- Email: [email protected]
Preparation and assay of monohydroxy-eicosatetraenoic acids
ANALYTICAL
BIOCHEMISTRY
Preparation
104, 259-267
(1980)
and Assay of Monohydroxy-eicosatetraenoic
J. M. BOEYNAEMS, A. R. BRASH, llepurtrnr~nt
of Phrrrmacolo~~.
School
J.
of ,Metiicinr~. Received
A.
Vuntierhilt
September
AND W. C.
OATES,
Unil~ersity.
Acids HUBBARD
Nosh~~ill~~.
7’c~nrrc~.s.rcr 37232
4. 1979
5-. 8-, 9-, 1 l-, 12-, and 15-hydroxy-eicosatetraenoic acids (HETEs) were prepared from arachidonic acid by reaction with H,O, in the presence of Cu’+ ions. They were separated by high-performance liquid chromatography on silica gel (PPorasil). using a linear solvent gradient from hexane to chloroform: only the 8- and 9-isomers were not resolved. Multimilligram quantities of highly purified HETEs could be easily generated by this method, which thus provides a useful tool to study the biological activity of these compounds. Octadeuterated analogs of HETEs prepared from octadeuterated arachidonic acid by this procedure were suitable for use as internal standards in stable isotope dilution assays, by combined gas chromatography and mass spectrometry. with selected ion monitoring. The detection limit of the HETEs was less than I ng.
Several mammalian tissues metabolize arachidonic acid to one or more isomeric hydroxy-eicosatetraenoic acids (HETEs).’ Human platelets produce 12-HETE (1,2), guinea pig lung 12-, 15, and ll-HETEs (3), rabbit polymorphonuclear leukocytes 5HETE (4), human neutrophils, 5, 12-. IS-, 8-, and 9-HETEs (5,6), human epidermis (7) and guinea pig spleen (8) I2-HETE, rat mast cells (9) and the VX, carcinoma (10) ll- and 15-HETEs. Initial reports indicate quite diverse biological activities of the hydroxy-eicosatetraenoic acids (HETEs) or their hydroperoxy precursors (HPETEs). I2-HPETE is an inhibitor of platelet thromboxane synthetase (1 l), whereas 15-HPETE blocks prostacyclin synthetase ( 12). 12- and IS-HPETEs, as well as prostaglandin endoperoxides, can stimulate a crude preparation of spleen guanylate cyclase (13). 12’ Abbreviations used: HETE, hydroxy-eicosatetraenoic acid: HPETE, hydroperoxy-eicosatetraenoic acid; hplc, high-performance liquid chromatography: gc-ms, gas chromatography-mass spectrometry; SIM, selected ion monitoring; D,, D,. unlabeled, octadeuterated compounds; ME, methyl ester: TMS, trimethylsilyl ether: C, equivalent chain length; M. molecular ion; ETYA, eicosatetraynoic acid; BSTFA. bis-trimethylsilyl-triflouro-acetamide.
HETE is chemotactic for polymorphonuclear leukocytes ( 14,15). Furthermore, the existence of stimulatory effects of arachidonic acid, which are blocked by the lipoxygenase inhibitor eicosatetraynoic acid (ETYA) but are unaffected by the cyclooxygenase inhibitor indomethacin, suggests that HETEs or HPETEs could play a role in several biological processes: enzyme secretion by neutrophils (16). histamine secretion by mast cells (17), lymphocyte mitogenesis (18--20), protein iodination in the thyroid (21). Progress in this field will depend on the availability of large quantities of highly purified HETEs for the evaluation of their biological activities and of sensitive and specific assays for the assessment of HETEs production itr iYt*o and in vitro. We have now developed a convenient chemical method to generate multimilligram quantities of highly purified HETEs and octadeuterated analogs of HETEs suitable for use as internal standards in stable isotope dilution assays. MATERIALS Prrpumtion
AND METHODS
o$HETEs.
acid (100 pmol, 2 &i) 259
[“HlArachidonic and CuCl, (50 pmol)
0003.2697/80/080259-09$02.00/O CopyrIght Q 1980 by Academx Pres, Inc. 411 right5 of reproduction in any form reverved
260
BOEYNAEMS
were dissolved in methanol (10 ml) and the pH was brought to 7 by addition of Tris buffer 0.2 M. pH 8.5 (2.5 ml). H,O, (1.8 mmol, 0.2 ml of a 305%solution) was added and the reaction mixture left for 30 min at room temperature. After extraction with ethyl acetate at pH 3 and solvent evaporation, the residue was dissolved in methanol and reduced with NaBH, (10 mg). After a second extraction, the reaction products were submitted to high-performance liquid chromatography. Reference I_(-HETE was prepared by incubation of arachidonic acid with soybean lipoxidase in 0.1 M borate buffer, pH 9, as described (22). HiSh-prrjbr1,lrtnc.c
liquid
c.llrotnrltoRrrr-
~11~. Chromatography was performed with a Waters Associates instrument (injector U6K, programmer model 600 and 2 pumps 6000A). Conventional (3.9 mm X 30 cm) and semipreparative (7.8 mm x 30 cm) columns of silica gel (PPorasil from Waters) were used. Linear solvent gradients from hexane (with 0.8% acetic acid) to chloroform (with 0.85%acetic acid) were delivered. Drri~,crti:ation utitl cutai~tic hytlrogetitrtion. Methylation and silylation were per-
formed by standard procedures using ethereal diazomethane (generated from nitrosomethylurea) and bis-trimethylsilyl-trifluoro-acetamide (BSTFA). For catalytic hydrogenation, about 20 pg of HETE was dissolved in 0.5 ml ethanol: after addition of I mg platinum oxide. H, was bubbled for 2 min. after which saturated reduced HETE was extracted in ethyl acetate. Gtrs c,hrot,lLrtogrcrpll?I. Gas-liquid chromatography was performed on a Varian 2100 instrument with flame ionization detection. A column of 3% OV-1 on GasChrom Q (2 m x 2 mm) was used isothermally at 230°C. Muss sprctromctry. Mass spectra scanning and selected ion monitoring were performed on a Hewlett-Packard combined gas chromatograph-quadrupole mass spectrometer (Model 5982A). Samples were
