolved in 50~1 chloroform. Portions of the chloroform extract (up to 10~1) were applied to plastic-backed silica gel

JUNE 1980 VOL. 19 NO. 6 843

PROSTAGLANDINS

!LC plates (!dorck siXca gel G, O.&m thick) and the plates developed in a solvent system consistin g of the organic phase of ethyl acetate: iso-octane: acetic acid: water (11:5:2:1Ov/v). Prostaglandins and netabolites were then locsted using a Packard Model 7201 Radio- chromatogram Scanner, with pure radiolzbelled FG's as standards. In the case of metabofites for which no pure radiolnbelled stadards were avasable the Rf vaiues of the pure netabolites (supplied by Dr. J. Pike, UiJohn Company) were determinea and compared with Rf values for reaction products. Fortions of the TLC pliLtes corres- ponding to FGE2 were cut out an2 placed ?drectly into a dioxane- based sc5ntillation fluid and the radioactivity determined by scintillation cou-ltin,rr as aescribed by r'lcaer an& MX.ure (1975). The rcca-ining ra&icactivi.T for each sam$e was similarly determined and express.ed as !I;e+zbolites. * sampl.e 172s calculated as the $

Percer+%ge metabolism for each rediosctivity converted to products

havtig Xfferent af valGes to that of PGZ2.

PG-Eetabolising activity has been observed in a3.1 specimens so far edned. In every case, two major products (9 and P2) can be demcnstrated by rzd%ochromatogram scanning (Fig. 1).

Fig. 1

I 0 .

Origin PGE2 15-K-D-PGEz

I Solvent Front

Fig. 1 - shows radiochromatoLqam scan showin, b FG metabolising activity of high speed supernatants of human colonic mucosa.

lCO,OOOg supernatant (4.2mg protein) of muccsa from patient with ulcerative colitis was incubated with IUD 2.5d, pGE2 in a total vplume of 4OOpl for 30 min at 37OC. FG's aa metabolites were extracted as describe& in the text.

a44 JUNE 1980 VOL. 19 NO. 6

PROSTAGLANDINS

Of these two compounds, Pp PC's and reaction products

Table 1 Rf' values of pure

Substance

PGF2

PGR2

predominates. The Rf velues of authentic are shomn in Table 1.

prostaglandins ma reaction proau0t.9.

Rp

0.10

0.17

15-k&o 13, U+ dShydro PGE2 0.37

l5-keto pGE2 0.25

Product Pl 0.24

Product P2 0.38

From the Rf values ihe products were tentatively identified: P as 15-keto PGE2 and P2 as 15-keto 13, I.4 dihydro PGE2. No furt er i; identification of the products has been attempted. The conversion of PCS2 to metabolites was strictly NAD dependent; neither MDH or N!iDP acted as cofactors for the reaction. The reaction proceeded in a time dependent manner (see Fig. 2).

Fig. 2.

IO 20 30 40 50 60 70 80 90 100 NO 120

Minutes

2 - reveals percentage conversion of PGE2 to metabolites by mucosal supernatants accord%ng to incubation time. Experi- mental conditions as described in the text. POi.&S represent mean values for quadruplicate determinations.

Frcm time curve experiments, 30 minutes was chosen as an optimal incubation time. Roiled protein, heat inactivated enzyme, and buffer blanks when incubated with PGE2 resulted in only small (less than $) conversion to other products. The pH requirements for metabolism were wide, with optimal activity in the range pH5 to pH8.

JUNE 1980 VOL. 19 NO. 6 845

PROSTAGLANDINS

Table 2

Specimen

Specific prostagla.ndin-metabolising supernatants of huxaz colonio mucosa conditions as described in text).

No. Diagnosis Site of Specimen

activity in high speed (experimental

FG metabolism nmoles FGE metabolised per mg pro z* ezn per 30 min.

1 Ulcerative Sigmoid Colon 0.25 Colitis

2 Ulcerative Sigmoid Colon 0.19 Colitis

3

4

Crobn's Disease

'Korm2.l' Colon

liid-Transverse 0.06 Colon

Ascending Colon 0.57

These values are substantially lower in magnitude to those determined for tuxan gastric LWCOS~ by Peskar er,a Peskzr (1976). From the table it can be seen ttat the specimen from a petient with Crohn's disease shows i: lower specific activiiy for PG me "Lxbolism thsx the normal or . . colltxc xcos&. It is, hcxcver, not psssibZe at tiis time to cement 3x2 the relevance of this redxed ictivitjr in relation to the under- lying pathophysioloey of the disease, i? ?G metabolism may be considerable

sixe person to person variation o r variations in metabolism in

different sites of the colon nzy occur.

846 JUNE 1980 VOL. 19 NO. 6

PROSTAGLANDINS

bexiett, A,

Fru:ta&ntis, PkysloIlogical, Phar;r.acolcgical ad Pathological hspecx.

(S .I$.!,:. krti, ed,) KTP Press. London 1976 p 247

Blovzer, R.J. and McClure, 8.0. (1975)

A simpie isotope derivative assay for prostagladins and prosta- &n&n metabolites.

Arxd.ytical Biochemistry 68 436

GoulS, S.R. (1975)

Prostab aC -l- dins, ulcerative colitis acd sulpfiasalazine.

Lancetg 98

Barris , D.X., Smith, P.R. and Swan, C.H.J. (1978)

Deterdation of prostagladin synthetase activity .i.n rectal biopsy sacerial and its signiiicance in colonic disease.

Gut2 E75

I-I.mt., R.X., Dilsx-ri, J.B. ar.6 Misiewicz, J,J. (1975)

I?e O_'feCt Of ktrsvenous prostaglandin P2 and E2 OI? the motility Or' the signoid colon.

Gutl& 47

Peskm, B.3LI. and Peskar, E.A. (1976)

On the meTabo:olism of prosta@ntis by kmu? gastric funduz mucoss.

Biochemica et.Biophyica Acta J+24:430

JUNE 1980 VOL. 19N0.6 847