A simplified radioimmunoassay for plasma 11-deoxycortisol

Clin. Biochem. 14 (2) 47-50 (1981)

A Simplified Radioimmunoassay for Plasma 11-Deoxycortisol PHILIP R. WALSH, MING-CHUNG WANG, and JEAN C. SU Department of Chemistry, Laboratory Procedures East, Inc., Subsidiary of The Upjohn Company, King of Prussia, PA 19406

A simple, specific, accurate, precise and sensitive radioimmunoassay procedure developed for plasma ll-deoxycortisol is described. I. The assay employs an anti-ll-deoxycortisol serum generated against ll-deoxycortisol-3-(0-carboxymethyl) oxime coupled to bovine serum albumin, crystalline 11-deoxycortisol as standard, and [3HI 11-dcoxycortisol as the radioactive ligand. 2. Cross-reactivity studies performed with structurally related steroids indicated cross reactivites with 17a-hydroxyprogresterone, deoxycorticosterone and progesterone of 2.0%, 1.3% and 0.4% respectively; cortisone, corticosterone, cortisol, testosterone, <0.1%; and estrone, 17-f~-estradiol, estriol, and metyrapone <0.001%. Due to the high specificity of the anti-11deoxycortisol serum, the method is simplified by the lack of need for chromatographic purification of the organic solvent extract of the plasma prior to the radioimmunoassay. The procedure was validated by comparing values for plasma 11-deoxycortisol with and without preliminary purification by chromatography on Sephadex LH-20 ® columns (y ----0.99_x -{- 4.0, r = .98). Pretreatment of the plasma with n - h e x a n e was found to eliminate interferences from high concentrations of 17a-hydroxyprogesterone or progesterone. 3. Parallel dose-response curves were demonstrated between dilutions of plasma with elevated 11-deoxycortisol concentrations and the standard reference preparation. A non-specific binding <4% of the total ['~H] ll-deoxycortisol was routinely observed. The detection limit of the assay was approximately 1O pg of ll-deoxycortisol which corresponds to a plasma concentration of approximately 0.7 ~g/L. 4. The analytical recovery of ll-deoxycortisol added to human plasma varied from 88 to 108%, with a mean recovery of 100%. The inter-assay variation was determined by assaying (n ---- 30) three different quality control pools. The following d a t a were obtained: T 1 = 3.8 _+ 0.6 ~g/L (CV : 15.8%); T 2 = 18.5 +_ 2.0 ,g/L (CV = 10.8%); ~-3 ---- 43.0 _+ 3.7 pg/dl (CV = 8.6%). All of the reagents are available commercially.

T H E PROPER INTERPRETATION OF THE METYRAPONE challenge test introduced in 1959 by Liddle et al. (1) requires an accurate method for the quantification of plasma 11-deoxycortisol. The accurate measurement of this steroid represents an analytical challenge for clinical laboratories as a result of the presence of structurally related steroids such as cortisol, progesterone Correspendence: P.R. Walsh Presented at the Joint Meeting of the American Association for Clinical Chemistry and the Canadian Society of Clinical Chemistry, Boston, Massachusetts, July, 1980.

