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Spectrophotometric estimation of estradiol-17β, progesterone, and testosterone
BIOCHEMICAL
14, 104-108 (1975)
MEDICINE
Spectrophotometric
Estimation
Progesterone,
and
of Estradiol-17P, Testosterone’
H. KHAYAM-BASHI Department
of Laborutory
Medicine, Unil~ersity of Culifirniu School Medicine. at the Clinical Laboratorie.~. Strn Francisco Grnerrrl Sun Francisco, Ctrlijiwrlirr
of
Sun Frnncisco.
Hospital.
AND
M. BOROUMAND Department
qf Biochemistry.
School
of Pharmacy.
Utlivvrsity
of l.sfahan,
I.s,fuhon.
Iron
Received August 4. 197.5
The quantitation of various hormones in body fluids for the clinical evaluation of the endocrine system has been given considerable attention over the past decade. As a result, the estimation of steroid hormones for screening or quantitative purposes contributes significantly to the workload of modern clinical laboratories. Rapid quantitation of steroids in various pharmaceutical preparations, as well as other reagents containing these chemicals that are used for biochemical studies (I), also requires considerable attention in many chemical or pharmaceutical laboratories. Quantitation of these compounds has been attempted by various means (2), including the application of specific receptors in competitive binding systems (3, 4). as well as chemical (5). double isotope technique (6), and radioimmunological (7) procedures. These techniques are necessary for the most accurate determination when low concentrations are involved. Automated systems may be necessary in order to analyze large numbers of specimens (8). However, these procedures often are laborious, and require sophisticated methodologies and expensive instrumentation. When relatively large amounts of steroid hormones are present, as in concentrated urine specimens or in pharmaceutical preparations, simple spectrophotometric measurements of each steroid can provide a rapid quantitative procedure effectively. With the application of chromatographic separation techniques, such as Sephadex LH-20 (9. lo), these compounds can be separated by major groups, or even as individual steroids, after which a spectrophotometric assay can be performed. In this study, 1 Portions of this study were completed at the University of Isfahan. Isfahan, Iran 104 Copyright All rights
@ I975 by Academic Press, Inc. of reproduction in any form resewed.
SPECTROPHOTOMETRIC
ESTIMATION
OF
STEROIDS
the relationship between ultraviolet light absorption and concentration three steroids is studied, and the results presented.
105
of
METHODS AND MATERIALS Steroids were dissolved in absolute ethanol (75 pg/ml). Dilutions from the stock solution were made using absolute ethanol as the solvent-diluent. Steroids used were donated by the World Health Organization (WHO), and purchased from Sigma Biochemical Corp. (St. Louis, MO., U.S.A.). Each steroid was recrystallized and checked for purity by thin layer chromatography on silica gel G, according to Cohn and Pancake (ll), to assure purity and homogeneity. After elution with cyclohexane: ethylacetate (1: 1, 90 min), the plates were sprayed with 12% phosphomolybdic acid to locate the spots. Elution with benzene : ethanol (9: 1 for 30 mitt), as described by Randerath (12), was found to be satisfactory for estradiol- 17p. A Zeiss Spectrophotometer PMQII (Carl Zeiss, Oberkochen/Wuertt, West Germany) was used for the light absorption, or optical density (OD), measurements. Quartz cells (l-ml capacity, l-cm light path with cover) were used in this study. All steroids were scanned to determine the optimum wavelength for maximum absorption. As a result of this study, estradiol-17fl and progesterone were measured at 230 nm, and testosterone at 240 nm. RESULTS A stock solution containing 75 pg/ml of each steroid was found to be satisfactory for this investigation. Although several solvents were used, it was noted that ethanol offered the least blank-light absorption for the uv range. For this reason, it was chosen as the solvent for use in these experiments. A solution containing 12.5 pg/rnl of each steroid was scanned for maximum absorption from 180-300 nm. It was noted that maximum absorption occurred at 230 nm for progesterone and estradiol17p, and at 240 nm for testosterone. These values were chosen for further investigation into the quantitation of the steroids described here. Absorption data for each of the steroids studied are described in Table 1, and are shown graphically in Fig. 1. The figure shows the absorption data for small concentrations from 1.25 to 12.5 pg/ml. The data for higher concentrations are summarized in Table 1. A straight line relationship (Beer’s Law) between concentration and light absorption (OD) is evident. When very large concentrations of the steroids were reached (e.g., OD for 75 kg/ml), it was evident that some quenching was taking place, such that the values fell below the line and a curved relationship appeared. In small concentrations, however (Fig. l), Beer’s Law was followed, and a straight line relationship was apparent. It was also evident that the respective optical densities for estradiol-l7P were not as high as those for the other two steroids. Consequently, it appeared that
106
KHAYAM-BASH1
AND
BOROUMAND
O.B-
T. (O.D. 240 nm)
0.7 P. (O.D. 230 nm)
E2 (0.D. 230 nm)
BR STEROID/ml ETHANOL FIG. 1. Relation between concentration of each steroid in ethanol and optical density (OD). For estradiol (E,) and progesterone Cp) absorption measurements were made at 230 nm, and for testosterone (T) at 240 nm, at a room temperature of 22°C.
