In vivo formation of leukotriene E5 by murine peritoneal cells

PROSTAGLANDINS

IN V I V O F O R M A T I O N OF L E U K O T R I E N E M U R I N E P E R I T O N E A L CELLS

E 5 BY

J. Whelan, K.S. Broughton, B. Lokesh, and J.E. Kinsella Lipids Research Laboratory, Institute of Food Science, Cornell University, Ithaca, NY 14853 ABSTRACT Resident mouse peritoneal cells, stimulated in vivo with opsonized zymosan, produced leukotriene C 4 and E 4, with LTE 4 being the major (80-90%) product. When mice were placed on diets containing increasing amounts of fish oil, four additional sulfidopeptide leukotrienes (SP-LT), LTCs, LTEs, 11-trans LTC s and I I-trans LTEs, were identified. The identity of LTE 5 was confirmed by spectrophotometric, chromatographic and enzymatic methods. When equivalent amounts of n - 6 and n-3 polyunsaturated fatty acids (PUFA) were included in the diet, the stimulated peritoneal cells (in viv0) produced higher quantities of LTE 5 (30.2 *__5.4 ng/106 cells) than LTE 4 (22.8 +_ 7.3 ng/106 cells). In addition, in vitro studies demonstrated a 60% reduction in LTC 4 (42.0 +_ 10.8 ng/10 e cells to 16.7 +_6.2 rig/10 e cells) and the appearance of LTC 5 (2.1 +_. 0.9 ng/106 cells) in resident macrophages (stimulated with A23187) from mice maintained on a fish oil diet compared to mice fed the control diet. This study demonstrated that formation of the pentaenyl SP-LT in vivo, in particular LTE s, by peritoneal cells can significantly contribute to the endogenous SP-LT pool in response to an inflammatory stimulus following a dietary regimen containing fish oil. INTRODUCTION The 4-series sulfidopeptide leukotriene(s) (SP-LT), LTC4, LTD 4 and LTE4, formed from arachidonic acid (AA, 20:4n-6), are collectively known as the slow reacting substances of anaphylaxis and are involved in the inflammatory response (I-4). At sites of inflammation, leukocytes such as neutrophils, lymphocytes and macrophages, can produce significant quantities of SP-LT upon stimulation. SP-LT have potent inotropic properties, can increase postcapillary venule permeability, are potent stimulators of airway smooth muscle cells, and are mediators of pulmonary asthma (5-8). The leukotrienes E are, in general, approximately 30-100 times less biologically potent than leukotrienes D and 10 times less potent than leukotrienes C in a bioassay utilizing guinea pig ileum (9). Pentaenyl (5-series) leukotriene(s) (LT) are formed from 5, 8, 11, 14, 17eicosapentaenoic acid (EPA, 20:5n-3), a polyunsaturated fatty acid (PUFA) derived from fish oils. Most attention has focused on the pentaenyl LT LTBs, with limited interest in the SP-LT, particularly LTE 5. While a few studies have reported LTE s production in vitro (9-11), there is little evidence which demonstrates that in viv0 formation of LTE s significantly contributes to the endogenous SP-LT pool (12). Since murine peritoneal cells are efficient producers of LTE 4 in vivo (13), and it has been demonstrated that EPA is a good substrate for the 5-1ipoxygenase (14,15), it is conceivable that LTE s could be a major metabolic component of the lipoxygenase

