42
ASSAYS
[5]
urine, together with a fractional elimination comparable to that of 2,3dinor-TxB2, make these measurements an extremely convenient approach to monitoring thromboxane production both in healthy and disease conditions. The unique chemical properties of 11-dehydro-TxB2 are associated with greater than usual specificity of the immunological recognition, which combined with adequate chromatographic procedures, can allow reliable assessment of metabolite levels in complex biological fluids. Acknowledgments The expert editorial assistance of G. Protasoni and M. L. Bonanomiis gratefullyacknowledged.Our studies were supportedby grants from ConsiglioNazionaledelleRicerche (Progetto Finalizzato MedicinaPreventivae Riabilitativa).
[5] M e a s u r e m e n t o f T h r o m b o x a n e M e t a b o l i t e s b y G a s Chromatography-Mass Spectrometry
By FRANCESCACATELLA and GARRET A. FITZGERALD Thromboxane (Tx)A2 is a potent vasoconstrictor and proaggregatory substance that, by virtue of these properties, may play a role in human syndromes of vascular occlusion. Consistent with this concept, aspirin (acetylsalicylic acid) which inhibits TxA2, has proved to be beneficial in syndromes associated with platelet activation, such as unstable angina. Biochemical assessment of thromboxane biosynthesis complements the use of pharmacological probes to elucidate the biological importance of this compound. Unfortunately, due to its chemical instability, TxA2 cannot be directly measured in biological fluids. Its hydration product, TxB2, is more stable, but its assessment in plasma is confounded by artifactual formation during blood withdrawal. 1-3 Although urinary excretion of TxB2 may be used as an index of systemic TxA2 formation in certain settings, such as platelet activation, it largely reflects renal production under physiological conditions. i G. A. FitzGerald, A. K. Pedersen, and C. Patrono, Circulation 67, 1174 (1983). 2 E. G r a n s t r o m , P. W e s t l u n d , K. K u m l i n , and A. N o r d e n s t r o m , Adv. Prostaglandins Thromboxane Leukotriene Res. 15, 67 (1985). 3 F. Catella, D. Healy, J. L a w s o n , and G. A. FitzGerald, Proc. Natl. Acad. Sci. U.S.A. 83, 5861 (1986).
METHODS IN ENZYMOLOGY,VOL. 187
Copyright© 1990by AcademicPress, Inc. All rights of reproduction in any form reserved.
[5]
43
MEASUREMENT OF THROMBOXANE METABOLITES OH
OH II-OH- DEHYDROGENASE
OH II-DEHYDRO-TxB 2
"~o"""'~~COOH H O / ' ~ ' - , - O ~ OH TxB 2
Ifl-OXIDATION OH z
~
" " ~
COOH
H0 .,,,"'--0 ~ OH
2,5-DINOR-TxB 2 Fro. 1.2,3-Dinor-TxB2 and 11-dehydro-TxB2 are indexes of the two major pathways of metabolism of TxB2.
Roberts e t a l . 4 identified twenty compounds formed via two major and one minor pathway of metabolism, by infusing [3H]TxB2 in a healthy volunteer. Indexes of the two major pathways are 2,3-dinor-TxB2, metabolized via/3-0xidation, and l l-dehydro-TxBz, resulting from dehydrogenation of the hemiacetal alcohol group at C-11 (Fig. I). More recently, these metabolites have also been shown to be the major metabolites of TxA2 itself in the monkey. 5 The small concentrations of these metabolites in plasma render lldehydro-TxB2 the only realistic target for analysis in plasma with available methodology. 3 However, measurement of either metabolite in urine has proved to offer a reliable index of in vivo TxA2 formation by platelets, although the kidney is likely to possess the capacity to form both the dinor and dehydro derivatives.6 Urinary excretion of both metabolites is almost, but not completely, inhibited following chronic administration of aspirin at a dose which completely inhibits platelet cyclooxygenase. 7 Furthermore, their excretion is significantly elevated in syndromes associated with platelet activation, such as peripheral vascular disease, unstable angina, thrombolysis, and systemic sclerosis. 4 L. J. Roberts, B. J. Sweetman, and J. A. Oates, J. Biol. Chem. 256, 8384 (1981). 5 p. Patrignani, H. Morton, M. Cirino, A. Lord, L. Charette, J. Gillard, J. Rokach, and C. Patrono, Clin. Res. 37, 341A (1989). 6 A. Benigni, C. Chiabrando, N. Perico, R. Fanelli, C. Patrono, G. A. FitzGerald, and G. Remuzzi, Am. J. Physiol. 257, F77 (1989). 7 F. Catella and G. A. FitzGerald, Thromb. Res. 47, 647 (1987).