small concentrations of estradiol did not produce OD changes that were large enough to allow for a very accurate measurement of this steroid. Nevertheless, values larger than 10 pgiml could be estimated accurately and reproducibly. However, the absorption data for progesterone and testosterone indicated that OD changes at very low concentrations (1.25 pg/ml) were large enough for an accurate measurement of these steroids. The values represented in Table 1 that are above 1.5 OD units were obtained by dilution of the corresponding concentration, so that all readings were around 1.2 OD units. This dilution was necessary for progesterone and testosterone, but not for estradiol-17j.3, which was linearly evaluated up to 75 kg/ml without dilution (Table 1). For this estrogen, OD values ranging from 0.254 to 1.430 were obtained, and
TABLE RELATIONSHIP
12.5 25.0 37.5 50.0 62.5 75.0
I
BETWEEN ULTRAVIOLET LIGHT AND STEROID CONCENTRATION
ABSORPTION
Progesterone OD 230 nma
Testosterone OD 240 nm*
Estradiol- 17B OD 230 nm”
0.606 1.065 1.972 2.610 2.905 3.235
0.755 1.440 2.046 2.925 3.420 4.060
0.254 0.502 0.728 0.975 I.175 I .430
I’ For OD values larger than 1.50. proper dilutions were made so that the OD reading was measured below 1.2 optical density unit.
SPECTROPHOTOMETRIC
ESTIMATION
OF
STEROIDS
107
showed a linear relationship between concentration and light absorption, with the possibility of some quenching at the 75 pg/ml level. DISCUSSION Urinary and serum estrogens and progesterone excretion have been correlated with many physiological changes, and can be used to evaluate the pathological status of the endocrine system. It has been reported that estradiol excretion increases in women bearing female infants, and estrio1 decreases in pregnancies ending in stillbirth (13, 14), while progesterone increases in late pregnancy plasma (4). Progesterone also has been reported to inhibit enzyme action in vitro (1). Several techniques involving gas chromatographic (13), calorimetric (IS), and fluorometric (2) procedures have been used to measure urinary excretion of estradiol during normal or complicated pregnancy. Other methods using radioimmunoassay techniques (7) and competitive protein binding systems (3, 4) have been used successfully for the accurate quantitation of steroids. Most of these procedures are elaborate. They require sophisticated technology and equipment, involve radioactive isotopes, and are inconvenient and/or expensive for screening purposes. By the application of a simple spectrophotometric analysis, the rapid screening test described here could be used to obtain valuable data, especially when the urgency of obtaining a laboratory result is critical. This approach could easily be made accessible to most laboratories where a quick estimate of steroids is desired. It has been reported that, during pregnancy, urinary estradiol can reach as high as 300 ,ug/24 hr, and that estriol of between 32-44 mg/24 hr and pregnanediol of 51.0-62.5 mg/24 hr, might be observed (13). These values are within the range appropriate for spectrophotometric analysis after fractionation on Sephadex LH-20 (4, 9) or other extraction, such as in chloroform (16), has been performed. Analyzing plasma estriol in late pregnancy, when values for total estriol (free and conjugate) reach 250 rig/ml (14), might not be possible by this method unless 5-10 ml plasma were used. For steroids or metabolites that are in very low concentrations, or when sample size and volume is very small, other microtechniques should be used. Although spectroscopy of steroids and steroid conjugates for identification and structural studies has been conducted extensively, no simple quantitative system has emerged from them. The studies by Scot (17) and by Smith and Bernstein (IS), together with present applied system discussed here, form the basis for a quantitative assay that can be utilized successfully in any laboratory. For a variety of conditions, including those discussed here, important and valuable screening data can be obtained economically in a short time, without the use of sophisticated instruments.
