ANALYTICALBIOCHEMISTRY

187,151-l%

(19%))

Immunoaffinity Purification of 11 -Dehydro-thromboxane B2 from Human Urine and Plasma for Quantitative Analysis by Radioimmunoassay Yoko Hayashi, Fumiaki Shone,’ Shozo Yamamoto,2 Keiko Watanabe,? Kouwa Yamashita,? and Hiroshi

Wataru Takasaki,* Miyazakitv3

Akihiko

Nakagawa,*

Department of Biochemistry, Tokushima University School of Medicine, Kuramoto-cho, Tokushima 770, *Analytical Metabolic Research Laboratories, Sankyo Co. Ltd., Shinagawa-ku, Tokyo 140; and TResearch Laboratories, Pharmaceutical Division, Nippon Kayaku Co. Ltd., Kita-ku, Tokyo 115, Japan

Received

May

and

12,1989

1 l-Dehydro-thromboxane Bz is now considered to be a reliable parameter of thromboxane AZ formation in vivo. An immunoaffinity purification method was developed for radioimmunoassay of this compound contained in human urine and plasma. Monoclonal anti- 1 ldehydro-thromboxane Bz antibody was prepared and coupled to BrCN-activated Sepharose 4B. Human urine or plasma was applied to a disposable column of the immobilized antibody. After the column was washed with water, 1 l-dehydro-thromboxane B2 was eluted with methanol/water (95/5) with a recovery of more than 90%. The purified extract was subjected to a radioimmunoassay utilizing ll-[3H]dehydro-thromboxane Bz methyl ester and the monoclonal anti- 1 l-dehydrothromboxane B2 antibody. The detection range of the assay was lo-600 fmol (ICE0 = 90 fmol). The cross-reactivities of the antibody with thromboxane Bz, 2,3-dinor-thromboxane Bz , and other arachidonate metabolites were less than 0.05%. These compounds were efficiently separated from 1 1-dehydro-thromboxane Bz by the immunoaffinity purification. This procedure also allowed the separation of 1 l-dehydro-thromboxane Bz from unidentified urinary and plasma substances which interfered with the radioimmunoassay. Validity of the results obtained by the radioimmunoassay was confirmed by GC/MS employing selected ion monitoring for quantification. 0 1990 Academic Press, Inc.

’ On leave from Department of Pharmacy, Tokushima Hospital. * To whom correspondence should be addressed. 3 Present address: The Second Department of Internal Showa University, School of Medicine, Shinagawa-ku, Japan. 0003.2697/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form

University

Medicine, Tokyo 142,

In biological samples such as urine or plasma various types of prostaglandin (PG)* and thromboxane (TX) are found in extremely small quantities (pg-rig/ml). Therefore, a quantitative assay of these arachidonate metabolites requires techniques to extract these compounds from plasma or urine and to concentrate them to a level which meets the detectability of the assay to be employed. Furthermore, these arachidonate metabolites must be separated from miscellaneous unidentified compounds which crossreact with the antibody and disturb the assay. For these purposes several steps of extraction and purification are usually performed. However, such multistep procedures often result in the loss of the sample in unpredictable yields. TXA2 is a proaggregatory and vasoconstrictive compound relevant to the pathogenesis of thrombosis, vasospasm, and arteriosclerosis (1). Since TXA2 is a very labile compound (2) and cannot be measured as such, its hydrolysis product (TXB2) has been widely utilized as an index of the TXAz biosynthesis. However, no reliable value of blood TXB2 level could be determined due to technical difficulties during blood collection (3). 11-Dehydra-TXB, has recently been identified as a major metabolite of TXBz infused in human, and is now considered as the most appropriate analytical parameter to follow the endogenous synthesis of TXAz. It has a longer life than TXBz in the circulating blood, and its amount is not influenced artificially in the blood-sampling procedures (43). Recently we prepared a monoclonal antibody with a high affinity and specificity for ll-dehydroTXB2. We attempted to utilize the monoclonal antibody for immunoaffinity purification of 11-dehydro-TXB, from biological materials and for its immunoassay with 4 Abbreviations

used: PG, prostaglandin;

TX,

thromboxane. 151

Inc. reserved.

152

HAYASHI

a high sensitivity and selectivity. Part of the study has been presented earlier in a preliminary form (6). EXPERIMENTAL

