- Email: [email protected]
[20] 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. 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).
[20]
HPLC OF LIPOXINS
169
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).
170
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
I
L
i
0
I0
20
3O
Time (rain)
FiG. 2. Reversed-phase HPLC chromatogram of lipoxins obtained from neutrophiis. Neutrophils(30 = 106cells; 1 mltotalincubation volume) were exposed to 15-HETE (30p, M} and ionophore A23187 (2.5/xM; 20 rain, 37°). The incubation was stopped by addition of alcohol and the products were extracted and chromatographed as described in the text.
min). The flow rate is 1.0 ml/min with an initial pressure of l l0 bar. Representative chromatograms are shown in Fig. 3A and a topogram of the same spectral data is given in Fig. 3B. The HPLC system consisted of an LKB dual-pump gradient HPLC equipped with a solvent controller and photodiode array rapid spectral detector linked to an AT&T PC 6300. Post-HPLC run analyses were performed utilizing either a 2140-202 Wavescan program or Wavescan EG 2146-002 program (Bromma, Sweden) and a Nelson Analytical 3000 series chromatography data system (Paramus, N J). The lipoxins can be quantitated by computer-aided manipulation from their UV spectra recorded on-line following HPLC. Standard curves are generated for compounds from each series utilizing known quantities of synthetic standards. This system permitted detection of -0.1 ng of lipoxin/107 human neutrophils following computer-aided analysis at a single wavelength (300 nm). The two trans isomers of LXB4,8-trans-LXB4 and (14S)--trans-LXB4, coelute on HPLC as free acids, while they can be separated as methyl esters on reversed-phase HPLC. The 7-cis-1 l-trans-LXA4 can be separated from the aU-trans-LXA4 isomers as its free acid. However, these
172
ASSAYS
[20]
¢=g
C1-C3~
1.oo 1.00
5.~o 5.~0
lOZOO 20~00
15~oo 15~00
20~00 20~00
25~00 ZSiO0
33.00 33.00 [mln]
FIG. 3. (A) Reversed-phase HPLC chromatograms. A representative profile of authentic standards following a single injection of a mixture of leukotrienes and lipoxins into a gradient system (see text for description). The profiles obtained at 270 nm (upper chromatogram [C 1] ) and 300 nm (lower chromatogram [C2] ) were recalled from stored UV spectral data moni-
[20]
HPLC OF LIPOXINS
173
three compounds [7-cis-ll-trans-LXA4, (6S)-ll-trans-LXA4, and lltrans-LXA4] coelute when chromatographed as the methyl esters (see Table I). Although some of these products may coelute in different HPLC systems, and they share similar prominent ions in their mass spectra, determination of the C value (equivalent chain length) of their methyl ester trimethylsilyl derivatives has proved to be a useful criterion for identifying the individual isomers. 3'!2 In general, the lipoxins that contain all-trans geometry of their conjugated tetraene display higher C values than either LXA4, LXB4, or other cis-containing compounds such as 7-cis-1 l-transL X A 4 (Table I). Determination of the C value is particularly useful, since LXA4 and its isomers give very similar mass spectra, as does L X B 4 and its isomers. 1'3'~2 This situation is unlike that observed with the mass spectra of LTB4 and its isomers [i.e., (5S, 12S)-DHETE, 6-trans-, and 12-epi-6trans-LTB4]. The C values for the lipoxins were determined by the method of Bergstrom et al. ~5as originally described for prostaglandins and related factors. Briefly, mixtures of methyl esters of fatty acids (20-26 carbons) are injected in the G C - M S and their retention times plotted on a logarithmic scale as a function of the number of carbon atoms present in each fatty acid on a normal scale. This procedure is useful because it provides a calibration plot for various GC columns and temperature programs or offers an index for eicosanoids and related products. Concluding Remarks As is the case when identifying other eicosanoids, lipoxins should be identified from biological sources by fulfilling several criteria that may include: (1) coelution of the product with authentic materials in more than one HPLC system (retention time); (2) coelution of the derivatized material with that of authentic standard (determination of C value); (3) UV spectral analyses of the isolated material; (4) the presence of diagnostic ions in the mass spectra of several derivatives (i.e., n-butyl boronate15 S. Bergstr6m, R. Ryhage, B. Samuelsson, and J, Sj6vall, J. Biol. Chem. 238, 3555 (1963).
