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Gut, 1983, 24, 1176-1182

Biosynthesis of lipoxygenase and cyclo-oxygenaseproducts from ['4C]-arachidonic acid by human colonicmucosa

N K BOUGHTON-SMITH, C J HAWKEY, AND B J R WHITTLE

From the Department ofProstaglandin Research, Wellcome Research Laboratories, Beckenham, Kent, andDepartment of Therapeutics, University Hospital, Nottingham

SUMMARY The human colon synthetises several prostanoids which may have a role ininflammatory bowel diseases. As lipoxygenase products of arachidonate metabolism have beenimplicated in inflammatory processes, we have now investigated the formation of bothlipoxygenase and cyclo-oxygenase metabolites from [14C]-arachidonic acid ([14C]-AA) by humancolonic tissue. Homogenates of human colonic mucosa were incubated with [14C]-AA and afterextraction into diethyl ether, separated by thin layer chromatography using two solvent systemsthat allowed resolution of cyclo-oxygenase and lipoxygenase products. The predominantcyclo-oxygenase products, as identified by their chromatographic mobility, were

PGE2>PGF2.>PGD2>TXB2>6-keto-PGF1,,. The formation of these products was inhibitedboth by indomethacin (1-10 uM) and the dual pathway inhibitor, BW755C (1-30 uM). Thepredominent lipoxygenase products formed, which had the chromatographic mobility of 11-, 12-,15-HETE (which ran together) were inhibited by BW755C (19 uM) but not by indomethacin (3,uM). Further resolution of this TLC band, performed using normal phase HPLC, indicated thatboth 12-HETE and 15-HETE were major lipoxygenase products formed by human colonichomogenate. The present findings indicate that human colonic tissue can convert [14C]-AA intolipoxygenase as well as cyclo-oxygenase products and support the suggestion that lipoxygenaseproducts may have a role in inflammatory bowel disease.

Previous studies of aracjiidonic acid metabolism inhuman colonic mucosa have concentrated onproducts of the cyclo-oxygenase (prostaglandinsynthetase) pathway. The synthesis of prostanoidssuch as prostaglandin E2 (PGE2), PGF2I,, prosta-cyclin, its breakdown product 6-keto PGF1,, andthromboxane B2 (TXB2) is increased during relapsein inflammatory bowel disease. 1-5 These compoundsmay play a role in the inflammation - for example,by enhancing vasodilatation and oedemaformation.6 7 They have little or no effect, however,in activating human leucocytes: indeed, prostacyclin(PGI2) in low concentrations has been shown toprevent polymorphonuclear leucocyte chemotaxis. 8Prostaglandin synthesis alone is therefore unlikely

Address for correspondence: Dr B J R Whittle. Department of ProstaglandinResearch. Wellcome Research Laboratories, Langley Court, Beckenham,Kent BR3 3BS.Received for publication 11 March 1983

to lead to the accumulation of leucocytes duringrelapse in inflammatory bowel diseases. Indeed,specific potent inhibitors of prostaglandin synthesissuch as indomethacin or flurbiprofen have not beenshown to be of benefit in ulcerative colitis.911The metabolism of arachidonic acid via

lipoxygenase enzymes has been recently describedin leucocytes and platelets.7 12T14he productsformed include the leukotrienes and 5-hydroxy-eicosatetraenoic acid (5-HE1t) from the 5-lipoxygenase pathway, and 12- and 15-HETEformed by the 12- and 15-lipoxygenase enzymes. Inaddition non-prostaglandin products of the cyclo-oxygenase pathway such as 11-HETE and 12-hydroxy-heptadecatrienoic acid (HHT) can beformed. In contrast with prostaglandins, some ofthese lipoxygenase products can stimulatelocomotion, lysosomal enzyme release, andsuperoxide production by human leuco-cytes.7 12 1517 As the synthesis of lipoxygenase

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Arachidonate products from human colon

products by solid tissues such as skin, pericardium,pleura, and peritoneum have also been shown18 19and because of the potential importance of suchcompounds in inflammatory bowel diseases, wehave investigated whether human colonic mucosaalso possesses the ability to metabolise radiolabelledarachidonic acid by lipoxygenase pathways.

