Biosynthesis and biological activity of leukotriene B5

PROSTAGLANDINS

BIOSYNTHESIS A N D BIOLOGICAL ACTIVITY OF L E U K O T R I E N E BS* Takashi Terano, 3ohn A. Salmon and Salvador Moncada Dept. of Prostaglandin Research Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR] 3BS. ABSTRACT Several studies indicate that increased intake of eicosapentaenoic acid (EPA) in the diet may lead to decreased incidence of thrombotic events. Most investigators agree that this is achieved by competitively inhibiting the conversion of arachidonic acid (AA) to thromboxane A? in the ptatelets. The effect of high EPA-intake on the formation of prosZacyclin is less clear. However, EPA is a good substrate for lipoxygenase enzymes which results in formation of hydroperoxy- and hydroxy-acids, and, in some cases, leukotrienes. The biological activities of the leukotrienes derived from arachidonic acid suggest that they mediate or modulate some symptoms associated with inflammatory and hypersensitivity reactions. In order to clarify the possible effect of dietary manipulation on inflammatory processes, leukotriene B S (LTB 5) was prepared and its biological activities assessed. L T B ~ , was biosyhthesised by incubating EPA with glycogen-elicited polymorpho'nuclear neutrophils (PMN) from rabbits in the presence of the divalent cation ionophore, A23187. The LTB~ was extracted from the incubate using minireverse phase extraction eolur6ns (Sep-pak) and purified by reverse-phase high pressure liquid chromatography (RP-HPLC). The purity of the product assessed by repeat RP-HPLC and straight phase (SP) HPLC was greater than 95%. Ultra-violet spectrophotometry of the product confirmed its purity and also provided assessment of the yield. The biological activity of LTB 5 was assessed and compared with that of LTB 4 in the following tests; aggregation of rat neutrophits, chemokinesis of human PMN, lysosomal enzyme release from human PMN and potentiation of bradykinin-induced plasma exudation. In all these tests, LTB 5 was considerably less active (at least 30 times) than LTB 4. INTRODUCTION Epidemiological surveys suggest a causal relationship between high intake of eicosapentaenoic acid (EPA) in the diet and a reduced incidence of thrombo-embolic episodes (t,2,3). Subsequent studies with animals and human volunteers fed an EPA-rich diet confirmed that EPA reduces ptatelet aggregation, increases cutaneous bleeding times, lowers plasma lipid levels and reduces whole blood viscosity (4,5,6,7). Some of these changes have been attributed to the synthesis of metabo|ites of EPA which are less active than *Preliminary data were presented at the Symposium on Prostanoids held in Cracow, Poland during September 1983.

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PROSTAGLANDINS similar compounds formed from arachidonic acid (AA) (8,9). However, most investigators acknowledge that EPA is a poor substrate for the cyclooxygenase enzyme (9,lO) and therefore one of the main mechanisms by which EPA exerts its anti-thrombotic activity is by direct competition with the Thus, high intake of EPA leads to a conversion of AA by the cycle-oxygenase. decrease in the amount of pro-thrombotic thromboxane A2 (TXA2) formed during platelet aggregation. However, EPA is a good substrate for the lipoxygenase enzymes (11,121 which convert some polyunsaturated fatty acids to hydroperoxy intermediates. 5-HPETE, is of particular interest One such product of AA metabolism, The because it can be further metabolised to leukotrienes (13,141. leukotrienes C4 and D4 (LTC , LTD ) are the major constituents of the “slow SRS-A) and probably contribute to the reacting substance of anaphy 4 axis+!t development of symptoms associated with asthma and similar hypersensitivity reactions (15,161. Another leukotriene, LTB4, is a potent chemokinetic and chemotactic agent for polymorphonuclear leukocytes (PMN) (17,18,19) and it has been suggested that LTB4 plays a role in the recruitment of PMN to sites of inflammation. Recently it has been demonstrated that both LTB and LTC can be biosynthesized by murine mastocytoma cells from EPA-ric ’ ?-I oils (202 indeed, Murphy et al. (20) indicated that the efficiency of this conversion to LTB5 is similar to the synthesis of LTB4 from arachidonic acid. Therefore, the intake of an EPA-rich diet may result in the formation of leukotrienes of the ‘5’ series. However, since the biological activities of these leukotrienes have not yet been described, it is unclear what influence a high intake of EPA would have on inflammatory and hypersensitivity reactions. In this paper, we report the biosynthesis of LTB5 from EPA by rabbit PMN and the comparison between the biological activities of LTB5 and LTB4. MATERIALS