PROCEDURES

11-Dehydro-TXB, and other arachidoMaterials. nate metabolites used in this work were gifts from Ono Pharmaceutical Company (Osaka). [3H]Methyl iodide (85 Ci/mmol), [5,6,8,9,11,12,14,15-3H]TXBz (180 Ci/ mmol), 6-[5,8,9,11,12,14,15-3H]keto-PGF,, (120 Ci/ mmol), [5,6,8,9,11,12,14,15-3H]PGFz, (160 Ci/mmol), [5,6,8,11,12,14,15-3H]PGE2 (143 Ci/mmol), [5,6,8,9,11, 12,14,15-3H]arachidonic acid (155 Ci/mmol), and [l14C]linoleic acid (56 mCi/mmol) werepurchased, and ll[5,6,8,9,12,14,15-3H]dehydro-TXB, (120 Ci/mmol) was kindly donated from Amersham International (Amersham). The procedure devised by Strife and Murphy (7) was modified to prepare 11-[‘80]dehydro-TXB,. The preparation was analyzed by GC/MS, and was found to be a mixture of 1803-, ‘s02-, and l80,-labeled variants in a ratio of 47.7:43.6:8.5. The content of nonlabeled lldehydro-TXB, was less than 0.3%. The 180-labeled lldehydro-TXBz was present in the lactonized form. Crown ether (dicyclohexane-18-crown-6) was purchased from Aldrich (Milwaukee), and Ultroser G (serum substitute for cell culture) from Reactifs IBF Sot. Chim. (Villeneuve-la-Garenne, France). Materials used to prepare monoclonal antibody (8) and to derivatize ll-dehydro-TXBz for GC/MS employing selected ion monitoring (9) were obtained as described previously. Sepharose 4B was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden), and Sepacol-Mini-PP column from Seikagaku-Kogyo (Tokyo). All other chemicals were of reagent grade. Production and purification of antibody. As will be described later, two forms of 11-dehydro-TXBz are in an equilibrium. As analyzed by HPLC, the compound in ethanol was found to be predominantly in a closed form (93%), the remaining 7% being in an open form. After evaporation of the solvent, the carboxyl group of ll-dehydro-TXBz (200 pug)was coupled to an amino group of bovine serum albumin (2 mg) by the N-succinimidyl ester method in the presence of phosphate buffer, pH 7.4 (10). The conjugate thus prepared was stored in the same buffer. Three female BALB/c mice 6 weeks of age were first immunized by intraperitoneal injection of the conjugate (5.5 pg of 11-dehydro-TXB,, 80 pg of protein), which was emulsified with an equal volume of complete Freund’s adjuvant. Two more injections were given every 2 weeks in the same way except that incomplete Freund’s adjuvant was used. The last immunogen was given in 0.2 ml of saline without adjuvant. The antibody was titrated using ll-[3H]dehydro-TXB, methyl ester, which was dissolved in phosphate buffer (pH 7.4) and therefore in an open form. Three days after the last antigen injection, the spleen was removed from a mouse with the highest titer of anti-

ET AL.

body. Fusion of the spleen cells with an aminopterinsensitive myeloma cell line (SP2/0-Ag14) was performed using 50% polyethylene glycol-1000 by the method of Goding (11). After a 12-day incubation in 96-well culture plates, the medium containing hypoxanthine, aminopterin, and thymidine was replaced by a medium containing hypoxanthine and thymidine in which Ultroser G substituted for serum. After a 2-day culture in this serum-free condition, an aliquot of the medium was removed for titration of antibody by radioimmunoassay using ll-[3H]dehydro-TXB, methyl ester. Selected hybridoma cells were cloned twice in soft agar according to the method of Kennet (12). A clone producing anti-11-dehydro-TXB, antibody was injected into the peritoneal cavity of BALB/c mice previously treated with pristane. The antibody in the ascites fluid was collected with ammonium sulfate at 50% saturation, and was further purified by the use of a protein A-Sepharose column (13). About 20 mg of IgG were obtained from one mouse. Preparation of labeled antigen. When we started this work, ll-[3H]dehydro-TXBz was not commercially available. Therefore, we prepared the [3H]methyl ester of 11-dehydro-TXBz by the method of Moonen et al., which was described for various PGs (14). Later Amersham International kindly provided us with a sample of 11-[5,6,8,9,12,14,15-3H]dehydro-TXB,, which was used in part of this work. 11-Dehydro-TXBz (20 pg) was dissolved in the dimethylformamide solution (20 ~1). A toluene solution of [3H]methyl iodide (85 Ci/mmol, 100 &i in 10 ~1) was added to the solution of 11-dehydro-TXBz. The mixture was incubated at 45°C for 30 min. 11-[3H]Dehydro-TXBz methyl ester (retention time, 27.0 min) was separated from the free acid form of unlabeled lldehydro-TXB2 (6.0 min) by reverse-phase HPLC using a PBondapak Cl8 column (3.9 X 330 mm) connected to a Waters dual-pump (Model 6000A) and a Waters injector (Model U-6K). The solvent system was a mixture of acetonitrile/water/acetic acid (35/65/0.1, v/v/v). The pH of the mixture was adjusted to 6.5 with NH40H. The flow rate was 1 ml/min. Unlabeled 11-dehydro-TXB2 methyl ester was prepared from 11-dehydro-TXBz using trimethylsilyldiazomethane by the method of Hashimoto et al. (15), and was used as a standard marker in the HPLC analysis. ll-[3H]Dehydro-TXB2 methyl ester thus prepared was kept in phosphate buffer at pH 7.4, 25°C for 1 week, and then analyzed by HPLC. Essentially all the radioactivity was recovered at a retention time corresponding to 11-dehydro-TXB, methyl ester. The results indicated that the methyl ester was stable under the preincubation condition prior to radioimmunoassay. Interconversion of the open and closed forms of 11-dehydro-TXB2. Two forms of 11-dehydro-TXBp are in equilibrium: the closed ring form (d-lactone) and the

