The Discovery of the Leukotrienes

BENGT SAMUELSSON

Department of Medical Biochemistry and Biophysics, Division of Chemistry II, Karolinska Institutet, Stockholm, Sweden

	INTRODUCTION

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In a study of the transformation of polyunsaturated fatty acids in rabbit polymorphonuclear leukocytes in 1976 it was discovered that arachidonic acid is oxygenated at C-5 (1). Subsequently a number of derivatives, including leukotriene B₄ (LTB₄), were identified (2). Extension of these studies led in 1979 to the discovery of the pivot epoxide intermediate, LTA₄ (5), and the elucidation of the structure of SRS-A (slow-reacting substance of anaphylaxis) as a group of cysteinyl-leukotrienes, namely LTC₄, LTD₄, and LTE₄ (6).

Earlier work had shown that the prostaglandins are formed by oxygenation and further transformation of arachidonic acid and other polyunsaturated fatty acids (11). The first intermediate in the formation of prostaglandins, the endoperoxide PGG₂, was isolated and identified in 1974 (12). At the same time we introduced the name cyclooxygenase for the enzyme catalyzing the transformation of arachidonic acid into PGG₂ (12). However, the endoperoxides, PGG₂ and PGH₂, had some biological effects that could not be explained by their conversion to the known prostaglandins (12). This finding eventually led to the discovery of thromboxane A₂, an unstable platelet-aggregating and vasoconstrictor substance (13). Subsequent work showed that the endoperoxide can also be converted into a derivative, prostacyclin (PGI₂), with opposite biological effects (14). Prostaglandins E₂ and I₂ have strong proinflammatory effects.

Aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs) inhibit the enzyme (cyclooxygenase) responsible for conversion of arachidonic acid into prostaglandins and thromboxanes (15). An induced form of the cyclooxygenase (COX-2) seems to play an important role in inflammation, thus opening the possibility of developing antiinflammatory NSAIDs that lack the side effects of the previous generation of such drugs (16, 17).

In 1975 antiinflammatory steroids were proposed to inhibit prostaglandin formation by blocking of the release of the precursor acid from phospholipid stores (18). Since steroids and NSAIDs have significantly different antiinflammatory effects it seemed conceivable that some of these differences might be explained by the formation of proinflammatory derivatives of arachidonic acid by cyclooxygenase-independent reactions. Our studies of the metabolism of arachidonic acid in leukocytes unraveled a new metabolic pathway and led to the discovery of the leukotrienes (8). These compounds play a role in allergy and asthma and have pronounced proinflammatory effects (19, 20).

	DISCOVERY OF THE 5-LIPOXYGENASE PATHWAY AND THE LEUKOTRIENES

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When peritoneal leukocytes from rabbits were incubated with ¹⁴C-labeled arachidonic acid, the major metabolite was found to be a new lipoxygenase product, that is 5(S)-hydroxy-6,8,11, 14-eicosatetraenoic acid (5-HETE) (1). However, more polar products were also formed. Structural studies demonstrated that they consisted of 5(S),12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid (leukotriene B₄, the major product), two additional 5(S),12-dihydroxy-6,8,10-trans-14-cis-eicosatetraenoic acids, epimeric at C-12, and two isomeric 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acids (Figure 1) (2, 4).

View larger version (21K): [in this window] [in a new window]   Figure 1.   Formation of dihydroxy derivatives via unstable intermediates.

The formation of two acids with an all-trans-conjugated triene, epimeric at C-12, and one major isomer [12(R)] with different configuration of the triene, raised the question of the mechanism of formation (4). Oxygen was used to demonstrate that the oxygen of the alcohol group at C-5 originated in molecular oxygen, whereas the oxygen of the alcohol group at C-12 was derived from water (Figure 1) (5). We therefore developed the hypothesis that leukocytes generated an unstable intermediate that would undergo nucleophilic attack by water, alcohols, and other nucleophiles. When leukocytes were incubated for 30 s with arachidonic acid before addition of 10 volumes of methanol, or 0.2 volume of 1 N HCl, we obtained the products shown in Figure 1.

Two new, less polar compounds, present in equal amounts, were formed after trapping with methanol. Their ultraviolet spectra were identical to those of compounds I and II (Figure 1), indicating the presence of three conjugated double bonds. Infrared spectrometry indicated that the conjugated double bonds have trans geometry. Gas chromatographic-mass spectrometric analyses of several derivatives of the two compounds showed that they were isomeric and carried hydroxyl groups at C-5 and methoxy groups at C-12. Steric analyses demonstrated that the alcohol groups had (S) configuration and that the compounds were the C-12 epimers of 5(S)-hydroxy-12-methoxy-6,8,10,14,(E,E,E,Z)-eicosatetraenoic acid (Figure 1).