AND

METHODS

All cis-5,8,11,14,17-eicosapentaenoic acid (EPA; 96% pure) was provided by NissuiPharmaceutical Company, Yuki, Ibaragi-pref, Japan. Chemically synthesised LTB4 was obtained from Professor E.J. Corey, Harvard University, Cambridge, Mass., USA. Oyster glycogen (grade II), HEPES buffer, N, O-Bis-(trimethylsilyl)trifluoroacetamide (BSTFA), cytochalasin B, phenolphthalein-8-D-glucuronic acid, lysozyme and micrococcus luteus were all purchased from Sigma Chemical Co., St. Louis, MO, USA. Other materials were obtained from the suppliers indicated: Ficoll-Paque (Pharmacia Fine Chemicals, Uppsala, Sweden), A arose (L’Industrie Biologique Francais, S.A., Gennevilliers, France), ~1 3 D-human serum albumin (Amersham International, Amersham, Bucks, England), Hanks balanced salt solution (Wellcome Diagnostics, Dartford, Kent, England), A23187 (Calbiochem, Bishops Stortford, Hefts, England), Sep-pak ODS columns (Waters Associates, Northwich, Cheshire, England), microtitre plates (Falcon Plastics Inc., Oxnard, CA, USA). Analytical Dorset) and respectively.

218

grade and HPLC solvents were purchased from Rathburn Chemicals (Walkerburn, Peebleshire,

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1.

Collection

of cells

Rabbit peritoneal PMN were collected as described by Borgeat et al. Briefly, 0.2% glycogen in sterile saline (200 ml) was injected (14,211. intraperitoneally (i.p.) into New Zealand white rabbits (2.5 - 4 kg), 4 h later saline containing 10 U/ml heparin (100 ml) was injected i.p. and the peritoneal exudates were withdrawn and centrifuged at 250 x g for 10 min. The cell pellets were suspended in HEPES buffered @H 7.4) Hank’s balanced salt solution (HBSS) and kept in this medium at 4 C until required but was used within 2 h of preparation. The proportion of PMN in this cell suspension was > 95%. 2.

Incubation

conditions

The cell suspension (lo8 cells/ml) was pre-incubated at 37’C for 10 min. The divalent cation ionophore, A23187 (2 PM) and EPA (300 ~_IM)were added in ethanol and 6 mM sodium carbonate respectively. The final concentration of ethanol in the incubation was 0.1%. After 2 min incubation at 37’C under a normal atmosphere, the reaction was stopped by addition of 2 volumes of ethanol. 3.

Extraction

of LTB5

The extraction of LTB5 from the incubation mixture was performed using ODS-silica mini-columns and was based upon the report by Powell (22). The ethanolic incubation mixture was centrifuged at 1500 x g and the supernatant removed. The pellet was washed with an additional volume of ethanol and, after centrifugation, the ethanolic supernatant was combined with the first extract. The pooled extracts were concentrated under reduced pressure to approximately the original incubation volume and then diluted with 2.3 volumes of water. The aqueous-ethanol mixture was acidified with 1N HCl to pH 3.0 and then applied to an ODS-silica cartridge (Cl&iep-Pak). The cartridge was successively washed with 15% aqueous ethanol (20 ml), water (20 ml), petroleum ether (40-60; 20 ml) and finally spectroscopic grade ethyl acetate (6 ml). The ethyl acetate fraction was passed through a 5 urn filter (Millex-SR; Millipore Corporation, Bedford, MA), evaporated under nitrogen and redisolved in 100 ~1 high pressure liquid chromatography (HPLC) solvent (methanol-water-acetic acid; 65:35:0.01 v/v/v/adjusted to pH 5.7 with ammonium hydroxide). 4.

