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Development and Review of Radioimmunoassay of 12-S- Hydroxyheptadecatrienoic Acid Harald John,*² Karl Cammann,² and Werner Schlegel 1 * *Klinik und Poliklinik fu ¨ r Geburtshilfe und Frauenheilkunde, Westfa ¨ lische Wilhelms-Universita ¨t Mu ¨ nster, Germany; ²Anorganisch-Chemisches Institut, Analytische Chemie, Westfa ¨ lische Wilhelms-Universita ¨t Mu ¨ nster, Domagkstr. 11, D-48149 Germany For more than 25 years 12-S-hydroxyheptadecatrienoic acid (HHT) has been known to be a product of thromboxanesynthase (TX-Syn) when synthesized with thromboxane A 2 (TXA 2 ). Although there are some hints that HHT has anti-aggregatory effects, to date, it has neither been shown to have any specific pathological relevance nor is there much information about its physiological role. This review presents a summary of the physicochemical properties of HHT, its chemical syn- thesis, the impact of various biological systems on its enzymatic and non-enzymatic production and its physiological function and metabo- lization, as well as a survey of the most important methods for ana- lyzing this unsaturated hydroxy-fatty acid. Due to the low antibody- raising potency expected in HHT, no immunological system for HHT quantification has been developed so far. In our report we present the development and validation of a sensitive and reliable, competitive radioimmunoassay (RIA) suitable for the quantitative determination of HHT. HHT was produced by an enhanced enzymatic method using platelet-rich plasma (PRP). With an effective and modified liquid-liq- uid and solid-phase extraction method we were able to produce highly purified HHT (97% purity by GC/MS) in sub-milligram ranges. These fractions were used for the synthesis of BSA-antigen-conjugates and for immunization of rabbits. The tritiated tracer was synthesized us- ing prostaglandin H synthase for the production of prostaglandin H 2 1 Address correspondence to: Prof. Dr. W. Schlegel, Klinik und Poliklinik fu ¨r Geburtshilfe und Frauenheilkunde, Westfa ¨ lische Wilhelms-Universita ¨ t Mu ¨ nster, Domagkstr. 11, D - 48149, Germany, Phone/Fax: 149 –251 835-6114. Prostaglandins & other Lipid Mediators 56:53–76, 1998 © 1998 by Elsevier Science Inc. 0090-6980/98/$19.00 655 Avenue of the Americas, New York, NY 10010 PII S0090-6980(98)00043-4

Development and review of radioimmunoassay of 12-S-hydroxyheptadecatrienoic acid

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Page 1: Development and review of radioimmunoassay of 12-S-hydroxyheptadecatrienoic acid

Development and Review ofRadioimmunoassay of 12-S-Hydroxyheptadecatrienoic Acid

Harald John,*† Karl Cammann,† and Werner Schlegel1*

*Klinik und Poliklinik fur Geburtshilfe und Frauenheilkunde,Westfalische Wilhelms-Universitat Munster, Germany;†Anorganisch-Chemisches Institut, Analytische Chemie, WestfalischeWilhelms-Universitat Munster, Domagkstr. 11, D-48149 Germany

For more than 25 years 12-S-hydroxyheptadecatrienoic acid (HHT) hasbeen known to be a product of thromboxanesynthase (TX-Syn) whensynthesized with thromboxane A2 (TXA2). Although there are somehints that HHT has anti-aggregatory effects, to date, it has neitherbeen shown to have any specific pathological relevance nor is theremuch information about its physiological role. This review presents asummary of the physicochemical properties of HHT, its chemical syn-thesis, the impact of various biological systems on its enzymatic andnon-enzymatic production and its physiological function and metabo-lization, as well as a survey of the most important methods for ana-lyzing this unsaturated hydroxy-fatty acid. Due to the low antibody-raising potency expected in HHT, no immunological system for HHTquantification has been developed so far. In our report we present thedevelopment and validation of a sensitive and reliable, competitiveradioimmunoassay (RIA) suitable for the quantitative determinationof HHT. HHT was produced by an enhanced enzymatic method usingplatelet-rich plasma (PRP). With an effective and modified liquid-liq-uid and solid-phase extraction method we were able to produce highlypurified HHT (97% purity by GC/MS) in sub-milligram ranges. Thesefractions were used for the synthesis of BSA-antigen-conjugates andfor immunization of rabbits. The tritiated tracer was synthesized us-ing prostaglandin H synthase for the production of prostaglandin H2

1Address correspondence to: Prof. Dr. W. Schlegel, Klinik und Poliklinik furGeburtshilfe und Frauenheilkunde, Westfalische Wilhelms-Universitat Munster,Domagkstr. 11, D - 48149, Germany, Phone/Fax: 149–251 835-6114.

