The effect of prostaglandins on the bioconversion of arachidonic acid in cervical tissue in early human pregnancy

PROSTAGLANDINS

THE EFFECT OF PROSTAGLANDINSON THE BIOCONVERSION OF ARACHIDONIC ACID IN CERVICAL TISSUE IN EARLY HUMAN PREGNANCY

N.J. Christensen* and M. Bygdeman** *Department of C l i n i c a l Chemistry and **Department of Obstetrics and Gynecology, Karolinska Hospital, S-I04 01 Stockholm, Sweden

ABSTRACT The aim of the present study was to investigate in late f i r s t t r i m e s t e r and early second t r i m e s t e r patients whether whole c e l l homogenates of cervical tissue incubated with 1"C-arachidonic acid was affected by pretreatment for 12 to 14 hours with PGEo and 9-deoxo-16,16-dimethyl-9methylene PGE~ (9-methylene PGEp). After e x t r a c t i o n , p u r i f i c a t i o n and separation, i d e n t i f i c a t i o n of t~e compounds found during incubation was achieved using radio-gas l i q u i d chromatography and g a s - l i q u i d chromatography - mass spectrometry. Treatment with 9-methylene PGE2 accomplished a reduced production of z4C-labelled PGF^ , -PGE^ and TxB2, while pretreatment with PGE^ induced increase in ~ e production of 1"C-6-ketoPGF1_when c e r v i c a l Z t i s s u e homogenates were compared with specimens obtaih~d from non-pretreated patients. Recently we reported a s i g n i f i c a n t l y increased formation of so far u n i d e n t i f i e d metabolite(s) in homogenates of human cervical tissue specimens obtained at or near term when compared with corresponding specimens obtained during early pregnancy. With both types of prostaglandin pretreatment there was a tendency of increased formation of these metabolites. I t seems possible that the influence on the biochemistry of cervical tissue induced by PGEp and 9-methylene PGE2 is mediated via the endogenous arachidonic acid cascade towards nonprostaglandin compound(s).

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PROSTAGLANDINS INTRODUCTION The physiological changes in the human cervix towards the end of pregnancy involves c o l l a g e n o l y s i s , a marked reduction of collagen fibres and an increase in the amount of ground substances ( I ) . These phenomena are s i m i l a r to those seen a f t e r i n t r a c e r v i c a l administrat i o n of PGE2 in vivo (2). In a recent study we found a s i g n i f i c a n t l y increased formation of a so far u n i d e n t i f i e d metabolite of arachidonic acid in homogenates of cervical tissue specimens obtained at or near term as compared with corresponding specimens obtained during early pregnancy (3). I t could be that t h i s compound is involved in the physiological ripening of the cervix. I t may be speculated that the effects on the cervix f o l l o wing treatment with PGE2 and E2-analoges in late f i r s t and early second t r i m e s t e r abortion patients (4, 5) are not d i r e c t effects of the drugs themselves, but i n d i r e c t effects mediated through the above mentioned compound. The present study was i n i t i a t e d in order to i n v e s t i g a t e whether the formation of t h i s u n i d e n t i f i e d compound(s) as well as d i f f e r e n t prostaglandins in whole cell homogenates of cervical tissue was affected by 12 to 14 hours' pretreatment with PGE2 or 9-deoxo-16,16dimethyl-9-methylene PGE2 (9-methylene PGE2). MATERIAL AND METHODS The tissue samples were taken from healthy patients admitted to the hospital for late f i r s t t r i m e s t e r or early second t r i m e s t e r abortion through vacuum a s p i r a t i o n . Three groups of patients were studied. The patients in group A received no pretreatment before vacuum a s p i r a t i o n . The patients in groups B and C received 0.5 mg PGE2 i n t r a c e r v i c a l l y (4) and 3.0 mg 9-methylene PGE2 v a g i n a l l y , r e s p e c t i v e l y , 12 to 14 hours before operation. None of the patients in the three groups had taken non-steroid-antiinflammatory drugs (NSAIDs) during the preceding week. The tissue was obtained t r a n s v a g i n a l l y from the midline of the a n t e r i o r l i p of the c e r v i x two to three cm proximal of p o r t i o . After dissecting the mucosa and the s u p e r f i c i a l one to two mm of the cervical t i s s u e , samples were taken from the middle t h i r d of the cervix. The samples were obtained before the s t a r t of the vacuum a s p i r a t i o n . In connection with operation a l l three groups received 0.1 mg fentanyl (Leptanal®) i . v . and 20 ml 0.5% p r i l o c a i n hydrochloride (Citanest®) l o c a l l y into the paracervical tissue. The degree of cervical d i l a t a t i o n was measured by the size of the largest Hegar's probe which could be i n serted through the cervical canal without resistance. Patient characte r i s t i c s are shown in Table I. All patients gave t h e i r informed consent and the study was approved by the Ethical Committee of the Karolinska Hospital.

