2302633код для вставки
BIOMEDICAL A N D ENVIRONMENTAL MASS SPECTROMETRY, Vol. 15, 157-161 (1988) A Gas Chromatographic/Mass Spectrometric Assay for Catechol Estrogens in Microsomal Incubations: Comparison with a Radiometric Assay D. J. Porubek and S. D. Nelson? Department of Medicinal Chemistry BG-20, School of Pharmacy, University of Washington, Seattle, Washington98195, USA A gas chromatographichass spectrometric assay for quantifying two catechol estrogens, 2-hydroxyestradiol and 4-hydroxyestradiol, in microsomal preparations is described. The assay employs deuterium-labeled analogs of the catechol estrogens as internal standards and permits quantification of catechol estrogens, in microsomal incubations, at low (1-2) p~ concentrations. The compounds are analyzed as their trimethylsilyl derivatives following separation by capillary gas chromatography. INTRODUCTION EXPERIMENTAL Aromatic hydroxylation of estradiol by microsomal oxygenases generates the catechol estrogens 2-hydroxyestradiol and 4-hydroxyestradiol (Fig. 1). Catechol estrogen formation is a major metabolic route for endogenous and exogeneous estrogens in animals and man.'-3 While the liver is primarily responsible for metabolism of estrogens into catechol estrogens, a number p l a ~ e n t a and ~'~ of other organs including the kidney"" possess metabolic activity. While the estrogens retain some of the physiological properties of the classic estrogen, estradiol, they possess certain activities that are unique (see reviews, Refs 12-14). The catechol estrogens are further metabolized in vivo to their corresponding 0-methyl ethers by the enzyme catechol-0-methyl transferase (COMT)." Because COMT is an abundant enzyme in many tissues, including red blood cells, and because catechol estrogens are good substrates for COMT,I6 only very low levels of catechol estrogens circulate freely. The catechol estrogens are also labile compounds that spontaneously auto-oxidize under neutral and basic condition^.'^ As a result of these technical difficulties a number of analytical methods have been developed to measure catechol estrogens in ~ i v o "and ~ ~in~vitro.2'-2'Among the methods that were developed the radiometric method of Fishman ef al." appeared to circumvent many problems. Because the method measures the process of radiolabel release from appropriately tritiated substrates, further biotransformation or degradation of the catechol estrogens should not influence the analytically sensitive step. Recently however, the accuracy of the radiometric method has been questioned. Non-specific and/or peroxidative radiolabel release have been suggested as possible complications.24~25 In order to compare the radiometric assay with direct product isolation assay, we developed a gas chromatographic/mass spectrometric assay which measures the catechol estrogens under conditions which allow their simultaneous quantification by the radiometric assay. Materials Chemicals purchased from Aldrich Chemical Company (Milwaukee, Wisconsin) included isopropenyl acetate, bromine, potassium tert-butoxide, acetone-& (99.5 at% D), methanol-d, (99.5 at% D), choloroformd, (98 at% D), sodium borodeuteride (98 at% D), potassium nitrosodisulfonate, L-ascorbic acid, and silica gel (60-200 mesh). Chemicals from Sigma Corporation (St Louis, Missouri) included estrone, estradiol, NADP, glucose-6-phosphate (G-6-P), glucose-6-phosphate dehydrogenase, bovine serum albumin and dimethyldichlorosilane. Phenol reagent (Fohn-Ciocalteau) used in the Lowry protein assay was supplied by Fisher Scientific (Fair Lawn, New Jersey) The derivatization reagents, N, 0-bis(trimethylsily)trifluoroacetamide (BSTFA) and silylation grade pyridine, were purchased from Pierce Chemical Company (Rockford, Illinois). Concentration tubes were 12 ml Pyrex (Corning Glass works, Corning, New York). Aquasol I1 scintillation cocktail was purchased from New England Nuclear (Boston, Massachusetts). All other solvents and chemicals were of reagent grade from commercial suppliers. ?" @ HO OH 4- ti" d r o x v r s t r a d i 0 1 t Author to whom correspondence should be addressed. 