ET AL
injected on a l-m x 2-mm column of 3% OV-1 on Gas-Chrom Q, with helium as carrier gas (flow rate: 30 mlimin). The temperature of the column was 22O”C, the injection port temperature was 250°C. Electron energy was 70 eV. HETE .vt~rtldrrr.~/i;.rrtiotl. HETEs were standardized either by uv spectrophotometry. using a Beckman Model 25 instrument, or by gas-liquid chromatography with flame ionization detection, employing methyl ester-trimethylsilyl ether derivatives (METMS) and lignoceric acid as internal standard. MMrrtc~riuls. Arachidonic acid (purity >99%) was purchased from Nu-Check and c’H]arachidonic acid (60 Ci/mmol) was obtained from New England Nuclear. H,O, (30%,) was purchased from Fisher and the organic solvents were obtained from Burdick and Jackson. OV-I (3q) on GasChrom Q was obtained from Applied Science Incorporated. BSTFA was purchased from Pierce and nitrosomethylurea from ICN Pharmaceuticals. Soybean lipoxidase (type I) and lignoceric acid were purchased from Sigma. Gctadeuterated arachidonic acid was a generous gift of Dr. D. Taber (Vanderbilt University). RESULTS When the products of the reaction between arachidonic acid, Cu”’ and H,O, were submitted to hplc on PPorasil using a linear gradient from 100% hexane-acetic acid ( 100:0.8, v/v) to 100% chloroform-acetic acid (100:0.8. v/v), several components were resolved (Fig. IA). They included unreacted arachidonic acid, several uncharacterized products, and compounds which were identified respectively as the &lactone of 5-HETE. a mixture of 12- and 15HETEs. ll-HETE, a mixture of 8- and 9-HETEs, and the free acid form of 5-HETE. Complete separation of 12-, 15, and 1I-HETEs was achieved using a shallower gradient of hexane to chloroform (Fig. IS). whereas
PREPARATION
AND
ASSAY
OF MONOHYDROXY-EICOSATETRAENOIC
the mixture of 8- and 9-HETEs remained unresolved under similar conditions. HETEs were identified using the following criteria: ultraviolet spectrophotometry. retention time in gas-liquid chromatography. fragmentation pattern under electron impact. and fragmentation pattern after catalytic hydrogenation. Ultraviolet spectrophotometry revealed an absorption maximum at 335 mm, indicating the presence of a conjugated diene. Furthermore. the concentrations of these compounds measured either by the uv assuming a molar extinction absorption, coefficient of 37,000 (23), or by gas-liquid chromatography. using flame ionization detection and lignoceric acid as internal standard, closely agreed. Gas-chromatographic analysis of the methyl ester-trimethylsilyl ether derivatives of these various compounds showed peaks with the same equivalent chain length of C-21.3 (3cI OVI ). This value is similar to those reported for HETEs of biological origin (1.3,4,9,10). The mass spectra of the ME-TMS derivatives of HETEs generated by this chemical reaction exhibited ions at W/Z 406 (M), 391 (M-15: loss of ‘CH,,) and 375 (M-31: loss of .OCH,,) (Figs. 2a-e, A). 12-HETE was identified on the basis of the presence of a major ion at m/r: 295 and other ions at tnk 229, 205, and 173, which is similar to the fragmentation pattern reported for the METMS derivative of L-12-hydroxy-5,8,10,14eicosatetraenoic acid produced by platelets (1). 5-HETE was identified on the ground of a fragmentation pattern showing characteristic ions at 4,: 305, 255. and 203, similar to the one of the ME-TMS derivative of D-5-hydroxy-6,8,11,14-eicosatetraenoic acid formed by polymorphonuclear leukocytes (4). IS-HETE was identified on the basis of a mass spectrum showing characteristic ions at tnk 335 and 225 (3): the fragmentation patterns of 15HETE produced by this chemical reaction and of L- I.5-hydroxy-5.8.11.13-eicosatetraenoic acid formed by the soybean lipoxidase were identical. I I-HETE-ME-TMS was char-
ACIDS
261
20,000 z ‘3
l0,000
FIG. I. (A) Separation of the products of the reaction between arachidonic acid. Cu’+ and H,O,. After extraction and reduction hy NaBH,, they were submitted to hplc on a semipreparative FPorasil column (7.8 mm ‘i 30 cm). A linear gradient from hexaneacetic acid (100:0.8. v/v) to chloroform-acetic acid (1OtkO.8 v/v) was delivered in 2 h. The flow rate was 3 mlimin and j-ml fractions were collected. AA. arachidonic acid. (B) Rechromatography of fractions containing 12.. IS-. and I I-HETEs obtained in (A) on a conventional FPPorasil cohmn (3.9 mm x 30 cm). A linear gradient from 25 to 5OV of chloroform--acetic acid ( 100:0.8, v/v) in hexane-acetic acid (100:0.8, v/v) was delivered in I h. The flow rate waq I .O mUmin and 0.5-ml fractions were collected.
acterized by one major ion at m/z 225, as it has beenreportedfor 11-hydroxy-5,8,12,14eicosatetraenoic acid produced by the guinea pig lung (3), the rat mast cells (9), and the VX, carcinoma (10). 8- and 9-HETEsME-TMS were tentatively identified on the basis of a mass spectrum exhibiting major ions at ml,: 265 and 255, respectively, in addition to ions at tni: 406, 391, and 375.
