Leukotriene A4: Enzymatic conversion to leukotriene C4

Vol.

96, No.

October

4, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

31, 1980

LEUKOTRIENE A4:

1679 - 1687

ENZYMATIC CONVERSION

TO LEUKOTRIENE C4 Olof

Radmark,

Department

Curt

September

and Bengt

of Chemistry, S-104

Received

Malmsten

Samuelsson

Karolinska

01 Stockholm,

Institutet,

Sweden

5,198O

SUMMARY: An unstable epoxide, leukotriene A4 (5(S)-trans-5,6-oxido-7,9-1,14-cis-eicosatetraenoic acid), was earlier proposed to be an intermte in theconversion of arachidonic acid into the slow reacting substance (SRS), leukotriene C4. In the present work synthetic leukotriene A4 was incubated with human leukocytes or murine mastocytoma cells. A lipoxygenase inhibitor, BW755C, was added in order to prevent leukotriene formation from endogenous substrate. Leukotriene C4 and 11-trans-leukotriene C4 were the main products with SRS activity. It was not emished whether the 11-trans-compound was formed by isomerization at the leukotriene A4 or C4 stage. INTRODUCTION Studies cytes

of the metabolism

demonstrated

in which

oxygen

isolation

(2,3).

traenoic

acid

introduced

(leukotriene

considerations

the acid

The structures are

important

mediators

been elucidated

was also

the

was first

precursor reported

(4)..

intermediate

in

leuko-

pathway

resulted

conjugated

Mechanistic

basis

in the

triene

immediate This

chromo-

in 1979

(Fig.

experiments

and

1) (5).

of anaphylaxis reactions

was based

in the forma-

to be 5,6-oxido-7,9,11,14-

hypersensitivity

on the

The structure

(9,lO).

of the oxygen

intermediate

of trapping

substances

work

of SRS-A (8).

studies

was proposed A4, LTA4)

reacting

(7).

peritoneal

catalyzed work

of an unstable

On the

(leukotriene

recently

SRS-A

LTBq)

(5).

of slow

Additional

characteristic

presence

acids

in rabbit

was 5(S),12(R)-dihydroxy-6,8,10,14-eicosate-

64, the

dihydroxy

eicosatetraenoic

with

product

acid

of a new lipoxygenase

at C-5 (1).

acids

indicated

of the

other

is

The main

incorporation tion

the occurrence

of dihydroxy

phores

of arachidonic

surmise

of a naturally

The material

was isolated

(SRS-A), (6),

which

have

that

LTA4

occurring from murine

0006-291X/80/201679-09$01.00/0 1679

Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

96, No.

4, 1980

mastocytoma

BIOCHEMICAL

cells

AND

and was found

cysteinylglycine same time from

acid

and properties

(10).

various

Both

sources

(13,14,15),

stereochemistry

11,14-cis-eicosatetraenoic -

acid)

LTA4 from thetic

of synthetic

LTA4 into

pig

compounds

described

at the

been identified

cells

(11,12),

in SRS-A

rat

monocytes

(15,16).

was established

by demonstrating

LTB4 and by isolation

(17,18). with

lung

The

of LTA4 (5(S)-trans-5,6-oxido-7,9-trans-

LTA4 into

human leukocytes

leukemia

(9,lO).

corresponding

D4, LTD4) was also

basophilic

the

conversion

i.e.

LTC4 and LTD4 have subsequently as rat

COMMUNICATIONS

C4, LTC4)

