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A system for separation of cortisol, 11-deoxycortisol, 17-hydroxyprogesterone and progesterone in a single chromatographic step and its application to radioimmunoassay
Clinica Chimica Acfa, 123 (1982) 211-219 Elsevier Biomedical Press
211
CCA 2201
A system for separation of cortisol, 1 1-deoxycortisol, 17-hydroxyprogesterone and progesterone in a single chromatographic step and its application to radioimmunoassay Sean
K. Cunningham
* and T. Joseph
McKenna
Department of Endocrinology, St. Vincent’s Hospital, Elm Park, Dublin 4 (Republic of Ireland) (Received
September
23rd. 1981; revision March 22nd, 1982)
Summary A procedure
for accurate, sensitive and highly specific measurement of cortisol, 17-hydroxyprogesterone and progesterone is described. We have developed a chromatographic system whereby the steroids may be separated in a single passage over celite microcolumns. This permits the specific radioimmunoassay of the principal glucocorticoid in man and its A6pregnene precursors and thus facilitates investigation of the physiological control and pathological disorders of glucocorticoid biosynthesis. 11-deoxycortisol,
Introduction Accurate measurement of steroid concentrations in blood is fundamental to the elucidation of both physiological and pathological processes affecting the adrenal glands and gonads. A variety of assay techniques have previously been used for this purpose. Calorimetric and fluorometric methods are neither sensitive nor specific, and are subject to interference, particularly from drugs [l-3]. High pressure liquid chromatography with ultraviolet detection provides sufficient sensitivity for cortisol [4] but not for normal plasma levels of its precursors. Competitive protein binding methods have been used [5,6], but lack specificity and have more recently been replaced by radioimmunoassay [7]. Although radioimmunoassay is the most sensitive of readily available techniques, antibodies lack complete specificity for any one steroid and react with related steroids. However, specificity may be greatly enhanced by extraction and chromatographic isolation of the desired steroid prior to quantifi-
* All correspondence Vincent’s Hospital,
should be directed to: Dr. Sean Cunningham, Elm Park, Dublin 4, Republic of Ireland.
0009-8981/82/0000-0000/$02.75
0 1982 Elsevier Biomedical
Press
Department
of Endocrinology,
St.
212
cation [7,8]. The use of celite as an efficient chromatographic agent for the isolation of steroids has been widely advanced by Abraham and colleagues [8]. We have developed and expanded this methodology to achieve separation of cortisol, 1 l-deoxycortisol, 17-hydroxyprogesterone and progesterone in a single chromatographic procedure. This system is particularly useful when a meticulous examination of cortisol biosynthesis is undertaken and should prove to be a valuable research tool. Experimental Materials Tritiated steroids ([ 1,2,6,7-3H]progesterone, 80 Ci/mmol; 17-hydroxy [7(n)‘HIprogesterone, 15 Ci/mmol; 1 I-deoxy [ 1,2- 3H]cortisol, 47 Ci/mmol; and [ 1,2,6,73H]cortisol, 81 Ci/mmol) were obtained from the Radiochemical Centre, Amersham and were purified by celite chromatography prior to use. Antiserum reacting with 1 I-deoxycortisol (Catalogue No. 152, Lot No. 3-Rl No. 1) was obtained from Radioassay Systems Laboratories, Inc., Carson, CA, USA. Antisera for cortisol (S6 No. 3), progesterone and 17-hydroxyprogesterone (S49 No. 6) were obtained from the Professional Association, Harbor General Hospital, Torrance, CA, USA. ‘Assay buffer’ consisted of borate buffer (0.05 mol/l, pH 8.0) to which bovine gamma globulin (333 mg/l) and bovine serum albumin (1 g/l) were added immediately before use. Stock dextran-coated charcoal consisted of Norit A activated washed charcoal (28 g/l) and dextran T-70 (3.75 g/l) in borate buffer. For use, the stock dextran-coated charcoal was diluted with borate buffer 1 in 25 for progesterone and 17-hydroxyprogesterone, 1 in 11 for 1 I-deoxycortisol and 1 in 13 for cortisol. Extraction Aliquots of plasma samples (ranging in volume from 1 ml for normal basal or suppressed values, down to 50 ~1 for levels in patients with untreated congenital adrenal hyperplasia) together with known standard concentrations of the steroids to be measured, were transferred to Pyrex glass tubes and the volume was adjusted to 1 ml with distilled water if necessary. Tritiated progesterone, 17-hydroxyprogesterone, 1 I-deoxycortisol and cortisol(2000 dpm for each) were added, mixed with the plasma or standards for assay and allowed to equilibrate for at least 30 min. Steroids were extracted twice with diethyl ether (10 ml) by mixing vigorously for at least 30 seconds. Both ether extracts were then combined. Chromatography (a) Preparation of columns. The stationary phase, ethylene glycol (Merck, Darmstadt, FRG): water = 3 : 1, v/v, was mixed well with celite (Sigma, London, UK) in the proportion 0.5 ml/g. The columns (5-ml disposable pipettes, Corning, Horseheads, NY) were filled with this mixture to 2 cm from the top, wetted with 1.5 ml iso-octane and allowed to soak for at least 40 min before packing tightly with a teflon tipped rod. The height of the packed material was .approximately 5 cm.