When ethanol or ethylene glycol was used for trapping, corresponding derivatives were formed. These results showed that a metabolite of arachidonic acid in leukocytes can undergo a facile nucleophilic reaction with alcohols. Analysis of samples obtained from trapping experiments performed under varous conditions always indicated inverse relationships between the amount of the 12-hydroxy- and that of the 12-O-alkyl derivatives. This finding suggested that the 5,12-dihydroxy-6,8,10- trans derivatives were formed nonenzymatically from the same intermediate that gave rise to the 12-O-alkyl derivatives.

The stability of the intermediate was studied by incubating rabbit polymorphonuclear leukocytes with arachidonic acid for 45 s before adding 1 volume of acetone (to stop enzymatic activity). Portions of the mixture were transferred at different time intervals to flasks containing 15 volumes of methanol. The relative amounts of the metabolites were determined by reversed-phase high-pressure liquid chromatography (RP-HPLC). The half-life of the intermediate, measured as the 12-O-methyl derivative at pH 7.4 and 37° C, was 3 to 4 min. Simultaneously with the decrease in the concentration of the intermediate, the concentrations of the 5,12-dihydroxy-6,8, 10-trans-derivatives and the 5,6-dihydroxy-7,9,11-trans- derivatives increased whereas the concentrations of LTB₄ and 5-HETE remained constant. This suggested that the epimeric 5,6- and 5,12-dihydroxy acids (compounds I-V) are formed nonenzymatically by hydrolysis of a common unstable intermediate, whereas LTB₄ arises by enzymatic hydrolysis of the same intermediate (Figure 1). Similar experiments performed at acid and alkaline pH showed that the intermediate was acid labile and stabilized by alkaline pH. The structure 5,6-oxido-7,9,11,14-eicosatetraenoic acid (Figure 1) was proposed as the intermediate on the basis of these data. The hydrolysis of epoxides is acid catalyzed, and the opening of allylic epoxides is favored at allylic positions (C-6 in this case). The findings that two 5,6-dihydroxy derivatives are formed nonenzymatically from the same intermediate as the enzymatic product, 5(S),12(R)-dihydroxy-eicosatetraenoic acid, and that ¹⁸O from molecular oxygen was exclusively retained at the C-5 position of these derivatives whereas ¹⁸O from water was introduced at C-6 or C-12, were of crucial importance in assigning the 5,6-oxido structure to the unstable intermediate (Figure 1). The formation of the dihydroxy acids from the epoxide intermediate is shown in Figure 1. Except for LTB₄ these are formed by chemical hydrolysis of the epoxide through a mechanism involving a carbonium ion. The latter added a hydroxyl anion, preferentially at C-6 and C-12, to yield four isomeric products that contain the stable conjugated triene structure. LTB₄ is formed enzymatically from the intermediate since it is not racemic at C-12 and because it is formed only by nondenatured cell preparations.

The structure 5,6-oxido-7,9,11,14-eicosatetraenoic acid (leukotriene A₄) was proposed as the intermediate (5). Chemical synthesis confirmed this structure and elucidated the stereochemistry (21). The enzymatically formed 5(S),12(R)-dihydroxy acid was previously shown to contain one cis and two trans double bonds in the conjugated triene. The location of the cis double bond at the ⁶-position was determined by means of a synthetic approach (22).

The mechanism suggested for the biosynthesis of the epoxide from arachidonic acid (Figure 1) involves initial formation of 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE). The epoxide is formed from 5-HPETE by abstraction of a hydrogen at C-10 and elimination of a hydroxyl anion from the hydroperoxy group.

	SLOW-REACTING SUBSTANCE OF ANAPHYLAXIS

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In 1938 Feldberg and Kellaway introduced the name slow- reacting substance (SRS) for a smooth muscle-contracting factor that appeared in the perfusate of guinea pig lung treated with cobra venom (23). Subsequent studies indicated that SRS is important as a mediator in asthma and other types of immediate hypersensitivity reactions (24, 25). Immunologically generated SRS is usually referred to as SRS-A (slow-reacting substance of anaphylaxis). SRS-A is considered to be released together with other mediators (e.g., histamine and chemotactic factors) after interaction between immunoglobulin E (IgE) molecules bound to membrane receptors and antigens such as pollen. Structural work on SRS was previously limited by the lack of pure preparations of SRS. However, it has been characterized as a sulfur-containing polar lipid with ultraviolet absorption (26). Previous studies indicated that labeled arachidonic acid was incorporated into SRS (29, 30).