44
ASSAYS
[5]
Gas Chromatography-Mass Spectrometry versus Immunoassay
Measurement of 2,3-dinor-TxBz and 1l-dehydro-TxB2 can be accomplished by gas chromatographic (GC)-mass spectrometric (MS) techniques or by radioimmunoassays (RIA). Enzyme immunoassays, already available for l l-dehydro-TxB2, are currently being developed for 2,3dinor-TxB2. The immunoassays can usually be performed on a larger scale in a relatively short time and do not require such expensive equipment. On the other hand, these methods may generate misleading results. This is generally due to lack of specificity resulting from inappropriate purification or from the use of antisera with high cross-reactivity with other metabolites of related compounds. To avoid these problems, any new immunoassays should be validated by characterization of the thin-layer chromatography (TLC) pattern of distribution of the extracted eicosanoid-like immunoreactivity, by using multiple antisera and by comparison with GC-MS determinations. RIA results which are highly comparable with GC-MS determinations have indeed been attained when the metabolites have been properly purified from biological matrixes and a highly specific antiserum has been employed, s'9 On the other hand, even highly validated immunoassays have practical limitations. Recently, for example, fish oils, rich in the oJ-3 fatty acid, eicosapentaenoic acid (EPA), have been extensively investigated for their potential antithrombotic properties. Dietary supplementation with fish oil results in decreased formation of TxA2 (derived from arachidonic acid; AA), and increased TxA3 (derived from EPA and reported to be less thrombogenic than TxA2). Commonly available immunoassays cannot discriminate between eicosanoids derived from AA and those derived from EPA, which differ only by one double bond. By contrast, GC-MS techniques can easily discriminate between these two series of compounds, l0 Another limitation of immunoassay methods overcome by GC-MS is their poor sensitivity. It was suggested that analysis of eicosanoids in plasma could facilitate the investigation of their temporal formation in vivo: this would be desirable when phasic eicosanoid formation might be of interest, such as during ischemic episodes in patients with unstable s C. Patrono, G. Ciabattoni, F. Pugliese, I. A. Blair, and G. A. FitzGerald, J. Clin. Invest. 77, 590 (1986). 9 G. Ciabattoni, J. Maclouf, F. CateUa, G. A. FitzGerald, and C. Patrono, Biochim. Biophys. Acta 918, 293 (1987). l0 H. R. Knapp, I. A. G. Reilly, P. Alessandrini, and G. A. FitzGerald, N. Engl. J. Med. 314, 937 (1986).
[5]
45
MEASUREMENT OF THROMBOXANE METABOLITES
100%=43952
I
100%=25206784
1~0 2~0 300 400 560 ~0
t~
/
RIC=511
RIC=515
7~0 860 9~0 1000~-~00 1200 1300 1400 1500
FIG. 2. Selected-ion monitoring of 11-dehydro-TxB2 (m/z 511) and its tetradeuterated internal standard (m/z 515). The plasma concentration of this sample corresponds to 1.5 pg/ml. (By permission, Ref. 3.)
angina. As far as TxA2 is concerned, plasma concentrations of its hydration product, TxB2, are readily confounded by e x v i v o platelet activation. 1-3 However, 11-dehydro-TxB2, a long-lived enzymatic metabolite in plasma, H has been shown to minimize this problem. 3 Its measurement in plasma requires a detection limit of less than 1 pg/ml. This can be attained by capillary gas chromatography-negative-ion chemical ionization mass spectrometry (GC-NCI-MS) (Fig. 2), but it is far below the limit of sensitivity achieved by most commercially available immunoassays. Combined analysis of 11-dehydro-TxB2 and 2,3-dinor-TxB2, indexes of the two major pathways of thromboxane metabolism in humans, permits one to distinguish between altered metabolism and increased biosynthesis of TxA2.7 Moreover, combined analysis of metabolites in plasma and urine can be utilized to identify alterations in the volume of distribution and renal clearance of TxA2, as might occur due to drug administration or disease. On the other hand, although the methods have been refined, GC-MS remains a time-consuming approach. Samples have to undergo extraction, purification, and derivatization prior to injection onto the instrument. 11 j. Lawson, C. Patrono, G. Ciabattoni, and G. A. FitzGerald, Anal. Biochem. 155, 198 (1986).
46
ASSAYS
[5]
Internal Standards The first step in quantitative analysis by GC-MS is to spike the samples with an internal standard. This permits accurate quantitation, irrespective of losses during purification or due to incomplete derivatization. The most satisfactory standards are analogs incorporating stable isotopes, because they have physiochemical properties almost identical to their natural isomers. 12 Some stable isotopes are commercially available (Biomol, Plymouth Meeting, PA; Cayman Chemicals, Ann Arbor, MI). The remainder must be chemically or biologically synthesized. Deuterated 11-dehydro-TxB2 can be biologically synthesized by incubation of deuterated TxB2 with the high-speed supernatant of guinea pig liver homogenate in the presence of NAD. ~t Deuterated 2,3-dinor-TxB2 can be produced by prolonged incubation of the tetradeuterated TxB2 in a culture of Mycobacterium rhodochrous, t3 Another example of biological generation of standards is the formation of the dinor metabolite of tetradeuterated 6-keto-PGFt~ by cultured hepatocytes, t4 Alternatively, 2,3-dinor-TxB2 and 11-dehydro-TxB2 can be labeled by exchange of the two carboxylic oxygen atoms with 180 using labeled water as a donor, t5 These exchange reactions can be catalyzed by acid, base, or enzymatically via esterases as described by Murphy and Clay. t6 A limitation to the use of tso-labeled compounds is the back-exchange loss of the label that can occur rapidly in plasma and tissues containing esterases. This undesired enzymatic exchange does not represent a problem in urine, due to the lack of esterase. In other biological matrixes, it can be avoided easily by lowering the pH of the medium to approximately 3.5 before adding the taO-labeled standard. Extraction
The selective extraction of 2,3-dinor-TxB2 can be achieved by relying on the chemical properties of the thromboxane ring; in a pH-dependent equilibrium, it exists with an open ring form. At pH 5, the open form, stabilized by derivatization as the methoxime, results in a hydroxyl con12 W. A. Garland and M. L. Powell, J. Chromatogr. Sci. 19, 392 (1981). 13 F. F. Sun, B. M. Taylor, F. H. Lincoln, and O. K. Sebek, Prostaglandins 20, 729 (1980). ~4 M. Balazy, E. P. Brass, J. G. Gerber, and A. S. Nies, Prostaglandins 36, 421 (1988). x5 H. J. Leis, E. Malle, R. Moser, J. Nimpf, G. M. Kostner, H. Esterbauer, and H. Gleispach, Biomed. Environ. Mass Spectrom. 13, 483 (1986). 16 R. C. Murphy and K. L. Clay, this series, Vol. 86, pp. 547.