JANUARY 1991 VOL. 41 NO. 1

29

PROSTAGLANDINS pathway when polyethylenic acids of the n-3 series are incorporated into the dietary regimen. In this present study, we investigated in vivo and in vitro LTE 5 production in murine peritoneal cells from mice placed on diets containing fish oils. MATERIALS AND METHODS Leukotrienes C4, C~, D4, D6, E 4, E 5 and PGB 1 were purchased from Cayman Chemical (Ann Arbor, MI). Carboxypeptidase A and 7-glutamyl transpeptidase (GGTP) were purchased from Sigma (St. Louis, MO). Animals: Male CD-I mice (Charles River, Wilmington, MA), 18-20g, were housed in a temperature and humidity controlled room on a 12 hour light/dark cycle upon arrival. The mice were randomly divided into six groups. Four of the groups (diets 1-4) were used for in vivo studies and the other two groups of animals (diets I and 4) were used for in vitr9 experiments. After the mice were received, they were placed on a maintenance diet of Prolab Chow (Agway, RMF 1000, Syracuse, NY). Because the chow diet contained 18:3 n-3 and our experimental diets contained low to moderate levels of n-3 PUFA (Table 2), the mice were placed on a fat free diet for one week prior to the introduction of the experimental diets. Diet,: The mice were fed one of three experimental diets containing 10 wt% fat with projected n - 3 / n - 6 PUFA ratios of 0.20, 0.40 and 1.0 (diets 2, 3 and 4, respectively) (Table I). The control diets (diet 1) contained safflower oil with linoleic acid (18:2 n-6) being the sole source of PUFA, while the experimental diets contained both refined sardine oil as the fish oil (n-3 PUFA) source and safflower oil as the n-6 fatty acid source. The filler oil was composed primarily of oleic (18:1 n-9) and palmitic (16:0) acids. Diets were prepared in bulk and prepackaged in separate WhirI-Pak bags under nitrogen at 4°C. Water and food were provided ad libitum for two weeks. To minimize exposure to air prior to consumption, fresh diets were provided daily with uneaten food being discarded. Gas chromatographic analysis demonstrated that storage of the diets did not alter their fatty acid composition. Table I COMPOSITION OF THE DIETS Ingredient

g/Kg diet

Fat free diet Vitamin free casein Alphacel Sucrose Salt mixture USP XIV ICN vitamin mix with choline chloride

188 148 523 36 5

Dietary fat (gee Table 2)

100

The fatty acid composition of the diets were determined as described below (Table 2).

30

JANUARY 1991 VOL. 41 NO. 1

PROSTAGLANDINS Table 2 FATTY ACID COMPOSITION OF DIETARY FATS Dietary Groups Fatty Acid

1

2

3

4

0.10 3.98 0.12 0.39 3.21 i.55 0.03 0.06 0.34 0.05 0.16 0.41

0.22 3.07 0.34 0.30 2.62 1.55 0.04 0.17 0.99 0.11 0.48 1.19

g/100g diet 14:0 16:0 16:1 18:0 18:1 18:2 18:3 n-3 18:4 n-3 20:5 n-3 22:5 n-3 22:6 n-3 n-3/n-6

3.21 0.32 3.15 3.27

0.06 4.30 0.06 0.42 3.30 1.51 0.02 0.03 0.17 0.02 0.08 0.21

Leukotriene production in vivo: Opsonized zymosan was prepared by the methods of Maroussem et al. (16) with the following modifications. Zymosan at 10 m g / m l in distilled water was heated to 100°C for one hour. Following heating, the zymosan was cooled and pelleted and the water was decanted. The zymosan was then resuspended in rabbit serum at 10 mg/ml and incubated for 30 minutes at 37°C. Following incubation, the zymosan was pelleted and the serum was decanted. The zymosan was then twice washed with saline (0.9% NaCI) and resuspended in saline at 2 mg/ml. Following two weeks on the control and experimental diets, opsonized zymosan (1 mg in 0.5 ml saline) was injected intraperitoneally to induce an inflammatory response (13). After 30 min the animals were sacrificed by ether inhalation and the peritoneal cavity was washed with 3 ml saline (0.9%) containing 1 mM EDTA and 100 ng of prostaglandin B 1 (PGBI) as an internal standard, followed by a second wash of E D T A saline (2 ml). The peritoneal exudate cell populations consisted primarily of macrophages (95%) and some mast cells (<5%). The cells were differentiated according to standard morphological criteria using cytospin preparations stained with may-grlJnwald-giemsa stain. The peritoneal washes were pooled, an aliquot was resuspended in a cell counting medium containing 120 mM NaCI and 5.8 mM sodium citrate and the cells were quantitated using a Model PCA II Coulter Counter (Coulter Electronics, Inc., Hialeah, FL). The remaining peritoneal wash was centrifuged (700 x g for 4 min at 25°C) to pellet the cells. The supernatant was made up to a final volume of 10 ml containing 20% methanol and 3 mM formic acid and analyzed for leukotriene formation. The pelleted cells were analyzed for phospholipid fatty acid composition. Leukotriene production in vitro; Resident peritoneal cells were removed from the peritoneal cavity in phosphate-buffered saline containing 10 IU heparin/ml and cell numbers were determined as described previously. The cells were suspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. The