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1-z4C[-arachidonic acid (50 mCi/mmole), 15,6,8,9,11,12,14,15-3HI PGF^ (257 Ci/mmole) and I5,6,8,11,12,14,15-3HI-PGE 2 (165 Ci/mmole) was purchased from New England Nuclear Inc. (Boston, Mass., USA). The radiochemical p u r i t i e s were in excess of 99 per cent as judged by radio thin layer chromatography (radio-TLC).

Standards' of synthetic PGF2~, PGE2, thromboxane B~ and 6-keto-PGF1~ were kindly supplied to us By drs. Udo Axen and J~hn Pike (The Upjohn Company, Kalamazoo, Mich., USA). The solvents used were all of analytical grade (E. Merck & Co, Darmstadt, West Germany). Incubation and extraction The tissue was immediately placed in ice-cold 0.1M potassium phosphate buffer (pH 7.40). Following transportation for 5 to 10 minutes the tissue was weighed and homogenized in 5 ml potassium phosphate buffer using a Polytron homogenizer (type 10/35, Kinematica Gmbh, Luzern, Switzerland) at maximal speed for 30 seconds. Immediately before homogenization 1-14C-arachidonic acid was added (dissolved in 0.1 ml 0.1M K2HP04 through sonication for 10 seconds) giving a final concestration of 5 ~g/100 mg wet tissue. The mixture was incubated at 37 C for 30 minutes, a c i d i f i e d to pH 3.0 with 2 N HCI and extracted with 95 ml of chloroform-methanol 2/I ( v / v ) . After f i l t r a tion 20 ml of 0.58% NaCl was added and the resulting organic phase was isolated and washed with 7 ml chloroform/methanol/water: 3/48/47 ( v / v / v ) . The total r a d i o a c t i v i t y recovered in the organic phase, water phase and wash phase was calculated. Separation of products formed during incubation The organic extract was evaporated and subjected to s i l i c i c acid chromatography (see below). The toluene and toluene-ethyl acetate f r a c t i o n s , in which arachidonic acid and hydroxy acids appeared, were evaporated ( P ) . The residue was treated with ethereal diazomethane and applied o~ a TLC plate which was developed with system I I (Table I I ) . The ethyl acetate-methanol fraction (PIT)' where prostaglandins appeared, was evaporated and subjected to TLC using system I (Table I I ) . Since PGE~ and TxB9 are not separated well in this system these compounds ~ere extracted from the s i l i c a gel and applied on reversed phase p a r t i t i o n column (RPPC) using system C-45 (see below). Quantitation of product s formed during incubation Following TLC separation of labelled products the r a d i o a c t i v i t y was detected with a TLC scanner. The areas of each peak of r a d i o a c t i v i t y in the graphs obtained were used to calculate the percentage amount of applied r a d i o a c t i v i t y that had been converted into each compound.