metabolite,

(leukotriene

and human and guinea

Recently,

(leukotriene

of an expected

derivative

RESEARCH

to be 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-

trans-11,14-e-eicosatetraenoic preparation

BIOPHYSICAL

In the

SRS-A activity

present

work

of the

enzymatic highly

the conversion

has been studied

unstable of syn-

in human

leukocytes. MATERIALS

AND METHODS

Synthetic LTA4 (5(S)-trans-5,6-oxido-trans-7,9-cis-ll,l4-eicosatetraenoic acid) was kindly provTZd-by Prof. E.J.rey andcolleagues at Harvard University, Cambridge, Mass., USA. BW755C (3-amino-l-(m-trifluoromethyl)phenyl)-2-pyrazoline), was a gift from Dr. F. Kuehl, Merck Institute for Therepeutic Research, Rahway, New Jersey, USA. FPL 55712 was kindly supplied by Fisons Limited, Lougborough, England. Dextran T-500 was purchased from Pharmacia Fine Chemicals, Uppsala, Sweden, and Lymphoprep from Nyegaard & Co., As., Oslo, Norway. Lipoxygenase, type I, was supplied by Sigma Chemicals, co., St. Louis, MO., USA. CXBG mice and mouse mast cell tumor (CXBGABMCl-), were kindly provided by Dr. Hans Bennich, Uppsala, Sweden. Preparation of cell suspensions, human polymorphonuclear leukocytes. Concentrates of human leukocytes were obtained from blood component preparation at the Karolinska Hospital, Stockholm, Sweden. PMNL-suspensions were prepared, essentially as outlined in ref. 8. Using the leukocyte concentrate, the initial centrifugation, to remove platelet rich plasma could be omitted. Instead the cells were directly mixed with dextran in 0.9% sodium chloride and allowed to sediment at t4oC. The sedimented white cells were collected, centrifuged at 250 g and resuspended in a buffered ammonium chloride solution, to induce lysis of remaining red cells. After lysis at 370C for 7 min, Lymphoprep was carefully added to the tubes, and these were spun at 400 G for 40 min. The pellets consisting of neutrophil and some eosinophil granulocytes (8) were resuspended in Dulbecco's PBS, pH 7.4 (30 x 106 cells/ml) and incubated immediately in batches of 100 ml. This preparation is referred to as human PMNL. For large scale incubations the Lymphoprep-sedimentation was omitted, and the whole leukocyte fraction incubated. Murine mastocytoma cells. A suspension of these tumor cells were previously described (9). A total of 6 x 109 cells were suspended of buffer, (10 x 106 cells/ml) for incubation.

1680

prepared as in 600 ml

Vol.

96, No.

4, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Incubations. The cell suspension, human or murine, was prewarmed to 370C. mvug/ml) was incubated for five minutes, before addition of LTA4 (19). LTA4 was added to a final concentration of 0.3-2.0 ug/ml and incubated for ten minutes (human PMNL), or 0.3 ug/ml, 20 min (murine cells). Four volumes of ethanol were added to stop the incubations. Bioassay. The guinea in ref. 9.

pig

ileum

bioassay

for

SRS was performed

as outlined

Isolation and purification. The procedure outlined in ref. 9 was followed. Thde ce by filtration and the filtrate was saponified at pH 11, for 30 min at 370C. Following acidification (pH 3.5), the polar lipids were extracted using Amberlite XAD-8 resin (Rohm & Haas). The extract was further purified by chromatography on silicic acid (Silicar CC-7, Mallinkrodt, St. Louis, MO., USA) This column was eluted first with ethylacetate/ methanol, 90/10, v/v, followed by methanol, 100%. The methanol fraction, that contained the SRS-compounds was subjected to reverse phase HPLC. A semipreparative scale column (Whatman Partisil M9, Clifton, New Jersey) was eluted at 4 ml/min with methanol/water/acetic acid, 70/30/0.1, v/v/v, the pH of which was adjusted to 5.1 with ammonium hydroxide. Final purification was achieved by a second HPLC step on an analytical scale column (Nucleosil C18, 5 pm spherical particles, Macherey-Duren, West-Germany). This was eluted with methanol/water/acetic acid, 60/40/0.1, v/v/v, pH 5.1, at 1 ml/min. For HPLC a Waters 6000 A pump was used together with a Waters U6K injector (Waters Associates, Milford, Mass., USA) and a UV-detector (LDC-III, Riviera Beach, Florida, USA). The UV-detector was set at 280 nm. Lipoxygenase conversion. The substrate (2 nmoles) was dissolved in 1 ml of Tyrodes buffer, 100 ug lipoxygenase (Sigma type I) was added, and the spectral shift recorded with a Cary 219 instrument. Amino acid analysis. Samples (5 nmoles) were hydrolyzed with 6 M hydrochloric acld/O.S% phenol, in evacuated ampoules for 20 hours, at 1lOoC. The amino acid contents were analyzed on a Beckman 121 M instrument. Raney nickel desulfurization, GC-MS. Samples (10 nmoles), were refluxed in 11 et an0 wit Raney nickel (W-2) for 30 min. Following methylation and silylation, the sample was injected onto a OV-101 column, in an LDB 9000 mass spectrometer. The energy of the ionization beam was 22.5