Studies in our laboratory showed that treatment of human neutrophils with the calcium ionophore A23187 stimulated the synthesis of the 5,12-dihydroxy acid (LTB₄) (Table 1) (3). The stimulation was not only due to increased release of the precursor acid. These findings were of considerable interest since previous studies had shown that the ionophore also stimulates release of SRS from leukocytes (31). In addition, the ultraviolet (UV) absorbance of purified SRS was similar to that of the dihydroxy acids of leukocyte origin (4, 27, 32). On the basis of the effects of the ionophore, the UV absorbance data, and other considerations we developed the hypothesis that there was a biogenetic relation between the unstable allylic epoxide intermediate in neutrophils and the SRS generated in a variety of systems.

Since available methods for generation of SRS gave poor yields, we developed a new system (murine mastocytoma cells) for producing the substance (6, 33). The mouse mast cell tumors were propagated in the peritoneal cavities of syngeneic mice, and cells were removed and incubated with labeled arachidonic acid or labeled cysteine. Subsequently, SRS was generated by stimulating the cells with ionophore A23187. By this method we could increase the yield of spasmogenic material antagonized by the SRS antagonist FPL-55712 and the incorporation of isotopically labeled precursors (6). The isolation procedure involved precipitation of protein with ethanol, alkaline hydrolysis, separation on Amberlite XAD-8 and silicic acid, and two steps of RP-HPLC. The material thus obtained was essentially pure, showed an absorbance maximum at 280 nm, and caused a typical contraction of guinea pig ileum that was reversed by FPL-55712 (6). The UV spectrum resembled the spectra of the dihydroxy acids described above; however, the maximum wavelength was shifted 10 nm higher. This was consistent with a sulfur substituent in the  position. Experiments with labeled precursors showed that arachidonic acid and cysteine were incorporated into the products. When the isolated SRS was subjected to desulfurization with Raney nickel 5-hydroxyarachidic acid was formed. This indicated that the arachidonic acid derivative and cysteine were linked by a thioether bond. The presence of an alcohol group at C-5 in the fatty acid reinforced our hypothesis (3) of a biogenetic relation between the arachidonic acid metabolites we had found in leukocytes and SRS (Figure 2) (33a).

View larger version (15K): [in this window] [in a new window]   Figure 2.   Structure elucidation of SRS-A (33a).

The positions of the double bonds in SRS were determined by subjecting material biosynthesized from tritiated arachidonic acid to reductive ozonolysis. The isolation of labeled 1-hexanol among the products indicated that the ¹⁴-double bond of arachidonic acid had been retained. The method used for locating the conjugated triene was based on previous work in our laboratory. These studies had shown that arachidonic acid and related fatty acids containing two methylene-interrupted cis double bonds at the 6 and 9 positions are oxygenated to give derivatives with isomerization of the 6 double bond to 7 (34). Incubation of the isolated SRS with lipoxygenase resulted in isomerization of the ¹⁴-double bond toward the conjugated triene (forming a tetraene), since there was a bathochromic shift of 30 nm. This result indicated the presence of a ¹¹-cis double bond and additional double bonds at ⁷ and ⁹ in SRS. Thus STR was a derivative of 5-hydroxy-7,9,11,14-eicosatetraenoic acid with a cysteine-containing substituent in thioether linkage at C-6. Derivatization of cysteine was suggested by the failure to isolate alanine after desulfurization. The cysteine-containing substituent was therefore referred to as RSH in the reports of this work (6). Further studies demonstrated that in addition of cysteine, 1 mol of glycine and 1 mol of glutamic acid were present per mole of SRS. End-group (dansyl) method and hydrazinolysis) and sequence analyses (dansyl-Edman procedure) of the peptide showed that it was -glutamylcysteinyl-glycine (glutathione). The structure of the SRS from murine mastocytoma cells was therefore 5-hydroxy-6(S)-glutathionyl-7,9,11,14-eicosatetraenoic acid, LTC₄ (Figure 3) (9).

View larger version (16K): [in this window] [in a new window]   Figure 3.   The formation of leukotrienes by the 5-lipoxygenase pathway.

This was the first determination of the structure of an SRS-A (9). The preparation and some properties of the corresponding cysteinyl glycine derivative (LTD₄) and cysteinyl derivative (LTE₄) were reported at the same time (9). These compounds were isolated later from natural sources. The structure of LTC₄ was confirmed by comparison with the synthetic material and preparation of stereoisomers of LTC₄ (22, 35). Leukotriene C₄ is thus 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14,cis-eicosatetraenoic acid.