262
BOEYNAEMS 5-
a
100
1
ET AL. HETE
‘55
!03
50
0
8.+9-
i
HETEs
L
C65 .,
IO0
n
IO0
50
0 I
3
IO0
200
29
287
50
0
I
FIG. 2. Mass spectra of the methyl ester-trimethylsilyl ether derivatwes of the HE’TEs generated by the described reaction between arachidonic acid. CL?+ and H,O,. (A) native compound, (B) saturated compound obtained by catalytic hydrogenation. Abscissa: mass scale, m/z: ordinate: abundance in percentage of the base peak.
PREPARATION
AND
ASSAY
OF MONOHYDROXY-EICOSATETRAENOIC
ACIDS
263
343 I
The identity of the various products of the reaction as HETEs was further supported by the mass spectra obtained after catalytic hydrogenation of the compounds. Gaschromatographic analysis of the ME-TMS derivatives showed in each case a peak of equivalent chain length C-22 (3% OV-1). The fragmentation pattern showed common ions at m/z 399 (M-15), 383 (M-31), 367 (M47), and for each compound a pair of major ions, compatible with a fragmentation on both sides of the carbon atom bearing the OTMS function (Figs. 2a-e, B). The identity of the &lactone of 5-HETE was supported by the following criteria: uv absorption maximum at 235 mm, equivalent chain length C-21.6 (3% OV-1) and mass spectrum (M = 302) of the underivatized compound and conversion to 5-HETE after alkali treatment (12 h at pH 12). The total yield of HETEs was about 5% and was reproducible from one experiment to the other. The proportion of individual HETEs was 22% for 15- and 5-HETE and
the mixture of 8- and 9-HETEs, each, 13% for I I-HETE and the &lactone of 5-HETE, each, and 8% for 12-HETE. Each HETE was at least 95% pure as judged by gaschromatographic analysis. The steric analysis has not been performed (23). Octadeuterated (D,) analogs of HETEs were prepared from 5,6,8,9,11,12,14,15octadeuterated arachidonic acid by the same procedure. They were identified according to the criteria of uv absorption, retention time during gas chromatography, and mass spectra of the ME-TMS derivatives. The Dx analogs of 5-, ll-, and 15HETE were evaluated for their potential use as internal standards in stable isotope dilution assays employing gas chromatography-mass spectrometry, with selected ion monitoring. Methyl ester-trimethylsilyl ether derivatives were used and fragment ions retaining at least four of the eight deuterium atoms were chosen for selected ion monitoring. The blanks were less than 0.6% (Table 1). In the case of 15-HETE,
264
o-
, 0
I IO
,
“g Do PQD8
20
FIG. 3. Selected ion monitoring assay of 1 I-HETE as methyl ester-trimethylsilyl ether. (A) Ion current profiles measured at ~tr/; 229 and 225. A mixture of D,- (100 ng) and D,,- (100 pg) I I-HETE-METMS was injected. The signal at tni: 225 was amplified 500 times over the signal at n)/,- 229. Column: 3% OV-1: column temperature: 220°C: injector temperature: 250°C; electron energy: 70 eV. (B) Standard curve obtained by injecting mixtures of D,,- and D,-II-HETE-ME-TMS in various ratios. The gc-ms was operated as in (A). Each point represents the mean of at least three injections.
the pair of ions at tnh 335 and 343 was used instead of the more intense ions at m/z 225 and 229, because it gave a lower TABLE CC-MS
ASSAY
HETE”
Ions monitored”
5II15
305-313 225-229 335-343
I or HETES
(parts
Blank< per thousand) 5.9 1.6 3.1
u The methyl ester-trimethylsilyl ether derivatives of D,-HETEs were used. I, gc-ms was operated as described under Materials and Methods. c The blank is defined as the ratio of selected ion currents characteristic of the D, and D, compounds. respectively, generated by the deuterated standard.
blank. The selected ion current profiles were free from interfering compounds (Figs. 3A, 4AJA). Linear standard curves were obtained for each HETE tested with the injection of 100 ng of standard (Figs. 3B,4BSB). Quantities of 100 pg 11-HETE, 500 pg 5HETE, and 400 pg 15-HETE were easily detected. The extraction of the HETEs from aqueous solutions was dependent on the pH and the solvent used. 1 l-HETE could be recovered in good yield from a standard medium for tissue incubation, either using hexane at acid pH, or chloroform at acid or neutral pH, or ethyl acetate even at alkaline pH (Fig. 6). 5- and 15-HETEs could be extracted under identical conditions (not shown).
PREPARATION
AND
ASSAY
OF MONOHYDROXY-EICOSATETRAENOIC
ACIDS
5-HETE-ME-TMS P
20
4
305 1x100)
I
IO
0
/ 0
i IO
I
“9 @, MD,
20
FIG. 4. Selected ion monitoring assay of 5-HETE as methyl ester-trimethylsilyl ether. (A) Ion current profiles measured at ml: 305 and 313. A mixture of D,- (120 ng) and D,,- (1 ng) 5-HETE-ME-TMS was injected. The signal at WI/,- 305 was amplified 100.fold as compared to the signal at ~1/; 313. (B) Standard curve obtained by injecting mixtures of D,- and D,-5-HETEME-TMS in various ratios. Each point represents the mean of at least three injections.
DISCUSSION
Progress of the understanding of the biological functions of the various hydroxyeicosatetraenoic acids obviously requires the availability of reasonable amounts of these compounds for testing their biological activity and a specific and sensitive assay. A stable isotope dilution method using gas chromatography-mass spectrometry apFIG. 5. Selected ion monitoring assay of 15.HETE as methyl ester-trimethylsilyl ether. (A) Ion current profiles measured at m/z, 343 and 335. A mixture of Du- (130 ng) and D,,- (1 ng) IS-HETE-ME-TMS was injected. The signal at ml; 335 was amplified 100.fold over the signal recorded at m/c 343. (B) Standard curve obtained by injecting mixtures of D,- and D,- I5HETE-ME-TMS, in various ratios. Each point represents the mean of at least three injections.
