Journal

of Steroid

Bmhemwrry,

1978. Vol.

9, pp. 63-74.

Printed m Great Britain Pergamon Press.

SIMULTANEOUS RADIOIMMUNOASSAY OF PLASMA ALDOSTERONE, CORTICOSTERONE, 1 l-DEOXYCORTICOSTERONE, PROGESTERONE, 17-HYDROXYPROGESTERONE, 1 l-DEOXYCORTISOL, CORTISOL AND CORTISONE W. G. SIPPELL, F. BIDLINGMAER,H. BECKER,T. BRCJNIG, H. D~RR, H. HAHN, W. GOLDER, G. HOLLMANN and D. KNORR University of Munich Medical School, Children’s Hospital, Division of Pediatric Endocrinology and Experimental Surgical Laboratory, Lindwurmstr. 4, D-8000 Miinchen 2, Germany (Received

4 April 1977)

SUMMARY A method for the simultaneous determination of eight major corticosteroid hormones and precursors in 052.0 ml of plasma has been developed and is described in detail. After extraction of the unconjugated steroids from plasma to which tritiated steroid tracers had been added, progesterone (P), 1l-deoxycorticosterone (DOC), 17-hydroxyprogesterone (17-OH-P), corticosterone (B), 11-deoxycortisol (S), aldosterone (A), cortisone (E) and cortisol (F) are simultaneously separated using eight mechanized 60cm Sephadex LH-20 columns in parallel. Each of the isolated steroids is quantitated by radioimmunoassay after saving an aliquot for recovery counting. The combination of chromatographic separation and appropriate antiserum resulted in high assay specificity. Water or steroid-free plasma blanks were undetectable, with sensitivities of the assays ranging between 0.020 (A) and 0.370 (E) ng/ml. There was very satisfactory precision and accuracy, with inter-assay coefficients of variation below 16.5% and linear relationships between added and found, respectively. Normal values are reported for males, females (follicular phase) and a small group of prepubertal children, revealing sex differences between the adults studied with higher A, B and P levels in females and higher 17-OH-P and E levels in males. The mineralocorticoids A and DOC were considerably higher in children than in adults. The highly practicable method is particularly useful in pediatric and experimental endocrinology.

tion (e.g., biosynthetic

INTRODUCTION In the last seven years, numerous radioimmunological procedures have been reported for the determination of single corticosteroid hormones or their biosynthetic precursors in peripheral plasma or adrenal tissue extracts. Since the adrenal cortex secretes a complex mixture of steroids whose biological effects differ from

defects, essential

hypertension,

clinical drug trials), or whenever very little biological material is available, e.g. in pediatric or experimental endocrinology or when frequent plasma sampling is required during episodic secretion studies. The present paper reports for the first time a practical method for the simultaneous radioimmunoassay of all of the eight major adrenal 4-ene-3-ketosteroids aldosterone (A), corticosterone (B), 1l-deoxycorticosterone (DOC), progesterone (P), 17-hydroxyprogesterone (17-OH-P), 11-deoxycortisol (S), cortisol (F) and cortisone (E) in the same small sample of peripheral plasma. The method involves a mechanized Sephadex LH-20 multiple column chromatography developed by us whose technical details have been described elsewhere [17,18] that enables the convenient and reliable simultaneous separation of these steroids from the same plasma sample. Multisteroid radioimmunoassay procedures utilize selected antisera showing no or negligible cross-reactivity with possibly interfering steroids. They have been standardized as far as possible using special manifolds and automatic pipettors for the economical processing of the resulting large numbers of samples.

each other not only qualitatively but also quantitatively, a detailed assessment of adrenocortical function can only be made by studying directly as many of its biologically active hormones as possible. Preferably, these analyses should be made simultaneously on the same plasma sample. In recent years several reports using laborious chromatographic methods have been published on the simultaneous determination of up to six steroids [l-16] from the final branches of the adrenal biosynthetic pathway leading from progesterone to either aldosterone or cortisol, the biologically most potent mineralo- and glucocorticoid hormones, respectively. To date, however, no systematic approach has been made to measure simultaneously and simply all major intermediates and end products of the adrenal steroidogenesis. Such a method could be of great value in all studies of normal, abnormal or altered adrenal steroid produc63