and 17a-hydroxyprogesterone, which are at concentrations equal to or greater than 11-deoxycortisol in some clinical conditions. Over the past twelve years, a number of methods have been published for the quantification of plasma 11-deoxycortisol. These methods have included gasliquid chromatography (2), competitive-protein binding (CPB) with cortisol-binding globulin (CBG) as the binding reagent together with selective solvent extraction to isolate 11-deoxycortisol (3, 10), radioimmunoassay (RIA) procedures which have multiple solvent extractions (11) and/or column chromatography (12,15), and most recently, high-performance liquid chromatography (16,17). Although the R I A techniques have proven to be more specific and reproducible than the C P B methods for this measurement, m a n y of the R I A procedures require multiple solvent extractions and column chromatography to improve the specificity of the measurement. Here, we describe our experience with an R I A method for plasma 11-deoxycortisol which requires no chromatographic purification before quantification by RIA, but does require organic solvent extraction. The high specificity of the antibody for 11-deoxycortisol allows for the elimination of the chromatographic step. MATERIALS AND METHODS Phosphate buffer, 0.1 tool/L, pH 7.0, containing per liter, 1 g of gelatin (Difco Laboratories, Detroit, MI 48232), 8.5 g sodium chloride, and 0.1 g of sodium azide. ll-Deoxycortisol s t a n d a r d . We dissolved 25 mg of 11-deoxycortisol (Steraloids, Inc., Wilton, NH 03086) in 100 mL of methanol to give a stock solution of 250 mg/L. A working standard (2500 ng/L) was prepared by evaporating 1.0 mL of the 250 mg/L standard and redissolving the residue in 100 mL of the assay buffer. This solution was diluted with the phosphate buffer to obtain final concentrations of 1250, 625, 313, 156, 78, 39 and 19.5 ng/L.

Tritiated ll-deoxycortisol. 17a-[I,2-3H(N)]hydroxy-ll-deoxycorticosterone {40-60 Ci/mmol) in benzene/ethanol {9/1,by vol) was obtained from New England Nuclear, Boston, M A 02118. A working solution of the radioactive steroid was prepared for the RIA step by evaporating 50 ~L of the benzene/ethanol and redissolving the residue in the phosphate buffer to a count of 10,000/100 HL (about 100 pg of the tritiated 11-deoxycortisol per 100 ~L of phosphate buffer).Tritiated 11-deoxycortisol for the recovery step was prepared by dilutingthe above preparation ten-foldwith the phosphate buffer. Rabbit, anti-11-deoxycortisolserum, lot no. 3RL#I. The anti11-deoxycortisol serum prepared against 11-deoxycortisol-3-