JANUARY 1991 VOL. 41 NO. 1

31

PROSTAGLANDINS cells were dispensed into Falcon tissue culture wells (35 mm) and incubated at 37°C for 2 hr in a humidified chamber under an atmosphere of 5% CO 2 and 95% air. The adherent cells were washed three times and incubated for another 2 hr in serum-free DMEM. The cells were then stimulated with calcium ionophore A23187 (0.5 #M) for 4 hr. The internal standard PGB 1 was then added to the cells and the supernatant was immediately removed and prepared for LT analysis. The adherent cells, previously identified as macrophages (>95%) by esterase straining and phagocytosis (18), were recovered for phospholipid fatty acid analysis using a rubber policeman. Leukotriene analysis: The ieukotrienes were isolated by solid phase extraction using a C- 18 cartridge (Burdick and Jackson, Muskegon, MI), sequentially washed with distilled water, then hexane, and eluted off the cartridge with methanol. Following evaporation, the residue was reconstituted in the HPLC solvent system of methanol:water (65:35, v/v), pH 5.6, containing 5mM ammonium acetate and 1 mM EDTA. The leukotrienes were separated by RP-HPLC on a Whatman Partisphere C-18 column (6 mm x 12.5 cm) with a flow rate of 0.9 ml/min and quantified using the internal standard PGB1, with a Hewlett-Packard 1040A Diode Array scanning spectrophotometer, monitoring at 280 nm. All compounds were identified by their distinct U.V. absorption spectra and retention times were compared to known standards. Conversion of 5.6-LTE~ to 5.6-LTF~ as catalvzed bv "~-~lutamvl transoentidase: The direct formation of 5,6-LTF 5 from 5,6-LrrE s was performed based on a modified method described by Bernstrom and Hammarstr~Jm (19). The G G T P reaction mixture (200 #1) contained 100 mM Tris-HC1, oH 8.0, 1 mM glutathione, 20 U GGTP, and 40 #M LTE 5. The reaction was initiated by the addition of enzyme and incubated at 30°C for 2 min. The reaction was terminated by the addition of an equal volume of methanol containing 0.1% glacial acetic acid and an aliquot was directly subjected to RP-HPLC under conditions described above. Conversion of 5.6-LTC= to 5.6-LTF~ catalyzed bv carboxvoeotidas¢ A: Conversion of 5,6-LTC 5 to 5,6-LTF 5 bycarboxypeptidase A was performed as previously described (20). Briefly, the carboxypeptidase A reaction mixture (200 #1) contained 0.1 M TrisHCI, pH 7.5,200 U carboxypeptidase A and 40 #M 5,6-LTC 5. The reaction was initiated by the addition of an equal volume of methanol containing 0.1% glacial acetic acid and an aliquot was directly analyzed by RP-HPLC as previously described. Fatty acid analysis: The pelleted peritoneal cells were resuspended in 0.8 ml (0.9%) saline and the lipids were extracted sequentially with chloroform:methanol (1:2, v/v), chloroform:saline (1:1, v/v) and finally chloroform (2x). The pooled organic extracts were evaporated to dryness under nitrogen and redissolved in chloroform. The macrophage phospholipids were separated by thin layer chromatography (TLC) using a chloroform:methanol (8:1, v / v ) s o l v e n t system and visualized with 8 - h y d r o x y - l , 3 , 6 pyrenitrisulfonic acid trisodium salt. The phospholipids were recovered from the TLC plates by scraping the appropriate bands and resuspended in toluene. The methyl ester of pentadecanoic acid was added to each sample. The samples were then saponified with 0.5 N KOH in methanol for 8 min at 86°C. Following acidification with 0.7 N HCI and the addition of an equal volume of hexane the mixture was rapidly cooled until the aqueous layer was frozen. The hexane layer was quickly decanted and following evaporation the free fatty acids were methylated with ethereal diazomethane. Following evaporation, the fatty acid methyl esters were resuspended in hexane and analyzed by gas chromatography with a DB23 capillary column (0.25 m m x 30 m) (J&W Chromatography,

