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equilibrium labeling of all major arachidonate-containing phosphoglyceride molecular species within mammalian cells. Furthermore, they also pointed out how major discrepancies can arise when comparing mass and label to determine sources of arachidonate used for eicosanoid biosynthesis. Acknowledgment Work discussed in this chapter was supported by National Institutes of Health Granls AI24985 and A126771.

[20] High-Performance Liquid Chromatography Separation and Determination of Lipoxins B y CHARLES N . SERHAN

The lipoxins are a series of biologically active, acyclic eicosanoids which contain a conjugated tetraene structure as a characteristic feature. 1-3 The two main compounds are positional isomers: one is designated lipoxin A4 ( L X A 4 ) and the other lipoxin B4 (LXB4) (Fig. 1). Multiple pathways exist for lipoxin biosynthesis that are substrate-, cell type-, and species-specific. 4-9 One route, which involves the transformation of 15HETE and formation of a 5(6)-epoxytetraene by human leukocytes, is outlined in Fig. 1. Other routes can involve the transcellular metabolism of i C. N. Serhan, M, Hamberg, and B. Samuelsson, Proc. Natl. Acad. Sci. U.S.A. 81, 5335 (1984). 2 B. Samuelsson, S.-E. Dahi6n, J. A. Lindgren, C. A. Rouzer, and C. N. Serhan, Science 7,,37, 1171 (1987). 3 C. N. Serhan, K. C. Nicolaou, S. E. Webber, C. A. Veale, S.-E. Dahl6n, T. J. Puustinen, and B. Samuelsson, J. Biol. Chem. 261, 16340 (1986). 4 H. K~hn, R. Wiesner, L. Alder, B. J. Fitzsimmons, J. Rokach, and A. R. Brash, Eur. J. Biochem. 169, 593 (1987). 5 N. Ueda, S. Yamamoto, B. J. Fitzsimmons, and J. Rokach, Biochem. Biophys. Res. Commun. 144, 996 (1987). 6 p. Walstra, J. Verhagen, M. A. Vermeer, J. P. M. Klerks, G. A. Veldink, and J. F. G. Vliegenthart, FEBS Left. 228, 167 (1988). 7 C.N. Serhan, U. Hirsch, J. Palmblad, and B. Samuelsson, FEBS Lett. 217, 242 (1987). s B. K. Lam, C. N. Serhan, B. Samuelsson, and P. Y.-K. Wong, Biochem. Biophyx. Res. Commun. 144, 123 (1987). 9 C. Edenius, J. Haeggstr~m, and J. A.. Lindgren, Biochem. Biophys. Res. Commun. 157, 801 (1988).

METHODS IN ENZYMOLOGY, VOL. 187

Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

168

ASSAYS

[9-0]

COOH "... OH 15- HETE

OOH

°... OH

l

~

COOH °

~

HO

OH C~H

~

HO COOH

.e" HO

OH LXA4

"*. OH LXB4

FIG. 1. One biosynthetic pathway for lipoxin formation (transformation of 15-HETE by activated human leukocytes). 15-HETE, (15S)-hydroxy-5,8,1l-cis-13-trans-eicosatetraenoic acid; LXA4, (5S,6R,15S)-trihydroxy-7,9,13-trans-ll-cis-eicosatetraenoic acid; LXB4, (5S, 14R,15S)-trihydroxy-6,10,12-trans-8-cis-eicosatetraenoicacid.

various substrates including leukotriene A4, arachidonic acid, 5,15D H E T E , and 5 - H E T E . 4-9 LXA4 and LXB4 each display biological activities that can be distinguished from those o f other eiscosanoids. ~o Several isomers o f LXA4 and LXB4 have been identified, including a novel 7-cis~oS.-E. Dahl~n, L. Franz6n, J. Raud, C. N. Serhan, P. Westlund, E. Wikstr6m, T. Bj6rck, H. Matsuda, S. E. Webber, C. A. Veale, T. Puustinen, J. HaeggstrOm, K. C. Nicolaou, and B. Samuelsson, Adv. Exp. Med. Biol. 229, 107 (1988).

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11-trans-LXA4.3,t1,12 Since LXA4, L X B 4 , and their isomers differ in both biological actions and potencies, their identification within biologically derived materials is of interest. This chapter describes the isolation, reversed-phase high-performance liquid chromatography (RP-HPLC) separation, and determination of lipoxins of the four series. Procedure

Materials HPLC-grade solvents were from American Scientific Products, Burdick and Jackson (Muskegon, MI). Methyl formate was from Sigma Chemical Company (St. Louis, MO). Sep-Pak C18 cartridges were from Waters Associates (Milford, MA). Synthetic LXA4, LXB4, LTB4, and other eicosanoids used as reference materials were from Biomol Research Laboratories (Philadephia, PA). Synthetic 7-cis-11-trans-LXA4 as well as the all-trans isomers of LXA4 and LXB4 were prepared 3"12 and were provided by K. C. Nicolaou, Department of Chemistry, University of Pennsylvania (Philadelphia, PA).