Studies with rat basophilic leukemia cells demonstrated that the major SRS was less polar than LTC₄ (36). That the fatty acid part of this compound and LTC₄ were identical was indicated by their UV spectra, the product obtained after Raney nickel desulfurization, and the spectral change observed after treatment with soybean lipoxygenase. Amino acid analyses, however, showed that the less polar product lacked glutamic acid. Edman degradation indicated that glycine was C terminal. Incubation of LTC₄ with -glutamyltranspeptidase yielded additional proof of the structure. The product, 5(S)-hydroxy, 6(R)-S-cysteinyl-glycine-7,9-trans-11,14-cis-eicosatetraenoic acid (LTD₄), was identical with the less polar product form RBL-1 cells (Figure 3). LTD₄ is more potent than LTC₄ in the guinea pig ileum bioassay and the contraction is faster (36). After the structure of SRS from mastocytoma cells was determined and after LTC₄, LTC₄, and LTE₄ were synthesized, all of these cysteine-containing leukotrienes (Figure 3) were found in a variety of biological systems (36). Thus SRS-A is a mixture of the cysteine-containing leukotrienes, that is, the parent compound LTC₄ and the metabolites LTD₄ and LTE₄. The relative proportion of these leukotrienes depends on the procedure used to prepare the SRS-A.

The hypothesized role of the unstable epoxide intermediate (LTA₄) as precursor of SRS (LTC₄) was confirmed (6, 45). The direct conversion of LTA₄ into LTC₄ was demonstrated in both mastocytoma cells and human leukocytes treated with the inhibitor of arachidonic acid metabolism, BW755C (46). These experiments thus confirmed the originally proposed pathway for the biosynthesis of SRS (Figure 3), namely, formation of LTA₄ from arachidonic acid via 5-HPETE, followed by glutathione conjugation of LTA₄ with opening of the epoxide at the allylic position C-6 (6, 9).

Because of the significance of the biosynthetic pathways described and the cumbersome systematic names of the compounds involved we introduced a trivial name for these entities (7). The term "leukotriene" was chosen because the compounds were discovered in leukocytes and the common structural feature is a conjugated triene. Various members of the group have been designated alphabetically: leukotrienes A are 5,6-oxido-7,9-trans-11-cis; leukotrienes B, 5(IS),12(R)-dihydroxy-6-cis-8,10-trans; leukotrienes C, 5(S)-hydroxy-6(R)-S--glutamyl-cysteinyl-glycyl-7,9-trans-11-cis; leukotrienes D, 5(S)-hydroxy-6(R)-S-cysteinyl-glycyl-7,9-trans-11-cis; and leukotrienes E, 5(S)- hydroxy-6(R)-S-cysteinyl-7,9-trans-11-cis eicosapolyenoic acids. Since precursor acids containing the ^5,8,11 double bond system (that is, 5,8,11-eicosatrienoic acid, arachidonic acid, and 5,8,11, 14,17-eicosapentaenoic acid) can be converted to leukotrienes containing three to five double bonds, a subscript denoting this number is used (47). Leukotriene C₄ can be metabolized to LTD₄ by enzymatic elimination of glutamic acid by -glytamyltranspeptidase (47). The remaining peptide bond in leukotriene D₄ is hydrolyzed by a dipeptidase to give leukotriene E₄ (48).

Subsequent studies of the biological effects of pure leukotrienes prepared by biosynthetic or synthetic methods have been summarized in several reviews (19, 47, 49). This work confirmed and extended the results obtained with various SRS-A preparations. In addition, LTB₄ was shown to have pronounced proinflammatory effects. Our work on the 5-lipoxygenase pathway later led to the discovery of a novel group of related biologically active compounds, the lipoxins (19).

The discovery of the leukotrienes was presented at the Fourth International Prostaglandin Conference held in Washington, DC, on May 28, 1979 (8). Subsequently, several drug companies started to develop inhibitors of the 5-lipoxygenase pathway or antagonists of cysteinyl-leukotrienes for treatment of asthma. Such inhibitors and antagonists are now available as drugs.

	Footnotes

Correspondence and requests for reprints should be addressed to Bengt Samuelsson, Department of Medical Biochemistry and Biophysics, Division of Chemistry II, Karolinska Institutet, S-171 77 Stockholm, Sweden.

Acknowledgments: Supported by the Swedish Medical Research Council (03X-00217).

	References

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