Methods

PATIENTSHuman colon was obtained at operation from fivepatients undergoing resection of carcinomas in therectum, sigmoid, or descending colon and from twopatients undergoing colectomy for ulcerative colitis.The colonic mucosa of the patients with ulcerativecolitis had the histological appearances of severeinflammation. The mucosa from the other patients(control tissue) was taken >5 cm away from thetumour, and was histologically normal. Bothpatients with ulcerative colitis were untreated at thetime of operation, while none of the patients wasreceiving any drug known to affect arachidonic acidmetabolism.Mucosal tissue (which had been stripped from the

resected human colon and stored at -70°C) wasmanually homogenised in 50mM Tris (pH 8.0) usinga 'Dual' ground glass homogeniser, to give a 50-85mg/ml w/v final suspension. Aliquots (2 ml) wereincubated for 20 minutes at 37°C with 840 ng (160nCi) of [14C]-arachidonic acid ([14C]-AA).

INHIBITION OF [14C]-AA METABOLISMFresh stock solutions of indomethacin (200 ug/ml;5.7x10.4M dissolved in 50 mM Tris; pH 8.0) andBW755C (1 mg/ml; 3-8x 10-3M dissolved in distilledwater) were diluted in 50 mM Tris (pH 8.0) andincubated with tissue suspension for 20 minutes (at0°C) before incubation with [14C]-AA.

Solve sysem A.. .

Tc

-

AA

11-,12 ,15-HETE.HHT

5-HETE

VI~T. 4 .)

LTP' (YB)

PG's.+TXB2

.c ...Huma oten

Solven sydtem fli,

AA

NETET.

HHIT

TXB2

__ ~~~PF..__~~~~~~~C

.

ostonl

Fig. 1 A utoradiogram of radiolabelled metabolities of[4Cl-arachidonic acidformed by homogenates ofhumancolonic mucosa and separated on TLC plate. Solvent system'A' (see text) was used to separate lipoxygenase productsfrom the prostaglandins and solvent system 'B' used toseparate the different cyclo-oxygenase products, authenticstandards being simultaneously co-chromatographed. Theradiolabelled products formedfrom incubation of['4C-AAwith rabbit polymorphonuclear leucocytes are shown forcomparison. The inhibition ofprostanoidformation byindomethacin (2-8 ,M) and ofprostanoid and lipoxygenaseproductformation by BW755C (19 ,M) is also shown.

EXTRACTION AND THIN LAYER CHROMATOGRAPHYAfter acidification to pH 3.5 with citric acid (2.3M),the products were extracted twice into two volumesof diethyl ether (total volume 8 ml). Extractedsamples were dried under nitrogen, redissolved in75 ,ul of chloroform:methanol (2:1) and applied tomulti-lane thin layer chromatography plates(Whatman LK 5D). In each experiment duplicatethin layer chromatography plates were prepared andwere developed using two separate solvent systems(Fig. 1).SOLVENT SYSTEM 'A'This consisted of ether:hexane:acetic acid (60:40:1,vlv/v). The system separates TXB2 and prosta-glandins (which remain together on the origin) from

the lipoxygenase products. The Raa values(chromatographic mobility of products with respectto arachidonic acid) were for leukotriene B4 (0.15),5-HETE (0.44), HHT (0.63), and 11-, 12-, 15-HETE (which run together, 0.75).

SOLVENT SYSTEM 'B'This was the organic phase of ethyl acetate:trimethyl pentane:water:acetic acid (110:50:100:20,vlv/vlv). The system facilitated the separation of theindividual prostaglandins and TXB2. The Raavalues were: 6-keto PGF1,, (0.29), PGF2I (0.39),TXB2 (0.50), PGE2 (0.59), and PGD2 (0.73).

Radioactive products were localised using auto-radiography (three days' contact with Kodak NS2T

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photographic film). The silica gel zone corres-ponding to each radioactive band was scraped off,and the radioactivity was determined by liquidscintillation counting (Packard Tricarb liquidscintillation counter). The dpm for each band wascalculated and expressed as a percentage of the totaldpm on the thin layer chromatography plate. Aftersubtraction of boiled tissue blanks, the data werecalculated as percentage [`4Cl-AA conversion/100mg mucosa and expressed as percentage conversion.