[5]
47
MEASUREMENT OF THROMBOXANE METABOLITES
H O
H O "~OH ~ ..
- -
H
R
0 0 H
H O R
>
MOHCI
N 0" CH3
R
Ac-
H
R H
O-~,R /
METHOXIME FORMATION IN URINE /OH STAT SUPPORT IONARY _ N ~ N
~~ ~ / B ' ' O H
R +
R R
> R - B \/o
R
HO FIG. 3.2,3-Dinor-TxB2 exists in equilibrium with an open ring form which is derivatized as the methoxime. Following derivatization, the hydroxyl configuration permits condensation with bonded-phase phenylboronic acid to form a stable complex. (By permission, Ref. 17.)
figuration favorable to the formation of a stable complex with bondedphase phenylboronic acid (Fig. 3). ~7This procedure has been employed for the purification of prostaglandin F metabolites as well as for carbohydrate and catecholamine analysis. Reagents and Materials Methoxyamine Hydrochloride (MOHCI) (Sigma, St. Louis, MO) Phenylboronic acid bonded-phase (PBA) columns (Infolab, Fort Oglethorpe, GA) Prep-Sep columns (Fischer Scientific, Fair Lawn, N J) Procedure. To each 5-ml urinary sample, 125 mg MOHCI dissolved in 1.5 ml sodium acetate 1.5 M (pH 5) is added. Following vigorous mixing, the samples are allowed to equilibrate at room temperature for 20 min and then applied to PBA columns, which have been previously washed with 3 ml methanol and 3 ml of 0.1 N hydrochloric acid (HC1). The PBA columns are then washed with 2 ml of 1 part of 1 M NaC1 and 1 part of 0.1 N HCI, followed by another wash with 2.5 ml of methanol. The samples are then eluted with 4 ml of 1 part 0.1 M NaOH and l part methanol and subsequently diluted to 15 ml with water. The pH is then adjusted to approximately 3 with 10% formic acid and the sample applied to a C18 Prep-Sep that has been preconditioned with 3 ml of methanol and 3 ml of 17 j. A. Lawson, A. R. Brash, J. Doran, and G. A. FitzGerald, Anal. Biochem. 150, 463 (1985).
48
ASSAYS
[5l
distilled water. The Prep-Sep is then washed with 3 ml of water and eluted with 3 ml of ethyl acetate. This technique, although highly specific, requires extensive purification following extraction, prior to mass spectrometric analysis. This represents a major drawback to its widespread application. The cleanup time has been greatly reduced by the use of immunopurification techniques.18'19 Biological samples are applied to a reversed-phase cartridge, interfering substances are partly removed by specific solvents, and the compound can then be eluted and applied to a stationary phase (hydroxysuccinimidyl-SP500 silica gel) where polyclonal antibodies are chemically immobilized.
Reagents and Materials Polyvinylpyrrolidone (PVP-40) (Sigma, St. Louis, MO) Antibody columns prepared as in Ref. 18 Procedure. Urine samples are extracted by C18 Prep-Sep, eluted with ethyl acetate, dried under nitrogen, and redissolved in 0.5 ml of buffer consisting of 1 g/liter PVP-40, 0.15 M NaCl, 0.01 M Trizma hydrochloride, 0.1 M CaCl2, and 0.5 M MgESO4.This is applied to the antibody affinity columns and eluted with 4 ml of 4 parts acetone and 1 part 1% acetic acid. After drying the acetone under nitrogen, the samples are extracted into 4 ml ethyl acetate. The columns are washed with phosphate-buffered saline (PBS) and stored in PBS at 4°. These techniques allow selective and simultaneous extraction of two or more metabolites, have an extraction efficiency from urine of about 50%, and are highly reproducible [coefficient of variation (CV) < 10%]. 19 Purification Following extraction of the metabolites from biological matrixes, further purification is necessary to obtain an interference-freechromatogram. As far as 2,3-dinor-TxB2 is concerned, purification usually relies on two TLC steps ifa previous extraction has been performed with PBA columns. Only one TLC step is necessary following antibody column extraction. In the first case, samples are applied as the methoxime derivatives to the preadsorbent zone of silicic acid plates (Linear-K silica gel, Whatman) and developed in a mobile phase consisting of ethyl acetate : acetic acid : hexla H. L. Hubbard, T. D. EUer, D. E. Mais, P. V. Halushka, R. H. Baker, I. A. Blair, J. J. Vrbanac, and D. R. Knapp, Prostaglandins 33, 149 (1987). 19 C. Chiabrando, A. Benigni, C. Piccinelli, C. Carminati, E. Cozzi, G. Remuzzi, and R. FaneUi, Anal. Biochem. 163, 255 (1987).