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Chromatographic methods S i l i c i c acid chromatography was performed on 0.50 g S i l i c AR CC-4 (Mallinckrodt Chemical WorKs, St. Louis, USA) after activation for at least four hours at 130vC. The columns were eluted with toluene, toluene-ethyl acetate 8/2 ( v / v ) , methanol-ethyl acetate I/9 (v/v) and methanol; 25 ml of each solvent. Thin layer chromatography (TLC) was performed with s i l i c a gel G coated glass plates (Kiselgel 60G, E. Merck & Co, Darmstadt, West Germany) using the solvent systems shown in Table I I (6). Open column reversed phase p a r t i t i o n chromatography was performed using system C-38 or C-45 as described before (7). Gas l i q u i d chromatography was performed with simultaneous registration of mass and r a d i o a c t i v i t y (radio GLC) using a combination of a Pye Unicam model 204 gas chromatograph (Philips, Eindhoven, The Netherlands) and a r a d i o a c t i v i t y detector (type 505, ESI Nuclear, Reigate, England). The stationary phase was I% Se-30 Ultraphase on Gas-Chrom Q (Mesh 100-120, Supelco Inc., PA). The observed retention time was converted into C-values by comparison with methyl esters of normal f a t t y acids (8). Detection of r a d i o a c t i v i t y Radioactivity Sweden) using Company Inc., scintillation Radioactivity Scanner I I LB

was measured in an LKB 1215 Rackbeta I I (LKB, Bromma, Permablend (Permablend I I I , Packard, Packard Instrument I I I . , USA) dissolved in xylol-isopropanol 4/I (v/v) as f l u i d . Internal standard was used for quench correction on TLC plates was detected with a Berthold DUnnschicht 2723 (Berthold, Wildbad, West Germany).

Mass spectrometry Mass spectra were obtained with the combined gas chromatograph- mass spectrometer LKB 2091 (Bromma, Sweden) equipped with a I% Se-30 Ultraphase column on Gas Chrom Q (Mesh 100-120, Supelco Inc., PA). The electron energy was 22.5 eV and the trap current 100 I~A. Preparation of derivates for gas l i q u i d chromatography and mass spectrometry The methyl esters (Me) were prepared by treatment with ethereal diazomethane and O~methyl oxime (MO) and trimethyl s i l y l ether (TMS) derivatives as e a r l i e r described (9). S t a t i s t i c a l methods Mann-Whitney U t e s t , Two-tailed test.

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Table I. Patient characteristics. Mean and (range). Group A n=5

Group B n=5

Group C n=5

35.2 (25-41)

28.2 (20-34)

29.2 (19-36)

Pregnancy

3.6 (2-6)

2.2 (I-5)

3.0 (I-4)

Parity

1.8 (I-3)

0.8 (0-3)

1.0 (0-2)

12.5 (12-14)

11.2 (10-13)

179 (123-212)

210

Age (years)

Pregnancy week at operation

10

(9-11)

Tissue sampels wet weight (mg)

108 (69-132)

Dilatation in mm at operation

11.8 (10-13)

(165-270)

9.6 (7-12)

Group A: no pretreatment Group B: 0.5 mg PGE2 intracervically Group C: 3.0 mg 9-methylene PGE2 intravaginally

Table I I . Solvent system used in thin layer chromatography. Solvent system

Composition Ethyl acetate/acetic acid/2,2,4-tri-methylpentane/water 110/20/30/100 (v/v/v/v)

II

Ethyl acetate/2,2,4-trimethylpentane water 50/I00:I00 (v/v/v/v)

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PROSTAGLANDINS RESULTS The recovery of r a d i o a c t i v i t y in the extracts and in the d i f f e r e n t f r a c t i o n s from s i l i c i c acid chromatography are shown in Tables I I I and IV. The toluene and toluene-ethyl acetate f r a c t i o n s (called PI ) were treated with diazomethane and chromatographed on TLC using system I I . In a l l three patient groups there were two radioactive peaks on the radio-chromatograms (see Fig. I ) . These were called AA and CXT, The AA peak had the same Rf value as arachidonic acid methyl ester and was i d e n t i f i e d as such using radio-gaschromatography and mass spectrometry (12). Peak CXI had an Rf value of 0.52 s i m i l a r to a hydroxy-eicosatetraenoic acid. So f a r the s t r u c t u r e ( s ) of t h i s material is unknown . . . . There was a marked v a r i a t i o n in the formation of 14C-labelled CX, from one incubation to another in a l l three patient groups. Ther~ was a s t a t s t i c a l l y s i g n i f i c a n t difference in the production of CXI (p
Table I I I .