I5-HETE
ME- TMS
265
266
BOEYNAEMS
RECOVERY
% . ETHYL
100
ACETATE
CHLOROFORM
50
-
o-
, 3
FIG. 6. Extraction at various solved
pH.
7
5
of I I-HETE About
in a modified
9
with
20 ng of D,-I Krebs-Ringer
various
solvents
I-HETE
was
medium
contain-
ing ?o-mM 4-(2-hydroxyethylj-l-piperazineethanesulfonic acid buffer. pH 7.4. After acidification 0.1 N HCI or alkalinization with 1 N NaOH. either
hexane,
chloroform,
or ethyl
The extraction efficiency was tillation counting of radioactivity.
monitored
acetate
dis-
2 vol was
by liquid
with of
added. scin-
pears as the best choice for this assay: it requires the availability of deuterated standards. Until now, only 12- and ISHPETEs and HETEs have been reproducibly prepared in sufficient quantities for biological studies using human platelets and soybean lipoxidase, respectively (I 1, 13-15). An assay of I2-HETE by gas chromatography-mass spectrometry was made possible by the availability of a deuterated standard generated by human platelets (24). The tentative preparation of other HETEs and their deuterated analogs by biosynthesis could have several potential disadvantages: the need to develop a separate method for each HETE (neutrophils for 5-HETE, the VX2 carcinoma for llHETE, for instance), the potential contamination of deuterated arachidonic acid by the endogenous unlabeled substrate present in mammalian tissues (responsible for a high blank), the need of an extensive purification. No chemical synthesis is
ET
AL
presently available for these compounds, although very recently progress has been done in that direction ((25) and N. Nelson, personal communication). The method described in this paper was adapted from Smolen and Shohet (26) who used it to peroxidize liposomes prepared from erythrocyte lipids. This simple, rapid. and cheap procedure allows the preparation of multimilligram quantities of each HETE and of octadeuterated analogs suitable for use as internal standards in stable isotope dilution assays. A single step of high-performance liquid chromatography is sufficient to provide a high purity (>955%). The compounds produced by this procedure are identical to the genuine HETEs of biological origin, according to a variety of criteria (uv spectrum, gc retention time, mass spectra),
except
for
the
probable
lack
of
stereochemical purity. This could constitute a liability for biological studies. However, two distinct advantages of this procedure over biosynthesis are the possibility to generate ail six isomeric HETEs in one reaction and the low blank of the deuterated standards obtained. ACKNOWLEDGMENTS This and
work
BRSG-
was
supported
RR-05494
by NIH and
hy
Grants
PHS
GM
Fellowship
I5431 TW
09682. J. M. Boeynaems is Fellow of the Fogarty International Center and Aspirant of the Fonds National de la Recherche Scientifique (Belgium). J. A. Oates is the J. and M. Werthan cine. We are grateful gift ful
Professor to Dr.
of octadeuterated suggestions
of Investigative D. Taber for the
arachidonic throughout
this
acid
and
Medigenerous for
help-
study.
REFERENCES 1. Hamberg,
Nut. 2. Nugteren. 3. 4. 5.
M..
A@.
and
Samuelsson. B. (1974) Prr~.. 71. 3400-3404. t 1975) Biochim. Bit>/~lz?‘.~. Ac,/tr
SC;. USA D.
H.
380. 299-307. Hamberg, M.. and Samuelsson. chc~tn. Eioph~~. Rc. Crmnzrr,~. Borgeat. (1976) Borgeat.
Ac,trd.
B. (1974) 61, 942-949.
P.. Hamberg. M.. and Samuelsson. J. Biol. Chrrri. 251. 7816-7820. P.. and Samuelsson. B. t 1979) Proc,. SC;. USA 76, 2148-2152.
EioB. Nrrt.
PREPARATION
AND
ASSAY
OF MONOHYDROXY-EICOSATETRAENOIC
6. Goetzl, E.. and Sun. F. (1979) .I. fi.tp. flZf&. 150. 406-411. 7. Hammarstrom. S.. Hamberg, M., Samuelsson, B.. Duel]. E. A., Stawiski. M.. and Voorhees. J. J. (1975) Pm Ntrt. Acod. Sci. USA 72. 5 1305134. 8. Hamberg, M. t 1976) Bi~~c~/riv). Birtplix\. A~,ttr 331. 65 I-654. 9. Roberts. L. J.. Lewis. R. A.. Lawson, J. A.. Sweetman. B. J.. Austen. K. F.. and Oates. J. A. (1978) P~o.\tcrc/trridin.v 1.5. 717. IO. Hubbard. W. C.. Hough, A.. Watson. J. T.. and Gates. J. A. t 1978) Pr~j.\tlr,~/tr,~rlir~.\ 15, 721. I I. Hammarstrom. S.. and Fdlardeau. P. t 1977) Pro(,. Ntrr. A<,cld. SC,;. USA 74, 3691-3695. 1’7. Moncada. Vane,
S.. Gryglewski, R. J., Bunting. S.. and J. R. (1976) P~r,.vttr,g/rrridir~\ 12, 715-737.
13. Graff, G., Stephenson. J. H., Glass. D. B.. Haddox. M. K.. and Goldberg. N. D. t 1978) J. Hid. C/w/rl. 253, 7662-7776. 14. Turner. S. R., Tainer. J. A., and Lynn. (1975) h’dtr~rc, fL~~ri&vt) 257. 680-68 I.
W. S.
15. Goetzl, E. J.. Woods. (1977) J. C‘lir1. llri.t~\l.
R. R.
M.. and German. 59. l79- 183.