[5]
MEASUREMENT OF THROMBOXANE METABOLITES
49
ane : water (54 : 12 : 25 : 60). Sample Rf is identified by 2,3-dinor-TxB2 methoxime standard (5/zg) spotted on a separate plate and visualized by 10% phosphomolybdic acid in ethanol. The samples are then scraped from the appropriate area and extracted into ethyl acetate. A second TLC step, the only one following antibody column extraction, is performed following further derivatization as pentafluorobenzyl (PFB) ester. The developing solvent is the organic layer of isooctane" ethyl acetate : water (65 : 85 : 100). The Rf if 2,3-dinor-TxB2-methoximePFB derivative is visualized as described above. In the case of 11-dehydro-TxB2, urinary or acidified plasma samples are extracted by reversed-phase cartridge, allowed to stand at room temperature in 10% formic acid for 2 hr to ensure ring closure, and finally purified by two TLC steps before [Solvent A = ethyl acetate : acetic acid (48 : 1)] and after [Solvent B = ethyl acetate : heptane (75 : 25)] derivatization as PFB ester. Only the second TLC is necessary following antibody column extraction. The purification procedure for both metabolites can also be considerably facilitated by the use of highly selective triple-stage quadrupole MS, instead of the single-quadrupole MS.2° Derivatization The polar functions of 2,3-dinor-TxB2 and I 1-dehydro-TxB2 need to be derivatized in order to improve both their gas chromatograhic characteristics and mass spectrometric fragmentation patterns. Usually the ketone group of 2,3-dinor-TxB2 is derivatized to methoxime and, in both molecules, the hydroxyl groups are derivatized to trimethylsilyl (TMS) ethers and the terminal carboxyl group to methyl or pentafluorobenzyl (PFB) esters (Fig. 4). One way to perform methoxime derivatization has been already described as a prerequisite for PBA column extraction. Alternatively, methoxime derivatives can be formed by adding/~l of 0.5% methoxyamine hydrochloride in pyridine to the dried samples and allowing the reaction mixture to stand at room temperature overnight. The pentafluorobenzyl (PFB) ester is formed by allowing the samples to stand at room temperature for 30 min in l0/A diisopropylethylamine and 20/A of 12.5% PFB Br in acetonitrile. The PFB group is electron capturing and has the advantage of allowing analysis in the negative-ion, chemical ionization (NCI) mode. It also directs fragmentation, essentially re~o H. Schweer, C. Meese, O. Furst, P. G. Kuhl, and H. W. Seyberth, Anal. Biochem. 164, 156 (1987).
50
ASSAYS
[5]
(CH3)3 Si I 0
o.~N-"
CH3
O" V
S~(CH3~J
" , ~ V V
0 I
Si (CH3)3 FIG. 4. Derivatization of 2,3-dinor-TxB2 as the methoxime, TMS ether, PFB ester.
stricting it to the loss of the PFB group. Taken together, these factors enhance sensitivity by several orders of magnitude over electron-impact techniques. The trimethylsilyl (TMS) ether is formed by leaving the sample at room temperature for 1 hr in 10/zl dry pyridine and 10/.d N, O-bistrimethylsilyltrifluoroacetamide (BSTFA). Quantitafion Quantitation by GC-MS is generally performed in the selected-ion monitoring (SIM) mode by measuring the ratio of the integrated areas of the peaks corresponding to the ion for the endogenous material and the ion for the internal standard. This represents a highly specific technique, allowing discrimination between the target compound and other contaminant peaks with different GC retention times. Specificity is also enhanced by the use of capillary rather than packed columns. Capillary columns enhance specificity by increasing efficiency, thereby resolving the metabolites from contaminants originating in biological fluids. They also improve sensitivity by concentrating the compound into a sharp peak. These features are apparent in Fig. 2. In conclusion, measurement of thromboxane metabolites by GCMS provides a reliable, specific, and sensitive index of thromboxane biosynthesis. It has permitted considerable insight into the pathophysiological role of thromboxane A2 in human disease. Acknowledgments Supported by grants (HL 30400, GM15431) from the National Institutes of Health and Daiichi Seiyaku. Dr. Catella is the recipient of a Faculty Development Award from the Pharmaceutical Manufacturers Association Foundation. Dr. FitzGerald is an Established Investigator of the American Heart Association and the William Stokes Professor of Experimental Therapeutics.