0887-6 134/ 88/030 157-05 $05.00 @ 1988 by John Wiley & Sons Ltd Figure 1. Formation of catechol estrogens from estradiol. Received 12 January 1987 Revised 1 June 1987 158 D. J. PORUBEK A N D S. D. NELSON The synthesis of unlabeled 2-hydroxyestradiol and 4-hydroxyestradiol was accomplished by the method of Gelbke et al.26 The synthesis of (15,16,17-,H3)2hydroxyestradiol and ( 15,16,17-2H3)4-hydroxyestradiol was accomplished by the method of Knuppen et al.27 up through the preparation of ( 15,16,17-2H3)estradiol. Oxidation of ( 15,16,17-2H3)estradiol according to the method of Gelbke et a1.26 afforded the deuterated catechol estrogens. Mass spectrometric analysis of (15,16,17-'H3)2-hydroxyestradiol revealed a labeling pattern of 2.0% 'Ho, 14.9% 2H,, 10.3% 'H2, 72.2% 2H, and 0.5% 'H,. Analysis of (15,16,17-'H,)4-hydroxyestradiol revealed 0.8% 'Ho, 10.2% ,HI, 11.2% ,H2, 77.6% ,H, and 0.2% 'H4. The synthesis of the radiolabeled estrogrens (2,H)estradiol and (4-3H)estradiol was reported previouslyZ8with the verification of position of label and determination of radiochemical purity (98% ) being described therein. The specific activity of the substrates employed in the metabolic incubations was 32 mCi/mol for (2-3H)estradiol and 34 mCi/mmol for (43H)estradiol. Methods Microsomes. Microsomes and microsomal stock sol- utions were prepared according to Wheeler et ~ 1 . ' Male ~ Sprague-Dawley rats (180-200 g) were used. It was possible to store the microsomes at -80°C in 0.1 M phosphate buffer (0.2 mM EDTA, pH 7.4) containing 20% glycerol for at least a month. Therefore, frozen microsomes were used in these studies. Preparation of standards. Standard solutions of catechol estrogens were prepared by dissolving 500,250, 125 and 25 pg of 2-hydroxyestradiol and 62.5, 31.2, 15.6 and 7.8 pg of 4-hydroxyestradiol in 1-ml volumes of methanol-acetic acid-ascorbic acid (MAA) (10 ml: 1 ml :60 mg).I7 Standard solutions of the deuterated internal standards were similarly prepared by dissolving 125 pg ( 15,16,17-2H3)2-hydroxyestradioland 15.6 pg (15,16,17-2H3)4-hydroxyestradiol in 1-ml volumes of the MAA mixture. All stock solutions were stored in silanized glass vials at -80 "C in the dark and were stable over a perod of at least 3 m o n t h ~ . ' ~ Incubation conditions. Incubation mixtures were made up to 1 ml combining 610 pl buffer (0.1 M phosphate, 0.8 mM ascorbate, pH 7.4, prepared freshly), 100 FI microsomes (10 mg/ml), 250 p1 cofactor mix (10 pmol G-6-P, 3 pmol NADP, 3 units G-6-P dehydrogenase and 5 pmol MgCl,), 20 p1 substrate (5 mM estradiol in ethanol) and 20 p1 internal standard (deuterated 2- or 4-hydroxyestradiol). For the construction of standard curves, 2 0 4 of standard stock solutions of 2- or 4hydroxyestradiol were added in place of substrates. All incubations were conducted for 10 minutes at 37 "C with shaking in a Dubnoff Incu-Shaker (Lab-Line Instruments Inc., Melrose Park, Illinois). Incubations and subsequent work-ups were conducted with glassware that had been silanized. then centrifuged at 1000 x g for 10 min and the aqueous layer separated from the organic. The aqueous layer was successively extracted with chloroform-acetone (4 x 6 ml, see Radiometric assay) while 4-ml aliquots of the initial organic extracts were transferred to concentration tubes containing anhydrous sodium sulfate (- 1 g). After drying for 1 h the organic extracts were transferred to clean concentration tubes and the solvent removed under a stream of nitrogen. To the dry extracts were added 0.1 ml BSTFA and 0.01 ml pyridine. The tubes were capped, gently vortexed and heated at 60°C for 30 min. Gas chromatography mass spectrometry assay. Capillary gas chromatography was performed with a Hewlett-Packard (Avondale, Pennsylvania) 5700 gas chromatograph fitted with a wide-bore DB-5 bonded-phase (normal film thickness) fused silica column (30m) from J. & W. Scientific Inc. (Rancho