Per cent of incubated r a d i o a c t i v i t y (1-z4~-arachidonic acid) found in organic phase (Org), water phase (H~O) and wash phase (W) a f t e r e x t r a c t i o n . Mean and (range), Group A

Group B

Group C

Org

97.0 (95.5-98.6)

93.5 (91.5-97.0)

94.4 (92.2-95.9)

H20

2.7 (1.1-4.15)

5.9 (2.8-7.6)

4.9 (3.8-6.0)

W

0.3 ( 0 . I - 0 . 4 )

0.6 ( 0 . 2 - I . 0 )

0.8 (0.2-1.8)

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PROSTAGLANDINS Table IV. Percentage d i s t r i b u t i o n of applied r a d i o a c t i v i t y in the d i f f e r e n t f r a c t i o n s from s i l i c i c acid chromatography. Mean and (range) (100%=organic phase in Table I I I ) . Group A

Group B

Group C

95.8 (90.8-98.2)

94.6 (93.7-95.3)

95.0 (94.3-95.5)

PII**

3.4 (1.6-7.7)

4.9 (4.0-6.0)

4.6 (4.2-5.3)

Methanol

0.8 (0.2-1.6)

0.5 (0.3-0.7)

0.4 (0.2-0.6)

PI*

*

pooled eluate: toluene and toluene/ethyl acetate 8/2 (v/v)

** ethyl acetate/methanol 9/I (v/v)

Peak CX~ had the same Rf value as 6-keto-PGF.. This material was extracted from the TLC p l a t e , cold 6-keto-PG~1~ was added and converted into the MeMOTMSd e r i v a t i v e and subjected to radio-GLC. All radioactive material co-chromatographed with cold 6-keto-PGF I . There was considerable v a r i a t i o n in the bioconversion to 14C-~abelled 6-keto-PGF1~ from one incubation to another in a l l three groups. The material from CX3 had the same Rf value as PGF~ and was extracted from the TLC plates. Tritium labelled PGF2~ was added to part of the extracted material and applied on an RPPC ~olumn using system C-38. Only one radioactive peak containing both ~H and 14C appeared at a r e t e n t i o n volume of 160 ml, c h a r a c t e r i s t i c of PGF~ . Cold PGF~ was added to another part of the material in CX3 and ~ n v e r t e d i n ~ the MeTMS d e r i v a t i v e and subjected to radio-GLC. All radioactive material co-chromatographed with the cold PGF2a. Therefore the material in peak CX3 was PGF2a. Very l i t t l e v a r i a t i o n in the formation of z4C-PGF~ was seen in specimens from group C patients. On the other h a n ~ a considerable v a r i a t i o n was seen from one incubation to another in both group A and group B. There was a s t a t i s t i c a l l y s i g n i f i c a n t higher production of z"C-PGF2a in group B as compared with group C (p
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PROSTAGLANDINS

AA

I ~ Front

CXI

c~' ~ x ~ c° ~ • • E2 • Tx B 2

I

F 2~ 6 - k AppL

Fig. 1. Incubation of cervical tissue mucosa homogenate with 14Carachidonic acid (group B). Upper panel: Thin-layer radiochromatogram (system I I ) of material PT from s i l i c i c acid chromatography after t r e a t ment with ~therereal diazomethane. Lower panel: Thin-layer radiochromatogram (system I) of material from PII from s i l i c i c acid chromatography.

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PROSTAGLANDINS with the r e t e n t i o n volume 120 ml co-chromatographed with cold PGE2 upon radio-GLC of the MeMOTMSd e r i v a t i v e . Thus, the r a d i o a c t i v i t y in peak CXm was due to a mixture of PGEo and TxBo. Forty to 70 per cent of the gotal r a d i o a c t i v i t y in peak CXa was du~ to PGE2 ( c . f . Fig. 2). In specimens obtained from group C patients there was very l i t t l e v a r i a t i o n in the synthesis of both z4C-PGEp and z"C-TxB 2 from one incubation to another; t h i s was not the cage neither in group A nor in group B. Group C had the lowest bioconversion and there was a s i g n i f i c a n t difference in the production of both ~%-PGE2 and z"CTxB2 when group C was compared with group A and group B, respectively (p
% 100 mg

Group A

Group B

15-

Group C

1,5-

15

VA

15-

E 2 i.c,

1,0

10

rag.