IMMUNOAFFINITY

EXTRACTION

open acyclic form (dicarboxylic acid). As previously described (16), the ratio of the two forms varies depending on the pH of the medium. Higher pH favors the open form whereas lower pH favors the closed form. The two forms of 11-dehydro-TXBz were separated by HPLC after its incubation at various pH values for 24 h (16). The lactone form of ll-dehydro-TXB, was prepared by treatment of its solution with 0.05 N HCl (pH 1.5) for 16 h at room temperature. When the solution was kept at pH 10.8 in freshly prepared 0.2% Na2C03 for 2 h at 37°C the lactone was hydrolyzed to the open form of ll-dehydro-TXB,. The two forms of 11-dehydro-TXBz were clearly separated by reverse-phase HPLC using a TSKGEL column (type ODS-12OT, 4.6 X 250 mm). The solvent system was a mixture of acetonitrile/water/phosphoric acid (33/67/0.05, v/v/v). The flow rate was 1 ml/ min. Both the open form (prepared by alkali treatment) and the lactone form (prepared by acid treatment) produced single peaks with retention times of 14-16 min and 28-30 min, respectively. Preparation of immunoafinity column. Sepharose 4B was activated with cyanogen bromide (17). The activated Sepharose 4B (20-ml settled volume, corresponding to 5.7 g of dried powder) was suspended in 20 ml of 0.1 M NaHC03 (pH 9.0), and anti-11-dehydro-TXB, antibody (100 mg IgG in 10 ml of the same buffer) was added quickly. The mixture was rotated gently on a rotary evaporator at 4°C for 16 h. Remaining active groups were inactivated with 0.1 M Tris-HCl buffer (pH 8.0). The antibody-coupled Sepharose 4B was stored in physiological saline containing 10 mM sodium phosphate buffer, pH 7.4, 0.14 M NaCl, and 0.1% sodium azide (buffer A) and mixed with Sepharose 4B so that 0.8 ml of Sepharose 4B contained 300 pg IgG. A polypropylene column (Sepacol-Mini-PP, 9 X 65 mm) containing 300 pg of the antibody coupled to 0.8 ml of Sepharose 4B was used as a standard immunoaffinity column. Immunoafinity purification of 11-dehydro-TXB,. The standard immunoaffinity column was rinsed with buffer A (5 ml) before use. To urine, plasma, or authentic compound dissolved in buffer A was added 3H-labeled ll-dehydro-TXBz (ZOOO-20,000 cpm) as a tracer. The mixture was applied to the column. The column was washed with buffer A (10 ml) and water (5 ml). The eluates were discarded unless otherwise noted. The remaining water in the column was pushed off with gentle pressure. 11-Dehydro-TXB, was eluted with 10 ml of methanol/water (95/5, v/v). The solvent of the eluate was evaporated to dryness, and the residue was ready for radioimmunoassay. Urine was collected from a healthy male aged 41 years over a period of 24 h (1420 ml). The urine (10 ml) was mixed with 10 ml of water containing ll-[3H]dehydroTXB2 (2000 cpm, 28 fmol). The pH was adjusted to 10.5 with 2 N NaOH. After incubation at 37°C for 2 h to com-

OF

ll-DEHYDRO-THROMBOXANE

Bz

153

plete the hydrolysis of the lactone form of ll-dehydroTXBz, the pH was adjusted back to 7.8 with 2 N HCl. The sample thus prepared was passed through a 0.2~pm disposable filter. The filtrate was applied to the standard immunoaffinity column. Samples of human plasma from 18 healthy male individuals were combined. ll-Dehydro-TXBz was extracted from human plasma (10 ml) using a SEP-PAK Cis cartridge (Waters) according to the method of Powell (18). Methanol (100 ~1) was added to the dried residue of the SEP-PAK Cl8 extract, and the mixture was vortexed. Freshly prepared 0.2% Na2C03 (5 ml) was added, and the mixture was sonicated for homogenization. After incubation at 37°C for 2 h, the pH was adjusted to 7.8 with 0.1 N HCl. Buffer A (15 ml) was added. The sample thus prepared was applied to the standard immunoafinity column. A control experiment was carried out to determine the recovery of ll-dehydro-TXBz and to confirm that the compound was in the open form during the above-mentioned procedure. ll-[3H]Dehydro-TXBz (2.9 pmol, 211,000 cpm) dissolved in ethanol was a starting material. The solvent was evaporated, and the residue was dissolved in 5 ml of 0.2% Na&03. After incubation at 37°C for 2 h, the solution was brought to pH 7.8 by the addition of 2 N HCl. The recovery of radioactivity up to this step was 98%. The sample was applied to the standard immunoaffinity column as described above. The phosphate buffer used to wash the column and 95% methanol used to elute 11-dehydro-TXB, were counted for radioactivity. The recovery of the radioligand in the eluate was 94%. After evaporation of the solvent, the residue was taken up in 0.5 ml of phosphate buffer at pH 7.4 in a recovery of 90% over the starting material. After incubation at 24°C for 48 h, an aliquot of the sample (50 ~1)was applied to HPLC using the solvent system of acetonitrile/water/phosphoric acid (33/67/0.05, v/v/v). Radioactivity was detected in an elution time corresponding to the open form of 11-dehydro-TXB, in a recovery of 93% (84% over the starting material). No significant radioactivity was detected in the fraction corresponding to the closed form of ll-dehydro-TXB, and in any other fractions. The result indicated that lldehydro-TXB, was present in the open form and no significant amount of the compound was degraded during the extraction and purification procedures. For determination of the recovery of plasma ll-dehydro-TXB, at the step of SEP-PAK Cl8 extraction, ll[3H]dehydro-TXB, (11,300 cpm, 158 fmol) was added to 10 ml of human plasma. An aliquot of the methanol solution was counted for radioactivity, and the recovery of 11-dehydro-TXB, was calculated to be 92 + 2% (n = 10). After immunoaffinity purification of this SEPPAK Cis extract, the recovery of ll-dehydro-TXBz over the starting material was 85 * 3% (n = 10).