Prostaglandins & other Lipid Mediators 56:53–76, 1998© 1998 by Elsevier Science Inc. 0090-6980/98/$19.00655 Avenue of the Americas, New York, NY 10010 PII S0090-6980(98)00043-4

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(PGH2) followed by an aqueous reaction with Fe21-solution to rear-range PGH2 to HHT. The dynamic range of the assay was from 30–400 pg/tube, with a sensitivity of approximately 40 pg/tube. The eval-uation of the assay was performed by a HPLC-RIA method as well asby correlation with a quantitative HPLC method and correlation withTXB2 concentrations in a blood coagulation study. The assay may beuseful for the quantification of HHT in several tissues and body fluidsunder various physiological conditions and may also help to under-stand the possible physiological role of HHT in biological processes.

Keywords: 12-S-hydroxyheptadecatrienoic acid; thromboxane A2;thromboxanesynthase; radioimmunoassay

Introduction

In the early seventies, Hamberg, Samuelsson and Wlodawer (1,2) discov-ered HHT and classified it as an enzymatic product of arachidonic acid(AA) formed by prostaglandin H synthase derived from the vesiculargland of sheep and from human platelets. Since then, many studies haveattempted to characterize HHT and to determine its physiological func-tion. Although HHT occurs as an AA-metabolite of the cycloxygenase(COX) pathway along with the well known prostaglandins (PGs) theirphysicochemical properties, molecular structure and physiological signif-icance are quite different. These differences require modified methods forpreparation of HHT-containing samples as well as specific analyticalprocedures. Endogenous HHT is usually measured via time-consumingchromatographic methods compounding the screening of a large numberof biological samples. We present a new method for immunologicalmeasurement of HHT using a competitive radioimmunoassay that allowsfor measurement of HHT in pg-ranges.

Review of 12-S-HHT

Physicochemical Properties and Cchemical Synthesis

Naturally occuring 12-S-hydroxy-(5Z,8E,10E)-heptadecatrienoic acid(12OH-17:3, M 5 280.4 g/mol, CAS-No 54397–84-1) is light-sensitive andunstable if exposed to oxygen. The physicochemical properties of HHTare characterized on the one hand by its hydrophobic unsaturated C17-carbon chain and on the other by its hydrophilic hydroxy function at C12and its carboxylic function at C1, which, through dissociation, can trans-form HHT into its anionic form (Fig. 1). This property is of great signif-icance for its solubility in aqueous systems and thus for its physiologicaldisponibility, such as the passage through lipid membranes or its distri-bution properties in biological fluids. Studies in our laboratory (unpub-lished) have shown that HHT binds non-covalently to fatty-acid binding

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proteins (FABP) and albumins. Albumins are known for their high-affin-ity binding sites for anionic fatty acids with hydrophobic side chains(3–5). It is possible that they serve as carrier proteins for HHT in thevascular system.

FIGURE 1. Metabolization of arachidonic acid (AA) and prostaglandin H2 (PGH2).

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The pKa value of this weak acid has not been determined yet. In viewof the pKa values found in other fatty acids and prostanoids, the pKa rangeof HHT is probably between 4.5 and 5.0 (6–9). The conjugated C-C transdouble bonds at C8 and C10 in combination with the a-hydroxy functionat C12 results in an UV-absorbance which reaches its maximum at 232nm (eEtOH 5 33,000 M21 cm21) (10). This absorbance property, whichdiffers from prostaglandin absorbance capacity (lmax 5 192 nm) (11), isoften used to detect and quantify HHT in HPLC analysis. The complexoverall 9-step chemical synthesis was described in 1982 by Russell et al.(12) using an intermediate synthesized by Osbond et al. (13). By perform-ing the heterogeneous catalytic Lindlar-cis-hydrogenation of triple bondsand the Wittig-reaction to produce double bonds, they were able tosynthesize a racemic mixture of 20–50 mg 12-R,S-(5Z,8E,10E,Z)-HHT. Inhis 1996 doctoral thesis, Mundkowski (14) described a retro-biomimeticHHT synthesis. HHT was stereospecifically produced from 12-keto-hep-tadecatrienoic acid (KHT) with a chiral reduction using the (CBS)-OAB-catalyst developed by Corey, Bakshi, and Shibata (15). KHT was producedfrom 12-keto-heptadecadienoic acid (KHD) by PhSeOH, which itself wasobtained from 12-hydroxyheptadecadienoic acid (HHD) by oxidation withClCrO3. The educt for synthesizing HHD was the commercially available5-pentyl-dihydro-furane-2-one.

Due to the fact that the molecular structure of HHT is much simplerthan that of prostaglandins, it was previously believed that specific anti-bodies could not be raised in animals (16).