Cordova, California). Quantitative gas chromatographic/mass spectrometric measurements were conducted on a VG Analytical Micromass 7070H mass spectrometer (VG Analytical Inc., Manchester, UK) with a VG 2000 data system interfaced with a Hewlett-Packard 5700 gas chromatograph and capillary injector. Typically 1-pl aliquots of the derivatized extracts were directly injected into the gas chromatograph/mass spectrometer for analysis. The gas chromatographic conditions were: helium carrier gas head pressure of 15 psi, splitless injection, injector temperature of 280 "C, initial oven temperature of 1OO"C, initial heating rate of 30deg.C/min for 4min, a second heating rate of 5 deg. C/min until 280 "C was attained and a 5-min hold at 280°C. Under these conditions the trimethylsilyl ethers of 2-hydroxyestradiol and 4-hydroxyestradiol eluted at 14.4 and 15.0 min, respectively; whereas that of estradiol eluted at 12.6 min The capillary column was run directly into the mass spectrometer source with the direct inlet transfer line held at 280 "C. The mass spectrometer conditions included a source temperature of 200 "C, 70 eV electron energy (electron impact), 4 kV accelerating voltage, 200 pA emission current, 2400-3800 V multiplier setting. The instrument was run in the selected ion monitoring (SIM) mode and calibrated for the measurement of ions at 504.2898 and 507.3084 a.m.u. Deuterated analogs of the catechol estrogens were employed as internal standards and by using trideuterated analogs interfering ion signals from the multiple isotopes of silicon derivatives were minimized. Radiometric assay. Following extraction of the aqueous incubation media with chloroform-acetone, the samples were analyzed by transfer of 0.2-ml aliquots to scintillation vials containing 10 ml scintillation cocktail. Samples were counted for 10min with a Beckmann LS-7500 instrument (Beckmann Instruments, Inc., Fullerton, California) utilizing a counting program with an external standard to correct for quench in all samples. RESULTS AND DISCUSSION Sample preparation. Upon completion of incubation the reactions were terminated by addition of 6 ml chloroform -acetone (4: 1). The mixtures were vortexed It was possible to separate the catechol estrogens of estradiol from each other and estradiol itself by capillary AN ASSAY FOR CATECHOL ESTROGENS 159 b c FID RESPONSE I * I . . 2 I , . 6 4 . . 8 . . a 10 , ' . 12 a s 14 . . 16 18 TINE(min) Figure 2. Separation of estradiol(a), 2-hydroxyestradiol(b), and 4-hydroxyestradiol(c) as their tri-trimethylsilyl derivatives by capillary gas chromatography. gas chromatography (Fig. 2). Derivatization was required to protect the labile catechol estrogens and render them volatile Conversion of the catechol tri-trimethylsilyl derivatives by treatment with BSTFA proved to be very convenient. The method is rapid and requires a minimum amount of sample manipulation. Shown in Fig.3(a) is the mass spectrum of the tritrimethylsilyl derivative of 2-hydroxyestradiol above 300 a.m.u. The spectrum of the tri-trimethylsilyl derivative of 4-hydroxyestradiol is nearly identical in this1 region (Fig. 3(b)). The relative intensities of the molecular ions substantiate their use for quantification. The standard curves of peak area ratio versus weight ratio of the unlabeled and deuterated metabolites are presented in Fig. 4. For construction of the standard curve for 2-hydroxyestradiol the amount of internal standard (( 15,16,17-2H3)2-hydroxyestradiol) was held constant at 2500 ng ml-' of microsomal incubation and the amount of unlabeled metabolite varied between 250 ng ml-' and 25 000 ng m1-I. At both extremes of the standard curve significant deviation from linearity occurs, indicating the limits of reliable quantification for the assay (Fig. 4(a)). The corresponding standard curve for 4-hydroxyestradiol (Fig. 4(b)) was constructed by holding constant the amount of internal standard (( 15,16,17-2H3)4-hydroxyestradiol) at 625 ng m1-l of microsomal incubation and the amount of unlabeled metabolite varied between 62.5 ng and 6250 ng ml-'. Significant deviation from linearity again occurred at both extremes of the standard curve but restriction of quan- (a) RELATIVE ION INTENSITY "1432 0 r 446 489 , . . I, 7_ 620 608 480 460 448 640 MOLECULAR WEIGHT Figure 3a. Mass spectrum of the tri-trimethylsilyl derivative of 2-hydroxyestradiol above 300 a.m.u. 1 1'"l RELATIVE ION INTENSITY 5:1, . 