154

HAYASHI

ET AL.

Radioimmunoassay. All the reagents were dissolved umn chromatography. The derivative obtained from the plasma sample was further purified by another silica gel in buffer B (0.1 M sodium phosphate, pH 7.4, containing 0.1% NaN3), except for antibody, which was diluted with column chromatography using a mixture of n-hexane/ diethyl ether (8/2) as an eluting solvent. buffer B containing 0.1% ovalbumin. Authentic ll-dehydro-TXBz, ll-[3H]dehydro-TXB2 methyl ester, and the sample to be tested (the extract from human urine RESULTS or plasma described above) were preincubated sepaMonoclonal Anti-l 1-Dehydro-TX& Antibody rately at pH 7.4, 25°C for 48 h for complete hydrolysis A mouse was immunized with 11-dehydro-TXBz conof the lactone form. Then, the standard solution or the jugated to bovine serum albumin. The conjugate was sample to be tested (100 ~1) was mixed with 0.8% gelatin stored in phosphate buffer, pH 7.4. Therefore, the hap(50 PI), and incubated with ll-[3H]dehydro-TXBz ten 11-dehydro-TXB, was presumably in the open form methyl ester (7000 cpm, 137 fmol, 100 ~1) and anti-ll(see below). Spleen cells of the immunized mouse were dehydro-TXB, antibody (3.6 ng IgG, 100 ~1) at 4°C for fused with myeloma cells. At first a conventional me16 h. The immunocomplex was precipitated using polyethylene glycol, and radioactivity was determined essen- dium containing 20% fetal calf serum was used in the screening of antibody-producing hybridoma cells. Since tially as described for PGE2 (19). anti-11-dehydro-TXBz antibody could not be detected GC/MS. 11-Dehydro-TXB, was extracted from huin any well of the culture medium, we suspected the seman urine using an immunoaffinity column as described rum of containing a certain substance which disturbed above. A trace amount of ll-[3H]dehydro-TXB, (215 the reaction of the radiolabeled antigen with the anticpm, 3 fmol/ml urine) was added to urine as an internal body. Therefore, the medium was replaced by a serumstandard to estimate the recovery of the compound at free medium 2 days before the screening of antibody. the step of the extraction. The immunoaffinity-purified Anti-11-dehydro-TXB, antibody was now detected in 11 material was devided into two portions, and subjected to out of 165 wells (6.7% efficiency). A clone producing an radioimmunoassay and GC/MS. anti-11-dehydro-TXB, antibody was grown in the A mixture of authentic 11-dehydro-TXBz (0, 0.8, 1.6, mouse peritoneal cavity. The antibody contained in the 2.4, and 3.2 pmol) and 180-labeled 11-dehydro-TXBz (27 ascites fluid was purified to an immunoglobulin fraction. pmol) as an internal standard was added to the immuIn order to find which of the two forms of ll-dehydronoaffinity-purified material derived from 1 ml of human TXB2 (lactone and dicarboxylic acid) was recognized by urine and treated with ethereal diazomethane. The re- the antibody, we examined the pH-dependency of the sulting 11-dehydro-TXB, methyl ester was purified by antigen-antibody reaction. Prior to reaction with antisilica gel column chromatography using a mixture of n- body the 3H-labeled antigen was preincubated at various hexane/ethyl acetate (l/l) as an eluting solvent and pH values ranging from 4.5 through 8.5 at 25°C for 24 h. then converted to its dimethyl-i-propylsilyl ether derivaFigure 1 shows dilution curves of anti-11-dehydro-TXB, tive (20). The derivatized 11-dehydro-TXBz (the lactone reacting with the 3H-labeled antigen pretreated with form) was dissolved in n-hexane including 0.5% pyribuffers of various pH values. The amount of antibody dine, and then subjected to GC/MS employing selected which gave 50% of the maximum binding of the antigen ion monitoring as described previously (20). GC/MS was was plotted against pH values (inset in Fig. 1). Because carried out under a mass spectrometric resolution of of its higher affinity around neutral pH, the antibody 4000 (M/AM). For quantitation we used the ions of [Mwas considered to recognize the open form of ll-dehy43]+ at m/z 539.32 for 11-dehydro-TXBP and at m/z dro-TXBz on the basis of an earlier experiment by Kum543.33 for ll-[‘80]dehydro-TXBz as an internal stan- lin and Granstrijm (16). In our experiment the affinity of dard. A standard curve was prepared from the plots of antibody for ll-dehydro-TXBz decreased slightly when the peak area ratio (m/z 539.321543.33) against the the pH was raised from 7.5 to 8.5 (inset in Fig. 1). A simamount of 11-dehydro-TXBz (O-270 fmol). The detec- ilar observation was described in the paper of Kumlin tion limit of the method was about 5.4 fmol with a signal- and Granstrijm with the statement “The reason for the to-noise ratio of 5:l. apparent decrease in titer at the high extreme pH is not In separate experiments, urine or plasma was mixed clear” (16). As a control experiment, PGF2, was preincuwith lag-labeled internal standard and known amounts bated at various pHs and then incubated with antiof 11-dehydro-TXB,. The mixture was applied to a PGF2, antibody. However, the antibody did not show Chem-Elut and a SEP-PAK Cl8 cartridge, successively, such a pH dependency as described above for anti-llas described previously (20). The extract was treated dehydro-TXBB antibody. On the basis of these findings with diazomethane and then purified by silica gel col- we concluded that our antibody recognized the open umn chromatography. After derivatization of the resultform of 11-dehydro-TXBz. Therefore, ll-[3H]dehydroTXBZ methyl ester (the labeled antigen) and the saming methyl ester with dimethyl-i-propylsilyl-imidazole, the excess reagent was removed by Sephadex LH-20 col- ples containing 11-dehydro-TXBz were preincubated at