Enzymatic and Non-Enzymatic Production of HHT

HHT is a metabolite of PGH2 produced from arachidonic acid via theCOX-pathway (2,17–20). Under physiological conditions, PGH2 is unsta-ble, t1/2 5 5 min (17). It is regio- and enantioselectively converted intoTXA2, malondialdehyde (MDA) and HHT by the TX-Syn (21) (Fig. 1).Evidence of TX-Syn in human and equine platelets was described in 1976by Moncada and Needleman (22,23). Shen and Tai (24) raised specificmonoclonal antibodies against this enzyme and determined its molecularweight to be 53 kDa by SDS-gel electrophoresis. Furthermore, they dem-onstrated its irreversible self-inactivation during catalysis. This phenom-enon—also observed in the prostaglandin H synthase (25,26)—may becaused by the endogenous peroxy-radicals (27). Various models for themechanism which determines the enzyme activity have been describedin the literature (21,28). Anderson et al. (29) postulated a bimolecularreaction of two PGH2 molecules which are simultaneously convertedinto TXA2 and HHT. Hall and Tai (30) found that the production of HHTwas realized by at least two different mechanisms when using purifiedTX-Syn from lung microsomes. Using optical difference spectroscopy,Hecker and Ullrich (31) characterized the TX-Syn as a cytochrome P 450enzyme (32) following a radical mechanism. They detected one heme per

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polypeptide chain of the enzyme, resulting in a molecular weight of 58.8kDa. Although this work resulted in a reliable and precise model ofcatalysis, it did not explain the 1:1 ratio between the TXA2 formation andthe rearrangement to HHT usually found in biological systems. Finally,Ohashi et al. (33) were able to isolate two species of human TX-synthasecDNA, called TXS-I and -II. They differ by a 163-bp deletion near the39-end of the coding region. Wang et al. (34) reported a method forexpressing TXS-I and TXS-II as recombinant enzymes in a baculovirussystem following reverse transcription-polymerase chain reaction (PCR)(RT-PCR). In contrast to TXS-I, TXS-II does not synthesize either TXA2nor HHT.

The non-enzymatic hydrolysis (19,35) and reduction (2) of PGH2 pro-duces several prostaglandins in addition to HHT. Usually these non-enzymatic conversions are not stereospecific, resulting in varying mix-tures of 10E and 10Z isomers of HHT. One known exception is therearrangement induced by Ru(PPh3)3Cl in HCCl3 which produced thephysiological 10E form (36).

Biological Systems Synthesizing HHT

Extensive enzymatic HHT formation has been observed in platelets(30,37–41) and lung microsomes (30,42–44). In these biological systems,a 1:1 ratio of TXA2 and HHT was found. Changes in this equimolarproduction could be achieved in the presence of certain stimulating orinhibiting substances (discussed below). The following concentrations ofendogenous HHT have been determined in blood samples: more than 50ng/mL in platelet-rich plasma (PRP) (45,46), about 10 pg/mL in platelet-poor plasma (PPP) (45,47) and 20–400 ng/mL in serum (48,49). Theconcentration in PPP is thought to correspond to the endogenous level inblood. The wide concentration range of the serum level is caused byinter-donor variability in platelet activity and coagulation parameterssuch as time and temperature. HHT is also synthesized in alveolar mac-rophages (50), vascular tissue (51), clara cells (52), and placental homog-enates (53).

Stimulation and Inhibition of HHT Production

The amount of HHT synthesized from AA can be varied by manipulatingthe participated enzymes such as phospholipase A2 (PLA2), COX, TX-Syn,and the lipoxygenases (LOX), which induces the conversion of AA intoseveral hydroperoxyeicosatetraenoic acids (HPETE). The enzymatic con-version of AA can be inhibited by acetylsalicylic acid (34,50), indometh-acin (38), sulfonylurea agents (54), eicosapentaenoic acid (EPA) and 15-hydroperoxyeicosapentaenoic acid (15-HPEPE) (55), imidazole anddipyridamol (56), OKY 1581 (50,57) dicramin (58). Vitamin E is alsoknown for its potency to inhibit platelet aggregation by inhibiting COXactivity (59). Pace-Asciak et al. (60) reported on the inhibitory effects of

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the red wine phenolic trans-resveratrol in human platelets. A selectivedose-dependent inhibition of the TXA2 and HHT formation was observedwith 1-hydroxyphenanzine (OHP) (61). This naturally occuring substanceis produced in vivo by Pseudomonas aeruginosa found in the airways ofpatients with cystic fibrosis. HHT production in vitro can also be unspe-cifically minimized by selection of unfavourable incubation parameterssuch as temperature, ionic strenght (30), and pH (27).