ASSAYS
[5]
urine, together with a fractional elimination comparable to that of 2,3dinor-TxB2, make these measurements an extremely convenient approach to monitoring thromboxane production both in healthy and disease conditions. The unique chemical properties of 11-dehydro-TxB2 are associated with greater than usual specificity of the immunological recognition, which combined with adequate chromatographic procedures, can allow reliable assessment of metabolite levels in complex biological fluids. Acknowledgments The expert editorial assistance of G. Protasoni and M. L. Bonanomiis gratefullyacknowledged.Our studies were supportedby grants from ConsiglioNazionaledelleRicerche (Progetto Finalizzato MedicinaPreventivae Riabilitativa).
[5] M e a s u r e m e n t o f T h r o m b o x a n e M e t a b o l i t e s b y G a s Chromatography-Mass Spectrometry
By FRANCESCACATELLA and GARRET A. FITZGERALD Thromboxane (Tx)A2 is a potent vasoconstrictor and proaggregatory substance that, by virtue of these properties, may play a role in human syndromes of vascular occlusion. Consistent with this concept, aspirin (acetylsalicylic acid) which inhibits TxA2, has proved to be beneficial in syndromes associated with platelet activation, such as unstable angina. Biochemical assessment of thromboxane biosynthesis complements the use of pharmacological probes to elucidate the biological importance of this compound. Unfortunately, due to its chemical instability, TxA2 cannot be directly measured in biological fluids. Its hydration product, TxB2, is more stable, but its assessment in plasma is confounded by artifactual formation during blood withdrawal. 1-3 Although urinary excretion of TxB2 may be used as an index of systemic TxA2 formation in certain settings, such as platelet activation, it largely reflects renal production under physiological conditions. i G. A. FitzGerald, A. K. Pedersen, and C. Patrono, Circulation 67, 1174 (1983). 2 E. G r a n s t r o m , P. W e s t l u n d , K. K u m l i n , and A. N o r d e n s t r o m , Adv. Prostaglandins Thromboxane Leukotriene Res. 15, 67 (1985). 3 F. Catella, D. Healy, J. L a w s o n , and G. A. FitzGerald, Proc. Natl. Acad. Sci. U.S.A. 83, 5861 (1986).
METHODS IN ENZYMOLOGY,VOL. 187
Copyright© 1990by AcademicPress, Inc. All rights of reproduction in any form reserved.
[5]
43
MEASUREMENT OF THROMBOXANE METABOLITES OH
OH II-OH- DEHYDROGENASE
OH II-DEHYDRO-TxB 2
"~o"""'~~COOH H O / ' ~ ' - , - O ~ OH TxB 2
Ifl-OXIDATION OH z
~
" " ~
COOH
H0 .,,,"'--0 ~ OH
2,5-DINOR-TxB 2 Fro. 1.2,3-Dinor-TxB2 and 11-dehydro-TxB2 are indexes of the two major pathways of metabolism of TxB2.
Roberts e t a l . 4 identified twenty compounds formed via two major and one minor pathway of metabolism, by infusing [3H]TxB2 in a healthy volunteer. Indexes of the two major pathways are 2,3-dinor-TxB2, metabolized via/3-0xidation, and l l-dehydro-TxBz, resulting from dehydrogenation of the hemiacetal alcohol group at C-11 (Fig. I). More recently, these metabolites have also been shown to be the major metabolites of TxA2 itself in the monkey. 5 The small concentrations of these metabolites in plasma render lldehydro-TxB2 the only realistic target for analysis in plasma with available methodology. 3 However, measurement of either metabolite in urine has proved to offer a reliable index of in vivo TxA2 formation by platelets, although the kidney is likely to possess the capacity to form both the dinor and dehydro derivatives.6 Urinary excretion of both metabolites is almost, but not completely, inhibited following chronic administration of aspirin at a dose which completely inhibits platelet cyclooxygenase. 7 Furthermore, their excretion is significantly elevated in syndromes associated with platelet activation, such as peripheral vascular disease, unstable angina, thrombolysis, and systemic sclerosis. 4 L. J. Roberts, B. J. Sweetman, and J. A. Oates, J. Biol. Chem. 256, 8384 (1981). 5 p. Patrignani, H. Morton, M. Cirino, A. Lord, L. Charette, J. Gillard, J. Rokach, and C. Patrono, Clin. Res. 37, 341A (1989). 6 A. Benigni, C. Chiabrando, N. Perico, R. Fanelli, C. Patrono, G. A. FitzGerald, and G. Remuzzi, Am. J. Physiol. 257, F77 (1989). 7 F. Catella and G. A. FitzGerald, Thromb. Res. 47, 647 (1987).
