Urine Aldosterone Radioimmunoassay: Validation of a Method Without Chromatography RONALD D. BROWN, ANNA SWANDER, AND JOHN K. McKENZIE Departments of Medicine, Mayo Clinic, Rochester, Minnesota; Baylor College of Medicine, Houston, Texas; and the University of Manitoba, General Centre, Health Sciences Centre, Winnipeg, Manitoba, Canada steronism determined by this method agreed closely (r = 0.95, P < 0.01) with values obtained using a standardized chromatographic method. This simplified assay represents a significant advance in our capabilities for evaluating patients for abnormalities in aldosterone physiology. (J Clin Endocrinol Metab 42: 894, 1976)
ABSTRACT. A simplified radioimmunoassay of urinary aldosterone is reported. Acid-hydrolyzed urine was extracted with dichloromethane and the extract assayed without further purification. Urinary aldosterone values in patients with Cushing's syndrome, low and normal-renin essential hypertension, congential adrenal hyperplasia, and primary aldo-
D
URING the past five years several radioimmunoassay procedures for measuring urinary aldosterone have been reported. The majority require either chromatography steps (1-11) or the formation of a derivative of aldosterone (12-14) to gain sufficient specificity. A few procedures, however, do not include such preliminary purification steps (15-19). Here we report a method for measuring aldosterone in urine which requires no preliminary chromatography or derivative formation. Urine is preextracted with ethyl acetate, the 18-glucuronide metabolite of aldosterone is hydrolyzed, the free aldosterone is extracted and, without further purification steps, assayed utilizing a very specific aldosterone antiserum prepared by one of us (JKM), hereafter referred to as the "Canadian antialdosterone antiserum." This method gives valid estimates of urinary aldosterone in patients with a wide variety of adrenal and hypertensive diseases.
Received July 25, 1975. Supported in part by Baylor Clinical Research Center RR 00134, by funds from the Mayo Foundation (Doctor Brown), and by the Canadian Medical Research Council Grant No. MA 4044 (Doctor McKenzie). Reprints: R. D. Brown, M.D., Mayo Clinic, Rochester, Minnesota 55901.
Materials and Methods Subjects. Urine was collected from patients with hypertensive and adrenal disorders and stored at 4 C using 0.5% thymol in acetic acid as a preservative. Materials. All solvents were reagent grade. Dichloromethane (Matheson, Coleman, Bell) was further purified by passing it through a column of silica gel (120 x 5 cm). Other solvents were used without further purification. Norit A charcoal (Matheson, Coleman, Bell) was washed three times with 0 . 1 N HC1, then with distilled 1
The following abbreviations and trivial names have been employed: 18-OH-DOC (18-OH-deoxycorticosterone = 18,21-dihydroxypregn-4-ene-3,20-dione); 18OH-corticosterone (11/3, 18, 21-trihydroxypregn-4-ene3,20-dione); 18-oxo-DOC (18-oxo-deoxycorticosterone = 21-hydroxy-18-oxo pregn-4-ene-3,20-dione); 17-OHprogesterone (17a-hydroxypregn-4-ene-3,20-dione); deoxycortisol (17a,21-dihydroxypregn-4-ene-3,20dione); pregnenolone (3/8-hydroxypregn-5-en-20-one); 17-OH-pregnenolone (3j8,17a-dihydroxypregn-5-en20-one); dihydrotestosterone (17/3-hydroxy-5a-androstan-3-one); 16/8-OH-testosterone (16/3,17/3-dihydroxyandrost-4-en-3-one); androstanedione (5a-androstan3,17-dione); 16a-OH-testosterone (16a,17/3-dihydroxyandrost-4-en-3-one); androsterone (3 a-hydroxy-5aandrostan-17-one); androstenediol (androst-5-ene-3/3, 17/3-diol), 16a-OH-dehydroepiandrosterone (3/3,16adihydroxyandrost-5-en-17-one); 16/3-OH-dehydroepiandrosterone (3j3,16/3-dihydroxyandrost-5-en-17-one); 16-oxo-androstenediol (3/3,17/3-dihydroxy androst-5en-16-one); 11-oxo-etiocholanolone (3a-hydroxy-5/3androstan-11,17,-dione).