The stimulation of enzymatic HHT synthesis can be achieved byesculetin (6,7-dihydroxy-coumarin) (62), ozone-atmosphere (63), dexam-ethazone (64), tbutyl hydroperoxide (tBOOH) (65), Cd21 (51), or glutathi-one (19). Naturally occuring proteins such as collagen (45) and thrombin(66,67) increase the HHT production of platelets. HHT synthesis of plate-lets can also be stimulated by hydrodynamic stress (68). Capdevila et al.(20) showed that physiological concentrations of reduced glutathione(GSH) in the presence of the isoforms COX-1 and COX-2 can metabolizearachidonic acid to HHT with production rates of 88% and 78%, respec-tively. This study indicated that in addition to O-O bond reductivecleavage, GSH supports a presumably enzymatic C-C bond cleavagesimilar to that observed during TXA2 biosynthesis.

Furthermore, stimulation of COX-products can be achieved by inhib-iting the concurrent reaction for metabolizing AA via selective inhibitionof LOX by 5,8,11-eicosatriynoic acid (ETY) (69) or by high density lipopro-tein (70). Evidence of selective inhibition and stimulation of HHT-bio-synthesis involved in TXA2-synthesis has not yet been found. However,Hall et al. (27) demonstrated that multiple additions of PGH2 to immo-bilized TX-Syn from bovine lung dramatically reduced TXA2 synthesis,while HHT synthesis remained unaffected. They suggested that the twosubstances are produced at different sites on the enzyme.

The Synthesis Ratio Q of HHT and TXA2

HHT and thromboxane are produced by the same enzyme, although theirphysicochemical and physiological properties differ. To date, there is nosatisfactory explanation for the commonly observed 1:1 ratio of thesecompounds. Some studies, however, describe deviations from thisequimolar production (Table 1).

Physiological Function and Metabolization of HHT

Sadowitz et al. (72) reported that HHT can stimulate prostacyclin pro-duction in human umbilical venous endothelial cell cultures. HHT stim-ulated AA release from cell phospholipids and had a clearly observableeffect on vascular cyclooxygenase. Metabolization of AA in plateletsremained unaffected by HHT, led Sadowitz et al. to postulate that HHTmight be an important local modulator of platelet plug formation, andthat it plays a protective antithrombotic role in modulating local hemo-stasis. In addition, Hecker and Ullrich (10) reported that HHT inhibited

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the activity of 5-lipoxygenase, derived from human neutrophil polymor-phonuclear leukozytes (PMN), and soybean 15-lipoxygenase in vitro. It isas yet unclear whether the properties observed here are physiologicallysignificant.

HHT can be enzymatically converted into 12-keto-heptadecatrienoicacid (KHT) (10). This reaction is NAD1-dependent and is induced by theprostaglandin-15-hydroxydehydrogenase (PG-15-HDH, E.C. 1.1.1.141)from porcine kidney (73), human erythrozytes (10), HL-60 Leukemia cells(74), rabbit lung microsomes (75), and human placenta (76). KHT might bean effective inhibitor of the secondary, TXA2-mediated aggregation ofhuman platelets (10). This inhibition is mediated through an increase inintracellular cAMP concentration. These results suggest that HHT mightserve as a reservoir for enzymatic KHT production which affects aggre-gation processes. Determination of whether this hypothesis is based on areal physiological event will require further study.

Analytical Methods for the Determination of HHT

Some of the commonly used chromatographic methods for the determi-nation of endogenously and exogenously produced HHT that have beenapplied include: TLC (77,79), HPLC-methods (38,78,80–83), or GC-tech-niques—as GC/MS (2,21,44,73,84) and GC/FID (85). Sample extractionusing liquid-liquid (38,73,86) or solid phase (87,89) methods is oftennecessary. When radiolabeled precursors were used in in vitro studies,HHT was quantified via liquid scintillation counting (82,90–92) or radio-TLC-scanners (77,79). Although there exist many HPLC-methods forquantifying fluorescent and UV-absorbing derivates of prostaglandins(93–97), to date these methods have not been used for quantification ofHHT. In most cases, underivatized HHT is usually detected at 232 nm(11,98). The most sensitive and accurate analysis of HHT was achieved byGC-methods, which provided a high level of reproducibility and preci-sion. They were, however, time consuming and required costly equip-ment.

TABLE 1.

Compound Biological system Q Literature

Imidazole Lung microsomes 3.0 56Dexamethasone Lung microsomes 3.9 64Indomethacin latelets 3.7 71Imidazole Platelets 2.5 28Catalase or superoxide

dismutasePlatelets 1.7 71

Dipyridamol Lung microsomes ,1 56Dibutyryl cAMP Platelets ,1 71

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Radioimmunological methods have been succesfully introduced forthe determination of prostaglandins and thromboxanes (99–102). Modi-fications of immunoassays have been developed which eliminate radio-activity in such procedures as fluoroimmunoassay (FIA) (103), time-re-solved fluoroimmunoassay (Tr-FIA) (104, 105), enzyme-linkedimmunosorbent assay (ELISA) (106), and chemiluminescence immunoas-say (CLIA) (107). Highly specific antibodies have been raised for mosteicosanoids. Interestingly, there is no specific antibody available for mea-surement of HHT. We were able to induce highly specific antibodies intorabbits, which were useful for the establishment of a radioimmunoassaythat detects HHT in pg-levels. This assay may be a helpful tool forlarge-scale analysis of biological samples.