44
ASSAYS
[5]
Gas Chromatography-Mass Spectrometry versus Immunoassay
Measurement of 2,3-dinor-TxBz and 1l-dehydro-TxB2 can be accomplished by gas chromatographic (GC)-mass spectrometric (MS) techniques or by radioimmunoassays (RIA). Enzyme immunoassays, already available for l l-dehydro-TxB2, are currently being developed for 2,3dinor-TxB2. The immunoassays can usually be performed on a larger scale in a relatively short time and do not require such expensive equipment. On the other hand, these methods may generate misleading results. This is generally due to lack of specificity resulting from inappropriate purification or from the use of antisera with high cross-reactivity with other metabolites of related compounds. To avoid these problems, any new immunoassays should be validated by characterization of the thin-layer chromatography (TLC) pattern of distribution of the extracted eicosanoid-like immunoreactivity, by using multiple antisera and by comparison with GC-MS determinations. RIA results which are highly comparable with GC-MS determinations have indeed been attained when the metabolites have been properly purified from biological matrixes and a highly specific antiserum has been employed, s'9 On the other hand, even highly validated immunoassays have practical limitations. Recently, for example, fish oils, rich in the oJ-3 fatty acid, eicosapentaenoic acid (EPA), have been extensively investigated for their potential antithrombotic properties. Dietary supplementation with fish oil results in decreased formation of TxA2 (derived from arachidonic acid; AA), and increased TxA3 (derived from EPA and reported to be less thrombogenic than TxA2). Commonly available immunoassays cannot discriminate between eicosanoids derived from AA and those derived from EPA, which differ only by one double bond. By contrast, GC-MS techniques can easily discriminate between these two series of compounds, l0 Another limitation of immunoassay methods overcome by GC-MS is their poor sensitivity. It was suggested that analysis of eicosanoids in plasma could facilitate the investigation of their temporal formation in vivo: this would be desirable when phasic eicosanoid formation might be of interest, such as during ischemic episodes in patients with unstable s C. Patrono, G. Ciabattoni, F. Pugliese, I. A. Blair, and G. A. FitzGerald, J. Clin. Invest. 77, 590 (1986). 9 G. Ciabattoni, J. Maclouf, F. CateUa, G. A. FitzGerald, and C. Patrono, Biochim. Biophys. Acta 918, 293 (1987). l0 H. R. Knapp, I. A. G. Reilly, P. Alessandrini, and G. A. FitzGerald, N. Engl. J. Med. 314, 937 (1986).
[5]
45
MEASUREMENT OF THROMBOXANE METABOLITES
100%=43952
I
100%=25206784
1~0 2~0 300 400 560 ~0
t~
/
RIC=511
RIC=515
7~0 860 9~0 1000~-~00 1200 1300 1400 1500
FIG. 2. Selected-ion monitoring of 11-dehydro-TxB2 (m/z 511) and its tetradeuterated internal standard (m/z 515). The plasma concentration of this sample corresponds to 1.5 pg/ml. (By permission, Ref. 3.)
angina. As far as TxA2 is concerned, plasma concentrations of its hydration product, TxB2, are readily confounded by e x v i v o platelet activation. 1-3 However, 11-dehydro-TxB2, a long-lived enzymatic metabolite in plasma, H has been shown to minimize this problem. 3 Its measurement in plasma requires a detection limit of less than 1 pg/ml. This can be attained by capillary gas chromatography-negative-ion chemical ionization mass spectrometry (GC-NCI-MS) (Fig. 2), but it is far below the limit of sensitivity achieved by most commercially available immunoassays. Combined analysis of 11-dehydro-TxB2 and 2,3-dinor-TxB2, indexes of the two major pathways of thromboxane metabolism in humans, permits one to distinguish between altered metabolism and increased biosynthesis of TxA2.7 Moreover, combined analysis of metabolites in plasma and urine can be utilized to identify alterations in the volume of distribution and renal clearance of TxA2, as might occur due to drug administration or disease. On the other hand, although the methods have been refined, GC-MS remains a time-consuming approach. Samples have to undergo extraction, purification, and derivatization prior to injection onto the instrument. 11 j. Lawson, C. Patrono, G. Ciabattoni, and G. A. FitzGerald, Anal. Biochem. 155, 198 (1986).
46
ASSAYS
[5]
Internal Standards The first step in quantitative analysis by GC-MS is to spike the samples with an internal standard. This permits accurate quantitation, irrespective of losses during purification or due to incomplete derivatization. The most satisfactory standards are analogs incorporating stable isotopes, because they have physiochemical properties almost identical to their natural isomers. 12 Some stable isotopes are commercially available (Biomol, Plymouth Meeting, PA; Cayman Chemicals, Ann Arbor, MI). The remainder must be chemically or biologically synthesized. Deuterated 11-dehydro-TxB2 can be biologically synthesized by incubation of deuterated TxB2 with the high-speed supernatant of guinea pig liver homogenate in the presence of NAD. ~t Deuterated 2,3-dinor-TxB2 can be produced by prolonged incubation of the tetradeuterated TxB2 in a culture of Mycobacterium rhodochrous, t3 Another example of biological generation of standards is the formation of the dinor metabolite of tetradeuterated 6-keto-PGFt~ by cultured hepatocytes, t4 Alternatively, 2,3-dinor-TxB2 and 11-dehydro-TxB2 can be labeled by exchange of the two carboxylic oxygen atoms with 180 using labeled water as a donor, t5 These exchange reactions can be catalyzed by acid, base, or enzymatically via esterases as described by Murphy and Clay. t6 A limitation to the use of tso-labeled compounds is the back-exchange loss of the label that can occur rapidly in plasma and tissues containing esterases. This undesired enzymatic exchange does not represent a problem in urine, due to the lack of esterase. In other biological matrixes, it can be avoided easily by lowering the pH of the medium to approximately 3.5 before adding the taO-labeled standard. Extraction
The selective extraction of 2,3-dinor-TxB2 can be achieved by relying on the chemical properties of the thromboxane ring; in a pH-dependent equilibrium, it exists with an open ring form. At pH 5, the open form, stabilized by derivatization as the methoxime, results in a hydroxyl con12 W. A. Garland and M. L. Powell, J. Chromatogr. Sci. 19, 392 (1981). 13 F. F. Sun, B. M. Taylor, F. H. Lincoln, and O. K. Sebek, Prostaglandins 20, 729 (1980). ~4 M. Balazy, E. P. Brass, J. G. Gerber, and A. S. Nies, Prostaglandins 36, 421 (1988). x5 H. J. Leis, E. Malle, R. Moser, J. Nimpf, G. M. Kostner, H. Esterbauer, and H. Gleispach, Biomed. Environ. Mass Spectrom. 13, 483 (1986). 16 R. C. Murphy and K. L. Clay, this series, Vol. 86, pp. 547.