w. G. SII'PI.I.LC’f trl

64 EXPERIMENTAL

(a) Radioactice

steroids

[I,2-3H]-D-aldosterone (NET-105), [l,2-3H]-corticosterone (NET-182), [1,2-3H]-1 l-desoxy-corticosterone (NET-184) [1,2-“HI-progesterone (NET-208), (TRK-3 15), [7(n)-3H]-17-hydroxy-progesterone [1,2-3H]-1 I-deoxycortisol (NET-295), [1.2-3H]-hydrocortisone (NET-185) and [ 1,2-3H]-cortisone (NET-183) all with a specific radioactivity of 40-60 Ci/mmol were purchased from either New England Nuclear Corp., Boston, MA, U.S.A. or Radiochemical Centre, Amersham, U.K. The radiochemical purity was 97-98”,,. About 3 x 1O”c.p.m. of each steroid, i.e., the amount to be used in a three months period, were repurified by chromatography on a 4Ocm Sephadex LH-20 column using methylene chloride-methanol (98 : 2, v;v) as solvent. Purified radioactive steroids were kept in benzene-ethanol (9 : 1, v/v) at 4°C. (h) Chelnicals Methylene chloride p.a. (Merck No. 6050), methanol p.a. (Merck No. 6009), ethanol p.a. (Merck No. 970), benzene p.a. (Merck No. 1783), dioxane p.a. (Merck No. 3115), ethyleneglycol p.a. (Merck No. 9621) were used without further purification. Non-labeled steroids were obtained from either Merck, Darmstadt. or Ciba, Wehr/Baden, Germany. Suppliers of the following chemicals were: Dextran T70: Pharmacia, Uppsala. Sweden; Human Gamma Globulin: Kabi. Stockholm, Sweden ; Charcoal Norit A: Serva, Heidelberg, Germany: Napthalene and PPO: Zinsser, Frankfurt, Germany. 50mM borate buffer was prepared by dissolving 6.184 g boric acid p,a. (Merck No. 165) and 7.456 g KC1 p.a. (Merck No. 4936) in a final vol. of 2000 ml of bidistilled water, after adjusting pH to 7.8 with approx. 7.8 ml of 1 M NaOH. Gamma globulin buffer (0.06”/ w/v) was made by dissolving 300 mg gamma globulin plus 162.5 mg sodium azide (Merck No. 6688) in a final vol. of 5OOml of the borate buffer. It was stored at 4°C for up to three months. Dextrancoated charcoal suspension: 250mg dextran and 2SOg Norit A were mixed with gamma globulin buffer to a final vol. of 100 ml. A modified Bray’s solution containing 4.0 g 2,5-diphenyl-oxazol (PPO). 60.0 g naphtalene, 20.0 ml ethyleneglycol, 100.0 ml methanol and dioxane up to IOOOml was used as scintillation cocktail. (c) Instruments All glassware used was made steroid-free by previous heating to 500” for 3-5 h [19]. Disposable 10 x 40 mm round bottom polystyrene tubes were used for the radioimmunoassays. Incubations were carried out in a modified Rotary Evapo-Mix (Buchler Instruments Division. Fort Lee. NJ, U.S.A.) with ex-

changeable tube-racks each holding 90 RIA-tubes. Rapid and reproducible pipetting of the charcoal suspension was pcrformcd using an automatic magnetic valve pipettor with ;I 100 /tl head (Braun Fe 213. Metsungen. Germany). For centrifugations a refrigerated RC-2B lab centrifuge (I. Sorvatl Inc.. Newtown. CT, U.S.A.) with a HS-4 swing-out head and special adapters carrying up to 120 tubes was used. Radioactivity was counted in an Isocap 300 liquid scintillation spectrometer (Searle, Nuclear-Chicago Division, Amsterdam. Netherlands) M,ith an elliciencq of 65”,, for tritium. For calculations. a pdp I l/IO laboratory computer (Digital Equipments Corp., Maynard, MA. U.S.A.) was used.

To 0.5-1.0 ml of peripheral plasma approx. 2000 d.p.m. of each of the following steroids (dissolved in 150 ~1 of gamma globulin buffer) were added as internal standards: [1,2-3H]-D-aldosterone, [1,2-“HI-corticosterone. [1,2-3H]-deoxycorticosterone, [1,2-3H]-progesterone, [7-3H-17]-hydroxyprogesterone, [ 1,2-3H]-1 1-deoxycortisol, [l.2-3H]cortisol and [1,2-3H]-cortisone. After thorough mixing and an equilibration period of at least two h, the plasma was extracted twice with 5 ml of ice-cold methylene chloride and then the extract washed once with 3 ml of sterile distilled water. After gentle centrifugation (1500g x 5 min) at 4 C, separation of the upper (aqueous) from the lower (methylene chloride) phase was performed by suction. The final extracts were then evaporated to dryness under a gentle stream of nitrogen at 45 C.