Development and Validation of the Radioimmunoassay of HHT

Materials and Methods

Chemicals

AA, TXB2, 6-keto-prostaglandin F1a (6-keto-PGF1a), prostaglandin A2(PGA2), B2 (PGB2), D2 (PGD2), E2 (PGE2) F2a (PGF2a), 13,14-dihydro-15-keto-PGE2 (PGE-M) and 13,14-dihydro-15-keto-PGF2a (PGF-M), (6)-iso-proterenol hydrochloride, L-tryptophan, human hemoglobin, indometh-acin, and acetylsalicylic acid (ASA) were purchased from Sigma (St. Louis,MO, USA). 12-HHT, 12-hydroxyeicosatetraenoic acid (12-HETE),5-HETE, oleic acid, stearidonic acid, g-linolenic acid, 12-oxoeicosatetrae-noic acid (12-OxoETE), 13-hydroxyoctadecatrienoic acid (13-HOTrE), andleukotriene B4 (LTB4) were obtained from Cayman (Ann Arbor, MI, USA).Tritiated arachidonic acid (3H-AA, 240 Ci/mmol, 1 mCi/mL) was pro-vided by ARC (St. Louis, MO, USA). Solid-phase extraction (SPE) columns(Chromabond C18 ec, 500 mg in glass) were obtained from Macherey-Nagel (Duren, Germany). Acetonitrile (ultragradient grade) and water(HPLC grade) were obtained from J. T. Baker (Deventer, The Nether-lands). Thrombin (50 NIH-E/mg), charcoal, and all salts (reagent grade)used for the buffers were obtained from Merck (Darmstadt, Germany).Gelatin (Difco, Detroit, MI, USA) and dextran T70 (Pharmacia, Uppsala,Sweden) were used for the RIA. “Ultima Gold” from Camberra Packard(Groningen, The Netherlands) was used as scintillation cocktail. Allother chemicals were provided by Merck and were of reagent grade.

Preparation and Purification of HHT from Human Platelet-RichPlasma

600 mL platelet rich plasma (' 9 3 108 platelets/mL) - preincubated for3 min with 1.3 mM isoproterenol, 5 mM L-tryptophan, 5 u/mL thrombin,and 100 mM Et3PbCl were incubated in air-saturated buffer for 90 min at37°C with an emulsion of 10 mg AA in 0.15 M Tris/HCl-buffer, pH 8.0,

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according to the method developed by VanRollins (38). Incubation wasterminated by the addition of acetone; the incubation mixture was thenextracted using various methods (Fig. 2). The liquid-liquid extractionmethod of Miller and Pawlak (108) was modified by replacing hexanewith petrolether (PE) and by changing the pH from 4.5 to 3.5. This

FIGURE 2. Scheme for extracting and purifying HHT from platelet rich plasma incubation. PE,petroleum ether

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extraction procedure was repeated three times. The dried residue wasdissolved in 1 mL CH3CN-H2O, pH 3.5 (30:70 v/v). This solution wasapplied to a SPE-C18 column. The column was washed with 10 mL water(pH 3.5) following 20 mL of CH3CN-H2O, pH 3.5 (30: 70 v/v) and thendried with 4 mL PE. Desorption of HHT was achieved using 5 mL EtOHand 5 mL ethyl acetate. The resulting purified amount of HHT wascalculated using HPLC technology at 232 nm (eEtOH 5 33,000 M21 3cm21) (10) with an external standard (Cayman, Ann Arbor, MI, USA).Purity was determined using GC/MS technology with a bis-(trimethyl-silyl)-derivate of HHT. The GC (Varian 3400, Varian, Walnut Creek, CA,USA) equipment consisted of a cold inlet system, Gerstel 502 with a 25-mHPU 2 capillary column (Hewlett-Packard, Waldbronn, Germany) whichterminates at the MS-EI source, 70 eV, (Finnigan MAT 8230, Finnigan,San Jose, CA, USA).

Preparation of HHT-BSA Conjugate and Immunization

800 mg HHT were derivatized to a BSA-conjugate as described elsewhere(85,109). The immunogen in complete Freund’s adjuvant (Sigma, St.Louis, MO, USA) was used to immunize two female white New Zealandrabbits (approved by a local ethics committee G 19/96). The procedurewas carried out as described by Schlegel et al. (110).