213
Following washing sample extracts.
with 3 ml of iso-octane
the columns
were ready for application
of
(b) Chromatography of extracts. The ether extracts were dried down by applying a jet of filtered air to the surface of the ether while the tubes containing the extracts were standing in a water bath at 38’C. The residue was redissolved in 20% ethyl acetate in iso-octane (0.3 ml) and applied to the columns. The tubes were then washed out with iso-octane (0.7 ml) and the washings also applied to the columns. The steroids were then eluted from the columns with 3 ml of iso-octane, 5 ml of 10% ethyl acetate in iso-octane, 5 ml of 25% ethyl acetate in iso-octane and 4 ml of 50% ethyl acetate in iso-octane. The flow of eluant was set at approximately 10 drops per minute, controlled by adjustment of air pressure applied to the top of the column. The fractions routinely collected are shown in Table I. Procedure
for radioimmunoassay
(a) 11 -deoxycortisol and cortisol. The chromatography fractions were dried down and redissolved in l-4 ml of assay buffer depending on the expected concentration (1 ml was used for basal 1 I-deoxycortisol and post-metyrapone cortisol samples, and 3-4 ml for post-metyrapone 1 I-deoxycortisol and basal cortisol samples). An aliquot of each was counted to assess recovery of tritiated steroid. Duplicate 0. l- and 0.2-ml aliquots were transferred to 12 X 75 mm glass tubes. One set of eight standards in duplicate was prepared for each 10 samples to be assayed. Standard curves ranged from 3 1 to 4000 fmol/tube for 11-deoxycortisol and from 0.22 to 27.6 pmol/tube for cortisol. The volume in the assay tubes was adjusted if necessary to 0.2 ml with assay buffer and then tritiated steroid (approximately 20 X lo3 dpm) and antibody (sufficient to bind 30% of tritiated steroid in the absence of unlabelled steroid) were added as a cocktail in 0.4 ml assay buffer to all tubes. Following overnight incubation, the ‘bound’ and ‘free’ steroids were separated by addition of dextran-coated charcoal (0.5 ml) and centrifugation following 20-25 min incubation at 2°C. The bound fraction was decanted into vials and the radioactivity
TABLE
I
ELUTION
OF STEROIDS
FROM
CELITE
MICROCOLUMNS
Eluates
* Steroid
obtained
for assay
Void volume + first 2 ml iso-octane Third ml iso-octane First 3 ml of 10% ethyl acetate in iso-octane Fourth and fifth ml of 10% ethyl acetate in iso-octane First 3 ml of 25% ethyl acetate in iso-octane Fourth and fifth ml of 25% ethyl acetate in iso-octane First 4 ml of 50% ethyl acetate in iso-octane
progesterone * none - discarded 17-hydroxyprogesterone none - discarded 1 I-deoxycortisol * none - discarded cortisol *
*
214
was counted. Concentrations of steroid in sample aliquots were obtained by interpolation from the standard curve. Steroid concentrations in plasma were calculated from the aliquot concentrations, following correction for procedural losses using the percentage recovery of the tritiated steroid added prior to extraction. (b) Progesterone and I7-hydroxyprogesterone. These steroids were assayed following the method described for 1 I-deoxycortisol and cortisol with some modifications. The chromatographed sample extracts were redissolved in ethanol (1 ml). An aliquot (0.3 ml) was dried down and counted to assess recovery of the tritiated steroid. Aliquots for assay (0.1 and 0.2 ml) and standard curves (also in ethanol) were dried down at 45°C under 40 mm Hg, achieved using a vacuum oven. The volume of the tritiated steroid and antibody cocktail added to each tube was 0.5 ml. Antiserum S49 No. 6, which reacts with progesterone and 17-hydroxyprogesterone was used as ligand for both assays. Tritiated progesterone was used as label in both the progesterone and 17-hydroxyprogesterone assays as it resulted in a more sensitive standard curve than did use of labelled 17-hydroxyprogesterone. The standard curves ranged from 25 to 3200 fmol/tube for progesterone and from 31 to 4000 fmol/tube for 17-hydroxyprogesterone. The incubation time with dextran-coated charcoal was 20 minutes. Metyrapone test This test, which is used to evaluate the activity of the hypothalamic-pituitaryadrenal axis, was performed as described by Spark [9]. Metyrapone inhibits the conversion of 1 I-deoxycortisol to cortisol. Plasma 1 I-deoxycortisol was measured in blood samples taken between 8.30 and 9.30 a.m. on the morning following a single dose of metyrapone (30 mg/kg) taken at midnight with a snack. In patients with limited hypothalamic-pituitary-adrenal function the 1 I-deoxycortisol response is subnormal. When a subnormal 1 I-deoxycortisol response to metyrapone was obtained cortisol was measured in the same sample to check whether sufficient hypocortisolism had been achieved to provide adequate stimulus to ACTH release. Results Chromatographic separation of steroids The chromatographic system was evaluated by examination of the separation and recovery of tritiated progesterone, 17-hydroxyprogesterone, 1 I-deoxycortisol and cortisol(4000 dpm each) applied separately to columns. A typical elution profile for each of the four steroids may be seen in Fig. 1. The profiles have been superimposed, demonstrating the complete separation of these steroids which may be achieved using this system. The percentage recoveries of tritiated steroids applied to columns without prior extraction, in the fractions indicated in Table I, were 97.8 2 1.6 for progesterone, 98.5 * 2.5 for 17-hydroxyprogesterone, 92.7 k 7.0 for 1 I-deoxycortisol and 91.6 k 4.5 for cortisol (means of eight observations for each steroid * one standard deviation). The percentage recoveries of the tritiated steroids following extraction from plasma and chromatography were 95 * 14 for progesterone, 81 * 11
215
PROGESTERoNE F
I
ll&~OXYCORTISOL 174H-PROGESTERONE I 1 I I
CORTISOL r
x Ii
70
I
I\ ! i
bo
I i i i ! i
so
!