[5]
47
MEASUREMENT OF THROMBOXANE METABOLITES
H O
H O "~OH ~ ..
- -
H
R
0 0 H
H O R
>
MOHCI
N 0" CH3
R
Ac-
H
R H
O-~,R /
METHOXIME FORMATION IN URINE /OH STAT SUPPORT IONARY _ N ~ N
~~ ~ / B ' ' O H
R +
R R
> R - B \/o
R
HO FIG. 3.2,3-Dinor-TxB2 exists in equilibrium with an open ring form which is derivatized as the methoxime. Following derivatization, the hydroxyl configuration permits condensation with bonded-phase phenylboronic acid to form a stable complex. (By permission, Ref. 17.)
figuration favorable to the formation of a stable complex with bondedphase phenylboronic acid (Fig. 3). ~7This procedure has been employed for the purification of prostaglandin F metabolites as well as for carbohydrate and catecholamine analysis. Reagents and Materials Methoxyamine Hydrochloride (MOHCI) (Sigma, St. Louis, MO) Phenylboronic acid bonded-phase (PBA) columns (Infolab, Fort Oglethorpe, GA) Prep-Sep columns (Fischer Scientific, Fair Lawn, N J) Procedure. To each 5-ml urinary sample, 125 mg MOHCI dissolved in 1.5 ml sodium acetate 1.5 M (pH 5) is added. Following vigorous mixing, the samples are allowed to equilibrate at room temperature for 20 min and then applied to PBA columns, which have been previously washed with 3 ml methanol and 3 ml of 0.1 N hydrochloric acid (HC1). The PBA columns are then washed with 2 ml of 1 part of 1 M NaC1 and 1 part of 0.1 N HCI, followed by another wash with 2.5 ml of methanol. The samples are then eluted with 4 ml of 1 part 0.1 M NaOH and l part methanol and subsequently diluted to 15 ml with water. The pH is then adjusted to approximately 3 with 10% formic acid and the sample applied to a C18 Prep-Sep that has been preconditioned with 3 ml of methanol and 3 ml of 17 j. A. Lawson, A. R. Brash, J. Doran, and G. A. FitzGerald, Anal. Biochem. 150, 463 (1985).
48
ASSAYS
[5l
distilled water. The Prep-Sep is then washed with 3 ml of water and eluted with 3 ml of ethyl acetate. This technique, although highly specific, requires extensive purification following extraction, prior to mass spectrometric analysis. This represents a major drawback to its widespread application. The cleanup time has been greatly reduced by the use of immunopurification techniques.18'19 Biological samples are applied to a reversed-phase cartridge, interfering substances are partly removed by specific solvents, and the compound can then be eluted and applied to a stationary phase (hydroxysuccinimidyl-SP500 silica gel) where polyclonal antibodies are chemically immobilized.
Reagents and Materials Polyvinylpyrrolidone (PVP-40) (Sigma, St. Louis, MO) Antibody columns prepared as in Ref. 18 Procedure. Urine samples are extracted by C18 Prep-Sep, eluted with ethyl acetate, dried under nitrogen, and redissolved in 0.5 ml of buffer consisting of 1 g/liter PVP-40, 0.15 M NaCl, 0.01 M Trizma hydrochloride, 0.1 M CaCl2, and 0.5 M MgESO4.This is applied to the antibody affinity columns and eluted with 4 ml of 4 parts acetone and 1 part 1% acetic acid. After drying the acetone under nitrogen, the samples are extracted into 4 ml ethyl acetate. The columns are washed with phosphate-buffered saline (PBS) and stored in PBS at 4°. These techniques allow selective and simultaneous extraction of two or more metabolites, have an extraction efficiency from urine of about 50%, and are highly reproducible [coefficient of variation (CV) < 10%]. 19 Purification Following extraction of the metabolites from biological matrixes, further purification is necessary to obtain an interference-freechromatogram. As far as 2,3-dinor-TxB2 is concerned, purification usually relies on two TLC steps ifa previous extraction has been performed with PBA columns. Only one TLC step is necessary following antibody column extraction. In the first case, samples are applied as the methoxime derivatives to the preadsorbent zone of silicic acid plates (Linear-K silica gel, Whatman) and developed in a mobile phase consisting of ethyl acetate : acetic acid : hexla H. L. Hubbard, T. D. EUer, D. E. Mais, P. V. Halushka, R. H. Baker, I. A. Blair, J. J. Vrbanac, and D. R. Knapp, Prostaglandins 33, 149 (1987). 19 C. Chiabrando, A. Benigni, C. Piccinelli, C. Carminati, E. Cozzi, G. Remuzzi, and R. FaneUi, Anal. Biochem. 163, 255 (1987).