48

WALSH, WANG AND SU

(O-carboxymethyl) oxime-bovine serum albumin was obtained from Radioassay Systems Laboratories, Inc., Carson, CA 90745. The 1 mL of antiserum shipped was a 15-fold dilution of the original antiserum. We further diluted it to 100 mL with phosphate buffer and froze it (-60°C) in 10-ml aliquots. We used 0.1 mL aliquots of the 1500-fold dilution for the RIA procedure. Dextran-coated charcoal. We dissolved 62.5 mg of Dextran T70 (Pharmacia Fine Chemicals, Piscataway, NJ 08854) in 100 mL of the phosphate buffer, on ice, with constant stirring. After the dextran had dissolved, we slowly added 625 mg of Norit Neutral A Charcoal (Schwartz/Mann, Orangeburg, NY 10962) and allowed the mixture to stir at 4°C for 30 rain before use. Liquid scintillation fluid. "RIA-FLUOR" was used as obtained from New England Nuclear. Solvents. Isooctane, toluene, hexane and ethylacetate (distilled in glass) were used as obtained from Burdick and Jackson Laboratories, Muskegon, M149440. Acetone and methanol (certified ACS grade) were used as obtained from Fisher Scientific Co., King of Prussia, PA 19406. Nonradioactive steroids. Cortisol, cortisone, deoxycorticosterone, corticosterone, 17a-hydroxyprogesterone, progesterone, testosterone, estrone, 17fPestradiol and estriol were obtained from The Upjohn Co., Kalamazoo, MI 49001. Metyrapone was a gift from CIBA Pharmaceutical Co., Division of CIBA-GEIGY Corp., Summit, NJ 07901. Organic solvent extraction of ll-deoxycortisol. A 100-~L aliquot of each plasma specimen and control was transferred to a 16 X 125 mm glass disposable test tube followed by the addition of 100 ~L (10 pg) of [ZH] ll-deoxycortisol, to estimate recovery. The tubes were vortex-mixed gently for 3 to 5 s and incubated at room temperature for 15 min. Similar aliquots, in duplicate, were pipetted into scintillation vials for total counts (control). A 300-~L aliquot of deionized water was added to each tube, the tubes were subsequently vortex-mixed gently for 3 to 5 s, 5 mL of n -hexane was added to each tube and the capped tubes were rotated end-over-end on a "Roto-Rack" Model 343 (Fisher Scientific) at a setting of 8. The specimens were centrifuged at 1600 X g for 15 min at room temperature, the aqueous layer (lower) frozen in a dry ice/acetone bath and the _n-hexane discarded. This aqueous layer was allowed to thaw and the hexane wash was repeated. A 10-mL aliquot of ethylacetate/n -hexane (3/2, by vol) was added to each of the hexane-washed specimens and the tubes were rotated endover-end for 30 min. All of the specimens were then centrifuged at 1600 X g for 15 min at room t e m p e r a t u r e and the aqueous layers (lower) were frozen in dry ice/acetone. The individual solvent layers were quantitatively transferred to appropriately labelled clean 16 X 125 mm disposable glass tubes, evaporated at 45°C in a stream of air, reconstituted with 3 ml of phosphate buffer, and placed in a 37°C water bath for 10 min or an ultrasonic cleaner for 1 min to dissolve the residue. The recovery of [3H] 11-deoxycortisol through the above extractions varied from 68 to 75% (mean 71%). Radioimmunoassay procedure. Disposable polystyrene (12 X 75 mm) t e s t t u b e s were m a r k e d for t r i p l i c a t e zero (11-deoxycortisol-free assay tubes) and duplicate 1250, 625, 313, 156, 78, 39, 19.5 and 9.8 pg/tube standards. Additional duplicate tubes were marked for each control and unknown to be assayed. Aliquots (500 /~L) of the individual standards (prepared in phosphate buffer), unknowns and controls (also prepared in phosphate buffer) were added to the appropriately marked tubes, followed by the addition of 100 I~L of [3H] 11-deoxycortisol (about 100 pg). Triplicate total count and blank assay tubes were prepared by adding 100 ~L of the [3H] 11-deoxyeortisol and 600 IJL of the phosphate buffer to individual tubes. The contents of all of the tubes (except the total-count and blank tubes) were gently vortex-mixed for 3 to 5 s and 100 pL of the anti-11-deoxycortisol serum was added. All of the tubes were then gently vortex-mixed and incubated at 4°C for 18 h. Then all of the tubes (except the total-count tubes) were placed in an ice bath. A 200 ~L-aliquot of ice-cold dextran-coated charcoal suspension was then added to each tube in rapid succession, and the tubes' contents were gently vortex-mixed immediately and allowed to stand at 4°C for 30 rain. Then all of the tubes (except the total-count tubes) were