40
PERwlTA6E *co~Env PER 1 n FRAClIOil
!
!
i ! !
!
!
I)
20
10
I
0
LL(sm-
18 nl5
Of clvrnt
PERCEWTAIiE ETHYL ACETATE IN ISO-OCTANE Fig. 1. Typical elution profiles obtained using our system when radioactive steroids were applied to 0 ; 17-hydroxyprogesterone, l - - - - - -0; 1 I-deseparate celite microcolumns. Progesterone, 0 oxycortisol, A------A; and cortisol, X-.-.-X The results for the four steroids have been superimposed.
for 17-hydroxyprogesterone, 78 * 9 for 11-deoxycortisol, and 60 2 8 for cortisol (means of more than 80 observations for each steroid * one standard deviation). Sensitivity The sensitivities of the standard curves, calculated by the method of Abraham were 25 fmol/tube for progesterone, 31 fmol/tube for 17-hydroxyprogesterone 11 -deoxycortisol and 200 fmol/tube for cortisol.
[8], and
Specificity The extent to which other steroids (progesterone, 17-hydroxyprogesterone, 11 -deoxycortisol, corticosterone, cortisol, 1 I-deoxycorticosterone, pregnenolone and 17hydroxypregnenolone) interfered in the four assays was investigaed. Known amounts of these steroids were added individually to water and submitted to the entire assay procedure for each of the four steroids. The concentrations of steroids were approximately three times the upper limit of normal levels, except the concentration of 11-deoxycortisol, which was chosen to reflect the high levels normally found following metyrapone treatment. The results of these studies are detailed in Table II. There was no evidence of clinically significant interference with the specificity of any of these assays.
216 TABLE
11
INTERFERENCE Steroid
OF SOME STEROIDS
added (nmol/l)
IN THE FOUR Steroid
recovered
Pro&
Progesterone 17-OH-progesterone Cortisol Deoxycorticosterone Corticosterone Cortisone I I-Deoxycortisol Pregnenolone 1“I-OH-pregnenolone
286.0 22.5 2415.0 0.6 6.1 248.0 434.0 20.0 37.2
ASSAYS (nmol/l)
as:
17-OH-prog.
1.75 1.25 0.46 0.48 KO.4 0.37 0.5 co.27 co.27
co.5 co.5
I 1-deoxycortisol
cortisol
(0.22 10.22 0.36 KO.22 (0.22 < 0.22
(7
(7 (7 -CJ 29 -=7 (7 17
co.3 CO.3
Precision and accuracy The between-assay precision was determined by measuring progesterone, 17-hydroxyprogesterone, 1 I-deoxycortisol and cortisol on several occasions in three plasma pools. The mean values obtained for each of the four steroids, together with the coefficients of variation are detailed in Table III. The accuracy of the methods, determined by measuring known amounts of the steroids added to water, is given in Table IV. The overall results of assaying each
TABLE
III
BETWEEN-ASSAY
PRECISION
OF THE METHODS Number of observations
Progesterone
Mean value (nmol/l)
Coefficient of variation
9 7 8
2.09 2.37 9.23
16.7% 15.3% 11.8%
17-Hydroxyprogesterone
7 14 12
3.94 12.67 38.8
13.3% 11.8% 16.4%
1I-Deoxycortisol
7 IO 6
1.63 294.5 365.5
10.6% 10.0% 7.7%
Cortisol
10 6 10
175.9 279.0 664.0
15.2% 18.3% 13.7%
217
TABLE
IV
PERCENTAGE RECOVERY OF STEROIDS CHROMATOGRAPHY AND ASSAY Standard Progesterone (P) 17-OH-progesterone ( 17P) 1 I-Deoxycortisol (D) Cortisol (C)
90.3% 100.0% 98.3% 107.8%
I
ADDED
TO WATER
Standard
II
102.0% 105.7% 94.3% 105.5%
FOLLOWING
Standard _ 96.0% 86.5% 94.5%
III
EXTRACTION,
Overall
mean
96.2% 100.4% 93.0% 102.6%
Standard I=3 nmol/I P, 3 nmol/l 17P, 2.9 nmol/l D, 173 nmol/l C. Standard II=6 nmoI/l P, 6 nmol/I 17P, 5.8 nmol/l D, 345 nmoI/l C. Standard III = 15 nmol/l 17P, 14.5 nmol/I D, and 690 nmol/l C. Results are means of 6 determinations.