RESULTS Human polymorphonuclear ted

LTA4 into

two compounds

in the

reverse

absent

in control

an analytical triene obtained

phase

with

incubations

column,

with

compound

arachidonate

2 with

and murine

SRS properties,

HPLC chromatogram

C4, and compound from

leukocytes

compounds

in Fig.

boiled

mastocytoma

2. These

cells.

In following

incubations

(Fig.

of murine

1681

3).

conver-

1 and 2, as shown

compounds

1 was shown to co-chromatograph 11-trans-LTC4

cells,

were

totally

HPLC-runs,

on

with

leuko-

The standards

were

mastocytoma

cells

(9).

The

eV.

Vol.

96, No.

4, 1980

BIOCHEMICAL

ea

AND

BIOPHYSICAL

Arachidonic

hydiolysis

RESEARCH

COMMUNICATIONS

acid

H-mm

-

l Glutathiono /

CompounQ Ip 8nd P ‘4 Fig.

1. Metabolism morphonuclear of conjugated

Fig.

2.

of

arachidonic leukocytes. triene has

acid to the leukotrienes Asterisk indicates that not been determined.

in human configuration

poly-

Reverse phase HPLC chromatogram of products obtained from LTAqincubation of human PMNL. The incubation was stopped after ten minutes with ethanol, saponified and extracted with XAD-B-resin. The extract was purified by silicic acid chromatography. The fraction active on the guinea pig ileum bioassay was evaporated and injected onto the HPLC-column (Whatman. Partisil). This was eluted with methanol/water/acetic acid, 70/30/0.1, pH 5.1, at 4 ml/min. UV-detection, 280 nm.

1682

Vol. 96, No. 4, 1980

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

1H-truns-LTC‘.

-i -L 40

30

Fig.

20

W-spectra

of the are

These

the

compound

I was shifted data

in the

bility

as substrates

pounds

1 and 2, on the

with

for LTC4,

earlier

lipoxygenase.

the guinea the

pig

ileum

compound for

the structures

acid

produced

lo

Om

for

of compound

2 was slightly

which

contents

of com-

2 was unaffec-

determines

their

activities

determined.

potent.

the This

suitaof com-

Contractions,

The dose

was practically

that

is

response same as for in.agreement

(20). of compounds

of LTC4 (10)

1683

were

less

ll-trans-LTC4

maxima

LTC4 and ll-trans-LTC4,

SRS-pattern.

bioassay,

with

li-

buffer.

2. The spectrum

The contractile strip

by soybean

in Tyrodes

triplet,

that

bond,

characteristic

I in the

of the amino with

those

for

compound while

with

of A" -double

observations

Analyses accordance

P

recorded

compound

while

geometry

by FPL, showed

relationship

in these

leukotriene

lipoxygenase,

in accordance

only

standard

30

were

characteristic

by the

are

shifts

4. The spectra

1, and at 278 nm for

differ

inhibited

u,

and the

in Fig.

showed

at 280 nm for

ted.

compounds,

shown

compounds

pound

hill

3. Cochromatography of compounds 1 and 2 with LTC4 and ll-transLTCq respectively on reverse phase HPLC. The samples anmndards were purified individoal7y, hereafter combined in equal amounts and cochromatographed. Prostaglandin B2 (PGB2) was used as reference. An analytical scale column was eluted with methanol/water/ acetic acid, 60/40/0.1, ptl 5.1, 1 ml/min. UV-detection 280 nm.

poxygenase Both

lo

1 and 2 were

and 11-trans-LTC4

(20)

both

in

respectively.

Vol.

96, No.

4, 1980

BIOCHEMICAL

A

AND

BIOPHYSICAL

pemil

Canpandl

k+i

Fig.

4.

the

results

of

equal

amounts

Raney

tion

nickel

showed

(M-101,

loss

260

300

these of

nickel

indicated

cystein,

glycin

with

ions

of

a C-value

at m/e 399 (M-15),

5.