108
KHAYAM-BASH1
AND
BOROUMAND
SUMMARY
An ultraviolet spectrophotometric method is presented for the quantitative estimation of steroids which are in concentrations of more than 1.25 lug/ml. Estradiol-17j3, progesterone, and testosterone were used in these studies. Based on uv light absorption at 230 nm for estradiol and progesterone, and at 240 nm for testosterone. the relative concentration of each steroid could be estimated. This method is very simple and rapid. It is economical, requires no sophisticated instruments. and is very practical for estimating steroids in pharmaceutical preparations. chemicals. or biological specimens. ACKNOWLEDGMENTS The encouragement and advice of sistance in obtaining from WHO the edged. The editorial assistance of S. of the manuscript are also very much
Dr. R. C. Barnett throughout this work. and his assteroids used in this study. are gratemlly acknowlEastwood and the help of C. Fung in the preparation appreciated.
REFERENCES 1. Khayam-Bashi, H., Boroumand, M., Boroumand. A.. Hekmatyar. F. and Barnett. R. C. Nature (London) 230. 529 (1971). 2. Bush, I. E.. Ad\*. Chin. C/rem. 12, 57 (1969). 3. Murphy. B. E. P.. Nut/rre fLondon~ 201, 679 (1964). 4. Murphy. B. E. P., Nutrtrr (Nr)r Bid. 232, 21 (1971). 5. Ittrich, G.. Acta Endocrinol. 35, 34 (1960). 6. Baird, D. T.. J. C/i??. E/ldocrino/. Metcrb. 28, 244 (1968). 7. Parker. C. W., Prog. C/in. Palho/. (M. Stafanini, Ed.) 4, 103 (1977). 8. Moscrop, K. H., Antcliff. A. C.. Braunsberg. H.. James. V. H. T.. Goudie. J. H.. and Burnett, D.. Ch. Chim. Actn 56, 265 (1974). 9. Mikhail. G.. Wu, C. H.. Ferin. M. and VandeWiele. R. L., Steroids 15, 333 (1970). 10. Murphy. B. E. P., Actu Edoc~rirwl. 64 (Suppl. 147). 37 (1970). I I. Cohn. G. L. and Pancake. E.. Nufrrrr (Lot)clon) 201, 75 (1964). 12. Randerath. K.. in “Thin Layer Chromatography” (D. D. L,ibman. Trans.). p. 120. Academic Press, New York, (1963). 13. Larsen. A. E., Amer. J. Med. Tech. 37, 279 (1971). 14. Ahmed. .I. and Kellie. A. E., J. Strroid Biochrm. 4, I (1973). 15. Klopper. A. and Billewicz. W.. J. O/>src~r. Gyro. Brit. 70, 1024 (1963). 16. Van de Calseyde, J. F., Schlotis. R. J. H.. Schmidt. N, A., and Kuypers, A. M. J.. C‘lirt. Chim. Actu 25, 345 (1969). 17. Scot. A. I.. “Interpretation of Ultraviolet Spectra of Natural Products.” Pergamon Press. Oxford. 1964. 18. Smith. L.. L. and Bernstein, S., in “Physical Properties of the Steroid Hormones” CL. L. Engel, Ed.). p. 321. Pergamon Press. Oxford. 1963.