High pressure

liquid chromatography

The LTB5 in the extract was separated from other products by reversed phase (RP)-HPLC. Samples in HPLC solvent (see above) were applied to a spherisorb ODS 5 urn column (250 mm x 4.5 mm; Laboratory Data Control, Stone, Staffs, England) using a Model 6K injector (Waters Associates, Northwich, Cheshire, England). HPLC solvent was pumped at 0.8 ml/min through the column using a 6000 A delivery system (Waters Associates). The absorbance of the column effluent at 271 nm was continually monitored with a variable wavelength detector (Spectromonitor III, Laboratory Data Control).

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PROSTAGLANDINS In some cases, the absorption at 254 nm was also measured using a Model 440 Fractions (15 set; 200 ~1) were absorbance detector (Waters Associates). collected for 1 h. The absorbance of each fraction at 271 nm was determined In addition, the Model 25 spectrophotometer. using a Beckman immunoreactivity in the fractions was assessed using a radioimmunoassay for LTB (23; see below). Four main compounds were resolved by RP-HPLC (see Fig.4); these were provisionally identified as 5(S)lZ(R) -6 - trans -LTB (Rt 15.9 min), 5 (S), 12 (S) -6 - trans - LTB (Rt 17.8 min), LTB (18.8 min) a?d 5hydroxy-6, 8, 11, 14, 17-eicosapenta&oic acid (Rt 56.85 min). Fractions containing these components were combined separately with reference to the UV absorption and immunoreactive profiles (see Fig. 2); particular attention was paid to the elimination of the diastereomers of 6-trans-LTB5 from the sample of LTB . The combined fractions containing these products were taken the residues were redissolved in 77g a rotary film evaporator; to dryness USI spectroscopic grade methanol and these solutions were stored at -2O’C. The purity of the biosynthesised LTBE was assessed bv repeat RP-HPLC by straight phase (SP)(conditions as ‘above; see Fig. 3) and, additionally,

0.05

Standard lniection

LTB,

+

c

u:\ J

:

1

0

10

20

30

40

50

I

‘55

Elution time (min)

Fig. 1. MetaboZism of EPA by A23187 stimulated rabbit PMN: separation of products by RP-HPLC after Sep-Pak extraction.

220

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6

20

25

Elution time (mid

Fig.

2. Separationof LTB5 and diastereomersof 6-trans-LTB5 by RP-HPLC. The absorbanceat 2 71 nm in the HPLC etuate was monitored and is shown in the upper panel. Peaks I and II are the diastereomersof 6-trans-LTB5and peak III is LTB5. The touer panel iZZustratestheirrununorea&ivity (measuredin a RIA for LTB ; see Methods) in the eluate. 4 LTB,

t LTB, 1 ;; :: .: :. :: it

Injection

: ;, i:

I

I

0

5

10 15 20 Elution time (mid

25

Fig. 3. Purity of LTB5 as assessed by RP-HPLC. 100 ng LTB5 was injected onto a Spherisorb5 bM ODS column and was eluted with methanol-water-acetic acid )65:35:0.01,adjusted to pH 5.7 with avnnoniwn hydroxide)at a flow rate of 0.8 ml/min. A separate injectionof 100 ng LTB4 is shown for comparison.