3@oml

acid,

1 is

both

glutamic

compounds acid

1 and 2 both

1 and

(data

not

resulted

2 shown).

in forma-

(OV-101).

The mass spectrum

of this

383 (M-31),

367 (M-CH3CH30H),

313

and a base peak at 203 (MeSiO@=CH(CH2)3 prepared

In Fig,

and mass spectrum. of compound

that

compounds

by hydrogenation

of 5-HETE,

5 the mass spectrum

of the

shown.

200 Fig.

XXI

and

of 21.8

of .CH2(CH2)2COOCH3),

same C-value product

260

analyses

desulfurization

COOCH3). 5-Hydroxyarachidic the

Mml

UV-spectra and shifts in these produced by soybean lipoxygenase, of compounds 1 and 2. The sample was dissolved in Tyrodes buffer and a UV-spectrum recorded. Lipoxygenase, 100 ug, was added, and UV-spectra recorded repetetively. The shift in the spectrum of compound 1 was complete after 12 minutes, while the spectrum of compound 2 was unaffected after 50 min.

of a compound

compound

conpand2

t=l2 min

iI

contained

COMMUNICATIONS

aoml t=Omin

Thus,

RESEARCH

300

Mass spectrum of product obtained zation of compound 1. The C-value 101).

1684

ml vc

after Raney nickel desulfurifor the product was 21.8 (OV-

gave Raney

Vol.

96, No.

BIOCHEMICAL

4, 1980

The yield ranged

from

of compounds 5 to lo%,

of compound

human PMNL the amount from

the

graphy

with

resulting

acid

ted

was quantified

and reverse

murine

less

polar

phase by its

that

mastocytoma

from

RESEARCH

for

9:l

with

from

silicic

the

was purified

The yield

The ratio of

The water-phase ether.

This

acid

extract

chromatoon sili-

8. The LTB4 thus of LTB4 in this

of LTC4 and 11-trans-LTC4

was 6%. In the

cells

of compounds

LTA4 the yield

LTA4

by chromatography

in ref.

UV-absorption.

with

compounds.

estimated.

PHLC as outlined

with

COMMUNICATIONS

In one incubation

and extracted

fraction that

both

to 4:6.

was also

was acidified

in an LTB-pool

tic

was 5%, while

an E of 40,000

of LTB4 formed

the

BIOPHYSICAL

2 in human PMNL incubated

2 varied

XAD-extraction

was combined

1 plus

assuming

1 to compound

AND

isolaincubation

incubation 1 plus

of

2 was 10%.

DISCUSSION In this epoxy-acid

study

we have demonstrated

leukotriene

A4 to the

11-trans-LTC4,

enzymatic

slow

reacting

in human polymorphonuclear

conversion substances

leukocytes

of the

unstable

leukotriene

and murine

C4 and

mastocytoma

cells. The structures son with

standards

chromatography tained.

of the two isolated and also

glutamic

acid

spectra

analysis

in both

for

in the

acid

indicated

with

that

was excluded

incubations

Furthermore

19).

to be an enzymatic

process

boiled

cells.

the earlier

mediate

not

Thus only

in the

the were

products

followed

glycin

obtained

in accordance

by gasacid

of cysteine, shifts

were

derived

from

oband

in the UV-

with

endogenous

of a lipoxygenase

The transformation

by the absence proposal

biosynthesis

by compari-

earlier

ob-

(9,10,20).

by the presence

(c.f.

determined

5-hydroxy-eicosatetraenoic

lipoxygenase,

the

were

desulfurization,

the presence

LTC4 and 11-trans-LTC4

The possibility donic

of the

compounds.

upon treatment

servations

by Raney nickel

mass spectrometry

Amino acid

compounds

1685

BW755C,

of LTA4 to LTC4 was assured

(5,9,10),

firmed.

inhibitor,

of LTC4 in control

of LTB4,

arachiJ

but

incubations

of

that

LTA4 is

an inter-

also

of LTC4 has been con-

Vol.

96, No.