894
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ALDOSTERONE RADIOIMMUNOASSAY water and acetone. Dextran T40 (Pharmacia Fine Chemicals) was used as obtained from the supplier. Steroids (Steraloids, Sigma, Schwarz/ Mann) were re-crystallized at least once prior to use. The melting points obtained agreed with published data. The 18-OH-DOC,1 18-OH-corticosterone, and 18-oxo-DOC used in this study were obtained from CIBA-GEIGY Coip. The small quantities of these steroids available precluded re-crystallization in our laboratory. Aldosterone-1,2,3H (New England Nuclear Corp., SA 45 Ci/niM) was stored in absolute ethanol at — 20 C. The purity of this substance was checked using paper chromatography every 3-6 weeks. The glassware was acid-washed. Assay tubes (12 x 75 mm soda lime tubes, Kimble) were used as supplied. The Canadian anti-aldosterone antiserum was prepared by one of us (JKM) in rabbits, using aldosterone-3-oxime conjugated to rabbit serum albumin (20). The anti-aldosterone antiserum, obtained from the National Institutes of Health, has been used in one of our labs (RDB) since 1971 to measure urinary aldosterone. The method employing the NIH antiserum, following the acid hydrolysis and extraction steps, requires a cyclohexane partition and a paper chromatography step (toluene:methanol: water 60:13:6) to purify the aldosterone prior to radioimmunoassay (21). The results, using this radioimmunoassay method, agreed closely (r = 0.98, P < 0.001) with results using a doubleisotope dilution derivative technique to measure urinary aldosterone employed by us up to 1971. Assay procedures. An aliquot of a 24-hour urine was extracted with 5 volumes of ethyl acetate. The urine pH was lowered to 0.9-1.0 with 60% HC1 and incubated for 22-24 hours at room temperature. In preliminary studies, to determine the feasibility of directly measuring aldosterone in hydrolyzed urine, the pH of 5 ml of hydrolyzed urine was adjusted to 7.5 with 50% NaOH and a 100 /xl aliquot of the urine was serially diluted in 0.05M borate buffer. This urine was then assayed using the Canadian antiserum. For these experiments the standard curve was made by serially diluting standard aldosterone in the same buffer. The remainder of the hydrolyzed urine was processed as previously described for measuring urinary aldosterone using the NIH antiserum (21).
895
In subsequent experiments 5-10 ml of the hydrolyzed urine was pipetted into extraction tubes, approximately 5000 dpm of 3H-aldosterone was added, and the urine was extracted with 7 vol dichloromethane. The dichloromethane was washed with 0.2N NaOH, distilled water, 1% acetic acid, and then with 0.05M phosphate buffer (pH 7.0) to adjust the pH to 7.0. The dichloromethane was evaporated to dryness under an air stream in a 40 C water bath. The residue was dissolved in 1 ml absolute ethanol. A 300 (x\ aliquot was removed, the tritiumlabeled aldosterone counted, and the procedural losses calculated. A 100 /xl aliquot was diluted 1:10 with absolute ethanol. From this solution 25, 50, and 100 /xl aliquots were added to assay tubes in duplicate, and the ethanol was evaporated in vacuo at 40 C. A standard curve was prepared for each assay by serially diluting a standard aldosterone solution (2.0 ng/100 /xl) using ethanol. A solution containing 2% ethanol, 1% human serum (obtained from an indivudual receiving dexamethasone), and 30,000 dpm 3H aldosterone/ ml was prepared using borate buffer (0.05M pH 8.0) in which the Canadian aldosterone antiserum was diluted to 1:40,000. To each assay tube 0.5 ml of this solution was added. The tubes were incubated at 37 C for 20 min then at 4 C overnight. The bound and free aldosterone were separated (at 0 C) using 0.5 ml of the charcoal-dextran solution. The supernatant was decanted into scintillation vials, 10 ml Scintisol added (Isolab Corp), and the radioactivity counted in a liquid scintillation spectrophotometer. The final aldosterone value was calculated by averaging the results from the three dilutions of the urine extract assayed, corrected for procedural losses.