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PROSTAGLANDINS

HPLC. In the latter test, LTB5 was initially methylated with freshly prepared diazomethane and the methyl ester was applied to a Zorbax Sil column (DuPont Instruments, Wilmington, DE) which was eluted at 3 ml/min with n-hexane-&The purity of LTB5 based on these propanol-acetic acid (95:5:0.01 v/v/v). combined measurements was greater than 95%; contamination with either the diastereomers of 6-trans-LTB5 and 5(S), 12(S)-dihydroxy-8, 14, 17-e-6, lotrans-eicosapentaeno=id was negligible (the latter product could be formed by a dioxygenation reaction analogous to the formation of 5(S) 12(S) dihydroxy8,14-cis-6, lo-trans-eicosatetraenoic acid (24). The concentrations of the methanolic solutions of LTB5 and the 6-transThe UV LTB diastereomers were determined by UV spectrophotometry. spec?ra of these compounds were similar to those of LTB4 and isomers (21); maximum absorption by LTf35 occurred at 270 nm and there were shoulders at 260 and 281 nm (see Fig. 4). The quantitative measurements were based upon

nMfiig

c$?!! 51,000 (14). The for the nm with shoulders at 258 and 280 nm. Gas-liquid

chromatography

diastereomers

of 6-trans-LTB5

- mass spectrophotometry

was 268

(GLC-MS)

Aliquots (1 ug) of the purified compounds provisionally identified as and 5-hydroxy-eicospentaenoic acid diastereomers of 6-trans-LTB LTB5, were converted to methyl ester, trime sn ylsilyl ether derivatives by successive A portion of the derivatives reaction with diazomethane and BSTFA. (approximately 400 ng) was injected into a Hewlett Packard Model 5730A gas

Fig.

4.

of LTB4 (

222

.

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UZtravioZet spectra (! and LTB5

. ).

1984 VOL. 27 NO. 2

PROSTAGLANDINS

liquid chromatograph GLC conditions were

linked to a VG Micromass 16F mass spectrometer. as follows: a 1% OV 1 column was employed at

and a helium flow rate of approximately 25 ml/min. was operated in the electron impact mode at 22 eV; 180 pA and the source temperature was 22O’C. Assessment

The mass the filament

The 23O’C

spectrometer current was

of immunoreactivity

The cross-reaction of LTB5 in a radioimmunoassay (RIA) for LTB4 was determined. The specific antiserum against LTB4 employeg in this test was characterized previously (23). The inhibition of binding of C i-O-LTB4 (SA 210 Ci/mMol; Amersham International) to the antibody induced by LTE15 (5 pg -100 ng) was compared to that caused by standard LTB4,(5 pg - 2 ng) using the same assay conditions described previously (23). The immunoreactivity in HPLC fractions was also assessed in this RIA system (see above); the HPLC solvent was removed under nitrogen and the residue reconstituted in RIA buffer (tris buffer, 50 mM pH 8.6 containing 0.1% gelatin) prior to assay. Biological a)

activities

Aggregation

of LTB5 of rat peritoneal

PMN

Aggregation of rat peritoneal PMN was assessed according to the method described by Cunningham et al. (25) with minor modifications. Briefly, peritoneal exudate cells (approximately 80% PMN) were obtained from male Wistar rats (150-200 g) 17 h after i.p. injection of 0.2% oyster glycogen. Cells were harvested immediately following i.p. injection of HHBS (20 ml) containing 20 U/ml heparin. The cells were washed once in HHBS without heparin and then resuspended at a concentration of 107cells/ml. Aliquots (490 ~1) of the cell suspension were transferred to siliconized glass cuvettes; the suspension was warmed to 37’C and stirred at 800 rpm in a Payton dual-chann$ aggregometer. After 5 min preincubation, the agonists (LTB4 or LTBS; 10 -10-&l) were added to the cell suspension in 50 mM tris buffer (pH 8.0; 10 i.11) and the aggregation of the PMN was monitored by changes in light transmission. Aggregation was measured as the maximum height of the response (mm) on a W and W pen recorder. The result was corrected for the change in light transmission induced by buffer alone (approx. 20 mm; see Fig. 5). b)

