Non-Chromatographic Radiotransinassay for Cortisol: Application to Human Adult Serum, Umbilical Cord Serum, and Amniotic Fluid BEVERLEY E. PEARSON MURPHY* Reproductive Physiology Unit, Montreal General Hospital and Department Medicine, McGill University, Montreal, Canada ABSTRACT. Rapid (1-2 hour) non-chromatographic radiotransinassay using horse transcortin can be used to measure cortisol in umbilical cord serum (after hexane-carbon tetrachloride prewash and methylene chloride extraction), in amniotic fluid (after methylene chloride extraction only), and in adult serum (after
W
HILE non-chromatographic radiotransinassays1 using human and dog transcortin (2,3) have provided satisfactory estimations of cortisol under most clinical circumstances (4,5), they are not sufficiently specific when large amounts of competing steroids are present as in pregnancy or in congenital adrenal hyperplasia. For maternal blood, a petroleum ether wash can be used to remove progesterone (2), or horse transcortin, which has a low affinity for progesterone (6), can be used as the binding protein. However, these are not satisfactory for umbilical cord blood, where cortisol concentrations are extremely low (7) in the presence of large amounts of many other steroids. Chromatography using Sephadex LH-20 provides an excellent means of separating cortisol from these other steroids but adds considerably to the time required for assay (8). While they show much promise, antibodies to hapten conjugates have so far not shown characteristics superior to those of horse transcortin (9,10,11). Horse serum also has the considerable advantages of ready availability, good stability, and constant specificity. Received November 27, 1975. * Associate Medical Research Council of Canada. 1 The term transin is used here to denote ; indigenous extracellular binding protein(l).
of Experimental
dilution and boiling without extraction). Since horse transcortin has high affinity (2.4 x 10B 1/mol) for cortisol, only a few microlitres of sample is required. The assay appears to be highly specific for cortisol clinically except in congenital adrenal hyperplasia. (J Clin Endocrinol Me tab 41: 1050, 1975)
The present method investigates solvent heating, pre-washing, and extraction followed by binding to horse transcortin, in the presence of gelatin (12), to provide assays with high specificity and sensitivity for cortisol. Materials and Methods Tritiated corticosterone (SA 50 Ci/mmol) was obtained from the New England Nuclear Co., Boston. On arrival it was diluted to a concentration of 10 /xCi/ml redistilled ethanol, and stored at - 1 0 C . Purity was checked using Sephadex LH-20 chromatography (methylene chloride: methanol, 98:2). Unlabelled steroids were obtained from the Sigma Chemical Co., 3500 DeKalb St., St. Louis, Mo. They were dissolved in redistilled ethanol and stored at —10 C. Redistilled solvents (reagent grade) were obtained from A. and C. American Chemicals, Montreal; horse serum, tissue culture select, pooled with no preservative, from BBL, Division of Bioquist, Divison of Becton, Dickinson and Co., Cockeysville, Md.; gelatin from Knox Gelatin of Canada, Trenton, Ont.; and Florisil, 100-200 mesh, no. F100, from the Fisher Scientific Co. If necessary, Florisil was rinsed 3 times with ethanol, the fines discarded, and dried at room temperature (12). Forty mg was dispensed with a plastic spoon. Protein-tracer solution (PTS) was made up by evaporating 4 /xCi 3H-corticosterone, dissolving
1050
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RADIOTRANSINASSAY FOR CORTISOL it in 1.0 ml horse serum, and making up to 100 ml with gelatin solution 0.5 g/1. Amniotic fluid was obtained at hysterotorny or by amniocentesis and kept frozen until determinations were made. Cord blood was obtained at delivery, allowed to clot, centrifuged, and the serum was stored at 4 C for long periods. Adult blood was obtained from normal or pregnant volunteers or hospital patients. It was centrifuged and stored similarly. Aliquots of 0.01 and 0.02 ml cord serum, 0.002 and 0.004 ml adult serum, or 0.03 and 0.06 ml amniotic fluid were usually used. Non-chromatographic sample preparation
1051
scintillator and shaken thoroughly. Vials were counted to 4000 counts x 2. Results were plotted as mean time required to reach 4000 counts (i.e. the reciprocal of the per cent of tracer bound) versus ng cortisol. Less sensitive assay. Because the procedure outlined above was too sensitive for routine hospital use, a less sensitive modification for adult serum was used as follows: 0.01 ml samples of serum were prepared in duplicate as in above (C). 5% horse serum was used in the protein-tracer solution, and 0, 1, 2, 4, and 6 ng cortisol were used as standards.