centrifuged (1600 X g, 10 rain, 4°C) and the supernates were decanted into appropriately marked scintillation vials. Each scintillation vial then received 10 mL of RIA-FLUOR, was capped, inverted 10 times, and its radioactivity counted in an automatic liquid scintillation spectrometer (Isolab 300 Tracor Analytic, In'c., Elk Grove Village, IL 60007) for 3 min. Calculations. The logit-log data reduction method (18) was used to calculate the 11-deoxycortisol concentrations of controls and unknowns. The values computed were corrected for the percent recovery of [SH] 11-deoxycortisol from the controls and unknowns. The results are reported as micrograms of 11-deoxycortisol per L of plasma. Sephadex LH-2O Chromatography. One-thousand mg of dry Sephadex LH-20 (Pharmacia Fine Chemicals) was placed in a column (1 X 18.5 cm; Isolab, Inc., Akron, OH 44309) with a porous polyethylene filter disc positioned at its bottom. The column solvent, isooctane/toluene/methanol (8/1/1 by vol) was passed through the column, swelling the column so that its height was about 7 cm. To eliminate floating, a second polyethylene filter disc was positioned above the LH-20 bed. The average flow rate through the column was 0.35 ml/min. The individual 1.0 ml extracts, which were prepared by reconstituting the original e t h y l a c e t a t e / n -hexane (3/2, by vol) extracts with isooctane/toluene/methanol, were then applied to appropriately labelled LH-20 columns with a Pasteur pipette. Thirteen mL of the column solvent were subsequently added to each column and the effluents discarded. Then an additional 13 mL-aliquot of the column solvent was added to each column and the effluents (ll-deoxycortisol fraction) were collected in appropriately labelled 16 X 150 mm disposable glass test tubes. The solvent was evaporated from each tube under air at 60°C, the individual residues dissolved in 2.0 mL of phosphate buffer and allowed to stand at 37°C for 15 rain before the RIA step. Duplicate aliquots (0.3 mL of each specimen) were removed from the appropriate tubes at this step to estimate recovery. The recovery of [aH] 11-deoxycortisol through the n-hexane p r e w a s h e s , e t h y l a c e t a t e / h e x a n e e x t r a c t i o n and LH-20 chromatography varied from 50 to 55% (mean 53%). RESULTS S e n s i t i v i t y . Fig. 1 d e p i c t s a t y p i c a l s e m i - l o g a r i t h m i c s t a n d a r d c u r v e for t h e a s s a y . T h e d e t e c t i o n l i m i t of t h e assay, defined as the smallest amount of 1 1 - d e o x y c o r t i s o l w h i c h could r o u t i n e l y b e d i s t i n g u i s h e d f r o m t h e z e r o s t a n d a r d , w a s a p p r o x i m a t e l y 10 p g p e r t u b e . T h i s d e t e c t i o n l i m i t c o r r e s p o n d s t o a p l a s m a conc e n t r a t i o n of a p p r o x i m a t e l y 0.7 ~g/L. T h e p e r c e n t a g e of a n t i b o d y - b o u n d [3H] 1 1 - d e o x y c o r t i s o l a t z e r o u n l a b e l l e d 1 1 - d e o x y c o r t i s o l v a r i e d f r o m 39 t o 4 3 % of t h e t o t a l 100

i~

8o

?~o0~

6O

8~ ~"

2o

'

10

~'o

'

'

o'

100 500 1 O0 11-DEOXYCORTISOL, pgltube

15'o0

Fig. 1 Semi-logarithmic standard curve of the plasma 11-deoxycortisol assay. The percent antibody-bound [sl-I] 11-deoxycortisol is plotted as a function of the quantity of 11-deoxycortisol (pg/tube/. The data points represent the means and ranges (± 1 S.D) of 20 individual assays. -

-

SIMPLIFIED RADIOIMMUNOASSAY FOR PLASMA ll-DEOXYCORTISOL 0 • • 0 • •

11-DEOXYCORTISOL 17 a-HYDROXYPROGESTERONE CORTISONE PROGESTERONE CORTISOL TESTOSTERONE

• DEOXYCORTICOSTERONE ~, METYRAPONE O CORTICOSTERONE ~ ESTRONE e ~ 17~-ESTRADIOL ~ ESTRIOL

49

400

300

~8~

- ".......... "..........

m ~ . X~- x 60

""@-

2< :W0 mo 0 ~ 40

~

•

ow ~o a.,r

~o O.OS O'.,

1'.0

10 STEROIDS. ng/tube

100

1000

SOlOO

Fig. ~ -- Cross reactivity study of the anti-11-deoxycortisol serum. The percent antibody-bound [SH] 11-deoxycortisol is plotted as a function of the quantity of steroid added {ng/tube).