[5]
MEASUREMENT OF THROMBOXANE METABOLITES
49
ane : water (54 : 12 : 25 : 60). Sample Rf is identified by 2,3-dinor-TxB2 methoxime standard (5/zg) spotted on a separate plate and visualized by 10% phosphomolybdic acid in ethanol. The samples are then scraped from the appropriate area and extracted into ethyl acetate. A second TLC step, the only one following antibody column extraction, is performed following further derivatization as pentafluorobenzyl (PFB) ester. The developing solvent is the organic layer of isooctane" ethyl acetate : water (65 : 85 : 100). The Rf if 2,3-dinor-TxB2-methoximePFB derivative is visualized as described above. In the case of 11-dehydro-TxB2, urinary or acidified plasma samples are extracted by reversed-phase cartridge, allowed to stand at room temperature in 10% formic acid for 2 hr to ensure ring closure, and finally purified by two TLC steps before [Solvent A = ethyl acetate : acetic acid (48 : 1)] and after [Solvent B = ethyl acetate : heptane (75 : 25)] derivatization as PFB ester. Only the second TLC is necessary following antibody column extraction. The purification procedure for both metabolites can also be considerably facilitated by the use of highly selective triple-stage quadrupole MS, instead of the single-quadrupole MS.2° Derivatization The polar functions of 2,3-dinor-TxB2 and I 1-dehydro-TxB2 need to be derivatized in order to improve both their gas chromatograhic characteristics and mass spectrometric fragmentation patterns. Usually the ketone group of 2,3-dinor-TxB2 is derivatized to methoxime and, in both molecules, the hydroxyl groups are derivatized to trimethylsilyl (TMS) ethers and the terminal carboxyl group to methyl or pentafluorobenzyl (PFB) esters (Fig. 4). One way to perform methoxime derivatization has been already described as a prerequisite for PBA column extraction. Alternatively, methoxime derivatives can be formed by adding/~l of 0.5% methoxyamine hydrochloride in pyridine to the dried samples and allowing the reaction mixture to stand at room temperature overnight. The pentafluorobenzyl (PFB) ester is formed by allowing the samples to stand at room temperature for 30 min in l0/A diisopropylethylamine and 20/A of 12.5% PFB Br in acetonitrile. The PFB group is electron capturing and has the advantage of allowing analysis in the negative-ion, chemical ionization (NCI) mode. It also directs fragmentation, essentially re~o H. Schweer, C. Meese, O. Furst, P. G. Kuhl, and H. W. Seyberth, Anal. Biochem. 164, 156 (1987).
50
ASSAYS
[5]
(CH3)3 Si I 0
o.~N-"
CH3
O" V
S~(CH3~J
" , ~ V V
0 I
Si (CH3)3 FIG. 4. Derivatization of 2,3-dinor-TxB2 as the methoxime, TMS ether, PFB ester.
stricting it to the loss of the PFB group. Taken together, these factors enhance sensitivity by several orders of magnitude over electron-impact techniques. The trimethylsilyl (TMS) ether is formed by leaving the sample at room temperature for 1 hr in 10/zl dry pyridine and 10/.d N, O-bistrimethylsilyltrifluoroacetamide (BSTFA). Quantitafion Quantitation by GC-MS is generally performed in the selected-ion monitoring (SIM) mode by measuring the ratio of the integrated areas of the peaks corresponding to the ion for the endogenous material and the ion for the internal standard. This represents a highly specific technique, allowing discrimination between the target compound and other contaminant peaks with different GC retention times. Specificity is also enhanced by the use of capillary rather than packed columns. Capillary columns enhance specificity by increasing efficiency, thereby resolving the metabolites from contaminants originating in biological fluids. They also improve sensitivity by concentrating the compound into a sharp peak. These features are apparent in Fig. 2. In conclusion, measurement of thromboxane metabolites by GCMS provides a reliable, specific, and sensitive index of thromboxane biosynthesis. It has permitted considerable insight into the pathophysiological role of thromboxane A2 in human disease. Acknowledgments Supported by grants (HL 30400, GM15431) from the National Institutes of Health and Daiichi Seiyaku. Dr. Catella is the recipient of a Faculty Development Award from the Pharmaceutical Manufacturers Association Foundation. Dr. FitzGerald is an Established Investigator of the American Heart Association and the William Stokes Professor of Experimental Therapeutics.