Lysosomal

enzyme

release

from

human

PMN

Highly purified human PMN (greater than 95%) were prepared from heparinized blood of healthy volunteers using differential centrifugation, Ficoll-Paque gradients and ammonium chloride lysis of erythrocytes according to the method of Palmer et al. (17). The LTB-induced release of the azurophilic granule marker, B-glucuronidase, and the specific granule enzyme, lysozyme, were determined using methods similar to those published previously (26,27,28). Briefly, a suspension of human PMN (800 pl containing approximately 6.2 x 106 cells) was preincubated at 37’C with cytochalasin B (5

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PRQSTAGLANDINS

pg added in 0.1 ml HHBS) for 15 min before the addition of LTB4 (10-Ia 3.2 x 106 in 50 mM Tris buffer, pH 8.0) or LT% (ICJ’O3.2 x lO_6M in 50 mM Tris buffer pH 8.0). The incubations were terminated after 15 min by precipitating the cells at 10,000 x g for 30 set in an Eppendorf mini-centrifuge (Model 5412). The supernatants were removed and the concentrations of the enzymes were determined. &glucuronidase was assayed by measuring the liberation of phenolphthaiein from phenolphthalein-B-D-glucuronic acid colourimetrically (26,27). Lysozyme activity was assessed by lysis of a suspension of M. luteus which was monitored by observing changes of light transmission in a Payton In some experiments, lactate dual-channel aggregometer (26,28). dehydrogenase (LDH) was also measured by an established procedure (29). These data, for all enzymes, were expressed as the proportion of the total enzyme which was released; total content of enzyme in the cell suspensions was measured after lysis of cells with 0.2% triton X-100. c)

Chemokinetic

activity

The chemokinetic indices for LTB and LTB4 towards human PMN were determined using the agarose-microdrop 5 et technique as described by Palmer et al. (17). The chemokinetic index was defined as: Mean distance cells moved in presence of agonist divided by Mean distance cells moved in absence of agonist. d)

Vascular

permeability

The potentiation of bradykinin-induced vascular permeability by LTB4 and LTB5 were compared using a protocol similar to that described by Higgs et al. (30). Plasma exudation, which was assessed by determining the leakage of radioactivity from the circulation following i.v. injection of 02SI]-human serum albumin according to the method of Williams (31), was measured in rabbit skin 30 min after the intra-dern;al injection of 500 ng bradykinin together with LTB4 or LTB5 (l-100 ng/skin site). The resuits were expressed as the \.olume (~1) of plasma exudate per skin site. RESULTS Leukotriene B5 was biosynthesised from EPA by A23187-stimulated rabbit PMN; was 0.6% of added EPA. the yield of purified LTB The time on RP-HPLC structure of LTB5 was established by its s I?orter retention relative to LTB4 (Fig. l), its similar UV absorption profile to that of LTB4 (Fig. 4), its cross-reaction with an anti-LTB4 serum (17.8%; see Fig. 2) and by mass spectrometry. The methyl ester, trimethylsilyl ether derivative of LTB5 was eluted from the GLC at a retention time equivalent to C 23.7 (derivative The mass spectrum of derivatized eluted at same retention time). ig. 5) was similar to that of the corresponding derivative of LTB4 (14). fragmentation pathway of the LTB5 derivative yields ions whrch do not include the Cl3 -C 2. segment of the molecule and consequently the major the ions in both spectra are at m/z 383, 293, 229, 217, 203 and 129. However, ions which are specific to LTB5 and confirm the identity of the biosynthesized

224

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1984 VOL. 27 NO. 2

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compound are m/z 492, 477, 461 and 402 which correspond to the M+, M-15, M-31 and M-90 fragments respectively (cf. 494, 479, 463 and 404 in the spectrum of derivatized LTB4). Other dihydroxy derivatives of EPA were also produced during incubation with rabbit PMN and these were assigned the structures 5, (S) 12 (R) - 6 -trans by analogy with products formed - LTB and 5 (S), 12 (S) - 6- trans - LTB from a rachidonic acid and with reference t d the UV absorption (see above) and mass spectrometry data generated in the present study. The methyl ester, trimethylsilyl ether derivatives of the diastereomers of 6-trans-LTB were eluted from the GLC at a retention time equivalent to C24.8 and C24.3. The mass spectra were similar to those of the but were distinguished by characteristic mass spectra of LTB5; Fig. 5).

diastereomers ioils at m/z

exhibited

of 6-trans-LTB 477, 46xand 409

low cross-reaction

(21) (cf.

with

the

and 5(S), 12(S)-6-trans-LTB5 crossto LTB4; cf LTB cross-reacted 17.8%);

the RIA

LTB4 from

for LTB4

was previously

its 6-trans-isomers

demonstrated

to clear 3 y discriminate

(23).