483 , 440 , , , , 468 , I , I , 460 MOLECULAR , , Illl, 688 , ~ 628 , I . , 640 . I WEIGHT Figure 3b. Mass spectrum of the tri-trimethylsilyl derivative of 4-hydroxyestradiol above 300 a.rn.u. 160 D. J. PORUBEK AND S. D. NELSON i\i iciii it11 i o Figure 4a. Standard curve of weight ratio versus peak area ratio for 2 hydroxyestradiol with [2H,]2-hydroxyestradiol held constant at 2500 ng m1-l (b) / 10.0 PEAK AREA 1.0 RATIO 0.1 1.0 0.1 10.0 I E I G t i T RATIO Figure 4b. Standard curve of weight ratio versus peak area ratio for 4-hydroxyestradiol with [2H,]4-hydroxyestradiol held constant at 625 ng ml-'. tification of metabolites within these extremes proved reliable. The corresponding linear regression parameters for each metabolite are given in Table 1. The reproducibility and accuracy of the assay were tested by replicate analysis of microsomes spiked with known amounts of each metabolite (Table 2). As mentioned in the introduction the development of a gas chromatographic/mass spectrometric assay intrinsically compatible with the radiometric assay was desired. Such methodology would enable direct assessment of the accuracy of the radiometric assay. Additionally, the methodology could provide a n accurate means for the quantification of specific catechol estrogens in tissues that produce these products at moderate rates (0.1-10 nmol min-' mg-' tissue). Thus, the gas chromatographic/mass spectrometric assay was used in conjunction with the radiometric assay to Table 1. Liner regression parameters calculated from standard curves of peak area ratios versus weight ratios 2-Hydroxyestradiol Slope Y-int. r2 * 0.97 0.02 0.08 0.04 0.99 * 4-Hydroxyestradioi * * 0.94 0.02 0.16 0.04 0.98 A N ASSAY FOR C A T E C H O L ESTROGENS Table 2. Reproducibility and accuracy of quantification of 2hydroxyestradiol and 4-hydroxyestradiol in microsomes Amount added Amount recovered (ndmll (ng/mlla 2-OH-Estradiol (n=4) 1250 5000 4-OH-Estradiol (n=4) 156 625 CVb Accuracy‘ 1520 53 5198 f 319 3.5 6.1 21.6 4.2 5.6 4.5 176+11 672 43 6.3 6.4 12.7k7.1 9.0 5.6 * * Table 3. Rates of formation (w f SD, ng product mg protein-’ 10 min-’) of 2-hydroxyestradiol and 4-hydroxyestradiol by liver microsomes obtained from male rats. Multiple incubations from a single preparation of microsomes obtained from 3 rats Assay * * * MeaniSD. CV=coefficient of variation. c Mean percentage deviation of all concentrations from the theoretical value. a measure catechol estrogen formation by hepatic microsomes from male rats. The gas chromatographic/mass spectrometric assay and radiometric assay were run in tandem in the same incubations by replacing unlabeled substrate estradiol with radiolabeled estradiol ((2- or 4-3H)estradiol). The catechol estrogens were isolated, derivatized and analyzed as described herein and the aqueous incubation media were processed and analyzed for tritium content as previously described for the radiometric assay.’ A comparison of the rates of formation of 2- and 4-hydroxyestradiol appears in Table 3. As expected, both assays revealed a greater rate of 2-hydroxylation than 4-hydroxylation. Additionally, the values obtained for 2-hydroxylation agree quite favorably between assays. However, a major discrepancy between assays was observed for rates of 4-hydroxylation, in that the radiometric assay gave a value at least three times that afforded by the gas chromatographic/mass spectrometric assay. The origin of this major discrepancy is not yet known; however, non-specific and/or peroxidative 161 GC/MS Radiometric 2 Hydroxyestradiol * 6715 188 5539 f40 4 Hydroxyestradiol 617*60 1691 *17 release of tritium from estrogens has been previously suspected. 