tored by scanning between 220-340 nm +-- 0.9 nm; integration time was 0.5 sec. Post-HPLC analyses were performed with the Wavescan EG 2140-002 program and a Nelson Analytical 3000 series chromatography data system. The elution times were: 5.60 min (20-COOHLTB4), 6.11 min (20-OH-LTB4), 9.00 min (all-trans-LXB4), 10.20 rain (LXB4), 11.22 min (all-trans-LXA4), 13.27 min (LXA4), 17.87 min (PGB2), and 30.65 rain (LTB4). (B) Topogram of HPLC spectral data. The stored spectral data presented in (A) is plotted here from the Wavescan EG 2140-002 program. Wavelength range 230-330 nm; absorbance range 0.000.03 A, and time 1.0-33.00 rain. It can be seen that the lipoxin region is clearly separated from LTB4 and its to oxidation products.
174
ASSAYS
[9.0]
4 ~ g d d m
m m m m m m m Z <
N 0 a~
e
g
~o~
'0 e~
..~ ~
X
X
r
.~.~
~.~
[21]
GC-MS MEASUREMENTOF EET AND DHET
175
trimethylsilyl derivatives); and (5) bioassay of the isolated products and comparison with authentic materials. Acknowledgments The author thanks Ms. Kelly Sheppard for technical assistance and Mary Halm Small for skillful preparation of the manuscript. This work was supported in part by NIH grants #AI26714 and GM38765. C.N.S. is a recipient of the J.V. Satterfield Arthritis Investigator Award from the National Arthritis Foundation and is a Pew Scholar (1988).
[21] Quantitation o f E p o x y - a n d D i h y d r o x y e i c o s a t r i e n o i c Acids b y S t a b l e I s o t o p e - D i l u t i o n M a s s S p e c t r o m e t r y B y JOHN T U R K , W . THOMAS S T U M P , W E N D Y C O N R A D - K E S S E L , ROBERT R. SEABOLD, and BRYAN A. WOLF
Arachidonic acid may be converted to four regionally isomeric epoxyeicosatrienoic acids (EET) t by the action of a microsomal cytochrome-P450 monooxygenase in the presence of NADPH. 2 These compounds are hydrolyzed to the corresponding vicinal diols (dihydroxyeicosatrienoic acids or DHET) 3 by a cytosolic epoxide hydrolase.4 These metabolites are produced from exogenous arachidonate by intact hepatocytes5 and may be endogenous components of hepatocyte membranes.6 Exogenous EET have been reported to influence the function of endocrine,7'8 vascular, 9 and renal t° tissues, suggesting a possible mediator role for these compounds. E. Oliw, F. Guengerich, and J. Oates, J. Biol. Chem. 257, 3771 (1982). 2 M. Laniado-Schwartzman, K. L. Davis, J. C. McGill, R. D. Levere, and N. G. Abraham, J. Biol. Chem. 263, 2536 (1988). 3 E. Oiiw, J. Lawson, A. Brash, and J. Oates, J. Biol. Chem. 256, 9929 (1981). 4 N. Chacos, J. Capdevila, J. Falck, S. Manna, C. Martin-Wixtrom, S. Gill, B. Hammock, and R. Estabrook, Arch. Biochem. Biophys. 223, 639 (1983). 5 E. Oliw and P. Moldeus, Biochim. Biophys. Acta 721, 135 (1982). 6 j. Capdevila, B. Pramanik, J. Napol, S. Manna, and J. Falck, Arch. Biochem. Biophys. 231, 511 (1984). 7 G. Snyder, J. Capdevila, N. Chacos, S. Manna, and J. Falck, Proc. Natl. Acad. Sci. U.S.A. 80, 3504 (1983). 8 j. Capdivila, N. Chacos, J. R. Falck, S. Manna, A. Negro-Vilar, and S. Ojeda, Endocrinology 113, 421 (1983). 9 K. G. Proctor, J. R. Falck, and J. Capdevila, Circ. Res. 60, 50 (1987). 1o L. Lapuerta, N. Chacos, J. R. Falck, H. Jacobson, and J. H. Capdevila, Am. J. Med. Sci. 295, 275 (1988).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
HPLC OF LIPOXINS
167
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).