HPLC RESOLUTION OF THE II-, 12-, I5-HETEBANDBulk incubation of homogenates of mucosa fromcontrol and inflamed colon with [14C]-AA wereperformed as above, maintaining the same tissue/substrate ratio. The resulting 11-, 12-, 15-HETEband (resolved using solvent system 'A') wasscraped from the thin layer chromatography plateand eluted with methanol (20 ml). The eluant wasdried under nitrogen and the residue redissolved in50 ,u1 of solvent (see below) for injection into thehigh performance liquid chromatography (HPLC)column.The dissolved residue was injected into a straight-

phase Zorbax Sil Column (Dupont 4.6x250 mm)previously equilibrated (3 ml/min) with solvent(n-hexane:isopropyl alcohol:acetic acid, 992:7:1,v/v/v) and standardised using authentic monoHETEs (11-, 12-, and 15-). Elution of metaboliteswas monitored by ultraviolet absorbance (280 and254 nm). Fractions were collected every 30 secondsand radioactivity measured. Authentic 15-HETEwas added to extracted samples as an internalstandard.

DRUGS AND CHROMATOGRAPHIC STANDARDSThe arachidonic acid metabolites were characterisedby co-chromatography with the following standards:(1) Authentic standards of PGD2, PGE2, PGF2a,TXB2, and 6-keto PGF1, stored in ethanol at-200C. They were visualised on the thin layerchromatography plate by phosphomolybdic acidspray and heating to 1000C. (2) [14C]-AA cyclo-oxygenase and lipoxygenase metabolites formed byrabbit polymorphonuclear leucocytes20 (Fig. 1) andradiolabelled TXB2 formed by incubating humanwashed platelets with [1 C]-AA. (3) Thelipoxygenase products 5-, 11-, 12-, and 15-HETEprepared in the Chemical Research Laboratory,Wellcome Research Laboratories, Beckenham, byoxygenation of arachidonic acid at room tem-perature.15 21 The HETEs were prepared byreduction of the corresponding hydroperoxyderivatives with triphenyl phosphine followed byHPLC purification. The identity of the HETEs was

confirmed by GC-MS of their methyl ester,trimethylsilyl ether derivatives. The products werequantified by ultraviolet spectrophotometry at 237nm and stored in methanol (1 mg/ml) at -20°C.BW755C (3-amino-1[m-(trifluoromethyl)-

phenyl]-2-pyrazoline), as the hydrochloride (mol wt266), was synthetised by Drs F C Copp and C VDenyer of the Chemical Research Laboratories,Beckenham. Indomethacin (mol wt 358) was fromSigma Chemical Company and [1-14C]-arachidonicacid (58 mCi/mmol stored at -20°C in toluene) wasfrom Amersham International Ltd. All solvents andchemicals were of analytical grade.

STATISTICAL ANALYSISInhibition of the conversion of [14C]-AA to radio-active products by indomethacin or BW775C wascalculated as a percentage of its paired control andexpressed as mean ± SEM of (n) values. Statisticalsignificance was calculated using Student's t test forpaired data, where p<O0O5 was consideredsignificant.

Results

The homogenates of control human colonic mucosaconverted 6-4±0.9% (mean + SEM, n=5) of theadded [14C]-AA to radioactive products (Fig. 1).The predominant cyclo-oxygenase products formed,as characterised by their chromatographic mobilityusing solvent system 'B' (Fig. 2) werePGE2>PGF2,,>PGD2>TXB2>6-keto PGFl,.The formation of these cyclo-oxygenase products

was inhibited concentration-dependently by thecyclo-oxygenase inhibitor indomethacin (0.3-8 ,uM)and by the dual cyclo-oxygenase-lipoxygenaseinhibitor,20 22 BW775C (1-38 ,uM). Concentrationsof indomethacin (2.8 ,uM) and BW775C (19 uM)which were close to the IC50 values (concentrationcausing 50% reduction) for inhibition of thesecyclo-oxygenase products were chosen for furtherstudies, and the results are shown in Table 1.The predominant lipoxygenase products,

characterised using solvent system 'A' (Fig. 3), hada chromatographic mobility corresponding with 11-,12-, or 15-HETE (which ran together). Whereasindomethacin (2.8 ,uM) failed to reduce significantlythe formation of these products, BW775C (19 ,uM)inhibited their production by 75±9%, n=5(p<0001; Table 1).