steroid, expressed as a percentage of the known amount added, yielded the following values: progesterone 96%; 17-hydroxyprogesterone 100%; 1 I-deoxycortisol93%; and cortisol 103%. Reference
ranges for steroid levels in blood
Reference ranges were established using 20 male and 20 female healthy volunteers. Basal plasma progesterone values were below 3.2 nmol/l in men and follicular phase women and ranged from 9.5-79.0 nmol/l in women in the middle of the luteal phase. The basal 17-hydroxy-progesterone range was l-4.5 nmol/l in males and follicular phase women and the concentration following metyrapone ranged from 20-40 nmol/l. 11-Deoxycortisol and cortisol values were similar in men and women. The normal basal plasma 1 I-deoxycortisol and cortisol values were O-3.0 and 220-700 nmol/l, respectively. Following metyrapone the plasma 1 l-deoxycortisol concentration ranged from 170 to 550 nmol/l and plasma cortisol was below 250 nmol/l. Discussion In this paper we have reported a system whereby cortisol and its ACpregnene precursors are isolated in a single chromatographic procedure in preparation for highly specific radioimmunoassay. The sensitivity, precision and accuracy of these assays are comparable to or superior to previously reported radioimmunoassays for these steroids [S,lO-181. The specificities of the assays described here are superior to those assays which do not incorporate a chromatographic purification step [8,12,15,17]. Several authors [8,10,13,14,16-181 have used column chromatography but have not examined the interference of other steroids in the subsequent assay. Complete steroid isolation was assumed by these authors but not demonstrated and in these instances specificity has not been adequately examined. Considerable interference from cross-reacting steroids may occur when they are present in plasma in high concentrations, even if only a
218
small percentage of the original amounts are contained in the elution fraction to be submitted for measurement of another steroid. We have investigated the interference of pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, 1 I-deoxycortisol, cortisol, corticosterone and deoxycorticosterone added to water and carried through all the assay procedures. These steroids caused no significant interference in any of the four assays. The reference ranges obtained using the methods described here are broadly similar to those obtained previously for basal progesterone [8,10,16], 17-hydroxyprogesterone [ 10,151 and cortisol [ 171 and for 1I-deoxycortisol following metyrapone [ 12,17, IS]. However, the basal 11-deoxycortisol reference range which we obtained is lower than some of those previously reported [ 11,17.18] but similar to that obtained by Maroulis et al [14]. The lower values observed in the present study and by Maroulis et al [ 141 probably reflect great specificity for 1 I-deoxycortisol. The method described in this paper has been applied to the investigation and management of adrenal hypofunction [19], congenital adrenal hyperplasia [20], and hirsutism and amenorrhoea [21]. The chromatographic separation of progesterone, 17-hydroxyprogesterone, 11-deoxycortisol and cortisol and highly specific assay of these steroids in blood will prove particularly useful for research into cortisol biosynthesis. Acknowledgement This study was supported in part by a grant-in-aid Health, Number 5 ROl HD 12248.
from the National
Institutes
of
References 1 Sunderman FW, Sunderman FW Jr. Lipids and the steroid hormones in clinical medicine. Philadelphia USA: JB Lippincott Co., 1960: 110. 2 Goldzieher JW, Besch PK. Fluorescence and absorption spectra of some corticosteroids in sulphuric and phosphoric acids. Anal Chem 1958; 30: 962-969. 3 Chattoraj SC. Endocrine function. In: Tietz NW, ed. Fundamentals of clinical chemistry. Philadelphia USA: WB Saunders Company 1976: 699-823. 4 Van Den Berg JHM, Mol ChR, Deelder RS, Thijssen JHH. A quantitative assay of cortisol in human plasma by high performance liquid chromatography using a selective chemically bonded stationary phase. Clin Chim Acta 1977; 78: 165-172. 5 Pegg PJ, Keane PM. The simultaneous estimation of plasma cortisol and transcortin binding characteristics by competitive protein binding technique. Steroids 1969; 14: 705-709. 6 Murphy BEF. Some studies of the protein binding of steroids and their application to the routine micro and ultra micro measurement of various steroids in body fluids by competitive protein binding. J Clin Endocrinol 1963; 27: 973-981. 7 James VHT, Jeffcoate SL. Steroids. Br Med Bull 1974; 30: 50-54. 8 Abraham GE. Radioimmunoassay of steroids in biological materials. Acta Endocrinol 1974; suppl 183, 75: 7-42. 9 Spark RF. Simplified assessment of pituitary-adrenal reserve. Ann Intern Med 1971; 75: 717-723. 10 Sippell WG, Bidlingmaur F, Becker H et al. Simultaneous radioimmunoassay of plasma aldosterone, corticosterone, 1I-deoxycorticosterone, cortisol and cortisone. J Steroid Biochem 1978; 9: 63-74.