[20]
HPLC OF LIPOXINS
169
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).
170
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
I
L
i
0
I0
20
3O
Time (rain)
FiG. 2. Reversed-phase HPLC chromatogram of lipoxins obtained from neutrophiis. Neutrophils(30 = 106cells; 1 mltotalincubation volume) were exposed to 15-HETE (30p, M} and ionophore A23187 (2.5/xM; 20 rain, 37°). The incubation was stopped by addition of alcohol and the products were extracted and chromatographed as described in the text.
min). The flow rate is 1.0 ml/min with an initial pressure of l l0 bar. Representative chromatograms are shown in Fig. 3A and a topogram of the same spectral data is given in Fig. 3B. The HPLC system consisted of an LKB dual-pump gradient HPLC equipped with a solvent controller and photodiode array rapid spectral detector linked to an AT&T PC 6300. Post-HPLC run analyses were performed utilizing either a 2140-202 Wavescan program or Wavescan EG 2146-002 program (Bromma, Sweden) and a Nelson Analytical 3000 series chromatography data system (Paramus, N J). The lipoxins can be quantitated by computer-aided manipulation from their UV spectra recorded on-line following HPLC. Standard curves are generated for compounds from each series utilizing known quantities of synthetic standards. This system permitted detection of -0.1 ng of lipoxin/107 human neutrophils following computer-aided analysis at a single wavelength (300 nm). The two trans isomers of LXB4,8-trans-LXB4 and (14S)--trans-LXB4, coelute on HPLC as free acids, while they can be separated as methyl esters on reversed-phase HPLC. The 7-cis-1 l-trans-LXA4 can be separated from the aU-trans-LXA4 isomers as its free acid. However, these
172
ASSAYS
[20]
¢=g
C1-C3~
1.oo 1.00
5.~o 5.~0
lOZOO 20~00
15~oo 15~00
20~00 20~00
25~00 ZSiO0
33.00 33.00 [mln]
FIG. 3. (A) Reversed-phase HPLC chromatograms. A representative profile of authentic standards following a single injection of a mixture of leukotrienes and lipoxins into a gradient system (see text for description). The profiles obtained at 270 nm (upper chromatogram [C 1] ) and 300 nm (lower chromatogram [C2] ) were recalled from stored UV spectral data moni-
[20]
HPLC OF LIPOXINS
173
three compounds [7-cis-ll-trans-LXA4, (6S)-ll-trans-LXA4, and lltrans-LXA4] coelute when chromatographed as the methyl esters (see Table I). Although some of these products may coelute in different HPLC systems, and they share similar prominent ions in their mass spectra, determination of the C value (equivalent chain length) of their methyl ester trimethylsilyl derivatives has proved to be a useful criterion for identifying the individual isomers. 3'!2 In general, the lipoxins that contain all-trans geometry of their conjugated tetraene display higher C values than either LXA4, LXB4, or other cis-containing compounds such as 7-cis-1 l-transL X A 4 (Table I). Determination of the C value is particularly useful, since LXA4 and its isomers give very similar mass spectra, as does L X B 4 and its isomers. 1'3'~2 This situation is unlike that observed with the mass spectra of LTB4 and its isomers [i.e., (5S, 12S)-DHETE, 6-trans-, and 12-epi-6trans-LTB4]. The C values for the lipoxins were determined by the method of Bergstrom et al. ~5as originally described for prostaglandins and related factors. Briefly, mixtures of methyl esters of fatty acids (20-26 carbons) are injected in the G C - M S and their retention times plotted on a logarithmic scale as a function of the number of carbon atoms present in each fatty acid on a normal scale. This procedure is useful because it provides a calibration plot for various GC columns and temperature programs or offers an index for eicosanoids and related products. Concluding Remarks As is the case when identifying other eicosanoids, lipoxins should be identified from biological sources by fulfilling several criteria that may include: (1) coelution of the product with authentic materials in more than one HPLC system (retention time); (2) coelution of the derivatized material with that of authentic standard (determination of C value); (3) UV spectral analyses of the isolated material; (4) the presence of diagnostic ions in the mass spectra of several derivatives (i.e., n-butyl boronate15 S. Bergstr6m, R. Ryhage, B. Samuelsson, and J, Sj6vall, J. Biol. Chem. 238, 3555 (1963).