Smaller amounts of other products were alsoseparated using solvent system 'A'. These were (a)product II, a band with a chromatographic mobility(Raa, 0.83) slightly greater than the 11-, 12-,15-HETE band, whose production was inhibited byBW775C (19 ,M) but not by indomethacin (2.8

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1 00-

0.75-

('1.) conversion

0 50

0.25-

0.00-PGD2 PGE2 TXB2 PGF2. 6-keto

PGFl1TLC band

Fig. 2 Formation of radiolabelled cyclo-oxygenasemetabolites of['4C]-arachidonic acid by homogenates ofhuman colonic mucosa. Levels ofprostanoids, separated onTLC using solvent system 'B', are expressed as percentageconversion of[`4Cl-AA. The results are shown asmean ± SEM offive experiments using control tissue.

Table 1 Inhibition of[`4C1-arachidonic acid metabolismin homogenate ofhuman colonic mucosa by BW755C andindomethacin

% Inhibition

BW755C IndomethacinTLC band (19 gLM) (3 ,uM)

Product 11 56±13* 11±2811-, 12-,15-HETE 75±9t 30±17HHT 54±13* 66±6t'5-HETE' 89±3t 64±2tProduct VI + 'LTB4' 76±16* 70±9tPGs + TXB2 55±18* 64±10O

Results, shown as % inhibition of ['4C]-AA conversion to variousproducts separated on TLC, are mean ± SEM of five experimentwith control tissue.* p<0.05 t p<0.01 t p<0.001.

2 5-

2.0

1.5

('1.) conversion

1-0

0-5

011 11-,12-, HHT 5- VI. PG's.

15- HETE LTB4 TXB2HETE

TLC bond

Fig. 3 Formation ofradiolabelled lipoxygenase and cyclo-oxygenase products of['4CJ-arachidonic acid metabolismby homogenates ofhuman colonic mucosa. The levels of theproducts, separated into six bands according to theirchromatographic mobility on TLC with solvent system 'A'(which included uncharacterised products II and VI), areexpressed as percentage conversion of['4Cl-AA. Resultsare shown as mean ± SEM offive experiments usingcontrol tissue.

,uM) as shown in Table 1; (b) a product corres-ponding to HHT (a cyclo-oxygenase product) whosesynthesis was inhibited by both indomethacin andBW775C; (c) a product corresponding to 5-HETEwhich was greatly reduced by both inhibitors,although the inhibition by BW775C was significantlygreater (p<001; Table 1); (d) two other products,one with the chromatographic mobility ofleukotriene B4 (LTB4) and another of novelmobility (product VI, Raa=0.31). When scrapedand pooled together, these products comprised12±3% of total products formed (n=5) and wereinhibited by either indomethacin or BW755C (Table1). In two experiments in which the two productswere scraped separately they made up 8% and 4%of the total products formed, respectively.

CONVERSION OF [14C]-AA BY INFLAMED MUCOSAA similar profile of products, separated on thin layerchromatography, were synthetised by the inflamed

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tissue from the two patients with ulcerative colitisand that from control tissue from the five patientswith colonic carcinomas. There appeared to begreater conversion of [14C]-AA, however, by theinflamed mucosa both to the products separatedusing solvent system 'A' and to the cyclo-oxygenaseproducts separated using solvent system 'B' (Table2).

HPLCOFTHE II-, I2-, I5-HETE BANDThe radioactivity co-chromatographing with the 11-,12-, 15-HETE thin layer chromatography band wasfurther resolved using HPLC techniques (Fig. 4).The predominant product in two homogenates ofcontrol colonic tissue was 12-HETE, with only smallamount of 15-HETE, whereas in two other controltissues, both 12-HETE and 15-HETE could bedetected. Overall, 12-HETE comprised 40±10%and 15-HETE comprised 24±10% (n=4) of thetotal radioactivity in this thin layer chromatographyband. Both 12-HETE and 15-HETE were formed inapproximately equal quantities in homogenates ofinflamed mucosa from a patient with ulcerativecolitis (Fig. 4). Only very small quantities of11-HETE were formed by these tissues, andalthough a number of other products were alsoresolved by HPLC, their identity requires furtherclarification.