ACIDS
767
16. Naccache. P. H.. Showell. H. J.. Becker. E. L.. and Sha’afi. R. 1. t 1979) Rir)c,/rc’ni. Hioplix.\. R
BIOCHEMISTRY
Preparation
104, 259-267
(1980)
and Assay of Monohydroxy-eicosatetraenoic
J. M. BOEYNAEMS, A. R. BRASH, llepurtrnr~nt
of Phrrrmacolo~~.
School
J.
of ,Metiicinr~. Received
A.
Vuntierhilt
September
AND W. C.
OATES,
Unil~ersity.
Acids HUBBARD
Nosh~~ill~~.
7’c~nrrc~.s.rcr 37232
4. 1979
5-. 8-, 9-, 1 l-, 12-, and 15-hydroxy-eicosatetraenoic acids (HETEs) were prepared from arachidonic acid by reaction with H,O, in the presence of Cu’+ ions. They were separated by high-performance liquid chromatography on silica gel (PPorasil). using a linear solvent gradient from hexane to chloroform: only the 8- and 9-isomers were not resolved. Multimilligram quantities of highly purified HETEs could be easily generated by this method, which thus provides a useful tool to study the biological activity of these compounds. Octadeuterated analogs of HETEs prepared from octadeuterated arachidonic acid by this procedure were suitable for use as internal standards in stable isotope dilution assays, by combined gas chromatography and mass spectrometry. with selected ion monitoring. The detection limit of the HETEs was less than I ng.
Several mammalian tissues metabolize arachidonic acid to one or more isomeric hydroxy-eicosatetraenoic acids (HETEs).’ Human platelets produce 12-HETE (1,2), guinea pig lung 12-, 15, and ll-HETEs (3), rabbit polymorphonuclear leukocytes 5HETE (4), human neutrophils, 5, 12-. IS-, 8-, and 9-HETEs (5,6), human epidermis (7) and guinea pig spleen (8) I2-HETE, rat mast cells (9) and the VX, carcinoma (10) ll- and 15-HETEs. Initial reports indicate quite diverse biological activities of the hydroxy-eicosatetraenoic acids (HETEs) or their hydroperoxy precursors (HPETEs). I2-HPETE is an inhibitor of platelet thromboxane synthetase (1 l), whereas 15-HPETE blocks prostacyclin synthetase ( 12). 12- and IS-HPETEs, as well as prostaglandin endoperoxides, can stimulate a crude preparation of spleen guanylate cyclase (13). 12’ Abbreviations used: HETE, hydroxy-eicosatetraenoic acid: HPETE, hydroperoxy-eicosatetraenoic acid; hplc, high-performance liquid chromatography: gc-ms, gas chromatography-mass spectrometry; SIM, selected ion monitoring; D,, D,. unlabeled, octadeuterated compounds; ME, methyl ester: TMS, trimethylsilyl ether: C, equivalent chain length; M. molecular ion; ETYA, eicosatetraynoic acid; BSTFA. bis-trimethylsilyl-triflouro-acetamide.
HETE is chemotactic for polymorphonuclear leukocytes ( 14,15). Furthermore, the existence of stimulatory effects of arachidonic acid, which are blocked by the lipoxygenase inhibitor eicosatetraynoic acid (ETYA) but are unaffected by the cyclooxygenase inhibitor indomethacin, suggests that HETEs or HPETEs could play a role in several biological processes: enzyme secretion by neutrophils (16). histamine secretion by mast cells (17), lymphocyte mitogenesis (18--20), protein iodination in the thyroid (21). Progress in this field will depend on the availability of large quantities of highly purified HETEs for the evaluation of their biological activities and of sensitive and specific assays for the assessment of HETEs production itr iYt*o and in vitro. We have now developed a convenient chemical method to generate multimilligram quantities of highly purified HETEs and octadeuterated analogs of HETEs suitable for use as internal standards in stable isotope dilution assays. MATERIALS Prrpumtion
AND METHODS
o$HETEs.
acid (100 pmol, 2 &i) 259
[“HlArachidonic and CuCl, (50 pmol)
0003.2697/80/080259-09$02.00/O CopyrIght Q 1980 by Academx Pres, Inc. 411 right5 of reproduction in any form reverved
260
BOEYNAEMS
were dissolved in methanol (10 ml) and the pH was brought to 7 by addition of Tris buffer 0.2 M. pH 8.5 (2.5 ml). H,O, (1.8 mmol, 0.2 ml of a 305%solution) was added and the reaction mixture left for 30 min at room temperature. After extraction with ethyl acetate at pH 3 and solvent evaporation, the residue was dissolved in methanol and reduced with NaBH, (10 mg). After a second extraction, the reaction products were submitted to high-performance liquid chromatography. Reference I_(-HETE was prepared by incubation of arachidonic acid with soybean lipoxidase in 0.1 M borate buffer, pH 9, as described (22). HiSh-prrjbr1,lrtnc.c
liquid
c.llrotnrltoRrrr-
~11~. Chromatography was performed with a Waters Associates instrument (injector U6K, programmer model 600 and 2 pumps 6000A). Conventional (3.9 mm X 30 cm) and semipreparative (7.8 mm x 30 cm) columns of silica gel (PPorasil from Waters) were used. Linear solvent gradients from hexane (with 0.8% acetic acid) to chloroform (with 0.85%acetic acid) were delivered. Drri~,crti:ation utitl cutai~tic hytlrogetitrtion. Methylation and silylation were per-
formed by standard procedures using ethereal diazomethane (generated from nitrosomethylurea) and bis-trimethylsilyl-trifluoro-acetamide (BSTFA). For catalytic hydrogenation, about 20 pg of HETE was dissolved in 0.5 ml ethanol: after addition of I mg platinum oxide. H, was bubbled for 2 min. after which saturated reduced HETE was extracted in ethyl acetate. Gtrs c,hrot,lLrtogrcrpll?I. Gas-liquid chromatography was performed on a Varian 2100 instrument with flame ionization detection. A column of 3% OV-1 on GasChrom Q (2 m x 2 mm) was used isothermally at 230°C. Muss sprctromctry. Mass spectra scanning and selected ion monitoring were performed on a Hewlett-Packard combined gas chromatograph-quadrupole mass spectrometer (Model 5982A). Samples were