Synthesis and Purification of Tritiated HHT Tracer

0.1 mCi of 3H-AA was dissolved in 1 mL 0.1 M Tris/HCl-buffer, pH 8.0at 37°C containing 1.3 mM isoproterenol, 6.1 mM L-tryptophan and 0.18mg/mL human hemoglobin. 100 mL of a buffered prostaglandin H syn-thase solution (10,000 u/mL Tris/HCl-buffer, pH 7.4 containing 0.1%(w/v) Tween 20, Oxford Biomedical Research, Oxford, MI, USA) wereadded. After incubation for 30 s at 37°C the mixture was acidified with1.5 M citric acid and immediately mixed with 5 mL 2 mM FeCl2. Aftergentle shaking for 45 min at 4°C the solution was applied to a SPE-C18-column following the procedure described above. The EtOH-ethyl acetateeluate was evaporated to dryness and redissolved in 1 mL EtOH andstored at 230°C. The purity of this tracer was examined by reversed-phase HPLC-method.

High-Performance Liquid Chromatographic Method

The HPLC equipment consisted of a Rheodyne 7725i syringe loadinginjector (Cotati, CA, USA), a pump system 322, a diode-array detector 440(DAD), Kroma 2000 HPLC software from Kontron Instruments (Neu-fahrn, Germany), and a fraction collector Superrac 2211 from LKB(Bromma, Sweden). Separations were carried out on a 125 3 4.6 mm I.D.column packed with Nucleosil 120 C18 (particle size 5 mm) and a pre-column (26 3 6.0 mm I.D.) of the same material from Bischoff (Leonberg,Germany). The aqueous solvent systems were comprised of varying per-

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centages of acetonitrile. pH was adjusted to 3.5 using trifluoroacetic acid(TFA). The flow-rate was 1.0 mL/min, and the experiments were carriedout at 30°C. Temperature of the RP column was adjusted in a PrecithermPFV water bath (Boehringer, Mannheim, Germany). 20 mL of the HPLCsample solution were injected and chromatographed using an acetonitrilegradient as follows: CH3CN was 55% for the first 10 min, after which thepercentage of CH3CN rose to 85% within 2 min and remained at thatlevel for 11 min Equilibration of the column was achieved within 2 minat a decrease in the percentage of acetonitrile to 55%. HHT was detectedat 232 nm and fractions of 20 s (0.33 mL) were collected in glass tubes.

Competitive Radioimmunoassay for HHT

Assays were carried out in duplicates in Duran glass tubes (12 3 75 mm,Schott, Mainz, Germany). Standards (10–2500 pg/tube) and samples wereincubated for over night at 4°C with 100 mL tracer solution (20,000cpm/100 mL assay-buffer I, pH 7.4) and 100 mL antiserum No T23006dilution (1: 3000 in assay-buffer I). The assay buffer I used is a commonlyused 0.05 M Tris/HCl-buffer, pH 7.4 containing 0.1% (w/v) gelatin and0.01% (w/v) thimerosal. An 0.25% (w/v) charcoal suspension containing0.025% (w/v) dextran T70 in the assay buffer was used to separateantibody-bound from free-tritiated tracer. After centrifugation of the sam-ples, radioactivity of the supernatants was measured with a liquid scin-tillation counter (LSC) for 1 min (LSC 1409, Wallac, Turku, Finland).Resulting data were calculated with the RIA Calc LM-software (Wallac,Turku, Finland). Serologic specificity of the antiserum No T23006 wasdetermined by measuring cross-reactivities of PGA2, PGB2, PGD2, PGE2,PGE-M, PGF2a, PGF-M, 6-keto-PGF1a, TXB2, AA, oleic acid, stearidonicacid, g-linolenic acid, 12-HETE, 12-OxoETE, 5-HETE, 13-HOTrE, KHT,LTB4, and HHT.

Coagulation Study of Whole Blood

Blood of a human donor who had not taken any COX-inhibitor for the last2 weeks was collected as follows: One set of samples was immediatelymixed with ASA (1.9 mL final concentration). The second was mixedwith indomethacin (8.4 mL), and no COX-inhibitor was added to the third.The blood was allowed to coagulate for 0, 0.5, 1, 2, and 4 h respectivelyat 37°C. It was then centrifuged at 15003 g at 4°C for 20 min The serumwas decanted and extracted using a SPE-C18-column. The column waswashed using 10 mL 0.05 M Tris/HCl-buffer, pH 7.4 and 4 mL water; pH3.5. 4 mL PE were used to dry the column. Prostaglandins and HHT wereeluated with 5 mL EtOH and 5 mL ethyl acetate. This eluate was evap-orated to dryness and redissolved in EtOH. Dilution ratios required forHHT measurement by RIA were 1:2 and 1:40 respectively, whereas HPLCdetermination required fourfold concentration.