Discussion

The present data show that arachidonate products ofthe lipoxygenase metabolic pathway can be formedby homogenates of human colonic mucosa. Theproducts have been characterised both by theirchromatographic mobility, using thin layer chromat-ography and HPLC, and by their selective inhibitionwith BW755C. In contrast with non-steroid anti-inflammatory drugs such as indomethacin whichinhibit only the cyclo-oxygenase pathway, BW755Calso inhibits the lipoxygenase pathway20 22 and thuscan be used as a pharmacological tool to study thesepathways. The predominant arachidonic acidmetabolites separated by thin layer chromatographyhad the chromatographic mobilities of PGE2,PGF2m, and 11-, 12-, 15-HETE. The synthesis ofPGE2 and PGF2c. as major cyclo-oxygenase productsof arachidonic acid metabolism in human colon haspreviously been shown in both cultured biopsies andhomogenates of human colon.2-5 In our studies,separation of the 11-, 12-, 15-HETE band by HPLCindicated that 12-HETE was the major identifiablemono-HETE formed by uninflamed colonicmucosa. This supports the recent finding of Bennettet a123 who, using GC-MS techniques, identified aproduct formed by normal human colonic mucosa

Table 2 Comparison ofthe profile ofradiolabelledmetabolitesfrom ['4C-arachidonic acid, separated on TLCusing solvent system 'A' and 'B' formed by homogenates ofhuman colonic mucosafrom control tissue (n=S) andfrominflamed tissuefrom patients with ulcerative colitis (n=2).Results shown as percentage conversion, are the mean of (n)experiments

% Conversion

TLC band Uninflamed Inflamed Ratio

Solvent system 'A'1 1 0-48 0-91 1.911-, 12-, 15-HETE 2.25 6.38 2.8HHT 0-48 1-41 2-75-HETE 0-21 0.59 2-8VI, VII 0-76 2-35 3-1PGs + TXB2 2-24 6-24 2-8

Total 6-4 17-8 2-8

Solvent system 'B'PGD2 0-42 0-87 2-1PGE2 0-85 2-31 2-7TXB2 0-23 0-92 4-0PGF2 0-71 2-18 3-16-keto PGF1a 0-14 0-33 2.4

Total 2-3 6-6 2-8

100'r15-HETE

12-HETE 11- HETE

f1{v} 1B00o

600

400 -

200-

i oL:25

1O0~.B 1000a:r800

FGrl

400 _

200

0C 4 8 12 16 20 24

Fraction number

Fig. 4 Resolution ofmajor lipoxygenase productsfrom['4C]-arachidonic acid metabolism by homogenates ofhuman colonic mucosa using HPLC technique. Resultsshow products from control tissue andfrom inflamed tissueofpatient with colitis. The position ofauthentic 12-HETE,15-HETE, and JI-HETE are shown by arrows, whereashatched bar represents 15-HETE internal standard.

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Arachidonate products from human colon 1181

from endogenous precursor corresponding to 12-HETE, although they did not quantify itsproduction. The detection of 12-HETE by mucosalhomogenates in both studies, as with TXB2, mayreflect formation by entrapped platelets, which areknown to convert efficiently arachidonic acid to thislipoxygenase product as well as to TXB2,12 22 buttheir formation by other cell types from the colonicmucosa cannot be excluded.The production of 15-HETE by human colonic

mucosa is a novel finding. As patients withulcerative colitis rarely come to operation whileuntreated, only a single HPLC experiment could beperformed on the products formed by inflamedtissue. Thus any conclusions concerning thedifferences between inflamed and control mucosaare necessarily limited. The 15-HETE found inhomogenates of the inflamed and uninflamedmucosa may reflect lipoxygenase activity derivedfrom leucocytes which have infiltrated the mucosa.Although the major lipoxygenase product generatedby intact rabbit neutrophils is 5-HETE,13 in broken-cell preparations the predominant product formed is15-HETE.14 Furthermore, 15-HETE is the majormono-HETE formed in intact human polymorpho-nuclear leucocytes incubated with arachidonic acid24and in an enriched fraction of human eosinophils, 25as well as in rabbit peritoneal tissue26 and humanlung.27The increased capacity of inflamed colonic

mucosa to form prostaglandins and thromboxanehas been shown previously.5 We have nowconfirmed these observations, using the conversionof [14C]-AA as an index of cyclo-oxygenase activity,but in addition an increase in the formation ofradiolabelled lipoxygenase products was alsoobserved. Whether this increase is entirely due tothe greater number of inflammatory cells in theinflamed mucosa, or to an increase in the capacity ofother cells in the mucosa to metabolise arachidonicacid requires further investigation.We have not yet been able to establish whether