Results Specificity of the anti-aldosterone antisera. The displacement of tritium-labeled aldosterone from the two aldosterone antisera by various C-21, C-19, and C-18 steroids is compared on Table 1. Standard curve (Fig. 1). Using a 1/40,000 dilution of the Canadian antiserum 45 ± 5% of added 3H-aldosterone was bound. The
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BROWN, SWANDER AND McKENZIE
896
TABLE 1. Comparison of specificity of the anti-aldosterone antisera NIH
Steroids C-21 Steroids Aldosterone Tetrahydroaldosterone3-glucuronide 18-oxo-Deoxycorticosterone Deoxycorticosterone Cortisone 18-OH-Deoxycorticosterone Corticosterone Progesterone 17-OH-Progesterone Deoxycortisol Cortisol Pregnenolone 17-OH-Pregnenolone C-19 Steroids Dihydrotestosterone Testosterone 4-Androsten-3,17-dione 16/3-OH-Testosterone Androstanedione 16a-OH-Testosterone 4-Androsten-3,17/3-diol 5a-Androstan-3/}-ol-16-one 5-Androsten-3j8,16/3-diol 5c*-Androstan-3/3,16adiol-17-one Androsterone Androstenediol Dehydroepiandrosterone 16a-OH-Dehydroepiandrosterone 16/3-OH-Dehydroepiandrosterone 16-oxo-Androstenediol 11-oxo-Etiocholanolone C-18 Steroids Estrone Estradiol Estriol
antiserum % Crossreactivity*
100
Canadian antiserum % Crossreactivity*
100
ABSTRACT. A simplified radioimmunoassay of urinary aldosterone is reported. Acid-hydrolyzed urine was extracted with dichloromethane and the extract assayed without further purification. Urinary aldosterone values in patients with Cushing's syndrome, low and normal-renin essential hypertension, congential adrenal hyperplasia, and primary aldo-
D
URING the past five years several radioimmunoassay procedures for measuring urinary aldosterone have been reported. The majority require either chromatography steps (1-11) or the formation of a derivative of aldosterone (12-14) to gain sufficient specificity. A few procedures, however, do not include such preliminary purification steps (15-19). Here we report a method for measuring aldosterone in urine which requires no preliminary chromatography or derivative formation. Urine is preextracted with ethyl acetate, the 18-glucuronide metabolite of aldosterone is hydrolyzed, the free aldosterone is extracted and, without further purification steps, assayed utilizing a very specific aldosterone antiserum prepared by one of us (JKM), hereafter referred to as the "Canadian antialdosterone antiserum." This method gives valid estimates of urinary aldosterone in patients with a wide variety of adrenal and hypertensive diseases.
Received July 25, 1975. Supported in part by Baylor Clinical Research Center RR 00134, by funds from the Mayo Foundation (Doctor Brown), and by the Canadian Medical Research Council Grant No. MA 4044 (Doctor McKenzie). Reprints: R. D. Brown, M.D., Mayo Clinic, Rochester, Minnesota 55901.
Materials and Methods Subjects. Urine was collected from patients with hypertensive and adrenal disorders and stored at 4 C using 0.5% thymol in acetic acid as a preservative. Materials. All solvents were reagent grade. Dichloromethane (Matheson, Coleman, Bell) was further purified by passing it through a column of silica gel (120 x 5 cm). Other solvents were used without further purification. Norit A charcoal (Matheson, Coleman, Bell) was washed three times with 0 . 1 N HC1, then with distilled 1
The following abbreviations and trivial names have been employed: 18-OH-DOC (18-OH-deoxycorticosterone = 18,21-dihydroxypregn-4-ene-3,20-dione); 18OH-corticosterone (11/3, 18, 21-trihydroxypregn-4-ene3,20-dione); 18-oxo-DOC (18-oxo-deoxycorticosterone = 21-hydroxy-18-oxo pregn-4-ene-3,20-dione); 17-OHprogesterone (17a-hydroxypregn-4-ene-3,20-dione); deoxycortisol (17a,21-dihydroxypregn-4-ene-3,20dione); pregnenolone (3/8-hydroxypregn-5-en-20-one); 17-OH-pregnenolone (3j8,17a-dihydroxypregn-5-en20-one); dihydrotestosterone (17/3-hydroxy-5a-androstan-3-one); 16/8-OH-testosterone (16/3,17/3-dihydroxyandrost-4-en-3-one); androstanedione (5a-androstan3,17-dione); 16a-OH-testosterone (16a,17/3-dihydroxyandrost-4-en-3-one); androsterone (3 a-hydroxy-5aandrostan-17-one); androstenediol (androst-5-ene-3/3, 17/3-diol), 16a-OH-dehydroepiandrosterone (3/3,16adihydroxyandrost-5-en-17-one); 16/3-OH-dehydroepiandrosterone (3j3,16/3-dihydroxyandrost-5-en-17-one); 16-oxo-androstenediol (3/3,17/3-dihydroxy androst-5en-16-one); 11-oxo-etiocholanolone (3a-hydroxy-5/3androstan-11,17,-dione).