radioactivity. The non-specific binding varied from 3 to 4°/0 of the total counts. An excellent logit-log doseresponse curve was routinely observed over the range from 10 to 600 pg/tube. Specimens with ll-deoxycortisol concentrations g r e a t e r than 600 pg/tube were diluted and reassayed. Specificity. The results of the cross-reactivity studies conducted to evaluate the specificity of the a n t i - l l deoxycortisol serum are illustrated in Fig. 2. The following percentages of cross reactivities were calculated according to the method of Abraham(19): l l - d e o x y c o r t i s o l , 100o/0; 1 7 a - h y d r o x y p r o g e s t e r o n e , 2.0%; deoxycorticosterone, 1.3%; progesterone, 0.4%; cortisone, corticosterone, cortisol and t e s t o s t e r o n e , <0.1°/0, estrone, 17/3-estradiol, estriol and metyrapone, <0.001%. Assay Blank. The assay blank was d e t e r m i n e d by s u b s t i t u t i n g 0.15 mol/L NaCl for plasma and performing the assay as described. The blank value was indistinguishable from the zero standard. Sephadex LH-20 column chromatography. Fig. 3 shows the correlation (y = 0 . 9 9 x + 4.0; _r = 0.98; 9- < 0.01) obtained when 30 specimens were assayed by the proposed method, with and without a prior purification on Sephadex LH-20. Mean values for the specimens analyzed with and without Sephadex LH-20 purification were 87.7 and 91.2 pg/L, respectively. Therefore, the values obtained without LH-20 purification agreed very well with those obtained when specimen were purified by LH-20 column chromatography, prior to the RIA step. TABLE 1 PARALLELISMOFTHEPLASMAI1-DEOxYCORTISOLASSAY Specimen # 1

2

3

4

Dilution

ll-Deoxycortisol (~g/L)

~ 0Z < 100 o.

0

l 300

l 400

(Fg/L),

Fig. 3 -- Correlation of the plasma 11-deoxycortisol concentrations (~g/LI as determined by the proposed R I A method with those determined by R I A after purification of the extracts on Sephadex LI-I-~O.

Parallelism. Table 1 is a s u m m a r y of studies performed to determine the parallelism of the proposed method. The four plasma specimens assayed with no dilution, X2 and X4 dilutions demonstrated acceptable parallelism. A n a l y t i c a l Recovery. T a b l e 2 s u m m a r i z e s t h e recovery data obtained following the additions of known quantities of 11-deoxycortisol to three different plasma specimens. The recovery varied for 88 to 108°/o (mean 100O/o). I n t r a - and Inter-assay variations. Intra-assay variation was determined by assaying (n = 10) quality control pools with normal, intermediate and elevated concentrations on the same day. The following data were obtained: normal: Y 1 = 3 . 6 _+ 0.3 lag/L, C V = 8 . 3 % ; intermediate: Y 2 = 16.7 _+ 1.8 pg/L, CV = 10.8O/o; elevated: Y 3 = 61 + 5.5 pg/L, CV = 9.0O/o. The inter-assay variation was d e t e r m i n e d by assaying the same control pools over a period of 30 days. The following data were obtained: Y 1 = 3.8 _+0.6 ~g/L, CV = 15.8%; Y 2 = 18.5 _+ 2.0 ~g/L, CV = 10.8%; ~ .~= 43.0 +_ 3.7 ~g/L, CV = 8.60/0. TABLE 2 A N A L Y T I C A L R E C O V E R Y OF 11-DEOxYCORTISOL A D D E D

TO H U M A N PLASMA

ll-Deoxycortisol (pg) Specimen

99 58 30

1

-x2 x4

78 42 20

2

x2 x4

75 38 21

-

84 42 22

x2 x4

l 200

PLASMA 11-DEOXYCORTISOL LH-20 CHROMATOGRAPHY

x2 x4

--

l 100

3

Present (A)

Added

(B)

Assayed (C)