Extraction of Lipoxins The lipoxins can be extracted along with the prostaglandins (PG) and leukotrienes (LT) from in vitro incubations of various cell types suspended in phosphate-buffered saline (PBS) by utilizing a combination of techniques. 3A3 (Trivial names used in this article are consistent with the recent nomenclature proposal.t4) To terminate the incubations, ethanol (2 volumes to that of the incubation volume) and PGBz (as internal standard, or radiolabeled eicosanoids can be used) are added to the cell suspensions. The mixture is allowed to stand at 4° for at least 30 min. Following centrifugation (1200 g, 15 min), the supernatants are removed and saved. The pellets are then suspended in methanol (2 volumes). This step is repeated twice and the resulting ethanol- and methanol-containing fractions are pooled and dried by rotoevaporation under reduced pressure in a round-bottom flask. Materials coating the round-bottom flask are next suspended in methanol : water (1 : 45, v/v) by vortexing ( - 1-2 min) and then transferred into a 11 j. Adams, B. J. Fitzsimmons, Y. Girard, Y. Leblanc, J. F. Evans, and J. Rokach, J. Am. Chem. Soc. 107, 464 (1985). ~2 K. C. Nicolaou, B. E . Marron, C. A. Veale, S. E. Webber, S,-E. DahMn, B. Samuelsson, and C. N. Serhan, Biochim. Biophys. Acta 1003, 44 (1989). t3 W. S. Powell, J. Biol. Chem. 259, 3082 (1984). ~4 C. N. Serhan, P. Y.-K, Wong, and B. Samuelsson, Prostaglandins 34, 201 (1987).

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ASSAYS

[20]

glass syringe. The samples are rapidly acidified with HC1 to pH 3.5 and loaded onto cartridges containing ODS silica (Cls Sep-Pak). Since both LXA4 and LXB4 can undergo isomerization to their corresponding alltrans isomers, 3,~2 the acidification, cartridge loading, and washing with 10 ml water to obtain pH 6-7 are performed rapidly (i.e., within 60 sec of the addition of acid). To ensure that appropriate pH values are achieved, eluates from each sample are checked with colorpHast (EM Science, Cherry Hill, NJ). Next, the cartridges are eluted with hexane (10 ml) followed by methyl formate (10 ml). 13 The lipoxins, dihydroxyeicosatetraenoic acids, and tri-HETEs (i.e., LTB4, (5S,12S)-DHETE as well as their to products) are eluted with methyl formate. Materials eluting in this fraction can either be concentrated with a stream of argon suspended in mobile phase and injected directly on reversed-phase HPLC (vide infra) or they can be treated with diazomethane before reversedphase HPLC. Chromatography of the methyl esters will enable separation of the all-trans-LXB4 isomers [8-trans-LXB4 and (14S)-8-trans-LXB4 ].3 As in the case of 5-HETE, the lipoxins can easily form 1,5-y-lactones, which drastically changes their chromatographic behavior. Therefore, the UV absorbance of the extracted materials should be examined prior to HPLC in order to provide quantitative data on recovery of the compounds following analysis.

High-Performance Liquid Chromatography The lipoxins display strong absorbance in UV because of their conjugated tetraene structure. 1 The presence of the tetraene chromophore renders these compounds well suited for reversed-phase HPLC separation coupled with photodiode array rapid spectral detection (see later Fig. 3). An isocratic system which enables the separation of LXA4, LXB4, and 7-cis-1 l-trans-LXA4 from their all-trans isomers employs an Altex Ultrasphere-ODS (4.6 mm x 25 cm) column eluted with methanol:water:acetic acid (70:30:0.01) as mobile phase (flow rate 0.7 ml/ min). A representative chromatogram of the lipoxins obtained from human neutrophils is shown in Fig. 2. If the resolution of diene-, triene-, and tetraene-containing eicosanoids is needed within individual samples, the extracted materials can be injected, for example, into a gradient reversed-phase HPLC system equipped with a photodiode array detector. The column, an Altex Ultrasphere-ODS (4.6 mm x 25 cm), is eluted with a gradient solvent controller (LKB, Bromma, Sweden) using methanol : water : acetic acid (65 : 35:0.01, v/v/v) as phase one (injection to = 20 min) and a linear gradient with methanol : acetic acid (99.99 : 0.01, v/v) as phase two (30-50

[20]

HPLC or LIPOXINS isomers

trons-B

T

7-c/s

E

~

II - t f a n s

,trans-A

0 0

171

-

LXA 4

isomers

IILxB4 I / L X A4

Q) C) C E) ..0

.PGB 2

c,) ..Q

High-performance liquid chromatography separation and determination of lipoxins.

[20] HPLC OF LIPOXINS 167 equilibrium labeling of all major arachidonate-containing phosphoglyceride molecular species within mammalian cells. Furt...
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