219
11 Newsome HH Jr, Clements AS, Borum EH. The simultaneous assay of cortisol, corticosterone, 1 I-deoxycortisol and cortisone in human plasma. J Clin Endocrinol 1972; 34: 473-483. 12 Mahajan DK, Wahlen JD, Tyler FH, West CD. Plasma II-deoxycortisol radioimmunoassay for metyrapone tests. Steroids 1972; 20: 609-620. 13 Brown JR,. Cavanagh AH, Farnsworth WE. A simple radioimmunoassay for plasma cortisol and I I-deoxycortisol. Steroids 1977; 28: 487-498. 14 Maroulis GB, Manlimos FS, Garza R, Abraham GE. Serum cortisol and I 1-deoxycortisol levels in hirsute pre-menopausal women. Obstet Gynecol 1976; 48: 388-391. 15 Davilia N, Rudd BT, Morris R, Kandeel F, Bodden J, Landan DR. A simple procedure for the radioimmunoassay of 17-hydroxypro8esterone in serum: comparison with an immunological purification technique. Ann Chn Biochem 1980; 17: 31-37. 16 Grczyk GP, Hichens M, Orth G, Behrman HR. Progesterone and 2Off-dihydropro8esterone. in: Jaffe BM, Behrman HR, eds. Methods of hormone radioimmunoassay. New York: Academic Press, 1979: 701-713. 17 Vescei P. Glucocorticoids:
cortisol, cortisone, corticosterone, compound S and their metaboiites. In: Jaffe BM, Behrman HR, eds. Methods of hormone radioimmunoassay. New York: Academic Press, 1979: 767-796. 18 Demers LM, Ebright L, Derek DD. Improved radioimmunoassay for I 1-deoxycortisol (compound S) in plasma. Clin Chem 1979; 25: 1704-1707. 19 Cunningham SK, Moore A, McKenna TJ. Evaluation 1980; 149: 133. 20 McKenna TJ, Lacroix A, Burr IM, Liddle GW, Miller of control in congenital adrenal hyperplasia. Clin Res 21 Moore A, Cunnin~am S, Cuiliton M, McKenna TJ. and/or amenorrhoea. Irish J Med Science 1980; 149:
for adrenal
hypofunction.
Irish J Med Science
RB, Nix P. Steroid levels in plasma as indices 1978; 26: 310A. The pathogenesis and treatment of hirsutism 133.
211
CCA 2201
A system for separation of cortisol, 1 1-deoxycortisol, 17-hydroxyprogesterone and progesterone in a single chromatographic step and its application to radioimmunoassay Sean
K. Cunningham
* and T. Joseph
McKenna
Department of Endocrinology, St. Vincent’s Hospital, Elm Park, Dublin 4 (Republic of Ireland) (Received
September
23rd. 1981; revision March 22nd, 1982)
Summary A procedure
for accurate, sensitive and highly specific measurement of cortisol, 17-hydroxyprogesterone and progesterone is described. We have developed a chromatographic system whereby the steroids may be separated in a single passage over celite microcolumns. This permits the specific radioimmunoassay of the principal glucocorticoid in man and its A6pregnene precursors and thus facilitates investigation of the physiological control and pathological disorders of glucocorticoid biosynthesis. 11-deoxycortisol,
Introduction Accurate measurement of steroid concentrations in blood is fundamental to the elucidation of both physiological and pathological processes affecting the adrenal glands and gonads. A variety of assay techniques have previously been used for this purpose. Calorimetric and fluorometric methods are neither sensitive nor specific, and are subject to interference, particularly from drugs [l-3]. High pressure liquid chromatography with ultraviolet detection provides sufficient sensitivity for cortisol [4] but not for normal plasma levels of its precursors. Competitive protein binding methods have been used [5,6], but lack specificity and have more recently been replaced by radioimmunoassay [7]. Although radioimmunoassay is the most sensitive of readily available techniques, antibodies lack complete specificity for any one steroid and react with related steroids. However, specificity may be greatly enhanced by extraction and chromatographic isolation of the desired steroid prior to quantifi-
* All correspondence Vincent’s Hospital,
should be directed to: Dr. Sean Cunningham, Elm Park, Dublin 4, Republic of Ireland.
0009-8981/82/0000-0000/$02.75
0 1982 Elsevier Biomedical
Press
Department
of Endocrinology,
St.
212
cation [7,8]. The use of celite as an efficient chromatographic agent for the isolation of steroids has been widely advanced by Abraham and colleagues [8]. We have developed and expanded this methodology to achieve separation of cortisol, 1 l-deoxycortisol, 17-hydroxyprogesterone and progesterone in a single chromatographic procedure. This system is particularly useful when a meticulous examination of cortisol biosynthesis is undertaken and should prove to be a valuable research tool. Experimental Materials Tritiated steroids ([ 1,2,6,7-3H]progesterone, 80 Ci/mmol; 17-hydroxy [7(n)‘HIprogesterone, 15 Ci/mmol; 1 I-deoxy [ 1,2- 3H]cortisol, 47 Ci/mmol; and [ 1,2,6,73H]cortisol, 81 Ci/mmol) were obtained from the Radiochemical Centre, Amersham and were purified by celite chromatography prior to use. Antiserum reacting with 1 I-deoxycortisol (Catalogue No. 152, Lot No. 3-Rl No. 1) was obtained from Radioassay Systems Laboratories, Inc., Carson, CA, USA. Antisera for cortisol (S6 No. 3), progesterone and 17-hydroxyprogesterone (S49 No. 6) were obtained from the Professional Association, Harbor General Hospital, Torrance, CA, USA. ‘Assay buffer’ consisted of borate buffer (0.05 mol/l, pH 8.0) to which bovine gamma globulin (333 mg/l) and bovine serum albumin (1 g/l) were added immediately before use. Stock dextran-coated charcoal consisted of Norit A activated washed charcoal (28 g/l) and dextran T-70 (3.75 g/l) in borate buffer. For use, the stock dextran-coated charcoal was diluted with borate buffer 1 in 25 for progesterone and 17-hydroxyprogesterone, 1 in 11 for 1 I-deoxycortisol and 1 in 13 for cortisol. Extraction Aliquots of plasma samples (ranging in volume from 1 ml for normal basal or suppressed values, down to 50 ~1 for levels in patients with untreated congenital adrenal hyperplasia) together with known standard concentrations of the steroids to be measured, were transferred to Pyrex glass tubes and the volume was adjusted to 1 ml with distilled water if necessary. Tritiated progesterone, 17-hydroxyprogesterone, 1 I-deoxycortisol and cortisol(2000 dpm for each) were added, mixed with the plasma or standards for assay and allowed to equilibrate for at least 30 min. Steroids were extracted twice with diethyl ether (10 ml) by mixing vigorously for at least 30 seconds. Both ether extracts were then combined. Chromatography (a) Preparation of columns. The stationary phase, ethylene glycol (Merck, Darmstadt, FRG): water = 3 : 1, v/v, was mixed well with celite (Sigma, London, UK) in the proportion 0.5 ml/g. The columns (5-ml disposable pipettes, Corning, Horseheads, NY) were filled with this mixture to 2 cm from the top, wetted with 1.5 ml iso-octane and allowed to soak for at least 40 min before packing tightly with a teflon tipped rod. The height of the packed material was .approximately 5 cm.