A. Prewash and extraction. To an aliquot of Chromatographic sample preparations. Samsample in a 15 ml centrifuge tube was added 0.1 ples to which 2000 counts per minute (15 ml water, then 2 ml hexane-carbon tetrachloride pg) of various tritiated steroids had been added (1:1 or 1.5:1). After mixing on a vortex mixer, the were extracted before or after prewashing with 5 phases were allowed to separate, and the organic volumes x 2 of methylene dichloride. The phase was discarded. The aqueous phase was combined extracts were dried under air, extracted with dichloromethane 1.0 ml x 2 and redissolved in 0.2 ml column solvent (meththe combined extracts were evaporated to dry- ylene chloride:methanol, 98:2), and applied ness under air along with tubes containing 0, to the top of a Sephadex LH-20 column 0.25, 0.50, 1.0, and 2.0 ng cortisol to provide a (39 x 0.8 cm) packed using the same solvent. standard curve. After allowing the sample to enter the gel, 70 ml solvent was passed through the column and the B. Extraction only. As in (A) but omitting the eluate collected in 2 ml fractions. One ml of prewash. each fraction was used for counting while the other was dried under air and assayed as above. C. Without extraction. An aliquot of sample in an assay tube was diluted with 0.10 ml water. CorResults responding amounts of diluted, charcoal-treated (30 min at room temperature) serum to which 0, Standard curves. Figure 1 shows examples 0.25, 0.40,1.0, and 2.0 ng cortisol had been added of the standard curves obtained by plotting served as standards. All tubes were placed in a the reciprocal of the per cent of bound rack, heated in a boiling water bath for 3 min, tracer versus added cortisol. Initial binding and allowed to cool. (no cortisol added) averaged 45% for the 1% horse serum solution and 67% for the Assay procedure. To each tube, in a rack, was 5% solution. The affinity constant as calcuadded 1.0 ml PTS. The tubes were incubated lated from the initial slope using a Scatchfor 5 min in a water bath at 45 C and for 10 ard plot was 2.4 x 109 1/mol. min or longer in an agitated cold water bath at 4 C . While still in the cold water bath 40 mg Florisil was added to each tube. The Sensitivity. The smallest amount of cortisol rack containing the tubes was shaken vigor- which could be measured with 95% confiously horizontally on a Eberbach shaker for dence (i.e. 2 x SD at zero concentration) in 1 min and immediately returned to the cold the assay using 1% horse serum was 40 pg. water bath. After allowing the Florisil to settle (2-3 min) 0.5 ml of each supernatant was Specificity. The competition of various transferred to a counting vial containing 10 ml of steroids for cortisol-binding sites on horse
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 October 2015. at 14:38 For personal use only. No other uses without permission. . All rights reserved.