BIOCHEMICAL

4, 1980

An isomer in the

of LTC4,

incubations.

nonenzymatic tion

It

is

Quantitative pared

with

about

equimolar

fluencing

conceivable

procedure. place studies

LTB4 indicated

that

the

on the

formation

the

Additional

COMMUNICATIONS

obtained

together isomer

with

is

structure

wheather

incuba the

of glutathione.

of compounds in progress

of LTA4 by hydrolysis

by

the

of LTC4 and ll-trans-LTC4

is

LTC4

formed

during

been established

addition

two types work

RESEARCH

ll-trans

triene

has not

or after

that

amounts.

It

before

the transformation

transferase

was always

of the conjugated

and purification takes

BIOPHYSICAL

ll-trans-LTC4,

isomerization

isomerization

AND

were

as com-

formed

in

to study

factors

g lutath

ione-S-

versus

in-

reaction. ACKNOWLEDGEMENTS

We wish excellent

to thank

technical

and MS Carina This

study

(project

MS Inger assistance.

Palmberg

was supported

for

very

Tollman-Hansson We are also valuable

by grants

from

help

and MS Ulla grateful with

the Swedish

Andersson

to Dr. the

amino

Medical

for

Hans Jijrnvall acid

analyses.

Research

Council

03X-217). REFERENCES

1. Borgeat, P., Hamberg, M. and Samuelsson, B. (1976) J. Biol. Chem. -'251 7816-7820. Correction (1977) 252, 8772. 2. Borgeat, P. and Samuelsson, B.1979) J. Biol. Chem. 254, 2643-2646. 3. Borgeat, P. and Samuelsson, B. (1979) J. Biol. Chem. m, 7865-7869. 4. Samuelsson, B. and Hammarstrbm, S. (1980) Prostaglandx, 79, 645-648. 5. Borgeat, P. and Samuelsson, B. (1979) Proc. Natl. Acad. Sci. USA, 76, 3213-3217. 6. Austen, K. (1978) J. Immunol. 121, 793-805. 7. Samuelsson, B. (1980) Trends Pharm. Sci., May issue, 227-230. 8. Borgeat, P. and Samuelsson, 8. (1979) Proc. Natl. Acad. Sci. USA, 76, 2148-2152. 9. Murphy, R.C., Hammarstrcim, S. and Samuelsson, 6. (1979) Proc. Natl. Acad. Sci. USA, 76, 4275-4279. 10. Hammarstroz S., Murphy, R.C., Samuelsson, B., Clark, D.A., Mioskowski, C. and Corey, E.J. (1979) Biochem. Biophys. Res. Commun. 91, 1266-1272. S. and Samuelsson, B. (1980) Got. Natl. Acad. 11. Urning, L., Hammarstrom, Sci. USA, 77, 2014-2017. 12. Morris, H.K, Taylor, G.W., Piper, P.J., Samhoun, M.N. and Tippins, J.R. (1980) Prostaglandins, 19, 185-201. 13. Bach, M.K., Brashler, Jx., Hammarstrijm, S. and Samuelsson, B. (1980) J. Immunol. 125. 115-117 14. Bach, M.K., Brashler, J.R., Harmnarstrijm, S. and Samuelsson, B. (1980) Biochem. Biophys. Res. Commun. 93, 1121-1126.

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AND

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15. Lewis, R.A., Austen, K.F., Drazen, J.M., Clark, D.A., Marfat, A. and Corey, E.J. (1980) Proc. Natl. Acad. Sci. 77, 3710-3714. 16. Morris, H.R., Taylor, G.W., Piper, P.J. anhiippins, J.R. (1980) Nature, 285, 104-106. 77. Core , E.J., Clark, D.A., Goto, G., Marfat, A.. Mioskowski, C., Samuelsson, B. and HammarstrGm, S. (1980) J. AM. Chem. Sot. 102, 1436-1439. 18. Ridmark, O., Malmsten, C., Samuelsson, B., Clark, D.A., Goto, G., Marfat, A. and Corey, E.J. (1980) Biochem. Biophys. Res. Commun. 92, 954-961. 19. Radmark, O., Malmsten, C. and Samuelsson, B. (1980) FEBS 'IIE;tt. -'110 213-215. 20. Clark, D.A., Goto, G., Marfat, A., Corey, E-J., Hammarstrijm, S. and Samuelsson, B. (1980) Biochem. Biophys. Res. Commun. -94, 1133-1139.

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