IMMUNOAFFINITY

EXTRACTION

OF

ll-DEHYDRO-THROMBOXANE

155

B2

with an association constant of 6.3 X 10’ and 6.5 X 10’ M -l, respectively. Cross-reactivities of the antibody with other eicosanoids and their metabolites were examined in the presence of the standard amount of the radioligand. The following showed cross-reactivities of less than 0.05%: TXBz (Fig. 2, closed circles), 2,3-dinor-TXBz, PGBz, PGD2, PGEz, PGFz,, 6-keto-PGF,,, 15-keto-PGFza, 13,14-dihydro-15-keto-PGE,, and 13,14-dihydro-15keto-PGF,, . Efficiency and Specificity of Immunoafinity Extraction of 11 -Dehydro-TXB2

FIG. 1. Dilution curves of anti-11-dehydro-TXBz antibody. The indicated amounts of antibody were incubated with the standard amount of ll-[3H]dehydro-TXB, methyl ester which had been pretreated at ‘25°C for 24 h with buffers at various pHs as follows: pH 4.5, closed circles; pH 5.0, crosses; pH 5.5, inverted open triangles; pH 6.0, closed squares; pH 6.5, open squares; pH 7.0, open triangles; pH 7.5, open circles; pH 8.0, closed triangles; pH 8.5, inverted closed triangles. Polyethylene glycol used for immunoprecipitation was dissolved in 0.1 M Tris-HCl at pH 7.5. Inset: pH dependency of immunoreactivity of anti-11-dehydro-TXB, antibody with the labeled antigen. The amount of antibody which gave 50% of maximum binding of the antigen, was plotted against pH values.

pH 7.4 at 25°C for 48 h before the reaction with the antibody so that the lactone ring of 11-dehydro-TXBz could be opened by hydrolysis.

Human urine or blood plasma containing a trace amount of ll-[3H]dehydro-TXBP (the open form) was applied to the standard immunoaffinity column. The column was washed with 10 ml of buffer A and 5 ml of water, and then 11-dehydro-TXB, was eluted with organic solvent mixtures listed in Table 1. Radioactivity in the wash and the eluates was determined. Il-DehydroTXB2 was eluted efficiently with a recovery of greater than 90% with acetonitrile/water (80/20) or methanol/ water (95/5). The latter solvent was chosen for the standard condition since it contained a lesser amount of water, and methanol was evaporated more easily. With methanol/water (95/5) as a standard elution solvent, recovery of radioactive 11-dehydro-TXB, added to 10 ml of sample was 92.3 -+ 1.8% (n = 10) for urine and 91.9 f 1.9% (n = 10) for plasma. The use of 100% methanol did not give a reproducible elution.

Radioimmunoassay Binding of ll-[3H]dehydro-TXB2 methyl ester to the monoclonal antibody was investigated in the presence of various amounts of unlabeled free form of ll-dehydroTXBz. When the antibody was preincubated at different pHs (5.5, 6.5, 7.4, and 8.5), the sensitivity of the assay was maximal at pH 7.4 as shown in Fig. 2. Il-DehydroTXBz was detectable over the range of 10 to 600 fmol (90 and 10% of maximum binding, respectively). The I(& was 90 fmol. Three groups of 10 tubes containing 25, 100, and 400 fmol of 11-dehydro-TXB, were subjected to the standard radioimmunoassay, giving intraassay coefficients of variation of 6.9, 4.7, and 8.1%, respectively. Three groups of 5 tubes containing 25,100, and 400 fmol of lldehydro-TXB, were examined by radioimmunoassay, and reexamined four times during a period of 2 months, when interassay coefficients of variation were found to be 15.1, 13.7, and 17.2%, respectively. Affinity of the antibody for the antigen was estimated by Scatchard plots of the binding data with ll-[3H]dehydro-TXBP and its methyl ester. The monoclonal nature of the antibody was obvious since the plots were linear and the antibody had a single class of binding site

I I-Dehydro-TX&

( pmol )

FIG. 2. Calibration curves for 11-dehydro-TXB,. The standard radioimmunoassay was performed in the presence of various amounts of authentic ll-dehydro-TXB, which had been preincubated at pH 5.5 (closed triangles), 6.5 (open triangles), 7.4 (open circles), and 8.5 (crosses) as described in Fig. 1. The antibody was used in the amount which gave 50% maximum binding of the antigen as shown in Fig. 1. Various amounts of TXBp were also subjected to the standard radioimmunoassay at pH 7.4 (closed circles). B, bound radioactivity at various doses of 11-dehydro-TXB,; B,,, bound radioactivity at zero dose.