JCE & M • 1975 Vol 41 • No 6
MURPHY
1052 1.75 i
-1.2
1.5 -
L 1.0
1.25 -
1-0.8
1% HORSE
5% HORSE
SERUM
SERUM
miato reach 4000 cts
min. to reach
1.0 -
4000 cts t-0.4
0.75 -
0.5
F I G . 1. Examples of standard curves using 1% horse serum (• •) and 5% horse serum (A---A).
i- 0.2 500
1000
1500
CORTISOL
peg
2
2000
4
CORTISOL ng
transcortin are shown in Table 1, where it is compared with that of other cortisolbinding proteins. Competition of some other substances relative to cortisol for sites on horse transcortin were: 11-hydroxyprogesterone, 8%; 11-dehydrocorticosterone,
W
HILE non-chromatographic radiotransinassays1 using human and dog transcortin (2,3) have provided satisfactory estimations of cortisol under most clinical circumstances (4,5), they are not sufficiently specific when large amounts of competing steroids are present as in pregnancy or in congenital adrenal hyperplasia. For maternal blood, a petroleum ether wash can be used to remove progesterone (2), or horse transcortin, which has a low affinity for progesterone (6), can be used as the binding protein. However, these are not satisfactory for umbilical cord blood, where cortisol concentrations are extremely low (7) in the presence of large amounts of many other steroids. Chromatography using Sephadex LH-20 provides an excellent means of separating cortisol from these other steroids but adds considerably to the time required for assay (8). While they show much promise, antibodies to hapten conjugates have so far not shown characteristics superior to those of horse transcortin (9,10,11). Horse serum also has the considerable advantages of ready availability, good stability, and constant specificity. Received November 27, 1975. * Associate Medical Research Council of Canada. 1 The term transin is used here to denote ; indigenous extracellular binding protein(l).
of Experimental
dilution and boiling without extraction). Since horse transcortin has high affinity (2.4 x 10B 1/mol) for cortisol, only a few microlitres of sample is required. The assay appears to be highly specific for cortisol clinically except in congenital adrenal hyperplasia. (J Clin Endocrinol Me tab 41: 1050, 1975)
The present method investigates solvent heating, pre-washing, and extraction followed by binding to horse transcortin, in the presence of gelatin (12), to provide assays with high specificity and sensitivity for cortisol. Materials and Methods Tritiated corticosterone (SA 50 Ci/mmol) was obtained from the New England Nuclear Co., Boston. On arrival it was diluted to a concentration of 10 /xCi/ml redistilled ethanol, and stored at - 1 0 C . Purity was checked using Sephadex LH-20 chromatography (methylene chloride: methanol, 98:2). Unlabelled steroids were obtained from the Sigma Chemical Co., 3500 DeKalb St., St. Louis, Mo. They were dissolved in redistilled ethanol and stored at —10 C. Redistilled solvents (reagent grade) were obtained from A. and C. American Chemicals, Montreal; horse serum, tissue culture select, pooled with no preservative, from BBL, Division of Bioquist, Divison of Becton, Dickinson and Co., Cockeysville, Md.; gelatin from Knox Gelatin of Canada, Trenton, Ont.; and Florisil, 100-200 mesh, no. F100, from the Fisher Scientific Co. If necessary, Florisil was rinsed 3 times with ethanol, the fines discarded, and dried at room temperature (12). Forty mg was dispensed with a plastic spoon. Protein-tracer solution (PTS) was made up by evaporating 4 /xCi 3H-corticosterone, dissolving
1050
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 October 2015. at 14:38 For personal use only. No other uses without permission. . All rights reserved.
RADIOTRANSINASSAY FOR CORTISOL it in 1.0 ml horse serum, and making up to 100 ml with gelatin solution 0.5 g/1. Amniotic fluid was obtained at hysterotorny or by amniocentesis and kept frozen until determinations were made. Cord blood was obtained at delivery, allowed to clot, centrifuged, and the serum was stored at 4 C for long periods. Adult blood was obtained from normal or pregnant volunteers or hospital patients. It was centrifuged and stored similarly. Aliquots of 0.01 and 0.02 ml cord serum, 0.002 and 0.004 ml adult serum, or 0.03 and 0.06 ml amniotic fluid were usually used. Non-chromatographic sample preparation
1051
scintillator and shaken thoroughly. Vials were counted to 4000 counts x 2. Results were plotted as mean time required to reach 4000 counts (i.e. the reciprocal of the per cent of tracer bound) versus ng cortisol. Less sensitive assay. Because the procedure outlined above was too sensitive for routine hospital use, a less sensitive modification for adult serum was used as follows: 0.01 ml samples of serum were prepared in duplicate as in above (C). 5% horse serum was used in the protein-tracer solution, and 0, 1, 2, 4, and 6 ng cortisol were used as standards.