14, 104-108 (1975)
MEDICINE
Spectrophotometric
Estimation
Progesterone,
and
of Estradiol-17P, Testosterone’
H. KHAYAM-BASHI Department
of Laborutory
Medicine, Unil~ersity of Culifirniu School Medicine. at the Clinical Laboratorie.~. Strn Francisco Grnerrrl Sun Francisco, Ctrlijiwrlirr
of
Sun Frnncisco.
Hospital.
AND
M. BOROUMAND Department
qf Biochemistry.
School
of Pharmacy.
Utlivvrsity
of l.sfahan,
I.s,fuhon.
Iron
Received August 4. 197.5
The quantitation of various hormones in body fluids for the clinical evaluation of the endocrine system has been given considerable attention over the past decade. As a result, the estimation of steroid hormones for screening or quantitative purposes contributes significantly to the workload of modern clinical laboratories. Rapid quantitation of steroids in various pharmaceutical preparations, as well as other reagents containing these chemicals that are used for biochemical studies (I), also requires considerable attention in many chemical or pharmaceutical laboratories. Quantitation of these compounds has been attempted by various means (2), including the application of specific receptors in competitive binding systems (3, 4). as well as chemical (5). double isotope technique (6), and radioimmunological (7) procedures. These techniques are necessary for the most accurate determination when low concentrations are involved. Automated systems may be necessary in order to analyze large numbers of specimens (8). However, these procedures often are laborious, and require sophisticated methodologies and expensive instrumentation. When relatively large amounts of steroid hormones are present, as in concentrated urine specimens or in pharmaceutical preparations, simple spectrophotometric measurements of each steroid can provide a rapid quantitative procedure effectively. With the application of chromatographic separation techniques, such as Sephadex LH-20 (9. lo), these compounds can be separated by major groups, or even as individual steroids, after which a spectrophotometric assay can be performed. In this study, 1 Portions of this study were completed at the University of Isfahan. Isfahan, Iran 104 Copyright All rights
@ I975 by Academic Press, Inc. of reproduction in any form resewed.
SPECTROPHOTOMETRIC
ESTIMATION
OF
STEROIDS
the relationship between ultraviolet light absorption and concentration three steroids is studied, and the results presented.
105
of
METHODS AND MATERIALS Steroids were dissolved in absolute ethanol (75 pg/ml). Dilutions from the stock solution were made using absolute ethanol as the solvent-diluent. Steroids used were donated by the World Health Organization (WHO), and purchased from Sigma Biochemical Corp. (St. Louis, MO., U.S.A.). Each steroid was recrystallized and checked for purity by thin layer chromatography on silica gel G, according to Cohn and Pancake (ll), to assure purity and homogeneity. After elution with cyclohexane: ethylacetate (1: 1, 90 min), the plates were sprayed with 12% phosphomolybdic acid to locate the spots. Elution with benzene : ethanol (9: 1 for 30 mitt), as described by Randerath (12), was found to be satisfactory for estradiol- 17p. A Zeiss Spectrophotometer PMQII (Carl Zeiss, Oberkochen/Wuertt, West Germany) was used for the light absorption, or optical density (OD), measurements. Quartz cells (l-ml capacity, l-cm light path with cover) were used in this study. All steroids were scanned to determine the optimum wavelength for maximum absorption. As a result of this study, estradiol-17fl and progesterone were measured at 230 nm, and testosterone at 240 nm. RESULTS A stock solution containing 75 pg/ml of each steroid was found to be satisfactory for this investigation. Although several solvents were used, it was noted that ethanol offered the least blank-light absorption for the uv range. For this reason, it was chosen as the solvent for use in these experiments. A solution containing 12.5 pg/rnl of each steroid was scanned for maximum absorption from 180-300 nm. It was noted that maximum absorption occurred at 230 nm for progesterone and estradiol17p, and at 240 nm for testosterone. These values were chosen for further investigation into the quantitation of the steroids described here. Absorption data for each of the steroids studied are described in Table 1, and are shown graphically in Fig. 1. The figure shows the absorption data for small concentrations from 1.25 to 12.5 pg/ml. The data for higher concentrations are summarized in Table 1. A straight line relationship (Beer’s Law) between concentration and light absorption (OD) is evident. When very large concentrations of the steroids were reached (e.g., OD for 75 kg/ml), it was evident that some quenching was taking place, such that the values fell below the line and a curved relationship appeared. In small concentrations, however (Fig. l), Beer’s Law was followed, and a straight line relationship was apparent. It was also evident that the respective optical densities for estradiol-l7P were not as high as those for the other two steroids. Consequently, it appeared that
106
KHAYAM-BASH1
AND
BOROUMAND
O.B-
T. (O.D. 240 nm)
0.7 P. (O.D. 230 nm)
E2 (0.D. 230 nm)
BR STEROID/ml ETHANOL FIG. 1. Relation between concentration of each steroid in ethanol and optical density (OD). For estradiol (E,) and progesterone Cp) absorption measurements were made at 230 nm, and for testosterone (T) at 240 nm, at a room temperature of 22°C.