32

JANUARY 1991 VOL. 41 NO. 1

PROSTAGLANDINS Foisom, CA) with hydrogen as the carrier gas. The methyl ester of pentadecanoic acid was added to each sample following phospholipid separation by TLC. The fatty acid composition of the diets were determined using the same methods with the following exceptions: (1) pentadecanoate methyl ester was added prior to the extraction procedure, and (2) the TLC separation step was omitted. Statistical analysis: The leukotriene data from all the dietary groups were statistically evaluated by Duncan's Protected Least Significant Difference. Significance was determined at p < .05. RESULTS The n-3 PUFA from the fish oil diets were very effective in altering the membrane phospholipid (PL) fatty acid compositions of the murine peritoneal cells (Table 3). The long-chain n-3 PUFA levels in the PL progressively increased with increasing fish oil consumption. Eicosapentaenoic acid (EPA, 20:5 n-3), docosapentaenoic acid (22:5 n-3) and docosahexaenoic acid (22:6 n-3) accounted for all of the changes in the n-3 polyunsaturated fatty acids. The cell yields in the peritoneal exudates were not different between any of the dietary groups with an average cell yield of 2.79 x 106 cells, which is consistent with what has been reported elsewhere (17). The recovery of PGB 1 from the peritoneal cavity was determined to be approximately 60% with no differences between dietary groups. Table 3 THE FATTY ACID COMPOSITION OF THE PHOSPHOLIPIDS OF MOUSE PERITONEAL CELLS (IN VIVQ) AND RESIDENT PERITONEAL MACROPHAGES (IN VITRO) DIETARY GROUPS In Vivo Fatty Acid

1

2

In Vitro 3

4

1

4

(tool %) 14:0 16:0 16:1 18:0 18:1 18:2n-6 20:3n-9 20:3 n-6 20:4n-6 20:5n-3 22:4 n-6 22:5n-6 22:5n-3 22:6n-3

1.05+0.08 0.85+0.08 0.96+_0.11 30.59 +_ 1.25 26.71 +_ 0.59 26.17 +- 0.79 2.15+-0.15 3.63+_0.50 3.90+_0.35 14.75+_0.24 14.54+_0.25 15.64+0.52 16.45+_0.45 26.59+_1.0923.16+_1.69 10.36+0.57 8.33+_0.69 9.68+-0.53 1.17+0.04 0,57+_0.04 0.44+_0,08 1.68 +_0.06 1,27 +_0.06 0.90 +_0.09 13.92+-0.90 6.35+_0.22 5.16+_0.44 1.73+-0.08 2.66+_0.14 4.43 +_0.35 0,90 +_ 0.03 0.43 +_0,06 1.86+_0.11 0.07+_0.02 0.03+_0.02 0.36+_0.04 3.24+_0.09 5.31+_1,21 1.21 +_0.08 5.21 +_0.15 5.46+_0,36

1.12+0.10 1.52+0.35 2.20+_0.31 32.44 + 1.13 34.61 +_ 1.10 34.21 +_ 1.30 2.42+-0.25 0.28+_.0.16 0.96+_0.22 16.03+0.25 18.92+0.66 20.88+_0.27 15.83+0.37 15.86+1.18 14.95+0.42 7.52+_0.35 6.74+0.59 5.96+0.22 0.32+_0.07 0.72+_0.21 0.28+_0.15 0.75 +0.05 1.66 +_0.09 0.65 +_ 0.24 6.10+-0.42 11.47+-0.51 6.35+_0.15 4.07+_0.14 1.45+-0.08 0.46 +_0.05 4.38 +_0.29 0.56 +_ 0.21 0.10+_0.04 1.52+_0.14 5.84+_0.31 1.08+0.19 6.53+-0.62 6.92 +_0.19 1.25 +_0.17 5.01+_0.41

Results are mean +_SEM of 8 experimental values.

JANUARYl~1VOL.

41NO. 1

PROSTAGLANDINS

n6 DIET

a°

LTC4

I

I

I

I

I

b.

PGBI

I

LTE4

I

lit

1

I

I

I

I

I

I

I

I

I

I

I

i

I

I

I

n3 DIET

I

Ia

Ib

I

I

~o

I

I

lib

I

LEUKOTRIENE STANDARDS

C.