ET AL
injected on a l-m x 2-mm column of 3% OV-1 on Gas-Chrom Q, with helium as carrier gas (flow rate: 30 mlimin). The temperature of the column was 22O”C, the injection port temperature was 250°C. Electron energy was 70 eV. HETE .vt~rtldrrr.~/i;.rrtiotl. HETEs were standardized either by uv spectrophotometry. using a Beckman Model 25 instrument, or by gas-liquid chromatography with flame ionization detection, employing methyl ester-trimethylsilyl ether derivatives (METMS) and lignoceric acid as internal standard. MMrrtc~riuls. Arachidonic acid (purity >99%) was purchased from Nu-Check and c’H]arachidonic acid (60 Ci/mmol) was obtained from New England Nuclear. H,O, (30%,) was purchased from Fisher and the organic solvents were obtained from Burdick and Jackson. OV-I (3q) on GasChrom Q was obtained from Applied Science Incorporated. BSTFA was purchased from Pierce and nitrosomethylurea from ICN Pharmaceuticals. Soybean lipoxidase (type I) and lignoceric acid were purchased from Sigma. Gctadeuterated arachidonic acid was a generous gift of Dr. D. Taber (Vanderbilt University). RESULTS When the products of the reaction between arachidonic acid, Cu”’ and H,O, were submitted to hplc on PPorasil using a linear gradient from 100% hexane-acetic acid ( 100:0.8, v/v) to 100% chloroform-acetic acid (100:0.8. v/v), several components were resolved (Fig. IA). They included unreacted arachidonic acid, several uncharacterized products, and compounds which were identified respectively as the &lactone of 5-HETE. a mixture of 12- and 15HETEs. ll-HETE, a mixture of 8- and 9-HETEs, and the free acid form of 5-HETE. Complete separation of 12-, 15, and 1I-HETEs was achieved using a shallower gradient of hexane to chloroform (Fig. IS). whereas
PREPARATION
AND
ASSAY
OF MONOHYDROXY-EICOSATETRAENOIC
the mixture of 8- and 9-HETEs remained unresolved under similar conditions. HETEs were identified using the following criteria: ultraviolet spectrophotometry. retention time in gas-liquid chromatography. fragmentation pattern under electron impact. and fragmentation pattern after catalytic hydrogenation. Ultraviolet spectrophotometry revealed an absorption maximum at 335 mm, indicating the presence of a conjugated diene. Furthermore. the concentrations of these compounds measured either by the uv assuming a molar extinction absorption, coefficient of 37,000 (23), or by gas-liquid chromatography. using flame ionization detection and lignoceric acid as internal standard, closely agreed. Gas-chromatographic analysis of the methyl ester-trimethylsilyl ether derivatives of these various compounds showed peaks with the same equivalent chain length of C-21.3 (3cI OVI ). This value is similar to those reported for HETEs of biological origin (1.3,4,9,10). The mass spectra of the ME-TMS derivatives of HETEs generated by this chemical reaction exhibited ions at W/Z 406 (M), 391 (M-15: loss of ‘CH,,) and 375 (M-31: loss of .OCH,,) (Figs. 2a-e, A). 12-HETE was identified on the basis of the presence of a major ion at m/r: 295 and other ions at tnk 229, 205, and 173, which is similar to the fragmentation pattern reported for the METMS derivative of L-12-hydroxy-5,8,10,14eicosatetraenoic acid produced by platelets (1). 5-HETE was identified on the ground of a fragmentation pattern showing characteristic ions at 4,: 305, 255. and 203, similar to the one of the ME-TMS derivative of D-5-hydroxy-6,8,11,14-eicosatetraenoic acid formed by polymorphonuclear leukocytes (4). IS-HETE was identified on the basis of a mass spectrum showing characteristic ions at tnk 335 and 225 (3): the fragmentation patterns of 15HETE produced by this chemical reaction and of L- I.5-hydroxy-5.8.11.13-eicosatetraenoic acid formed by the soybean lipoxidase were identical. I I-HETE-ME-TMS was char-
ACIDS
261
20,000 z ‘3
l0,000
FIG. I. (A) Separation of the products of the reaction between arachidonic acid. Cu’+ and H,O,. After extraction and reduction hy NaBH,, they were submitted to hplc on a semipreparative FPorasil column (7.8 mm ‘i 30 cm). A linear gradient from hexaneacetic acid (100:0.8. v/v) to chloroform-acetic acid (1OtkO.8 v/v) was delivered in 2 h. The flow rate was 3 mlimin and j-ml fractions were collected. AA. arachidonic acid. (B) Rechromatography of fractions containing 12.. IS-. and I I-HETEs obtained in (A) on a conventional FPPorasil cohmn (3.9 mm x 30 cm). A linear gradient from 25 to 5OV of chloroform--acetic acid ( 100:0.8, v/v) in hexane-acetic acid (100:0.8, v/v) was delivered in I h. The flow rate waq I .O mUmin and 0.5-ml fractions were collected.
acterized by one major ion at m/z 225, as it has beenreportedfor 11-hydroxy-5,8,12,14eicosatetraenoic acid produced by the guinea pig lung (3), the rat mast cells (9), and the VX, carcinoma (10). 8- and 9-HETEsME-TMS were tentatively identified on the basis of a mass spectrum exhibiting major ions at ml,: 265 and 255, respectively, in addition to ions at tni: 406, 391, and 375.