small concentrations of estradiol did not produce OD changes that were large enough to allow for a very accurate measurement of this steroid. Nevertheless, values larger than 10 pgiml could be estimated accurately and reproducibly. However, the absorption data for progesterone and testosterone indicated that OD changes at very low concentrations (1.25 pg/ml) were large enough for an accurate measurement of these steroids. The values represented in Table 1 that are above 1.5 OD units were obtained by dilution of the corresponding concentration, so that all readings were around 1.2 OD units. This dilution was necessary for progesterone and testosterone, but not for estradiol-17j.3, which was linearly evaluated up to 75 kg/ml without dilution (Table 1). For this estrogen, OD values ranging from 0.254 to 1.430 were obtained, and
TABLE RELATIONSHIP
12.5 25.0 37.5 50.0 62.5 75.0
I
BETWEEN ULTRAVIOLET LIGHT AND STEROID CONCENTRATION
ABSORPTION
Progesterone OD 230 nma
Testosterone OD 240 nm*
Estradiol- 17B OD 230 nm”
0.606 1.065 1.972 2.610 2.905 3.235
0.755 1.440 2.046 2.925 3.420 4.060
0.254 0.502 0.728 0.975 I.175 I .430
I’ For OD values larger than 1.50. proper dilutions were made so that the OD reading was measured below 1.2 optical density unit.
SPECTROPHOTOMETRIC
ESTIMATION
OF
STEROIDS
107
showed a linear relationship between concentration and light absorption, with the possibility of some quenching at the 75 pg/ml level. DISCUSSION Urinary and serum estrogens and progesterone excretion have been correlated with many physiological changes, and can be used to evaluate the pathological status of the endocrine system. It has been reported that estradiol excretion increases in women bearing female infants, and estrio1 decreases in pregnancies ending in stillbirth (13, 14), while progesterone increases in late pregnancy plasma (4). Progesterone also has been reported to inhibit enzyme action in vitro (1). Several techniques involving gas chromatographic (13), calorimetric (IS), and fluorometric (2) procedures have been used to measure urinary excretion of estradiol during normal or complicated pregnancy. Other methods using radioimmunoassay techniques (7) and competitive protein binding systems (3, 4) have been used successfully for the accurate quantitation of steroids. Most of these procedures are elaborate. They require sophisticated technology and equipment, involve radioactive isotopes, and are inconvenient and/or expensive for screening purposes. By the application of a simple spectrophotometric analysis, the rapid screening test described here could be used to obtain valuable data, especially when the urgency of obtaining a laboratory result is critical. This approach could easily be made accessible to most laboratories where a quick estimate of steroids is desired. It has been reported that, during pregnancy, urinary estradiol can reach as high as 300 ,ug/24 hr, and that estriol of between 32-44 mg/24 hr and pregnanediol of 51.0-62.5 mg/24 hr, might be observed (13). These values are within the range appropriate for spectrophotometric analysis after fractionation on Sephadex LH-20 (4, 9) or other extraction, such as in chloroform (16), has been performed. Analyzing plasma estriol in late pregnancy, when values for total estriol (free and conjugate) reach 250 rig/ml (14), might not be possible by this method unless 5-10 ml plasma were used. For steroids or metabolites that are in very low concentrations, or when sample size and volume is very small, other microtechniques should be used. Although spectroscopy of steroids and steroid conjugates for identification and structural studies has been conducted extensively, no simple quantitative system has emerged from them. The studies by Scot (17) and by Smith and Bernstein (IS), together with present applied system discussed here, form the basis for a quantitative assay that can be utilized successfully in any laboratory. For a variety of conditions, including those discussed here, important and valuable screening data can be obtained economically in a short time, without the use of sophisticated instruments.