LTC=

I

I

INJECT 2

LTC,, PGB. LTE.

LTD.

LTE=

I

I

I

1

I

I

I

I

I

I

I

I

I

4

6

8

I0

12

14

16

18

20

22

24

26

28

TIME (rain) Fig. i RP-HPLC chromatograms of leukotrienes synthesized in vivo by murine peritoneal cells stimulated with opsonized zymosan in vivQ using animals maintained on diets without (a) or with (b) fish oil. Leukotriene standards, along with the internal standard PGB l, are shown in (c). Conditions for RP-HPLC: Whatman partisphere C-18 column (6 m m x 12.5 cm); mobile phase, methanol, water (65:35, v/v) containing 5 mM ammonium acetate and 1 mM EDTA, adjusted to pH 5.6 with glacial acetic acid; flow rate, 0.9 ml/min.

34

JANUARY 1991 VOL. 41 NO. 1

PROSTAGLANDINS Leukotriene formation in vivo: LTC4, LTE 4 and their 11-trans isomers (when observed) were the only SP-LT identified in viv9 from the stimulated peritoneal contents of mice maintained on the control (fish. oil-free) diet (Fig. la). Four additional peaks were observed, compounds Ia, Ib, lla and lib, during HPLC analysis of the peritoneal contents from mice consuming the fish oil diets (Fig. lb). Compounds la, lb, IIa and lib contained U.V. absorption spectra similar to the 4-series SP-LT, indicating the presence of conjugated trienes (21). The U.V. absorption spectra of compound la and IIa were very similar and possessed ~max at 280 nm (Fig. 2a and 2b). When compared to the retention times of the leukotriene standards on RPHPLC (Fig. lc), compound Ia corresponded to LTC 5, while compound lla possessed a similar retention time as that of LTE s. Compounds Ia and IIa were identified as LTC 5 and LTEs, respectively. The U.V. absorption maxima of compounds Ib and lib were identical with Arnax at 278 nm (Fig. 2). Compounds lb and lib appear to be the 1 l-trans isomers of compounds la and lla, as there was a 2 nm hypochromic shift associated with the U.V. spectral maxima along with slight increases in the RP-HPLC retention times (21).

70~ o C

Z;'Onm-...._.._ ~ \~ COMPOUND

2eonm

//

a. \\'l

,Pv

240

260 200 Nivilenith

LO0

b.

COMPOUNO

27Ohm ~

o 8B 4,

:,.1,, _

30Q (.m) ~

oo..oo.oI,
'-L

%

240

340

20Ohm

~- _1"4

220

320

\

260 NeVl

280 IInIih

(rim)

3OB

320

340

Fig. 2 U.V. absorption spectra of compounds la and Ib (a), and compounds lla and llb (b) (see Fig. 3b).

JANUARY 1991 VOL. 41 NO. 1

35

PROSTAGLANDINS

2

4

S (mln.)

Time

8

10

12

b.

1.O1"51~_.~ ~ 0.5 0.0 . . . . . . . . . . . . . 2 C.

'81

.~ Time

I

o,JJ

6

4

"'

" ....... 8

,

10

12

(mln.)

Authentic

LTF 5

2

~

4 6 Time (min.)

8

10

d.

,

2

~1

I/

4 Time

6 (min.)

.

.

.

,

8

10

8

IO

LTCs

IE

2

4

Time

S

(mln.)

Fig. 3 RP-HPLC chromatograms of (a) compound IIa prior to incubation with G G T P (b), the incubation of compound IIa with GGTP, (c) the reaction products of authentic LTE s and GGTP, (d) the reaction products of authentic LTC s with carboxypeptidase A and (e) authentic LTC 6. Conditions for HPLC: see Fig. 1.