156

HAYASHI ET AL. TABLE

1

TABLE

Elution of 11-Dehydro-TXB2 from the Immunoaffinity Column with Various Solvents Recovery

Hz0 Urine

Plasma

(%)

Organic

4 5 5 4 4 4 5

(80:20), (90:10), (95:5), (75:25), (85:15), (95:5), (95:5),

Loss 91 46 10 80 84 92 92

(%) 5 49 85 16 12 4 3

Note. The open form of ll-[3H]dehydro-TXBZ (19,620 cpm, 272 fmol) was added to urine (10 ml) or plasma (5 ml). The mixture was applied to the standard immunoaffinity column. Radioactivities in the washes with water and the eluates with organic solvents were determined in duplicate, and the mean values were presented.

Compound

The closed and open forms of radiolabeled ll-dehydro-TXB, were prepared by the acid and alkali treatments, respectively, and applied to the standard column at pH 7.4. As predicted from the above-mentioned result of the pH-dependency study of the antibody, when the closed form was applied to the column, more than 90% of the radioactivity was found in the water fraction (Fig. 3A). In contrast, more than 90% of the open form adsorbed to the column, and appeared in the methanol elu-

(A)

1

0

0.27

27

2700

Solvent

92 4 96 2 94 5 94 3 94 2 95 2 96 2

W’ MeOH/HxO Hz0 MeOH/HZO I-W MeOH/H20 Hz0 MeOH/HxO Hz0 MeOH/HxO Hz0 MeOH/H*O Hz0 MeOH/H*O

% Recovery 11-Dehydro-TXB, TXBx 6-Keto-PGF,, PGFze PGEx Arachidonic Linoleic

r

and Related Substances to Column

pm01 solvent

CHsCN/H20 CHsCN/H1O CHsCN/HZO CHs0H/H20 CHsOH/HxO CHsOH/H1O CHs0H/H20

2

Application of 11-Dehydro-TXB, Immunoaffinity

acid acid

3 93 95 4 94 4 92 4 93 3 94 3

5 92 95 4 93 5 95 2 94 3 92 5 91 4

Note. ll-[3H]Dehydro-TXB2 (open form, 17,100 cpm, 237 fmol) or each of the other tritiated substances dissolved in buffer A was applied to the standard immunoaffinity column, and the radioactivities in the wash with water and the eluate with methanol/water (95/5) were determined. Each value was a mean of duplicate determinations.

ate (Fig. 3B). Thus, only the open form of ll-dehydroTXBz was bound to and eluted from our immunoaffinity column. The 0.8-ml column containing 0.1 mg, 0.3 mg (the standard immunoaffinity column) and 1 mg of IgG could bind at least 4, 30, and 100 pmol of ll-dehydroTXBz , respectively. Several radiolabeled related substances other than 11-dehydro-TXBZ passed through our immunoaffinity column (Table 2). Radioimmunoassay of the Immunoafinity-Purified Urinary 11 -Dehydro-TXB,

I )L J&l=] 0 I.1 I IO 100 Charged I I-Dehydro-TXBP

L 1000

(pmol)

FIG. 3. Extraction of the closed and open forms of ll-dehydroTXBz. The closed form (A) and the open form (B) of the radioactive or unlabeled ll-dehydro-TXB, were prepared by acid and alkali treatments, respectively, as described under Experimental Procedures. Each solution was passed through the standard immunoaffinity column at pH 7.4. The radioactivity was determined for the wash with water (closed circles) and the eluate with methanol/water (95/5) (open circles).

When human urine as such was subjected to radioimmunoassay of 11-dehydro-TXB,, a linearity was not observed between the amount of urine and the value of 11-dehydro-TXBP (Fig. 4, open circles). The amount of 11-dehydro-TXBz per unit volume of urine increased as the amount of urine was raised. The observation suggested the presence of certain endogenous compound(s) which disturbed radioimmunoassay and gave an apparently higher value of 11-dehydro-TXBz. The urine was applied to the standard immunoaffinity column. The radioimmunoassay of 11-dehydro-TXB, was performed with the passthrough water fraction which should not contain 11-dehydro-TXBs. The result showed an apparent presence of a large quantity of immunoreactive material in the fraction (Fig. 4, closed circles). It seemed as if more than 0.4 pmol of ll-dehydro-

IMMUNOAFFINITY

Urine

EXTRACTION

(yl)

FIG. 4. Radioimmunoassay of ll-dehydro-TXB, contained in human urine and the purified urinary extract. Varying amounts of human urine (open circles), the immunoaffinity-purified urinary extract (crosses), and the passthrough fraction (closed circles) were subjected to the standard radioimmunoassay for 11-dehydro-TXB,. The amount of the extract was expressed in terms of the original volume of urine.