tored by scanning between 220-340 nm +-- 0.9 nm; integration time was 0.5 sec. Post-HPLC analyses were performed with the Wavescan EG 2140-002 program and a Nelson Analytical 3000 series chromatography data system. The elution times were: 5.60 min (20-COOHLTB4), 6.11 min (20-OH-LTB4), 9.00 min (all-trans-LXB4), 10.20 rain (LXB4), 11.22 min (all-trans-LXA4), 13.27 min (LXA4), 17.87 min (PGB2), and 30.65 rain (LTB4). (B) Topogram of HPLC spectral data. The stored spectral data presented in (A) is plotted here from the Wavescan EG 2140-002 program. Wavelength range 230-330 nm; absorbance range 0.000.03 A, and time 1.0-33.00 rain. It can be seen that the lipoxin region is clearly separated from LTB4 and its to oxidation products.
174
ASSAYS
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[21]
GC-MS MEASUREMENTOF EET AND DHET
175
trimethylsilyl derivatives); and (5) bioassay of the isolated products and comparison with authentic materials. Acknowledgments The author thanks Ms. Kelly Sheppard for technical assistance and Mary Halm Small for skillful preparation of the manuscript. This work was supported in part by NIH grants #AI26714 and GM38765. C.N.S. is a recipient of the J.V. Satterfield Arthritis Investigator Award from the National Arthritis Foundation and is a Pew Scholar (1988).
[21] Quantitation o f E p o x y - a n d D i h y d r o x y e i c o s a t r i e n o i c Acids b y S t a b l e I s o t o p e - D i l u t i o n M a s s S p e c t r o m e t r y B y JOHN T U R K , W . THOMAS S T U M P , W E N D Y C O N R A D - K E S S E L , ROBERT R. SEABOLD, and BRYAN A. WOLF
Arachidonic acid may be converted to four regionally isomeric epoxyeicosatrienoic acids (EET) t by the action of a microsomal cytochrome-P450 monooxygenase in the presence of NADPH. 2 These compounds are hydrolyzed to the corresponding vicinal diols (dihydroxyeicosatrienoic acids or DHET) 3 by a cytosolic epoxide hydrolase.4 These metabolites are produced from exogenous arachidonate by intact hepatocytes5 and may be endogenous components of hepatocyte membranes.6 Exogenous EET have been reported to influence the function of endocrine,7'8 vascular, 9 and renal t° tissues, suggesting a possible mediator role for these compounds. E. Oliw, F. Guengerich, and J. Oates, J. Biol. Chem. 257, 3771 (1982). 2 M. Laniado-Schwartzman, K. L. Davis, J. C. McGill, R. D. Levere, and N. G. Abraham, J. Biol. Chem. 263, 2536 (1988). 3 E. Oiiw, J. Lawson, A. Brash, and J. Oates, J. Biol. Chem. 256, 9929 (1981). 4 N. Chacos, J. Capdevila, J. Falck, S. Manna, C. Martin-Wixtrom, S. Gill, B. Hammock, and R. Estabrook, Arch. Biochem. Biophys. 223, 639 (1983). 5 E. Oliw and P. Moldeus, Biochim. Biophys. Acta 721, 135 (1982). 6 j. Capdevila, B. Pramanik, J. Napol, S. Manna, and J. Falck, Arch. Biochem. Biophys. 231, 511 (1984). 7 G. Snyder, J. Capdevila, N. Chacos, S. Manna, and J. Falck, Proc. Natl. Acad. Sci. U.S.A. 80, 3504 (1983). 8 j. Capdivila, N. Chacos, J. R. Falck, S. Manna, A. Negro-Vilar, and S. Ojeda, Endocrinology 113, 421 (1983). 9 K. G. Proctor, J. R. Falck, and J. Capdevila, Circ. Res. 60, 50 (1987). 1o L. Lapuerta, N. Chacos, J. R. Falck, H. Jacobson, and J. H. Capdevila, Am. J. Med. Sci. 295, 275 (1988).
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