262
BOEYNAEMS 5-
a
100
1
ET AL. HETE
‘55
!03
50
0
8.+9-
i
HETEs
L
C65 .,
IO0
n
IO0
50
0 I
3
IO0
200
29
287
50
0
I
FIG. 2. Mass spectra of the methyl ester-trimethylsilyl ether derivatwes of the HE’TEs generated by the described reaction between arachidonic acid. CL?+ and H,O,. (A) native compound, (B) saturated compound obtained by catalytic hydrogenation. Abscissa: mass scale, m/z: ordinate: abundance in percentage of the base peak.
PREPARATION
AND
ASSAY
OF MONOHYDROXY-EICOSATETRAENOIC
ACIDS
263
343 I
The identity of the various products of the reaction as HETEs was further supported by the mass spectra obtained after catalytic hydrogenation of the compounds. Gaschromatographic analysis of the ME-TMS derivatives showed in each case a peak of equivalent chain length C-22 (3% OV-1). The fragmentation pattern showed common ions at m/z 399 (M-15), 383 (M-31), 367 (M47), and for each compound a pair of major ions, compatible with a fragmentation on both sides of the carbon atom bearing the OTMS function (Figs. 2a-e, B). The identity of the &lactone of 5-HETE was supported by the following criteria: uv absorption maximum at 235 mm, equivalent chain length C-21.6 (3% OV-1) and mass spectrum (M = 302) of the underivatized compound and conversion to 5-HETE after alkali treatment (12 h at pH 12). The total yield of HETEs was about 5% and was reproducible from one experiment to the other. The proportion of individual HETEs was 22% for 15- and 5-HETE and
the mixture of 8- and 9-HETEs, each, 13% for I I-HETE and the &lactone of 5-HETE, each, and 8% for 12-HETE. Each HETE was at least 95% pure as judged by gaschromatographic analysis. The steric analysis has not been performed (23). Octadeuterated (D,) analogs of HETEs were prepared from 5,6,8,9,11,12,14,15octadeuterated arachidonic acid by the same procedure. They were identified according to the criteria of uv absorption, retention time during gas chromatography, and mass spectra of the ME-TMS derivatives. The Dx analogs of 5-, ll-, and 15HETE were evaluated for their potential use as internal standards in stable isotope dilution assays employing gas chromatography-mass spectrometry, with selected ion monitoring. Methyl ester-trimethylsilyl ether derivatives were used and fragment ions retaining at least four of the eight deuterium atoms were chosen for selected ion monitoring. The blanks were less than 0.6% (Table 1). In the case of 15-HETE,
264
o-
, 0
I IO
,
“g Do PQD8
20
FIG. 3. Selected ion monitoring assay of 1 I-HETE as methyl ester-trimethylsilyl ether. (A) Ion current profiles measured at ~tr/; 229 and 225. A mixture of D,- (100 ng) and D,,- (100 pg) I I-HETE-METMS was injected. The signal at tni: 225 was amplified 500 times over the signal at n)/,- 229. Column: 3% OV-1: column temperature: 220°C: injector temperature: 250°C; electron energy: 70 eV. (B) Standard curve obtained by injecting mixtures of D,,- and D,-II-HETE-ME-TMS in various ratios. The gc-ms was operated as in (A). Each point represents the mean of at least three injections.
the pair of ions at tnh 335 and 343 was used instead of the more intense ions at m/z 225 and 229, because it gave a lower TABLE CC-MS
ASSAY
HETE”
Ions monitored”
5II15
305-313 225-229 335-343
I or HETES
(parts
Blank< per thousand) 5.9 1.6 3.1
u The methyl ester-trimethylsilyl ether derivatives of D,-HETEs were used. I, gc-ms was operated as described under Materials and Methods. c The blank is defined as the ratio of selected ion currents characteristic of the D, and D, compounds. respectively, generated by the deuterated standard.
blank. The selected ion current profiles were free from interfering compounds (Figs. 3A, 4AJA). Linear standard curves were obtained for each HETE tested with the injection of 100 ng of standard (Figs. 3B,4BSB). Quantities of 100 pg 11-HETE, 500 pg 5HETE, and 400 pg 15-HETE were easily detected. The extraction of the HETEs from aqueous solutions was dependent on the pH and the solvent used. 1 l-HETE could be recovered in good yield from a standard medium for tissue incubation, either using hexane at acid pH, or chloroform at acid or neutral pH, or ethyl acetate even at alkaline pH (Fig. 6). 5- and 15-HETEs could be extracted under identical conditions (not shown).
PREPARATION
AND
ASSAY
OF MONOHYDROXY-EICOSATETRAENOIC
ACIDS
5-HETE-ME-TMS P
20
4
305 1x100)
I
IO
0
/ 0
i IO
I
“9 @, MD,
20
FIG. 4. Selected ion monitoring assay of 5-HETE as methyl ester-trimethylsilyl ether. (A) Ion current profiles measured at ml: 305 and 313. A mixture of D,- (120 ng) and D,,- (1 ng) 5-HETE-ME-TMS was injected. The signal at WI/,- 305 was amplified 100.fold as compared to the signal at ~1/; 313. (B) Standard curve obtained by injecting mixtures of D,- and D,-5-HETEME-TMS in various ratios. Each point represents the mean of at least three injections.
DISCUSSION
Progress of the understanding of the biological functions of the various hydroxyeicosatetraenoic acids obviously requires the availability of reasonable amounts of these compounds for testing their biological activity and a specific and sensitive assay. A stable isotope dilution method using gas chromatography-mass spectrometry apFIG. 5. Selected ion monitoring assay of 15.HETE as methyl ester-trimethylsilyl ether. (A) Ion current profiles measured at m/z, 343 and 335. A mixture of Du- (130 ng) and D,,- (1 ng) IS-HETE-ME-TMS was injected. The signal at ml; 335 was amplified 100.fold over the signal recorded at m/c 343. (B) Standard curve obtained by injecting mixtures of D,- and D,- I5HETE-ME-TMS, in various ratios. Each point represents the mean of at least three injections.