36

J A N U A R Y 1991 VOL. 41 NO. 1

PROSTAGLANDINS

Following HPLC separation, compound lla was incubated in the presence of GGTP (Fig. 3b). The reaction products were subjected to RP-HPLC and resulted in a more polar compound (compound III) whose retention time was slightly later than authentic LTC s (Fig. 3e), and corresponded to the LTF 6 standard produced following the incubation of authentic LTE 5 with GGTP (Fig. 3c) and authentic LTC s with carboxypeptidase A (Fig. 3d). (When the RP-HPLC conditions were changed, LTF s and LTC s were clearly delineated.) The U.V. absorption spectrum of compound III indicated a conjugated triene system similar to that found with other SP-LT with a >'max at 280 nm, suggesting an l l - c i s isomer. This evidence indicates that compound III is LTF 5 and was derived from LTE s by transpeptidation (19). Th~ ¢ffe~:ts of digtary fish oil on LTE s formation in vivo: The leukotrienes E accounted for 80-90% of the total SP-LT produced by the mouse peritoneal cells in viv0 (Fig. 4). When EPA was absent from the diet, no detectable levels of EPA were found in the tissue phospholipids and LTE~ was not produced following stimulation. However, as n-3 PUFA replaced n-6 PUFA in the tissue phospholipids, the 4-series LT levels decreased as the 5-series LT became the dominant SP-LT formed (Fig. 4). In animals maintained on the highest fish oil diet, LTE 5 accounted for more than 50% of all the SP-LT formed, 4- and 5-series combined. The leukotrienes C made only minor contributions to the total SP-LT pool, but the effects of dietary fish oil on LTC 4 and LTC 5 formation was very similar to that observed for the leukotrienes E (Fig. 4, inset).

100 (D

:a

_-

_ LTE4

-~

LTEs

8 --

9O 0 .i-I

80

• ,,.

70

~" ~0

60

'~

50

ILl

40

¢~ I=

30

•~

20

0

10

o

0

LTC5

-

\T \

O.00 ~.

o.oo

au

!4

\

/

[

0.40

0.60

O.BO

1.O£

Dietary n 3 / n 6 ratio

I

0.20

0.20

ab

~.

I

0.40

Dietary Fig. 4

- - - - --/TO___.

~,a \ \\ \ ab

"" ~

o

12

I

0.80

n3/n6

0.80

1.00

ratio

The effect of varying the n - 3 / n - 6 polyunsaturated fatty acid ratio on murine peritoneal cell leukotriene formation in vivo. Results are mean +_SEM. Intergroup comparisons with different letters are statistically different at p < .05.

JANUARY

1991 V O L . 41 N O . 1

37

PROSTAGLANDINS

50 O

40

N

30

Z

20

O

10

D ;a O LTC 4

LTC 5 Diet

1

LTC 4

LTC 5 Diet 4

Fig. 5 Leukotriene C 4 and C s production in vitro by resident peritoneal macrophage following stimulation with A23187 from mice maintained on the control diet (diet l) or a diet containing fish oil (diet 4). Results are mean +_SEM of eight experimental values. s Significantly different from the control group (diet 1) at p < .05. Leukotriene formation in vitro: LTC 4 and LTC s were the only SP-LT produced in vitro from isolated murine peritoneal macrophages following stimulation with A23187. Macrophage from animals fed the control diet produced 42.0_+10.8 ng/10 a cells of LTC4, while LTC s production was undetectable (Fig. 5). However, in stimulated macrophage obtained from animals fed the highest fish oil diet (diet 4), LTC 4 production dropped 60% to 16.7_+6.2 ng/10 e cells with only minimal amounts of LTC 6 detected (2.1+0.9 ng/106 cells). DISCUSSION Dietary fish oils tend to reduce the biosynthesis of 4-series LT and may result in the generation of 5-series LT, in particular LTB 5 (13,18,22-24). LTB 5 has received increasing attention because it is less biologically active than LTB 4 (25,26). However, little attention has been focused on the pentaenyi SP-LT. This is surprising considering that the 4- and 5-series SP-LT have been shown to be equipotent physiological mediators (6,10,12). Several investigators have reported in vitro production of SP-LT derived from exogenous sources of EPA (9-11 ), with only one report of 5-series SP-LT formation in vivo (12). However, analyses of the SP-LT were limited by incomplete HPLC resolution and constraints associated with radioimmunological assays (12).