213
Following washing sample extracts.
with 3 ml of iso-octane
the columns
were ready for application
of
(b) Chromatography of extracts. The ether extracts were dried down by applying a jet of filtered air to the surface of the ether while the tubes containing the extracts were standing in a water bath at 38’C. The residue was redissolved in 20% ethyl acetate in iso-octane (0.3 ml) and applied to the columns. The tubes were then washed out with iso-octane (0.7 ml) and the washings also applied to the columns. The steroids were then eluted from the columns with 3 ml of iso-octane, 5 ml of 10% ethyl acetate in iso-octane, 5 ml of 25% ethyl acetate in iso-octane and 4 ml of 50% ethyl acetate in iso-octane. The flow of eluant was set at approximately 10 drops per minute, controlled by adjustment of air pressure applied to the top of the column. The fractions routinely collected are shown in Table I. Procedure
for radioimmunoassay
(a) 11 -deoxycortisol and cortisol. The chromatography fractions were dried down and redissolved in l-4 ml of assay buffer depending on the expected concentration (1 ml was used for basal 1 I-deoxycortisol and post-metyrapone cortisol samples, and 3-4 ml for post-metyrapone 1 I-deoxycortisol and basal cortisol samples). An aliquot of each was counted to assess recovery of tritiated steroid. Duplicate 0. l- and 0.2-ml aliquots were transferred to 12 X 75 mm glass tubes. One set of eight standards in duplicate was prepared for each 10 samples to be assayed. Standard curves ranged from 3 1 to 4000 fmol/tube for 11-deoxycortisol and from 0.22 to 27.6 pmol/tube for cortisol. The volume in the assay tubes was adjusted if necessary to 0.2 ml with assay buffer and then tritiated steroid (approximately 20 X lo3 dpm) and antibody (sufficient to bind 30% of tritiated steroid in the absence of unlabelled steroid) were added as a cocktail in 0.4 ml assay buffer to all tubes. Following overnight incubation, the ‘bound’ and ‘free’ steroids were separated by addition of dextran-coated charcoal (0.5 ml) and centrifugation following 20-25 min incubation at 2°C. The bound fraction was decanted into vials and the radioactivity
TABLE
I
ELUTION
OF STEROIDS
FROM
CELITE
MICROCOLUMNS
Eluates
* Steroid
obtained
for assay
Void volume + first 2 ml iso-octane Third ml iso-octane First 3 ml of 10% ethyl acetate in iso-octane Fourth and fifth ml of 10% ethyl acetate in iso-octane First 3 ml of 25% ethyl acetate in iso-octane Fourth and fifth ml of 25% ethyl acetate in iso-octane First 4 ml of 50% ethyl acetate in iso-octane
progesterone * none - discarded 17-hydroxyprogesterone none - discarded 1 I-deoxycortisol * none - discarded cortisol *
*
214
was counted. Concentrations of steroid in sample aliquots were obtained by interpolation from the standard curve. Steroid concentrations in plasma were calculated from the aliquot concentrations, following correction for procedural losses using the percentage recovery of the tritiated steroid added prior to extraction. (b) Progesterone and I7-hydroxyprogesterone. These steroids were assayed following the method described for 1 I-deoxycortisol and cortisol with some modifications. The chromatographed sample extracts were redissolved in ethanol (1 ml). An aliquot (0.3 ml) was dried down and counted to assess recovery of the tritiated steroid. Aliquots for assay (0.1 and 0.2 ml) and standard curves (also in ethanol) were dried down at 45°C under 40 mm Hg, achieved using a vacuum oven. The volume of the tritiated steroid and antibody cocktail added to each tube was 0.5 ml. Antiserum S49 No. 6, which reacts with progesterone and 17-hydroxyprogesterone was used as ligand for both assays. Tritiated progesterone was used as label in both the progesterone and 17-hydroxyprogesterone assays as it resulted in a more sensitive standard curve than did use of labelled 17-hydroxyprogesterone. The standard curves ranged from 25 to 3200 fmol/tube for progesterone and from 31 to 4000 fmol/tube for 17-hydroxyprogesterone. The incubation time with dextran-coated charcoal was 20 minutes. Metyrapone test This test, which is used to evaluate the activity of the hypothalamic-pituitaryadrenal axis, was performed as described by Spark [9]. Metyrapone inhibits the conversion of 1 I-deoxycortisol to cortisol. Plasma 1 I-deoxycortisol was measured in blood samples taken between 8.30 and 9.30 a.m. on the morning following a single dose of metyrapone (30 mg/kg) taken at midnight with a snack. In patients with limited hypothalamic-pituitary-adrenal function the 1 I-deoxycortisol response is subnormal. When a subnormal 1 I-deoxycortisol response to metyrapone was obtained cortisol was measured in the same sample to check whether sufficient hypocortisolism had been achieved to provide adequate stimulus to ACTH release. Results Chromatographic separation of steroids The chromatographic system was evaluated by examination of the separation and recovery of tritiated progesterone, 17-hydroxyprogesterone, 1 I-deoxycortisol and cortisol(4000 dpm each) applied separately to columns. A typical elution profile for each of the four steroids may be seen in Fig. 1. The profiles have been superimposed, demonstrating the complete separation of these steroids which may be achieved using this system. The percentage recoveries of tritiated steroids applied to columns without prior extraction, in the fractions indicated in Table I, were 97.8 2 1.6 for progesterone, 98.5 * 2.5 for 17-hydroxyprogesterone, 92.7 k 7.0 for 1 I-deoxycortisol and 91.6 k 4.5 for cortisol (means of eight observations for each steroid * one standard deviation). The percentage recoveries of the tritiated steroids following extraction from plasma and chromatography were 95 * 14 for progesterone, 81 * 11
215
PROGESTERoNE F
I
ll&~OXYCORTISOL 174H-PROGESTERONE I 1 I I
CORTISOL r
x Ii
70
I
I\ ! i
bo
I i i i ! i
so
!