synthesis of 5-HETE or LTB4 (5, 12-di-HETE) canoccur in human colonic mucosa as the bandscorresponding to these products were inhibited byindomethacin as well as by BW755C. As the5-HETE band was inhibited less by indomethacin,however, than by BW755C, this band may consist ofa mixture of both lipoxygenase and cyclo-oxygenaseproducts. There were somewhat smaller amounts ofanother unidentified metabolite (product II) whichalso appeared to be a lipoxygenase product as itsformation was inhibited by BW755C but not byindomethacin. Although our findings onarachidonate metabolism by broken-cell prepara-tions of human colonic mucosa may give limited

information about its metabolism in intact mucosa insitu, such studies form a necessary basis for furtherinvestigation.The synthesis of lipoxygenase products by human

colonic mucosa has implications for the patho-genesis and treatment of human inflammatory boweldiseases. Sulphasalazine and 5-amino salicylic acidinhibit synthesis of 5,12-di-HETE (LTB4) by humanisolated neutrophils stimulated with calciumionophore.28 In our experiments, these products didnot appear to be major products of arachidonic acidmetabolism in human colonic mucosa although it ispossible that this is the result of using a broken cellpreparation,14 a procedure required to enable moreefficient conversion of [14C]-AA. The effect ofsulphasalazine and its degradation products on thesynthesis of 12- and 15-lipoxygenase products is atpresent under investigation.Both 12-HETE and 15-HETE have chemotactic

or chemokinetic properties7 12 15 and inducesecretion of specific granule contents from humanpolymorphonuclear leucocytes although they areless potent than 5-HETE and 5,12-di-HETE.17Furthermore, recent studies indicate that 15-HETEcan activate leukotriene biosynthesis in a mast cellline.29 These products therefore have the potentialto contribute to inflammatory processes in thehuman bowel. We have shown that indomethacin,which appears to be of no value as an anti-inflammatory agent in ulcerative colitis,9 10 does notinhibit synthesis of the lipoxygenase product byhuman colonic mucosa, whereas BW775C does. Itwill therefore be important to compare the potencyof sulphasalazine and its active constituents asinhibitors of the lipoxygenase pathways found inhuman colonic mucosa and to consider whether dualcyclo-oxygenase/lipoxygenase pathway inhibitorsmight be of value as anti-inflammatory agents inulcerative colitis and other inflammatory conditionsof the gastrointestinal tract.

References

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2 Harris AW, Smith PR, Swan CHI. Determination ofprostaglandin synthetase activity in rectal biopsymaterial and its significance in colonic disease. Gut1978; 19: 875-7.

3 Rampton DS, Sladen GE, Youlten LJF. Rectalmucosal prostaglandin E2 release and its relation todisease activity, electrical potential difference, andtreatment in ulcerative colitis. Gut 1980; 21: 591-6.



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4 Hawkey CJ, Truelove SC. Effect of prednisolone onprostaglandin synthesis by rectal mucosa in ulcerativecolitis: investigation by laminar flow bioassay andradio-immunoassay. Gut 1981; 22: 190-3.

5 Ligumsky M, Karmeli F, Sharon P, Zor U, Cohen F,Rachmilewitz D. Enhanced thromboxane A2 andprostacyclin by cultured rectal mucosa in ulcerativecolitis and its inhibition by steroids and sulfasalazine.Gastroenterology 1981; 81: 444-9.

6 Wedmore CV, Williams TJ. Control of vascularpermeability by polymorphonuclear leukocytes ininflammation Nature 1981; 289: 141-7.

7 Higgs GA, Palmer RMJ, Eakins KE, Moncada S.Arachidonic acid metabolism as a source of inflam-matory mediators and its inhibition as a mechanism ofaction for anti-inflammatory drug. Molec Aspects Med1981; 4: 275-301.

8 Weksler BB, Knapp JM, Jaffe EA. Prostacyclin (PGI2)synthesised by cultured endothelial cells modulatespolymorphonuclear leukocyte function. Blood 1977;50: suppl 1, 287.

9 Gilat T, Ratan J, Rosen P, Peled Y. Prostaglandins andulcerative colitis. Gastroenterology 1979; 76: 1083.

10 Campieri M, Lanfranchi GA, Bazzocchi G, BrignolaC, Penatti A, Boccia S, Labo G. Prostaglandins,indomethacin and ulcerative colitis. Gastroenterology1980; 78: 193.

11 Rampton DS, Sladen GE. Prostaglandin synthesisinhibitors in ulcerative colitis: flurbiprofen comparedwith conventional treatment. Prostaglandins 1981; 21:417-25.