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TXB2 was measured by a previously described radioimmunoassay insample dilutions of 1:50 (86).

High-Performance Liquid Chromatography (HPLC)-Radioimmunoassay (HPLC-RIA) of HHT

An aliquot of the ethanolic serum sample solution, equivalent to theextract of 1 mL serum, was dried under nitrogen and redissolved in 50 mLCH3CN-H2O, pH 3.5 (60: 40 v/v). RP-HPLC was carried out as describedabove. 200 mL of each fraction were dried in assay tubes using a speed vacSC110 (Savant, Farmingdale, NY, USA). Calculation of HHT levels wascarried out in single measurements of each fraction by redissolving theresidues in tracer and antiserum dilution and treating them as described.

Results

Preparation and Purification of HHT

Under the conditions described above, the activated platelets produced ahigh level of HHT (approximately 1 mg). Overall recoveries followingpurification and extraction was 800 mg HHT, with recovery ranges of95 6 2% for the ethyl acetate extraction and 90 6 2% for the solid-phaseextraction. The GC/MS chromatogram showed only one single peak,indicating a purity of nearly 97%. The MS of the bis-(trimethylsilyl)-derivate is shown in Figure 3.

Synthesis of 3H-HHT

PGH2 was unstable in aqueous solution and was converted into severalprostaglandins, especially PGF2a. This effect was observed in a time-dependent manner during incubation. The described solid-phase extrac-tion successfully separated HHT from these prostaglandins.

HHT-RIA

Antiserum No T23006 was useful in carrying out a highly sensitive andspecific radioimmunoassay; it was used in a dilution of 1:3000. Thedynamic range was between 30 and 400 pg per tube, with a sensitivity ofapproximately 40 pg. The intra- and inter-assay coefficients of variationranged from 9 to 13% in the region of interest. Serologic specificity of theantiserum is shown in Table 2. All tested ligands showed negligiblecross-reactivities. The highest values (2.5%) were found for KHT and13-HOTrE.

HPLC-RIA of Extracted Serum

Figure 4 shows the chromatogram of the serum extract after radioimmu-nological determination of HHT. One high peak could be detected with aretention similar to the HHT reference (tR 5 6.7 min) and a second one

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(16-fold lower than the HHT peak) was eluted after 19.5 min. Thisretention is similar to nonpolar fatty acids like AA.

Coagulation Study of Whole Blood

Recoveries for HHT and TXB2 extractions from serum by SPE-C18 were85 6 2% and 75% 6 3% respectively. The EtOH-ethyl acetate eluate wasa clear and colorless solution. HHT and TXB2 in serum increased in atime-dependent manner in almost the same concentrations (Fig. 6). Pro-duction of HHT and TXB2 was inhibited by ASA and indomethacin (Fig.6).

Discussion

Preparation and Purification of HHT, Antibodies, and Tracer

In order to produce HHT in sub-milligram ranges we used an enhancedplatelet incubation in air-saturated buffer that converted AA stereospe-cifically to HHT (17,38,41). This enzymatic reaction was induced by theaddition of Et3PbCl (41,111), thrombin (66,67), isoproterenol (25,112), andL-tryptophan (44,113). The extraction and purification procedure de-scribed by Powell (114) was modified by replacing EtOH and non-end-

FIGURE 3. Mass spectrum (70 eV) of bis-(trimethylsilyl)-derivate of HHT; molecular weight, 424.GC/MS analysis was done with a 25-m HPU 2 capillary column, MS-EI, 70 eV. Conditions forGC: carrier gas: He, injection volume: 1 mL, injection temperature: 60–310°C, oven tempera-ture, 80–300°C at 8°C/min for 24 min; m/e 424 (M1), 409 (M1- 15, loss of CH3), 353 (M1-71,loss of C5H11), 334 (M1-90, loss of Me3SiOH), 225 (M1- 199, loss of CH2-CH 5 CH-(CH2)3-COOSiMe3), 73 (Me3Si1) and 75 (Me2SiOH1).

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capped C18 material by CH3CN and endcapped C18 material. Selectiveelution of polar AA metabolites (TXs and PGs) was achieved using theSPE-C18 method following introduction of a clean-up solvent of CH3CN-H2O, pH 3.5 (30: 70 v/v). This procedure was highly effective and resultedin sufficient amounts of purified HHT. The bis-(trimethylsilyl)-derivateof HHT was synthesized in our laboratory for GC/MS analysis (Fig. 3).