38

JANUARY 1991 VOL. 41 NO. 1

PROSTAGLANDINS In a previous study, murine peritoneal cells were shown to be effective producers of LTE 4 in vivQ and that adding fish oil to the diet significantly reduced production; however, 5-series SP-LT formation was not reported (13). Similar results were observed with the production of LTC 4 in vitro from isolated murine peritoneal macrophages stimulated with the calcium ionophore A23187 (18,23). In this study, changing the RP-HPLC conditions resulted in better HPLC resolution and improved separation of products, thus allowing for the identification and quantitation of LTC s and LTE s. Leukotriene E s production in viv0 was confirmed by spectrophotometric, chromatographic and enzymatic analytical methods. In those animals fed a diet containing fish oil, a peak (compound IIa) corresponding to the retention time of authentic LTE 5 was present. U.V. spectral analysis revealed a conjugated triene chromophore with a )~max at 280 nm. The identification of the I I-trans isomer lends added support to LTE 5 formation. Additionally, animals fed diets which were devoid of fish oil, produced no peaks corresponding to compound IIa or authentic LTE s. Compounds Ia, Ib and llb were also absent from the stimulated peritoneal contents of animals fed the control diet. It has been demonstrated that LTF 4 can be produced by the action of G G T P on LTE 4 (19,27). Therefore, the identity of LTE 5 was confirmed when compound IIa was incubated with G G T P in the presence of reduced glutathione. This reaction resulted in the formation of a compound (compound III) possessing similar U.V. spectral characteristics as LTE s. Compound III was identified as LTF s by comparing its RP-HPLC retention time, which is slightly later than that of LTC6, with that of LTF s standard. Since LTF s standard was not commercially available it was independently synthesized from authentic LTE s and LTC 5 by the actions of G G T P and carboxypeptidase A, respectively (19,20,27). U.V. absorption spectral analysis indicated that compound III (Amnx, 280 nm) was not the l 1-trans isomer of LTC 5. It is important to note that the reaction involving G G T P had to be modified because of significant dipeptidase contamination in the commercially available G G T P preparation. If the reaction was allowed to proceed for 15 rain any glutamate added to LTE, as catalyzed by GGTP, was subsequently removed by the dipeptidase contaminant, regenerating LTE. Therefore, all reactions were run for no longer than 2 min in an attempt to maximize yields. Conversion of LTE 4 and LTE s standards to their respective LTF products have been as high as 30% (Fig. 3c). As the amount of dietary fish oil increased, LTE s formation increased and was greater than LTE 4 in animals consuming equivalent amounts of n-3 and n-6 PUFA (diet 4). In addition, the LTE~/LTE 4 ratio was highly correlated (r > 0.99) with the dietary n - 3 / n - 6 PUFA ratio, indicating the efficacy of dietary fish oil in altering the LT profile in vivo. Eicosapentaenoic acid has been shown to be an excellent substrate for the 5-1ipoxygenase purified from leukocytes of various species (14,15). Our data suggests that, at least in the mouse peritoneal system, EPA is effectively utilized in viv9 by that portion of the arachidonic acid cascade involved in SP-LT biosynthesis. Juan et al. (10) and Simmit et al. (1 I) have reported similar findings using isolated anaphylactic heart of guinea pig perfused with EPA and guinea pig lung parenchymal strips incubated with EPA. However, our data also indicate that fish oils, in general, have an inhibitory influence on the LT pathway. Total SP-LT production in vivo (4-series ÷ 5-series) was reduced by 36% when comparing diet 1 to diet 4, 93.8 ng/10 a cells vs. 60.3 ng/106 cells. We investigated whether resident peritoneal macrophages were responsible for LTE s formation from endogenous sources of EPA. However, following the isolation of the resident macrophages and stimulation with the calcium ionophore A23187, only the leukotrienes C were