20

29

217

I73

293

203 1’

477 383,i

4611

m/z

Fig. 5. Mass spectrum of the methyl ester, trimethyZsiZy2 ether derivative of LTB5.

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PROSTAGLANDINS

10- ‘(M)

6 xl .

0-w)

LW trance

*I

:

f :

LTB,

10-w) 15TL

IL

,Blank(tris

butter:

-Fig. 6. Changes in light transmittance of stirred suspensions of rat peritonea2 PMN (490 ~1 containing 5X106 cells) after addition of LTB and LTL35as indicated. The change of transmittance induce$ by vehzcZe alone is shown in the inset.

Fig. 7. Aggregation of rat peritoneal PMN by LTB4 f--O--l and LTB5 C--b-). Each point is the mean -+S.E.M. of between 5 and 10 separate observations.

226

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60

40 t

j 30 E

;

20-

E t w’

IO -

OL

Fig. 8. Release of lysosomal enzymes from cytochalasin B-treated . B-gZucuronidase human PMN. Lysozyme reZease:-o-LTB4+LTB release:******LTB,***A***LTB . Each point is t2e mean of duplicate observations usiig the sam2 preparation of PMN. In a second experiment the enzyme-release was less (31.0% total lysozyme and 11.4% total 8-glucuronidasereleased by 10m6M LTB4) but the doseresponse relationships for both LTB4 and LTB3 were similar to the experiment illustrated. Neither LTB nor LTB5, at the concentration tested, caused significant release I?! f LDH. 6,

6s

4B 1 .j

3

p g

2-

Leukotrimn B concentration (n(l.rn~.‘l

and Fig. 9. Human PMN chemokinetic activity of LTB4 I+) LTB5 c--b-). Each point is the mean f S.E.M. of 12 observations uszng 2 preparations of hwnan PMN.

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PROSTAGLANDINS In each of the biological tests the activity of LTB5 was compared with of LTq and the concentration of both compounds was established by UV $leOH 270 to be 51,000 (14) for both compounds. spectrophotometry assuming Leukotriene B5 was considerably less active than LTB4 in aggregating rat PMN (Figs. 6 & 7), Inducing degranulation and chemokinesrs of human PMN (Fig. 8 and Fig. 9, respectively) and potentiating bradykinin-induced plasma exudation was in rabbit skin (Fig. 10). A precise ratio of the potencies of LTB4 and LTB not calculated in most of these tests since full dose-response curves cou i5d not be constructed due to limited availability of LTB;. However, it is clear that LTB5 is at least 30 times less active than LTB4 in the in vitro tests (aggregation, degranulation, chemokinesis). that

These in .vitro data were supported by the in vivo observation that 10 and 100 ng LTB4 srgnrfrcantly potentiated the plasma exudation induced by intradermal injection of 500 ng bradykinin but LTB up to 100 ng failed to significantly potentiate this response least 10 times less active than LTB4.

(Fig.

10).

T?ws,

in vivo,

LTB5

was

at

OO-

40 -

-~_-__________________ z %

2 5

a?