9-me

1.0

•

10

,e.

O,S

05-

0.5.

5

4•

•

•

.4•

1" |

:

.,,-

% ¢ .a. 6k

m F2~

E2

Tx B 2

CX1

•

41.

m 6k

F2•

E2

TxB 2

CX1

6k

F2=

E2

TxB2

CX1

Fig. 2. Summary of the conversion of 14C-labelled arachidonate in percentage of incubated r a d i o a c t i v i t y . 100 mg wet t i s s u e , into 6-keto-PGF. (6k), PGF~a (F~a), PGE~ (E~), TxB^ and unknown compound's) (CXI) i~ who#e cell ~omoOenatesLof tissue obtained from f i r s t t r i m e s t e r patients (groups A, B and C) (Median value). (For d e t a i l s , see t e x t ) .

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DISCUSSION The objectives of t h i s i n v e s t i g a t i o n was to examine what products of the arachidonic acid cascade that could be formed in v i t r o in cervical tissue homogenates obtained during early pregnancy, when the patients had been treated for 12 to 14 hours with prostaglandins p r i o r to cervical biopsy. The results demonstrate that in homogenates of cervical tissue obtained from the three d i f f e r e n t patient groups 6-keto PGFI~, PGF~ , PGE^ and TxB^ were formed as well as nonpolar compound(s) of ~ h i t h e r t o u~known s t r u c t u r e . I n t r a c e r v i c a l administration of 0.5 mg PGEp causes cervical ripening without stimulation of myometrical a c t i v i t p (2,4,10). This process is associated with biochemical events (2) probably caused by the prostaglandin treatment. How the effect is accomplished i s , however, l a r g e l y unknown. Neither the degree of penetration of exogenous PGE2 into the cervical tissue nor the degree of metabolism in the tissue has been studied. Preincubation of cervical tissue homogenates in v i t r o with PGE~ (50 ~g/100 mg wet tissue for f i v e minutes) did ~ t a--hY6ge c the p r o f i l e of products formed from ~"C-arachidonic acid (Christensen and Green, unpublished observations). Application of 9-methylene PGEe in the posterior f o r n i x induces uterine contractions probably 9ia resorption from the vagina into the blood stream (11). I t is not known whether any local absorption of t h i s drug into the cervical tissue also occurs. The l i m i t e d v a r i a t i o n in the formation of 14C-labelled PGF~ , PGE~ and TxB2 seen in homogenates obtained from patients receiving 9-~ethylene PGE~ (group C), could implicate a drug e f f e c t . However, i t must be kept ~n mind that the amount of prostaglandins formed from exogenous arachidonic acid is sometimes very d i f f e r e n t from that formed from endogenous arachidonate (12). The method used in t h i s study only shows the bioconversion of the exogenous precursor. To what extent t h i s applies also to the bioconversion of arachidonic acid, e.g. via the lipoxygenase pathway, is not known. The technique used in t h i s study only shows the maximal capacity of the tissue to convert 14C-labelled arachidonic acid into labelled products at the time when the speciment is taken. I t does not reveal any biochemical events that have taken place in s i t u during the 12 to 14 hours having elapsed between the p r o s t a g l a ~ p l i c a t i o n (PGE2 or 9-methylene PGE2) and the time of tissue c o l l e c t i o n . Although the bioconversion of Z%-arachidonate varied widely in the homogenates studied, i t seems that vaginal a p p l i c a t i o n of 9-methylene PGE2 decreased the capacity of the cervical tissue to form d i f f e r e n t prostaglandins. When specimens obtained from prostaglandin pretreated patients (groups B and C) were compared with those obtained from the non-treated patients (group A), the formation of the so far unknown polar compound(s) tended to be higher.