108
KHAYAM-BASH1
AND
BOROUMAND
SUMMARY
An ultraviolet spectrophotometric method is presented for the quantitative estimation of steroids which are in concentrations of more than 1.25 lug/ml. Estradiol-17j3, progesterone, and testosterone were used in these studies. Based on uv light absorption at 230 nm for estradiol and progesterone, and at 240 nm for testosterone. the relative concentration of each steroid could be estimated. This method is very simple and rapid. It is economical, requires no sophisticated instruments. and is very practical for estimating steroids in pharmaceutical preparations. chemicals. or biological specimens. ACKNOWLEDGMENTS The encouragement and advice of sistance in obtaining from WHO the edged. The editorial assistance of S. of the manuscript are also very much
Dr. R. C. Barnett throughout this work. and his assteroids used in this study. are gratemlly acknowlEastwood and the help of C. Fung in the preparation appreciated.
REFERENCES 1. Khayam-Bashi, H., Boroumand, M., Boroumand. A.. Hekmatyar. F. and Barnett. R. C. Nature (London) 230. 529 (1971). 2. Bush, I. E.. Ad\*. Chin. C/rem. 12, 57 (1969). 3. Murphy. B. E. P.. Nut/rre fLondon~ 201, 679 (1964). 4. Murphy. B. E. P., Nutrtrr (Nr)r Bid. 232, 21 (1971). 5. Ittrich, G.. Acta Endocrinol. 35, 34 (1960). 6. Baird, D. T.. J. C/i??. E/ldocrino/. Metcrb. 28, 244 (1968). 7. Parker. C. W., Prog. C/in. Palho/. (M. Stafanini, Ed.) 4, 103 (1977). 8. Moscrop, K. H., Antcliff. A. C.. Braunsberg. H.. James. V. H. T.. Goudie. J. H.. and Burnett, D.. Ch. Chim. Actn 56, 265 (1974). 9. Mikhail. G.. Wu, C. H.. Ferin. M. and VandeWiele. R. L., Steroids 15, 333 (1970). 10. Murphy. B. E. P., Actu Edoc~rirwl. 64 (Suppl. 147). 37 (1970). I I. Cohn. G. L. and Pancake. E.. Nufrrrr (Lot)clon) 201, 75 (1964). 12. Randerath. K.. in “Thin Layer Chromatography” (D. D. L,ibman. Trans.). p. 120. Academic Press, New York, (1963). 13. Larsen. A. E., Amer. J. Med. Tech. 37, 279 (1971). 14. Ahmed. .I. and Kellie. A. E., J. Strroid Biochrm. 4, I (1973). 15. Klopper. A. and Billewicz. W.. J. O/>src~r. Gyro. Brit. 70, 1024 (1963). 16. Van de Calseyde, J. F., Schlotis. R. J. H.. Schmidt. N, A., and Kuypers, A. M. J.. C‘lirt. Chim. Actu 25, 345 (1969). 17. Scot. A. I.. “Interpretation of Ultraviolet Spectra of Natural Products.” Pergamon Press. Oxford. 1964. 18. Smith. L.. L. and Bernstein, S., in “Physical Properties of the Steroid Hormones” CL. L. Engel, Ed.). p. 321. Pergamon Press. Oxford. 1963.