The problem of generating antisera of adequate sensitivity arisesmainly from the instability of HHT. We succeeded in producing antiserausing the procedure of Schlegel et al. (110). Following antiserum chroma-tography and separation with protein G (106), the binding sites of thepurified protein were localized on a IgG-fraction. This rules out a non-specific protein binding. Radioimmunological measurements were car-ried out using 3H-HHT tracer produced with purified PGH synthase. Thisenzymatic conversion of AA into HHT has been described in detailelsewhere (1,25,44,108,115). PGH2 was rearranged by adding Fe (II) to theincubation buffer according to Hamberg and Samuelsson (2), resulting ina successfuly purified tracer (25% of applied 3H-AA).

Validation of Radioimmunoassay

The assay presented here shows the usual coefficients of variation (9–13%) and a large dynamic range of 30–400 pg/tube, with a detection limitof approximately 40 pg/tube. The antiserum No T23006 has a highserologic specificity (Table 2) and can distinguish between both the

TABLE 2.

Ligand Cross-reactivity [%]

Prostaglandins and thromboxane B2

PGA2

0.02PGB2, PGD2, PGE2, PGE-M,

PGF2a, PGF-M, 6-keto-PGF1a

,0.02

TXB2 0.03

Fatty acidsAA, oleic acid, stearidonic acid,

g-linolenic acid,0.02

Hydroxy fatty acids of the COX and LOX-pathway12-HETE, 5 HETE ,0.0212-OxoETE 0.0313-HOTrE 2.5KHT 2.5LTB4 ,0.02HHT 100

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12-hydroxy-function of HHT and the 12-oxo-function of KHT, as well asbetween the (CH2)3-COOH chain of HHT and the (CH2)4-COOH chain of13-HOTrE. Both ligands show small cross-reactivities of 2.5%. Accordingto methods described by Scatchard (116) and Odell et al. (117) avidity ofthe antiserum was 0.88 3 109 L/mol and 0.83 3 109 L/mol respectively.These values closely correspond to those already found for certain PGantisera (5–6 3 109 L/mol) (16). HHT and other AA metabolites wereco-extracted with other serum compounds. The immunological relevanceof these substances for the determination of HHT was demonstratedusing a combination of HPLC-RIA, which is an effective method for thedetermination of the immunological impact of sample matrix com-pounds (Fig. 4). All interfering substances were eluted with the HPLCgradient. The RIA-chromatogram of the serum extracts did not show any

FIGURE 4. HPLC-RIA of extracted serum sample. Extract from the solid-phase extraction ofserum was chromatographed on Nucleosil 120 C18 (125 3 4.6 mm I.D.), 5-mm particle size, ata flow rate of 1.0 mL/min with an acetonitrile gradient from 55 to 85% (v/v) at 30°C. Fractionsof 20 s (0.33 mL) were collected. 200 mL were evaporated to dryness and investigatedradioimmunologically.

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major matrix influence. Compared to the HHT peak, the second peak inFigure 4 was relatively small. Due to the fact that the samples needed tobe diluted to a minimum ratio of 1:10, the concentrations of the secondpeak far exceeded the dynamic range of the standard curve, and thereforehad no effect on HHT binding. However, in order to analyse effectsarising from unknown sample matrices, the HPLC-RIA method should beused to evaluate possible background interferences. The assay was furthervalidated according to the procedure described by Granstrom and Kindahl(16). Serum samples were spiked with HHT in physiological concentra-tion ranges (49) and measured using the RIA and the HPLC-method (232nm) described above. The results, which correlate well with each other,are shown in Figure 5. The fact that the expected physiological increaseof HHT during blood coagulation and its inhibition by COX-inhibitors(Fig. 6) was observed would also appear to validate the assay.

The measured HHT serum-concentrations correlated with the concen-trations of TXB2 in a 1:1 ratio (Fig. 6). The analysis of HHT by radioim-munoassay, HPLC and HPLC-RIA described here may be useful for de-termination of HHT levels in various biological fluids or tissues. Thiscould help to shed additional light on the physiological role of HHT inbiological processes.

FIGURE 5. Correlation of HHT-RIA and HPLC results of spiked serum samples. Extract from thesolid-phase extraction of serum was chromatographed on Nucleosil 120 C18 (125 3 4.6 mmI.D.), 5-mm particle size, at a flow rate of 1.0 mL/min with an acetonitrile gradient from 55 to85% (v/v) at 30°C (232 nm).

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Acknowledgments

We thank Prof. Dr. Dr. Sibrowski, Institut fur Transfusionsmedizin,Westfalische-Wilhelms-Universitat Munster, Germany for kindly provid-ing us with platelet-rich plasma. We also thank U. Mende for providing uswith KHT. The GC/MS analyses were carried out by Dr. H. Luftmann, B.Wippich, and B. Sommer, Institute of Organic Chemistry, Westfalische-Wilhelms-Universitat Munster. This study was carried out as part of thework toward the doctoral thesis of H. John.

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Editor: Dr. E. Granstrom Received: 12-05-97 Accepted: 03-30-98

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