40
PERwlTA6E *co~Env PER 1 n FRAClIOil
!
!
i ! !
!
!
I)
20
10
I
0
LL(sm-
18 nl5
Of clvrnt
PERCEWTAIiE ETHYL ACETATE IN ISO-OCTANE Fig. 1. Typical elution profiles obtained using our system when radioactive steroids were applied to 0 ; 17-hydroxyprogesterone, l - - - - - -0; 1 I-deseparate celite microcolumns. Progesterone, 0 oxycortisol, A------A; and cortisol, X-.-.-X The results for the four steroids have been superimposed.
for 17-hydroxyprogesterone, 78 * 9 for 11-deoxycortisol, and 60 2 8 for cortisol (means of more than 80 observations for each steroid * one standard deviation). Sensitivity The sensitivities of the standard curves, calculated by the method of Abraham were 25 fmol/tube for progesterone, 31 fmol/tube for 17-hydroxyprogesterone 11 -deoxycortisol and 200 fmol/tube for cortisol.
[8], and
Specificity The extent to which other steroids (progesterone, 17-hydroxyprogesterone, 11 -deoxycortisol, corticosterone, cortisol, 1 I-deoxycorticosterone, pregnenolone and 17hydroxypregnenolone) interfered in the four assays was investigaed. Known amounts of these steroids were added individually to water and submitted to the entire assay procedure for each of the four steroids. The concentrations of steroids were approximately three times the upper limit of normal levels, except the concentration of 11-deoxycortisol, which was chosen to reflect the high levels normally found following metyrapone treatment. The results of these studies are detailed in Table II. There was no evidence of clinically significant interference with the specificity of any of these assays.
216 TABLE
11
INTERFERENCE Steroid
OF SOME STEROIDS
added (nmol/l)
IN THE FOUR Steroid
recovered
Pro&
Progesterone 17-OH-progesterone Cortisol Deoxycorticosterone Corticosterone Cortisone I I-Deoxycortisol Pregnenolone 1“I-OH-pregnenolone
286.0 22.5 2415.0 0.6 6.1 248.0 434.0 20.0 37.2
ASSAYS (nmol/l)
as:
17-OH-prog.
1.75 1.25 0.46 0.48 KO.4 0.37 0.5 co.27 co.27
co.5 co.5
I 1-deoxycortisol
cortisol
(0.22 10.22 0.36 KO.22 (0.22 < 0.22
(7
(7 (7 -CJ 29 -=7 (7 17
co.3 CO.3
Precision and accuracy The between-assay precision was determined by measuring progesterone, 17-hydroxyprogesterone, 1 I-deoxycortisol and cortisol on several occasions in three plasma pools. The mean values obtained for each of the four steroids, together with the coefficients of variation are detailed in Table III. The accuracy of the methods, determined by measuring known amounts of the steroids added to water, is given in Table IV. The overall results of assaying each
TABLE
III
BETWEEN-ASSAY
PRECISION
OF THE METHODS Number of observations
Progesterone
Mean value (nmol/l)
Coefficient of variation
9 7 8
2.09 2.37 9.23
16.7% 15.3% 11.8%
17-Hydroxyprogesterone
7 14 12
3.94 12.67 38.8
13.3% 11.8% 16.4%
1I-Deoxycortisol
7 IO 6
1.63 294.5 365.5
10.6% 10.0% 7.7%
Cortisol
10 6 10
175.9 279.0 664.0
15.2% 18.3% 13.7%
217
TABLE
IV
PERCENTAGE RECOVERY OF STEROIDS CHROMATOGRAPHY AND ASSAY Standard Progesterone (P) 17-OH-progesterone ( 17P) 1 I-Deoxycortisol (D) Cortisol (C)
90.3% 100.0% 98.3% 107.8%
I
ADDED
TO WATER
Standard
II
102.0% 105.7% 94.3% 105.5%
FOLLOWING
Standard _ 96.0% 86.5% 94.5%
III
EXTRACTION,
Overall
mean
96.2% 100.4% 93.0% 102.6%
Standard I=3 nmol/I P, 3 nmol/l 17P, 2.9 nmol/l D, 173 nmol/l C. Standard II=6 nmoI/l P, 6 nmol/I 17P, 5.8 nmol/l D, 345 nmoI/l C. Standard III = 15 nmol/l 17P, 14.5 nmol/I D, and 690 nmol/l C. Results are means of 6 determinations.