24,25330 Another possibility is binding of a reactive metabolite of estrogens to tissue macromolecules in such a way that tritium is lost from the 4-p0sition.~’-~’ Alternatively, the discrepancy may in part lie in the fact that during incubation of (4-’H)estradiol with microsomes 2-hydroxylation as well as 4-hydroxylation is occurring. The formation of radiolabeled 2-hydroxyestradiol under such circumstances could lead to spurious release of tritium if a small portion of the metabolite decomposed or was further biotransformed. Such a process would not be expected to play a major role during the incubation of (2-’H)estradiol since the amount of radiolabeled 4-hydroxyestradiol thus formed would be negligible. In summary, a gas chromatographic/mass spectrometric assay suitable for measurement of catechol estrogen formation in tissues such as liver has been developed. The assay permits the direct quantification of metabolites and, while it was primarily developed for the purpose of assessing the accuracy of the radiometric method , it should also be suitable for routine measurements of catechol estrogen production where more specific measurements of 2- and 4-hydroxylation might be warranted. Acknowledgement D. J. Porubek was supported by NIH Training Grant no. GM 07750. REFERENCES 1. J. Fishman, J. Clin. Endocrinol. Metab. 23, 207 (1963). 2. P. Ball and R. Knuppen, Acta fndocrinol. Suppl. 232, 1 (1980). 3. J. Fishman, in Catechol Estrogens, ed. by G. R. Merriam and M. B. Lipsett, p. 1. Raven Press, New York (1983). 4. J. Fishman, F. Naftolin, I. J. Davies, K. J. Ryan and 2. Petro, J. Clin. Endocrinol. Metab. 42, 177 (1976). 5. P. Ball and R. Knuppen, J. Clin. fndocrinol. Metab. 47,732 (1978). 6. S . M. Paul, J. Axelrod and E. J. Diliberto, Endocrinology 101, 1604 (1977). 7. S. W. Smith and L. R. Axelrod, J. Clin. Endocrinol. Metab. 29, 85 (1969). 8. M. J. Namkung, D. J. Porubek, S. D. Nelson and M. R. Juchau, J. Steroid Biochem. 22, 563 (1985). 9. R. L. Barbieri, J. A. Canickand K. J. Ryan, Steroids 32,529 (1978). 10. A. R. Hoffman, S. M. Paul and J. Axelrod, Biochem. Pharmacol. 29, 83 (1979). 11. M. Numazawa. Y. Kiyono and T. Nambara, J. Steroid Biochem. 13, 1101 (1980). 12. N. J. MacLusky, F. Naftolin, L. C. Krey and S . Franks, J. Steroid Biochem. 15, 111 (1981). 13. N. J. Maclusky, E. R. Barnea, C. R . Clark and F. Naftolin, in Catechol Estrogens, ed. by G. R. Merriam and M. B. Lipsett, p. 151. Raven Press, New York (1983). 14. J. Fishman, Ann. Rev. Physiol. 45, 61 (1983). 15. H. Breur, W. Vogel and R. Knuppen, Hopper Seylers 2. Physiol. Chem. 327,217 (1962). 16. P. Ball, R. Knuppen, M.Haupt and H. Breuer, J. Clin. Endocrinol. 34,736 (1972). 17. H. P. Gelbke and R. Knuppen, J. Chromatogr. 71, 456 (1972). 18. J. Fishman, H. Guzikand L. Hellman, Biochemistry9,1593(1970). 19. P. Ball, G. Emons, 0. Haupt, H.-0. Hoppen and R. Knuppen, Steroids 31, 249 (1978). 20. R. Knuppen, 0. Haupt, W. Schramm and H.-0. Hoppen, J . Steroid Biochem. 11, 153 (1979). 21. J. Daly, J. K. lnscoe and J. Axelrod, J. Med. Chem. 8, 153 (1965). 22. M. Numazawa, Y. Kiyono and T.Nambara, Anal. Biochem. 104, 290 (1980). 23. R. M. Hersey, K. I. H. Williams and J. Weisz, Endocrinology 109. 1912 (1981). 24. P. H. Jellinck, B. Norton and J. Fishman, Steroids 35, 579 (1980). 25. J. Fishman and B. Norton, J. Steroid Biochem. 19. 219 (1983). 26. H. P. Gelbke, 0. Haupt and R. Knuppen, Steroids 21, 205 (1973). 27. R. Knuppen, 0. Haupt and H.-0. Hoppen, Steroids 39,667 (1982). 28. D. J. Porubek, M. J. Namkung, M . R. Juchau and S. D. Nelson, J. Labelled Compd. Radiopharm. 21, 703 (1984). 29. C. Wheeler, W. F. Trager and W. R . Porter, Biochem. Pharmacol. 30,1785 (1981). 30. P. H. Jellinck and J. Fishman, Steriods 43, 559 (1984). 31. H. A. Sasame, M. M. Ames and S. D. Nelson, Biochern. Biophys. Res. Commun. 78, 919 (1977). 32. Y. J. Abdul-Hajj, Biochem. Biophys. Res. Commun. 133, 1078 (1985). 33. P. W . LeQuesne, S. Abdel-Baky and A. V. Durga, Biochemstry 25, 2065 (1986).