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39

PROSTAGLANDINS produced. Those macrophage from animals fed the control diet produced only LTC4, whereas LTD 4 and LTE 4 were not detected. Similar results have been reported with cultured mouse peritoneal macrophage incubated with the phagocytic stimuli zymosan (28). The only SP-LT produced by macrophage from animals on the high fish oil diet (diet 4) were LTC 4 and LTC s (excluding their l l-trans isomers). Since it has been reported that murine peritoneal macrophage do not possess GGTP, this probably accounts for their inability to further metabolize leukotrienes C (29). Additionally, in vitro stimulation of peritoneal cell exudates with A23187 or zymosan produced only LTC 4 (17). Other cells or tissues associated with the peritoneum may be responsible for the production of leukotrienes E or are involved in the further metabolism of macrophage-derived leukotrienes C. The amount of LTC s produced in_ vitro by macrophage isolated from animals consuming the high fish oil diet (diet 4) was low, but was most likely the result of alterations in the macrophage phospholipid fatty acid composition of the cells following incubation. The EPA content of the "in vitro" cells isolated from animals fed diet 4 was significantly lower than the content observed in the "in vivo" cells from animals fed the same diet, 1.45 mol% vs. 4.05 mol% (Table 3). Also, the EPA phospholipid content of the peritoneal cells in vivo was highly correlated (r > 0.99) with LTC s production. Based on the equation generated from the linear regression analysis, the predicted level of LTC s production in vitro (1.98 ng/I 0 s cells) from the diet 4 animals was virtually identical to the observed value (2.01 n g / i 0 e cells), indicating that the low phospholipid EPA content was probably responsible for the LTC 6 levels. A follow-up experiment using similar animals and diets of identical composition produced the same phospholipid fatty acid profile in the peritoneal cells, ruling out possible biological variation to explain the in vitro fatty acid data. It has been shown that EPA is effectively and efficiently elongated to 22:5 n-3 by cultured peritoneal macrophage in vitro and may be partially responsible for the low EPA content (18). In summary, the capacity of murine peritoneal cells to produce the 4-series LT diminished with increasing levels of dietary fish oil along with a progressive and dose-dependent increase in LT derived from EPA. This study presented evidence for significant LTE s production in vivQ and demonstrated that the 5-series SP-LT significantly contributed to the endogenous SPLT pool. Finally, LT formation by resident peritoneal macrophages in vitro did not fully account for LT production in vivo. ACKNOWLEDGEMENT: This work is a result of research sponsored by the NOAA Office of Sea Grant, U.S. Department of Commerce, under Grant # N A 9 0 A A - D - S G 0 7 8 to the New York Sea Grant Institute. The U.S. Government is authorized to produce and distribute reprints for governmental purposes notwithstanding any copyright notation that may appear hereon.

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PROSTAGLANDINS 17)

Doherty, N.S., P. Poubelle, P. Borgeat, T.H. Beaver, G.L. Westrich, and N.L. Schrader. Intraperitoneal injection of zymosan in mice induced pain, inflammation and the synthesis of peptide leukotrienes and PGE 2. Prostaglandins 3__0:769. 1985.

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Lokesh, B.R., B. German, and J.E. Kinsella. Differential effects of docosahexaenoic acid and eicosapentaenoic acid on suppression of lipoxygenase pathway in peritoneal microphages. Biochim. Biophys. Acta 958:99. 1988.

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Lokesh, B.R., J.M. Black, J.B. German, and J.E. Kinsella. Docosahexaenoic acid and other dietary polyunsaturated fatty acids suppress leukotriene synthesis by mouse peritoneal macrophages. Lipids 23:968. 1988.

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Terano, T., J.A. Salmon, and S. Moncada. Effect of orally administered eicosapentaenoic acid (EPA) on the formation of leukotriene B5 by rat leukocytes. Biochem. Pharmacol. 33:3071. 1984.

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Lee, T.H., T. Sethi, A.E. Crea, W. Peters, J.P. Arm, C.E. Hortan, M.J. Walport, and B.W. Spur. Characterization of leukotriene Bs: Comparison of its biological activities with leukotriene B4 and leukotriene B 6 in complement receptor enhancement, lysozyme release and chemotaxis of human neutrophils. Clin. Sci. 7__44:467. 1988.

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Anderson, M.E., R.D. Allison and A. Meister. Interconversion of leukotrienes catalyzed by purified "/-glutamyl transpeptidase: Concomitant formation of leukotriene D 4 and 7glutamyi amino acids. Proc. Natl. Acad. Sci. USA 79:1088. 1982.

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Schade, U.F., H. Moll, and E.T. Rietchel. Metabolism of exogenous arachidonic acid by mouse peritoneal macrophages. Prostaglandins 34:401. 1987.

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Abe, M., and T.E. Hugli. Characterization of leukotriene C 4 synthase in mouse peritoneal exudate cells. Biochim. Biophys. Acta. 95__?:386. 1988. Editor:

42

L.J.

Roberts

Received:

2-22-90

Accepted:

10-3-90

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