%Recovery

42

20

42 42

50 I00

63 96 144

105 108 102

140 140 140

20 50 100

162 190

Ii0 I00

228

88

335 335 335

20 50 100

354 382

93 94

431

96

0/0 Recovery = C-A x 100 Average recovery = 99.60/0 B

WALSH, W A N G

50

Normal Range. The normal r a n g e was d e t e r m i n e d by collecting. E D T A plasma from 45 a p p a r e n t l y normal healthy adult volunteers (27 women and 18 men) between the ages of 18 and 54 years, who had fasted for at least 12 h. We found a mean value of 2.4 ~g/L, with a range of 2.0 to 5.0 pg/L. DISCUSSION The proposed method described in this r e p o r t has proven to be a sensitive, accurate, specific, precise and s i m p l e R I A for t h e q u a n t i f i c a t i o n of p l a s m a ll-deoxycortisol. • The simplification of the method was possible due to the high specificity of the a n t i - l l - d e o x o y c o r t i s o l serum which was prepared against the 11-deoxycor tisol-3-(O-carboxymethyl)oxime-BSA conjug a t e in the rabbit. Specificity studies performed and calculated according to the method of A b r a h a m (19) yielded the following (Fig. 2): l l - d e o x y c o r t i s o l , 100%; 17a-hydroxyprogesterone, 2.0%; deoxycorticosterone, 1.3%; progesterone, 0.4%; cortisone, corticosterone, cortisol, and testosterone, <0.1%; estrone, 17/3-estradiol, estriol, and m e t y r a p o n e , <:0.001%. The data (Fig. 3) comparing concentrations as d e t e r m i n e d by the proposed method with concentrations as determined by the p r e l i m i n a r y LH-20 column c h r o m a t o g r a p h y s u p p o r t the specificity of the proposed method, which does not require the tedious chromatographic purification prior to quantification by radioimmunoassay. The two n -hexane extractions, which are p a r t of the proposed method, were included to eliminate potential i n t e r f e r e n c e s from v e r y high c o n c e n t r a t i o n s of 17a-hydroxyprogesterone and/or p r o g e s t e r o n e in certain clinical conditions. We observed through the s e p a r a t e addition of [3H] 1 7 a - h y d r o x y p r o g e s t e r o n e (17a-PG) and [3HI p r o g e s t e r o n e (PG) to plasma t h a t the first n - h e x a n e extraction removed 55% and 92% of these t r i t i a t e d steroids, respectively. The corresponding combined e x t r a c t i o n efficiencies of [3HI 17a-PG and [3H] PG with two n - h e x a n e extractions were 82% and 97%. A p p r o x i m a t e l y 14% of the [3HI l l - d e o x y c o r t i s o l was e x t r a c t e d with the two n -hexane t r e a t m e n t s . Our experience with no n - h e x a n e extraction, one extraction and the two extractions indicated t h a t the proposed method which includes two extractions gave results w h i c h c o r r e l a t e d b e s t w i t h t h e LH-20 c o l u m n chromatographic method. Since the cross-reactivity data s u p p o r t low interferences from 17a-PG (2.0%) and PG (0.4%), the two n -hexane extractions are most likely r e m o v i n g a steroid(s) not included in our specificity study. Regardless, the data (Fig. 3) indicate t h a t two n-hexane e x t r a c t i o n s prior to the e t h y l a c e t a t e / h e x a n e (3/2) provide the specificity r e q u i r e d for this measurement. The normal range (2-5 p g / L ) of p l a s m a l l - d e o x y c o r t i s o l for the proposed method compares well with the normal r a n g e (0.4-4.0 ~g/L) o b s e r v e d for this steroid by gas-liquid c h r o m a t o g r a p h y (2). ACKNOWLEDGMENTS Thanks a r e e x t e n d e d to Dr. Charles D. H a w k e r for his r e v i e w of the manuscript and his many helpful suggestions. A special thanks is e x t e n d e d to Mrs. S a n d r a L. Hamilton for the p r e p a r a t i o n of the manuscript.

AND SU REFERENCES 1. Liddle, C.W., Estep, E.L., Kendall, J.W., Alexander, W., Townes, W. and Carter, Win. Jr. Clinicalapplication of a new test of pituitary reserve. J. Cli~ EndocrinoL Metab. 19, 875-894 (1959). 2. Mason, P.A., and Fraser, R. Estimation of aldosterone, 11-deoxycorticosterone, 18-hydroxy-11-deoxycorticosterone, eorticosterone, cortisol and 11-deoxycortisol in human plasma by gas-liquid chromatography with electron capture detection. J. EndocrinoL 64, 277-320 (1975).