Tril Control

:: 0-

I 1 10 Lwkotrime B dose(nflhkinsite)

I 100

Fig. 10. Va-scuZarpermeabiZity changes in rabbit skin induced by LTt4 i-i?) and LTB5 ( --A--) in the presence of bradykinin. Each poznt is th mean _+S.E.M. of 5 observations. * P < 0.05 (compared to bradykinin control). 228

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PROSTAGLANDINS

DISCUSSION Eicosapentaenoic acid is not a good substrate for the cycle-oxygenase, and it has been suggested that it is a competitive inhibitor of the conversion of arachidonic acid to prostaglandin endoperoxides by this enzyme (9). However, EPA is converted efficiently by lipoxygenase enzymes (11,12,20); indeed, pentadiene leukotrienes can be hiosynthesized in similar yields, both in vitro (11,lZ) and ex viva (20) to those leukotrienes derived from arachidonic acid. The relatively efficient conversion of EPA to LTB5 by A23187-stimulated rabbit PMN was confirmed in the present study. As yet little information is available on the biological activities of the leukotrienes of the “5” series (32). An evaluation of these activities is important since there is now considerable support for supplementing the diet with purified EPA or fish oil concentrates in order to reduce the incidence of myocardial infarction and other thromboembolic disorders (see 7). If the EPA content of neutrophil cell-membrane phospholipids can be increased by displacement of AA during dietary manipulation then this will lead to an increase in leukotrienes of the ‘5’ series relative to LTI3$ LTC f” LTDLl etc. since both EPA and AA are liberated efficiently by p ospho ipases (9). Leukotrienes derived from AA have biological activities which suggest that they may mediate or modulate and thus inflammatory and hypersensitivity reactions (15,16,17,18,19) formation of pentadiene leukotrienes could affect the pathology of these conditions. In the present study, we have demonstrated that LTB5 is, considerably less active (at least 30 times) than LTS4 in affecting neutrophil function g vitro (aggregation, degranulation and chemokinesis). Also, the potency of LTB in potentiating hradykinin-induced plasma exudation, which is probably attri 3 utable to its leukotactic activity (33) is at least 10 times lower than that of LTB4. Preliminary experiments performed in our laboratory demonstrate that significantly more LTB5 is synthesized by PMN from animals fed an EPA-rich diet compared to those from animals on a normal diet. If the amount of LTB formed is reduced, as well as LTB5 being increased, after feeding with EP 6t Thus, then the net result may be a decreased chemotactic response. inflammatory reactions in animals with a high intake of EPA could he characterized by reduced cell influx if LTB& ,is indeed an important mediator of this response. The concentration of LTB4 IS higher than normal in synovial fluid from patients with gout (34) and rheumatoid arthritis (35), in blister fluid from psoriatic skin (36) and in exudate from animals with experimentallyinduced inflammation (37). It is, therefore, necessary to study whether an EPA-rich diet could reduce chronic inflammatory reactions. This hypothesis is supported by the reports that Eskimos, who consume an EPA-rich diet, are almost free of the chronic degenerative diseases, including ulcerative colitis and rheumatoid arthritis, (38) and from the work of Prickett and colleagues who have recently demonstrated that feeding EPA prevents protinuria and prolongs survival in an animal model of human systemic lupus erythematosis (39).

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PROSTAGLANDINS

Acknowledqement The authors studies.

wish

Lo thank

Dr.

M.V.

Doig

for

performing

the

GLC-MS

REFERENCES

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Dyerberg, J. polyunsaturated

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Hirai, A., Hamazaki, T., Terano, T., Kumagai, A. & Sajiki, J. Eicosapentaenoic Japanese. Lancet, 1980. -ii:1132,

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11.

Jakschik, B.A., Sams, structural requirement -20:401, 1980.

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Yokoyama, C., Mizuno, K., Mitachi, H., Yoshimoto, T., Yamamoto S. & Pace-Asciak, C.R. Partial purification and characterization of arachidonate 12-lipoxygenase from rat lung. Biochim. Biophys. Acta., 750, 237, 1983.

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H. and

& Needleman, biosynthesis.

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P. Fatty acid Prostaglandins,

1984 VOL. 27 NO. 2

PROSTAGLANDINS

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Murphy, R.C., Hammarstrom, S. & Samuelsson, reacting substnace from murine mastocytoma Sci., 7&:4275, 1979.

14.

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