I5-HETE
ME- TMS
265
266
BOEYNAEMS
RECOVERY
% . ETHYL
100
ACETATE
CHLOROFORM
50
-
o-
, 3
FIG. 6. Extraction at various solved
pH.
7
5
of I I-HETE About
in a modified
9
with
20 ng of D,-I Krebs-Ringer
various
solvents
I-HETE
was
medium
contain-
ing ?o-mM 4-(2-hydroxyethylj-l-piperazineethanesulfonic acid buffer. pH 7.4. After acidification 0.1 N HCI or alkalinization with 1 N NaOH. either
hexane,
chloroform,
or ethyl
The extraction efficiency was tillation counting of radioactivity.
monitored
acetate
dis-
2 vol was
by liquid
with of
added. scin-
pears as the best choice for this assay: it requires the availability of deuterated standards. Until now, only 12- and ISHPETEs and HETEs have been reproducibly prepared in sufficient quantities for biological studies using human platelets and soybean lipoxidase, respectively (I 1, 13-15). An assay of I2-HETE by gas chromatography-mass spectrometry was made possible by the availability of a deuterated standard generated by human platelets (24). The tentative preparation of other HETEs and their deuterated analogs by biosynthesis could have several potential disadvantages: the need to develop a separate method for each HETE (neutrophils for 5-HETE, the VX2 carcinoma for llHETE, for instance), the potential contamination of deuterated arachidonic acid by the endogenous unlabeled substrate present in mammalian tissues (responsible for a high blank), the need of an extensive purification. No chemical synthesis is
ET
AL
presently available for these compounds, although very recently progress has been done in that direction ((25) and N. Nelson, personal communication). The method described in this paper was adapted from Smolen and Shohet (26) who used it to peroxidize liposomes prepared from erythrocyte lipids. This simple, rapid. and cheap procedure allows the preparation of multimilligram quantities of each HETE and of octadeuterated analogs suitable for use as internal standards in stable isotope dilution assays. A single step of high-performance liquid chromatography is sufficient to provide a high purity (>955%). The compounds produced by this procedure are identical to the genuine HETEs of biological origin, according to a variety of criteria (uv spectrum, gc retention time, mass spectra),
except
for
the
probable
lack
of
stereochemical purity. This could constitute a liability for biological studies. However, two distinct advantages of this procedure over biosynthesis are the possibility to generate ail six isomeric HETEs in one reaction and the low blank of the deuterated standards obtained. ACKNOWLEDGMENTS This and
work
BRSG-
was
supported
RR-05494
by NIH and
hy
Grants
PHS
GM
Fellowship
I5431 TW
09682. J. M. Boeynaems is Fellow of the Fogarty International Center and Aspirant of the Fonds National de la Recherche Scientifique (Belgium). J. A. Oates is the J. and M. Werthan cine. We are grateful gift ful
Professor to Dr.
of octadeuterated suggestions
of Investigative D. Taber for the
arachidonic throughout
this
acid
and
Medigenerous for
help-
study.
REFERENCES 1. Hamberg,
Nut. 2. Nugteren. 3. 4. 5.
M..
A@.
and
Samuelsson. B. (1974) Prr~.. 71. 3400-3404. t 1975) Biochim. Bit>/~lz?‘.~. Ac,/tr
SC;. USA D.
H.
380. 299-307. Hamberg, M.. and Samuelsson. chc~tn. Eioph~~. Rc. Crmnzrr,~. Borgeat. (1976) Borgeat.
Ac,trd.
B. (1974) 61, 942-949.
P.. Hamberg. M.. and Samuelsson. J. Biol. Chrrri. 251. 7816-7820. P.. and Samuelsson. B. t 1979) Proc,. SC;. USA 76, 2148-2152.
EioB. Nrrt.
PREPARATION
AND
ASSAY
OF MONOHYDROXY-EICOSATETRAENOIC
6. Goetzl, E.. and Sun. F. (1979) .I. fi.tp. flZf&. 150. 406-411. 7. Hammarstrom. S.. Hamberg, M., Samuelsson, B.. Duel]. E. A., Stawiski. M.. and Voorhees. J. J. (1975) Pm Ntrt. Acod. Sci. USA 72. 5 1305134. 8. Hamberg, M. t 1976) Bi~~c~/riv). Birtplix\. A~,ttr 331. 65 I-654. 9. Roberts. L. J.. Lewis. R. A.. Lawson, J. A.. Sweetman. B. J.. Austen. K. F.. and Oates. J. A. (1978) P~o.\tcrc/trridin.v 1.5. 717. IO. Hubbard. W. C.. Hough, A.. Watson. J. T.. and Gates. J. A. t 1978) Pr~j.\tlr,~/tr,~rlir~.\ 15, 721. I I. Hammarstrom. S.. and Fdlardeau. P. t 1977) Pro(,. Ntrr. A<,cld. SC,;. USA 74, 3691-3695. 1’7. Moncada. Vane,
S.. Gryglewski, R. J., Bunting. S.. and J. R. (1976) P~r,.vttr,g/rrridir~\ 12, 715-737.
13. Graff, G., Stephenson. J. H., Glass. D. B.. Haddox. M. K.. and Goldberg. N. D. t 1978) J. Hid. C/w/rl. 253, 7662-7776. 14. Turner. S. R., Tainer. J. A., and Lynn. (1975) h’dtr~rc, fL~~ri&vt) 257. 680-68 I.
W. S.
15. Goetzl, E. J.. Woods. (1977) J. C‘lir1. llri.t~\l.
R. R.
M.. and German. 59. l79- 183.
ACIDS
767
16. Naccache. P. H.. Showell. H. J.. Becker. E. L.. and Sha’afi. R. 1. t 1979) Rir)c,/rc’ni. Hioplix.\. R