3. Murphy, B.E.P., Engelberg, W. and Pattee, C. Simple method for determination of pla~ ~ corticoids. J. Cli~ EndocrinoL Metab. 23, 293-298 (1963). 4. Nugent, C.A., and Mayes, D.M. Plasma corticosteroids determined by use of corticosteroid-binding globulin and dextran-coated charcoal. J. Cli~ EndocrinoL Metab. 26, 1116-1120 (1966). 5. Strott, C.A., West, C.D., Nakagawa, K., Toshifumi, K., and Tyler, F.H. Plasma ll-deoxycorticosteroid and ACTH re .fonse to metyrapone J. Cli~ EndocrinoL Metab. 29, 6-11 (1969). 6. Meikle, A.W., Jubiz, W., Hitchings, M.P., West, C.D., and Tyler, F.H. A simplified metyrapone test with determination of plasma ll-deoxycortisol. J. Cli~ EndocrinoL Metab. 29, 985-987 (1969). 7. Spark, R.F. Simplified assessment of pituitary-adrenal reserve: Measurement of serum 11-deoxycortisol and cortisol after metyrapone. A n n Intern. Me& 75, 717-723 (1971). 8. Newsome, H.H., Jr., Clements, A.S., and Borum, E.H. The simultaneous assay of cortisol, corticosterone, ll-deoxycortisol and cortisone in human plasma. J. Cli~ EndocrinoL Metab. 34, 473-478 (1972). 9. Fisher, M., Curtis, G.C., Ganjam, V.K., Joshlin, L., and Perry, S. Improved measurement of corticosteroids in plasma and urine by competitive protein binding radioassay. Cli~ Chem. 19, 511-516 (1973). 10. Kolenowski, J. Simultaneous determination of cortisol, deoxycortisol and corticosterone in plasma and urine by competitive protein binding assay. Response of normal subjects to ACTH and metyrapone. J. Steroid Biochem. 5, 55-62 (1974). 11. Lee, L.M.Y., and Schiller, H.S. Non-chromatographic radioimmunoassay of plasma ll-deoxycortisol for use in the metyrapone test with polyethylene glycol as the precipitant. Clin. Chem. 21, 719-724 (1975). 12. Brown, J.R., Cavanaugh, A.H., and Farnsworth, W.E. A simple radioimmunoassay for plasma cortisol and ll-deoxycortisol (17, 21-dihyroxy-4-pregnene-3, 20-dione). Steroids 28, 487-498 (1976). 13. Maroulis, G.B., Maulimos, F.S., Carza, R., and Abraham, G.E. Serum cortisol and ll-deoxycortisol levels in hirsute premenopausal women. Obstet. GynecoL 48, 388-391 (1976). 14. Sippel, W.G., Bidlingmaier, F., Becker, H., Briinig, T., DSrr, H., Hahn, H., Golder, W., Hollmann, G., and Knorr, D. Simultaneous radioimmunoassay of plasma aldosterone, corticosterone, ll-deoxycorticosterone, progesterone 17-hydroxyprogesterone, ll-deoxycortisol, and cortisone. J. Steroid Biochem. 9, 63-74 (1978). 15. Demers, L.M., Elbright, L., and Clark, D.D. Improved radioimmunoaasay for ll-deoxycortisol (compound S) in plasma. Cli~ Chem. 25, 1704-1707 (1979}. 16. Canalis, E., Caldarella, A.M., and Reardon, G.E. Serum cortisol and ll-deoxycortisol by liquid chromatography: clinical studies and comparison with radioimmunoassay. Clin. Chem. 25, 1700-1703 (1979}. 17. DeVries, C.P., Tomecky-Janousek, M., and Popp-Snijders, C. Rapid quantitative assay of plasma ll-deoxycortisol and cortisol by high-performance liquid chromatography for use in the metyrapone test. J. Chromatogr. 183, 87-91 (1980). 18. Rodbard, D., Bridson, W., and Rayford, P.L. Rapid calculation of radioimmunoassay results. J. Lab. Cli~ Me& 74, 770-781 (1969). 19. Abraham, G.E. Solid-phase radioimmunoassay of estradio1-17/3. J. Cli~ EndocrinoL Metab. 29, 865-871 (1969).