12 Turner SR, Tainer JA, Lynn WS. Biosynthesis of-chemotactic molecules by the arachidonate lipoxy-genase system of platelets. Nature 1975; 257: 680-81.

13 Borgeat P, Hamberg M, Samuelsson B. Transforma-tion of arachidonic acid and homo-y-linoleic acid byrabbit polymorphonuclear leukocyte. J Biol Chem1976; 251: 7816-20.

14 Narumiya S, Salmon JA, Cottee FH, Weatherley BC,Flower RJ. Arachidonic acid 15-lipoxygenase fromrabbit peritoneal polymorphonuclear leucocytes. J BiolChem 1981; 256: 9583-92.

15 Palmer RMJ, Stepney R, Higgs GA, Eakins KE.Chemokinetic activity of arachidonic acid lipoxygenaseproducts on leukocytes from different species. Prosta-glandins 1980; 20: 411-18.

16 Ford-Hutchinson AW, Bray MA, Doig MV, ShipleyME, Smith MJH. Leukotriene B4, a potent chemo-kinetic and aggregating substance released from poly-morphonuclear leukocytes. Nature 1980; 286: 264-5.

17 Stenson WF, Parker CW. Monohydroxy-eicosatetraenoic acids (HETEs) induce degranulation

of human neutrophils. J Immunol 1980; 124: 2100-4.18 Hammarstrom S, Hamberg M, Samuelsson B, Duell

EA, Stawiski M, Voorhees JJ. Increased concen-trations of non-esterified arachidonic acid, 12L-hydroxy 5,8,10,14 eicosatetraenoic acid, prostaglandinE2 and prostaglandin F2o, in epidermis in psoriasis. ProcNatl Acad Sci USA 1975; 72: 5130-4.

19 Herman AG, Claeys M, Moncada S, Vane JR.Biosynthesis of prostacyclin (PGI2) and 12L hydroxy5,8,10,14 eicosatetraeynoic acid (HETE) bypericardium, pleura, peritoneum and aorta of therabbit. Prostaglandins 1979; 18: 439-52.

20 Randall RW, Eakins KE, Higgs GA, Salmon JA,Tateson JE. Inhibition of arachidonic acid cyclo-oxygenase and lipoxygenase activities of leukocytes byindomnethacin and compound BW755C. Agents Actions1980; 10: 553-5.

21 Porter NA, Wolfe RA, Yabro EM, Weene H. Theautoxidation of arachidonic acid: formation of theproposed SRS-A intermediate. Biochim Biophys ResComm 1979; 89: 1059-64.

22 Higgs GA, Flower RJ, Vane JR. A new approach toanti-inflammatory drugs. Biochem Pharmacol 1979; 28:1959-61.

23 Bennett A, Hensby CN, Sanger GJ, Stamford IF.Metabolites of arachidonic acid formed by humangastrointestinal tissues and their actions on the musclelayers. Br J Pharmacol 1981; 74: 435-44.

24 Borgeat P, Samuelsson B. Arachidonic acidmetabolism in polymorphonuclear leukocytes: effect ofionophore A23187. Proc Natl Acad Sci USA 1979; 76:2148-52.

25 Turk J, Maas RL, Brash AR, Jackson Roberts L,Oates JA. Arachidonic acid 15-lipoxygenase productsfrom human eosinophils. J Biol Chem 1982; 257:7068-76.

26 Claeys M, Coene M-C, Herman AG, Jouvenaz GH,Nugeteren DH. Characterization of monohydroxylatedlipoxygenase metabolites of arachidonic and linoleicacid in rabbit peritoneal tissue. Eur J Pharmacol 1982;713: 160-9.

27 Hamberg M, Hedqvist P, Radegran K. Identification of15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE)as a major metabolite of arachidonic acid in humanlung. Acta Physiol Scand 1980; 110: 219-21.

28 Stenson WF, Lobos E. Sulfasalazine inhibits thesynthesis of chemotactic lipids by neutrophils. J ClinInvest 1982; 69: 494-7.

29 Vanderhoek JY, Tare NS, Bailey JM, Goldstein AL,Pluznik DH. New role for 15-hydroxyeicosatetraenoicacid: activator of leukotriene biosynthesis in PG-18mast/basophil cells. J Biol Chem 1982; 20: 12191-5.



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