TXB2 was contained in the passthrough fraction derived from 100 ~1 of urine. In contrast, the immunoaffinitypurified material gave much lower values (0.15 pmol per 100 11 of human urine), and the amount of ll-dehydroTXBp determined by radioimmunoassay increased linearly depending on the amount of the purified material (Fig. 4, crosses or Fig. 5A). Radioimmunoassay was also performed with the purified material to which known amounts of authentic 11-dehydro-TXB, were added. There was a good correlation between the added (x) and the measured (y) values (y = 1.013~ + 1.44, r = 0.996) (Fig. 5B). The added 11-dehydro-TXB, was recovered with an average yield of 101.0 + 8.0% (mean f SD). The endogenous level of 11-dehydro-TXB, was 1.44 + 0.31 pmol/ml of urine (mean + 95% confidential limit) as calculated by the orthogonal polynomial equation (21). Validity of the radioimmunoassay of the immunoaffinity-purified urinary 11-dehydro-TXB, was confirmed by GC/MS. As presented in Fig. 5C, the authentic compound added in various amounts was recovered with an average yield of 104.5 f 5.0% (mean * SD), and a good correlation was observed between the added amount and the measuredvalue (r = 0.997). The endogenous level of 11-dehydro-TXB, was 1.82 + 0.26 pmol/ml of urine (mean f 95% confidential limit) as estimated from the intercept of the plots in Fig. 5C. This value was not far from the value of 1.44 + 0.31 pmol/ml determined by the radioimmunoassay (Fig. 5B). When the data of Figs. 5B and 5C were replotted, the values measured by radioimmunoassay (y) and GC/MS (x) correlated satisfactorily (y = 0.94r - 0.26 pmol/ml urine, r = 0.998). In the above-mentioned sample preparation for GC/MS, the 180-labeled internal standard was added after immunoafiinity purification as described under Experimental Procedures. As an alternative experiment, urine was

OF

ll-DEHYDRO-THROMBOXANE

Bz

157

mixed with internal standard and then subjected to successive chromatographies by Chem-Elut, SEP-PAK C!i8, and silica gel. In this method 11-dehydro-TXB, remained as the lactone form. The sample thus prepared was examined by selected ion monitoring and gave values 1.63 & 0.05 pmol of 11-dehydro-TXB,/ml of urine (n = 3). When TXB2 was infused in human, 15-keto-13,14dihydro-11-dehydro-TXBz was detected in plasma and urine in a quantity nearly equal to the amount of lldehydro-TXB, (4,5). It was possible that this compound was extracted from urine together with its precursor by immunoaffinity chromatography using the anti-ll-dehydro-TXBz antibody, which might cross-react with the 15-keto-13,14-dihydro derivative. Namely, we may have extracted and measured the two compounds together. The immunoaffinity-purified material was applied to HPLC using a solvent system of acetonitrile/water/ phosphoric acid (33/67/0.05, v/v/v). When we examined many fractions eluted from the column, our radioimmunoassay detected only one peak in a retention time corresponding to 11-dehydro-TXB,, and there was no immunoreactive material eluted later than ll-dehydroTXBz. According to a previous report, 15-keto-13,14-dihydro-11-dehydro-TXBz was detected in a longer retention time than 11-dehydro-TXBz upon HPLC using essentially the same solvent system (5).

Urine (ml)

2 2 Added I I-Dehydro- TX& ( pmol/ml urine 1

FIG. 5. Determination of the immunoaffinity-purified urinary lldehydro-TXBz by radioimmunoassay and GC/MS. To 200 ml of human urine was added ll-[3H]dehydro-TXB, (43,000 cpm, 602 fmol). Each lo-ml portion was applied to a standard immunoaffinity column. Eluates with 95% methanol from all the columns were combined, and the solvent was evaporated. The dried residue was taken up in ethanol. Two aliquots were removed for determination of radioactivity, The volume of the ethanol solution was adjusted so that the sample could be concentrated by lo-fold. The recovery of ll-[3H]dehydro-TXBz estimated with the two aliquots was 89.5 and 93.4%, namely, an average of 91.5%. (A) Varying amounts of the immunoaffinity-purified extract were subjected to the standard radioimmunoassay. The amount of extract was presented in terms of the volume of urine from which the sample derived. To the purified materials derived from 100 ~1 (B) and 2 ml (C) of urine were added known amounts of authentic ll-dehydroTXBz, and the mixtures were analyzed by radioimmunoassay (B) and GC/MS employing selected ion monitoring (C) as described under Ex11 _

penmentalr’rocedures.

158

HAYASHI

ET

AL.

by radioimmunoassay Cy)and GC/MS (x) correlated satisfactorily (y = 1.03x + 6.5 fmol/ml plasma, r = 0.993). DISCUSSION

FIG. 0. Radioimmunoassay and GC/MS of 11-dehydro-TXB, in human plasma. (A) Varying amounts of the material extracted by immunoaffinity chromatography (closed circles) and the material purified by immunoaffinity chromatography after SEP-PAK Cl8 extraction (open circles) were subjected to the standard radioimmunoassay. The amount of the material was corrected for the recovery mentioned under Experimental Procedures and expressed in terms of the original volume of plasma. (B) The material purified by immunoaffinity chromatography after SEP-PAK C,s extraction from 3 ml of plasma was mixed with known amounts of authentic ll-dehydro-TXB2. The mixtures were analyzed by radioimmunoassay. (C) To 20 ml of plasma were added known amounts of ll-dehydro-TXB, and “O-labeled internal standard, and 11-dehydro-TXB, contained in the mixture was purified and derivatized and analyzed by GC/MS as described under Experimental Procedures.