894
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ALDOSTERONE RADIOIMMUNOASSAY water and acetone. Dextran T40 (Pharmacia Fine Chemicals) was used as obtained from the supplier. Steroids (Steraloids, Sigma, Schwarz/ Mann) were re-crystallized at least once prior to use. The melting points obtained agreed with published data. The 18-OH-DOC,1 18-OH-corticosterone, and 18-oxo-DOC used in this study were obtained from CIBA-GEIGY Coip. The small quantities of these steroids available precluded re-crystallization in our laboratory. Aldosterone-1,2,3H (New England Nuclear Corp., SA 45 Ci/niM) was stored in absolute ethanol at — 20 C. The purity of this substance was checked using paper chromatography every 3-6 weeks. The glassware was acid-washed. Assay tubes (12 x 75 mm soda lime tubes, Kimble) were used as supplied. The Canadian anti-aldosterone antiserum was prepared by one of us (JKM) in rabbits, using aldosterone-3-oxime conjugated to rabbit serum albumin (20). The anti-aldosterone antiserum, obtained from the National Institutes of Health, has been used in one of our labs (RDB) since 1971 to measure urinary aldosterone. The method employing the NIH antiserum, following the acid hydrolysis and extraction steps, requires a cyclohexane partition and a paper chromatography step (toluene:methanol: water 60:13:6) to purify the aldosterone prior to radioimmunoassay (21). The results, using this radioimmunoassay method, agreed closely (r = 0.98, P < 0.001) with results using a doubleisotope dilution derivative technique to measure urinary aldosterone employed by us up to 1971. Assay procedures. An aliquot of a 24-hour urine was extracted with 5 volumes of ethyl acetate. The urine pH was lowered to 0.9-1.0 with 60% HC1 and incubated for 22-24 hours at room temperature. In preliminary studies, to determine the feasibility of directly measuring aldosterone in hydrolyzed urine, the pH of 5 ml of hydrolyzed urine was adjusted to 7.5 with 50% NaOH and a 100 /xl aliquot of the urine was serially diluted in 0.05M borate buffer. This urine was then assayed using the Canadian antiserum. For these experiments the standard curve was made by serially diluting standard aldosterone in the same buffer. The remainder of the hydrolyzed urine was processed as previously described for measuring urinary aldosterone using the NIH antiserum (21).
895
In subsequent experiments 5-10 ml of the hydrolyzed urine was pipetted into extraction tubes, approximately 5000 dpm of 3H-aldosterone was added, and the urine was extracted with 7 vol dichloromethane. The dichloromethane was washed with 0.2N NaOH, distilled water, 1% acetic acid, and then with 0.05M phosphate buffer (pH 7.0) to adjust the pH to 7.0. The dichloromethane was evaporated to dryness under an air stream in a 40 C water bath. The residue was dissolved in 1 ml absolute ethanol. A 300 (x\ aliquot was removed, the tritiumlabeled aldosterone counted, and the procedural losses calculated. A 100 /xl aliquot was diluted 1:10 with absolute ethanol. From this solution 25, 50, and 100 /xl aliquots were added to assay tubes in duplicate, and the ethanol was evaporated in vacuo at 40 C. A standard curve was prepared for each assay by serially diluting a standard aldosterone solution (2.0 ng/100 /xl) using ethanol. A solution containing 2% ethanol, 1% human serum (obtained from an indivudual receiving dexamethasone), and 30,000 dpm 3H aldosterone/ ml was prepared using borate buffer (0.05M pH 8.0) in which the Canadian aldosterone antiserum was diluted to 1:40,000. To each assay tube 0.5 ml of this solution was added. The tubes were incubated at 37 C for 20 min then at 4 C overnight. The bound and free aldosterone were separated (at 0 C) using 0.5 ml of the charcoal-dextran solution. The supernatant was decanted into scintillation vials, 10 ml Scintisol added (Isolab Corp), and the radioactivity counted in a liquid scintillation spectrophotometer. The final aldosterone value was calculated by averaging the results from the three dilutions of the urine extract assayed, corrected for procedural losses.
Results Specificity of the anti-aldosterone antisera. The displacement of tritium-labeled aldosterone from the two aldosterone antisera by various C-21, C-19, and C-18 steroids is compared on Table 1. Standard curve (Fig. 1). Using a 1/40,000 dilution of the Canadian antiserum 45 ± 5% of added 3H-aldosterone was bound. The
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BROWN, SWANDER AND McKENZIE
896
TABLE 1. Comparison of specificity of the anti-aldosterone antisera NIH
Steroids C-21 Steroids Aldosterone Tetrahydroaldosterone3-glucuronide 18-oxo-Deoxycorticosterone Deoxycorticosterone Cortisone 18-OH-Deoxycorticosterone Corticosterone Progesterone 17-OH-Progesterone Deoxycortisol Cortisol Pregnenolone 17-OH-Pregnenolone C-19 Steroids Dihydrotestosterone Testosterone 4-Androsten-3,17-dione 16/3-OH-Testosterone Androstanedione 16a-OH-Testosterone 4-Androsten-3,17/3-diol 5a-Androstan-3/}-ol-16-one 5-Androsten-3j8,16/3-diol 5c*-Androstan-3/3,16adiol-17-one Androsterone Androstenediol Dehydroepiandrosterone 16a-OH-Dehydroepiandrosterone 16/3-OH-Dehydroepiandrosterone 16-oxo-Androstenediol 11-oxo-Etiocholanolone C-18 Steroids Estrone Estradiol Estriol
antiserum % Crossreactivity*
100
Canadian antiserum % Crossreactivity*
100