A. Prewash and extraction. To an aliquot of Chromatographic sample preparations. Samsample in a 15 ml centrifuge tube was added 0.1 ples to which 2000 counts per minute (15 ml water, then 2 ml hexane-carbon tetrachloride pg) of various tritiated steroids had been added (1:1 or 1.5:1). After mixing on a vortex mixer, the were extracted before or after prewashing with 5 phases were allowed to separate, and the organic volumes x 2 of methylene dichloride. The phase was discarded. The aqueous phase was combined extracts were dried under air, extracted with dichloromethane 1.0 ml x 2 and redissolved in 0.2 ml column solvent (meththe combined extracts were evaporated to dry- ylene chloride:methanol, 98:2), and applied ness under air along with tubes containing 0, to the top of a Sephadex LH-20 column 0.25, 0.50, 1.0, and 2.0 ng cortisol to provide a (39 x 0.8 cm) packed using the same solvent. standard curve. After allowing the sample to enter the gel, 70 ml solvent was passed through the column and the B. Extraction only. As in (A) but omitting the eluate collected in 2 ml fractions. One ml of prewash. each fraction was used for counting while the other was dried under air and assayed as above. C. Without extraction. An aliquot of sample in an assay tube was diluted with 0.10 ml water. CorResults responding amounts of diluted, charcoal-treated (30 min at room temperature) serum to which 0, Standard curves. Figure 1 shows examples 0.25, 0.40,1.0, and 2.0 ng cortisol had been added of the standard curves obtained by plotting served as standards. All tubes were placed in a the reciprocal of the per cent of bound rack, heated in a boiling water bath for 3 min, tracer versus added cortisol. Initial binding and allowed to cool. (no cortisol added) averaged 45% for the 1% horse serum solution and 67% for the Assay procedure. To each tube, in a rack, was 5% solution. The affinity constant as calcuadded 1.0 ml PTS. The tubes were incubated lated from the initial slope using a Scatchfor 5 min in a water bath at 45 C and for 10 ard plot was 2.4 x 109 1/mol. min or longer in an agitated cold water bath at 4 C . While still in the cold water bath 40 mg Florisil was added to each tube. The Sensitivity. The smallest amount of cortisol rack containing the tubes was shaken vigor- which could be measured with 95% confiously horizontally on a Eberbach shaker for dence (i.e. 2 x SD at zero concentration) in 1 min and immediately returned to the cold the assay using 1% horse serum was 40 pg. water bath. After allowing the Florisil to settle (2-3 min) 0.5 ml of each supernatant was Specificity. The competition of various transferred to a counting vial containing 10 ml of steroids for cortisol-binding sites on horse
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 October 2015. at 14:38 For personal use only. No other uses without permission. . All rights reserved.
JCE & M • 1975 Vol 41 • No 6
MURPHY
1052 1.75 i
-1.2
1.5 -
L 1.0
1.25 -
1-0.8
1% HORSE
5% HORSE
SERUM
SERUM
miato reach 4000 cts
min. to reach
1.0 -
4000 cts t-0.4
0.75 -
0.5
F I G . 1. Examples of standard curves using 1% horse serum (• •) and 5% horse serum (A---A).
i- 0.2 500
1000
1500
CORTISOL
peg
2
2000
4
CORTISOL ng
transcortin are shown in Table 1, where it is compared with that of other cortisolbinding proteins. Competition of some other substances relative to cortisol for sites on horse transcortin were: 11-hydroxyprogesterone, 8%; 11-dehydrocorticosterone,