Radioimmunoassay of the Immunoafinity-Purified Plasma 11 -Dehydro-TX& When 11-dehydro-TXBz was extracted from human plasma only by the use of an immunoaffinity column, the extract still contained certain substance(s) which disturbed the radioimmunoassay and gave a significantly high level of 11-dehydro-TXBz as shown in Fig. 6A (closed circles). The interfering substance could be removed by SEP-PAK Cl8 extraction prior to the immunoaffinity purification. There was a linearity between the amount of the material thus purified and the value measured by radioimmunoassay (Fig. 6A, open circles). When ll-dehydro-TXB:, in varying amounts was added to a given amount of the purified plasma extract, and the mixtures were assayed by radioimmunoassay, the results showed a good correlation between the added and the measured values (y = 0.97x + 13.8, r = 0.993) (Fig. 6B). From the intercept of the plots the 11-dehydro-TXB2 content of the plasma was estimated to be 13.8 fmol/ml. For GC/MS analysis of plasma sample “O-ll-dehydro-TXBz was added to plasma,, and the mixture was purified by a series of chromatography as described under Experimental Procedures. As presented in Fig. 6C, the added and measured amounts of 11-dehydro-TXB, showed a good correlation (y = 0.91x + 7.6, r = 0.986). The endogenous level of 11-dehydro-TXB, was 7.6 fmol/ ml plasma as calculated by the orthogonal polynomial equation. This value was not far from the value determined by radioimmunoassay (Fig. 6B). When the data of Figs. 6B and 6C were replotted, the values measured

Purification of bioactive arachidonate metabolites from urine or plasma for their quantitative determination generally includes solvent extraction or silica solid-phase extraction followed by one or more chromatographic steps such as TLC and HPLC (22). Such multiple-step purification procedures are laborious and time-consuming. Sometimes the recovery of the compound is low and varies from experiment to experiment. As a simple purification method of arachidonate metabolites we attempted to develop an immunoaffinity purification of 11-dehydro-TXBz using a monoclonal antibody raised against this compound. Several papers have recently reported immunoaffinity purification of arachidonate metabolites from human urine or plasma: TXBP (23,24), 2,3-dinor-TXB, (23), 6-keto-PGF,, (25), and chemically stable PGIz analogues (26,27). All these works employed GC/MS analysis, which itself includes the sample separation by gas chromatography. Our efforts were directed toward the cleanup of the urinary and plasma 11-dehydro-TXBz for subsequent radioimmunoassay which is a more convenient method for routine laboratory assay. One of the important components of the radioimmunoassay system is the labeled antigen with high specific radioactivity. When we started this work, the labeled lldehydro-TXB, was not commercially available. Therefore, we prepared ll-[3H]dehydro-TXBz methyl ester using [3H]methyl iodide. The radiolabeled ligand can be prepared by this method at a very low cost (about one thousandth the price of the now commercially available [3H]ll-dehydro-TXBP). The longer half life of 3H as compared with that of 1251,is another advantage for the use of the tritiated ligand. This preparation is useful for the radioimmunoassay of a large number of samples. In the radioimmunoassay using this labeled ligand and our monoclonal antibody, the detection limit (10 fmol, 3.3 pg) was within the same order as the values reported by other radioimmunoassays: 4.1 fmol by the use of a polyclonal antibody and biologically prepared ll- [3H] dehydro-TXB2 (16,28), and 14 fmol using 11-[1251]dehydro-TXB, tyrosine methyl ester (29). The values of endogenous ll-dehydro-TXB2, which we determined by radioimmunoassay, were 1.44 pmol/ ml of human urine (2.1 nmol/day) and 13.8 fmol/ml of human plasma. The basal level of 11-dehydro-TXB, in human urine and plasma has been reported in several papers: 1.4-2.7 nmol/day (urine) and less than 14 fmol/ ml of plasma (detection limit) by radioimmunoassay of an aliquot of urine or plasma without purification (16); 16 pmol/h (urine) and less than 14 fmol/ml of plasma (detection limit) by radioimmunoassay (28); 3.2 nmol/

IMMUNOAFFINITY

EXTRACTION

OF

day (urine) and 2.2-6.8 fmol/ml of plasma by GC/MS (30); 2.4-4.9 fmol/ml of plasma by GC/MS (31); 1.4 f 0.8 nmol/g creatinine (urine) by GC/MS with sample cleanup on phenylboronate cartridges (32). In Refs. (28,30,31), SEP-PAK Cl8 extraction followed by TLC was employed for purification of ll-dehydro-TXBz. The values we obtained were not far from these literature values. Extensive work is required to determine the normal range of urinary and plasma levels of 11-dehydro-TXB, in healthy subjects. Previous investigators have reused the immunoaffinity column many times, and checked the change in extraction efficiency and binding capacity of the column throughout the repeated uses (23-27). Our monoclonal antibody against 11-dehydro-TXB, has a constant specificity and affinity, and can be supplied in unlimited quantity. As an advantage of the use of monoclonal antibody, we can prepare disposable immunoaffinity columns. About 70 standard immunoaffinity columns (300 pg IgG/column) can be prepared with 20 mg IgG, which is produced in the peritoneal cavity of one mouse. ACKNOWLEDGMENTS We are grateful to Dr. Michael McCabe for his critical reading of this manuscript. S.Y. was supported by Grants-in-Aid for scientific research from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan, and grants from the Japanese Foundation of Metabolism and Diseases, Takeda Science Foundation, the Mochida Memorial Foundation, and the Tokyo Biochemical Research Foundation.

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