steroid, expressed as a percentage of the known amount added, yielded the following values: progesterone 96%; 17-hydroxyprogesterone 100%; 1 I-deoxycortisol93%; and cortisol 103%. Reference
ranges for steroid levels in blood
Reference ranges were established using 20 male and 20 female healthy volunteers. Basal plasma progesterone values were below 3.2 nmol/l in men and follicular phase women and ranged from 9.5-79.0 nmol/l in women in the middle of the luteal phase. The basal 17-hydroxy-progesterone range was l-4.5 nmol/l in males and follicular phase women and the concentration following metyrapone ranged from 20-40 nmol/l. 11-Deoxycortisol and cortisol values were similar in men and women. The normal basal plasma 1 I-deoxycortisol and cortisol values were O-3.0 and 220-700 nmol/l, respectively. Following metyrapone the plasma 1 l-deoxycortisol concentration ranged from 170 to 550 nmol/l and plasma cortisol was below 250 nmol/l. Discussion In this paper we have reported a system whereby cortisol and its ACpregnene precursors are isolated in a single chromatographic procedure in preparation for highly specific radioimmunoassay. The sensitivity, precision and accuracy of these assays are comparable to or superior to previously reported radioimmunoassays for these steroids [S,lO-181. The specificities of the assays described here are superior to those assays which do not incorporate a chromatographic purification step [8,12,15,17]. Several authors [8,10,13,14,16-181 have used column chromatography but have not examined the interference of other steroids in the subsequent assay. Complete steroid isolation was assumed by these authors but not demonstrated and in these instances specificity has not been adequately examined. Considerable interference from cross-reacting steroids may occur when they are present in plasma in high concentrations, even if only a
218
small percentage of the original amounts are contained in the elution fraction to be submitted for measurement of another steroid. We have investigated the interference of pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, 1 I-deoxycortisol, cortisol, corticosterone and deoxycorticosterone added to water and carried through all the assay procedures. These steroids caused no significant interference in any of the four assays. The reference ranges obtained using the methods described here are broadly similar to those obtained previously for basal progesterone [8,10,16], 17-hydroxyprogesterone [ 10,151 and cortisol [ 171 and for 1I-deoxycortisol following metyrapone [ 12,17, IS]. However, the basal 11-deoxycortisol reference range which we obtained is lower than some of those previously reported [ 11,17.18] but similar to that obtained by Maroulis et al [14]. The lower values observed in the present study and by Maroulis et al [ 141 probably reflect great specificity for 1 I-deoxycortisol. The method described in this paper has been applied to the investigation and management of adrenal hypofunction [19], congenital adrenal hyperplasia [20], and hirsutism and amenorrhoea [21]. The chromatographic separation of progesterone, 17-hydroxyprogesterone, 11-deoxycortisol and cortisol and highly specific assay of these steroids in blood will prove particularly useful for research into cortisol biosynthesis. Acknowledgement This study was supported in part by a grant-in-aid Health, Number 5 ROl HD 12248.
from the National
Institutes
of
References 1 Sunderman FW, Sunderman FW Jr. Lipids and the steroid hormones in clinical medicine. Philadelphia USA: JB Lippincott Co., 1960: 110. 2 Goldzieher JW, Besch PK. Fluorescence and absorption spectra of some corticosteroids in sulphuric and phosphoric acids. Anal Chem 1958; 30: 962-969. 3 Chattoraj SC. Endocrine function. In: Tietz NW, ed. Fundamentals of clinical chemistry. Philadelphia USA: WB Saunders Company 1976: 699-823. 4 Van Den Berg JHM, Mol ChR, Deelder RS, Thijssen JHH. A quantitative assay of cortisol in human plasma by high performance liquid chromatography using a selective chemically bonded stationary phase. Clin Chim Acta 1977; 78: 165-172. 5 Pegg PJ, Keane PM. The simultaneous estimation of plasma cortisol and transcortin binding characteristics by competitive protein binding technique. Steroids 1969; 14: 705-709. 6 Murphy BEF. Some studies of the protein binding of steroids and their application to the routine micro and ultra micro measurement of various steroids in body fluids by competitive protein binding. J Clin Endocrinol 1963; 27: 973-981. 7 James VHT, Jeffcoate SL. Steroids. Br Med Bull 1974; 30: 50-54. 8 Abraham GE. Radioimmunoassay of steroids in biological materials. Acta Endocrinol 1974; suppl 183, 75: 7-42. 9 Spark RF. Simplified assessment of pituitary-adrenal reserve. Ann Intern Med 1971; 75: 717-723. 10 Sippell WG, Bidlingmaur F, Becker H et al. Simultaneous radioimmunoassay of plasma aldosterone, corticosterone, 1I-deoxycorticosterone, cortisol and cortisone. J Steroid Biochem 1978; 9: 63-74.
219
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