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PROSTAGLANDINS The data presented demonstrate that treatment with prostaglandins does not cause any dramatic changes in the bioconversion of exogenous arachidonic acid into prostaglandin compounds in cervical tissue homogenates. However, s l i g h t differences in t h i s respect between treated and untreated tissue could be demonstrated. Therefore, i t seems possible that the influence on the biochemistry of cervical tissue induced by PGEp and 9-methylene PGEe is mediated via the endogenous arachidonic acld cascade towards noh-prostaglandin compound(s). ACKNOWLEDGEMENTS This i n v e s t i g a t i o n received f i n a n c i a l support from the Special Programme of Research, Development and Research Training in Human Reproduction, World Health Organization, Geneva, Switzerland. 9-deoxo-16,16-dimethyl-9-methylene-PGE2 and PGE2 were supported from Dr. K. Kirton, The Upjohn Company, Kalamazoo, Mich., USA. We are also grateful to Maria Lindel~f for typing the manuscript and to Anna-Lisa SjUkvist for s k i l f u l technical assistance, and to our research nurses for supervising the patients. REFERENCES 1) Danforth, D.N., A. Veis, M. Breen, H. Weinstein, J.C. Buckingham, and P. Manalo. The e f f e c t of pregnancy and labor on the human cervix: changes in collagen, glycoprotein and glycosaminoglycans. Am. J. Obstet. Gynecol. 120:641. 1974. J

.

2) Uldbjerg, N., G. Ekman, A. MalmstrUm, B. Sporrong, U. Ulmsten, and L. Wingerup. Biochemical and morphological changes of the human cervix a f t e r local application of prostaglandin E2 in pregnancy. Lancet 1:268. 1981. 3) Christensen, N.J., P. Belfrage, M. Bygdeman, J. Floberg, N. Mitzuhashi, and K. Green. Bioconversion of arachidonic acid in human pregnant uterine cervix. Submitted to Acta Obstet Gynecol Scand. 4) Wingerup, L., U. Ulmsten, M. Bygdeman, G. Ekman, L. Hamberger, V. LundstrUm, and A. NorstrUm. A new prostaglandin E2-gel for pretreatment of the cervix in nulliparous patients having a late f i r s t t r i m e s t e r termination of pregnancy. Arch. Gynecol. 231:1. 1981. 5) Bygdeman, M., K. Bremme, N. Christensen, V. LundstrUm, and K. Green. A comparison of two stable prostaglandin E analogues for termination of early pregnancy and for cervical d i l a t a t i o n . Contraception 22: 471. 1980. 6) Green, K., and B. Samuelsson. Prostaglandins and related factors: XIX. T h i n - l a y e r chromatography of prostaglandins. J. Lipid Res. 5:117. 1964.

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7) Green, K., M. Hamberg, B. Samuelsson, M. Smigel, and J.C. Fr~lich. Measurement of prostaglandins, thromboxanes, prostacyclin and t h e i r metabolites by gas liquid chromatography - mass spectrometry. In: Advances in Prostaglandin and Thromboxane Research. (J.C. Fr~lich, ed.) Raven Press, New York, 1978.p. 15. 8) Green, K. Metabolism of prostaglandin E2 in rat. Biochem. 10:1972. 1971. 9) Green, K. Gas chromatography - mass spectrometry of O-methyl-oxime derivatives of prostaglandins. Chem. Phys. Lipids 3:254. 1969. 10) Forman, A., U. Ulmsten, Lo Wingerup, and Z. BAnyai. Effects of intracervical PGE2-gel on myometrical a c t i v i t y and cervical state in f i r s t trimester pregnancy. Prostaglandins 24:303. 1982. 11) Green, K., O. Vesterquist, M. Bygdeman, N.J. Christensen, and S. Bergstr~m. Plasma levels of 9-deoxo-16,16-dimethyl-9-methylenePGE2 in connection with i t s development as an abortifacient. Prostaglandins 24:451. 1982. 12) Dimov, V., N.J. Christensen, and K. Gr6en. Analysis of prostaglandins formed from endogenous and exogenous arachidonic acid in homogenates of human reproductive tissues. Biochim. Biophys. Acta 754:38. 1983.

Editor:

302

Peter

W.

Ramwell

Received: Accepted:

3-14-84 11-26-84

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