Plasma extracts were redissolved in 0.250 ml of the solvent system [20] consisting of methylene chloridemethanol (98:2. v.!v) and then submitted to our automated Sephadex LH-20 chromatography using eight 60 cm columns running in parallel. Thus. as has been described in detail elsewhere [ 171, a complete separation (Fig. I) of B (4348; 5 ml), S (52-58; 6 ml), A (58-65: 7ml), E (67 ~75; 8ml) and F (114-126; 12ml) could be obtained. whereas P. DOC and 17-OH-P, because of incomplete resolution in this system, were collected between 27 and 34 ml as a combined 7 ml fraction. While the 60cm columns were still being eluted. the combined progestin + DOC’ fraction was separated (Fig. 2) into P (19.-24; 5 ml), DOC (28-35; 7 ml) and 17-OH-P (46 56; IO ml) on eight 40 cm LH-20 columns using a solvent system consisting of n-heptane~chloroform+thanol (50:50:0,25, by vol.) plus water to saturation [ 18.201. The fractions each containing one of the eight isolated steroids were evaporated to dryness, redissolved in 2.0 ml of absolute ethanol (15”) and were then rapidly divided into two different aliquots at a con-

65

Simultaneous RIA of 8 plasma steroids SA

TB

P DOC 17-OH-P

F

E

,fi I(II,;,

II I

60

60

110

120

130

ml eluate Fig. 1. Chromatogram of tritiated steroids eluted from mechanized 60-cm Sephadex LH-20 columns using 40ml/h of methylene chloride-methanol (98:2, v/v) as solvent. Peaks eluted: (P) progesterone, (DOC) 11-deoxycorticosterone, (17-OH-P) 17-hydroxyprogesterone, (T) testosterone, (B) corticosterone, (S) 11-deoxycortisol, (A) aldosterone, (E) cortisone and (F) cortisol [17]. stant temperature

of 15°C: For determination

of inter-

nal tracer recovery, one aliquot was transferred into scintillation vials containing 0.150 ml of gamma globulin buffer and 10ml of Bray’s solution. After thorough mixing, vials were allowed to stand in the dark for at least two h before counting radioactivity up to 10,000 counts. The other aliquot was used in duplicate for steroid quantification by radioimmunoassay. During preliminary experiments, no systematic difference was found in the recoveries whether the aliquot was taken from the RIA buffer solutions containing the antibody or from the ethanolic fraction introduced into the RIA tubes, provided dried steroids were very thoroughly dissolved in the gamma globulin buffer solution. 3000

DOC

/I

0%

I

IO

I

I

I

I

1

20

30

40

50

0

I 60

ml emote Fig. 2. Chromatogram of tritiated progesterone (P), lldeoxvcorticosterone (DOC) and 17-hydroxyprogesterone (17~dH-P) on 40-cm Sephadex LH-20 columns using a solvent system of n-heptane-chloroform (1: 1, v/v) plus 0.25% of ethanol plus water to saturation [18].

(c) Steroid radioimmunoassays (RIA) RIA of akfosterone (A). Of the 2.0 ml ethanolic A fraction, 400 ~1 (= 20%) were taken for recovery and 750 ~1(= 37.5%) in duplicate for RIA. Standards were prepared in duplicate (zero samples in quadruplicate) by evaporating respectively 0, 20, 50, 100, 200, 400 and 800 pg of A in ethanolic solution using a vacuum oven at 45”. To compensate for contingent unspecific blanks that might be introduced into the RIA-tubes by the column eluate containing the unknown steroid, an appropriate vol. of eluate collected during prerinsing of the chromatographic columns was dried down and redissolved in 17.5 ml of absolute ethanol. 750 4 of this solution were evaporated in each standard tube in addition to the standard solution. To unknowns and standards, 100~1 of gamma globulin buffer, containing 12.000 d.p.m. of [“HIaldosterone and 500~1 of an aldosterone antiserum diluted to 1:10,000 with gamma globulin buffer were added. It had been raised in rabbits after immunization with aldosterone-3-(O-carboxymethyl)-oxime (CMO) linked covalently to bovine serum albumin (BSA). All tubes were incubated with gentle shaking at 37°C for 20min and then allowed to stand at 4°C for two h. Then 100~1 of thoroughly stirred dextran-coated cl&coal suspension were added to each tube using an automatic pipettor. After horizontal shaking for exactly 15 s, the tube rack was replaced into the ice bath and bound from free fractions were separated exactly 15 min later by centrifugation (1500 g x 20 min) at 4°C. The supernatants (containing the bound fraction) were decanted directly into scintillation-vials containing 10 ml of Bray’s solution. Radioactivity was counted with a statistical error of 2% or less. Standard curves were constructed by computer, using a modified Gaussian regression of the third order for reciprocal standard values [21]. Unknown sample concentrations were then obtained by using the parameters of the calculated standard curve.

RI A

ofco,_fico.strror~e (B), I I -r/co.\-~~cor~i.so/ (S), ~vrti-

sol (F) LUICI cortisor~ (E). Essentially. these RIAs were performed as described for aldosterone. For B and S. the same aliquots of the dry residues from the column eluatc fractions as for A were taken for recovery (=20”,,) and RIA (37.5”,,). For both F and E, of the 2.0 ml ethanolic fraction 2 x 750 111(= 75”,,) were used for recovery and 2 x 100 ptl (= 5”J for RIA. Standards covering the range between 50 and 2000 pg of B and S, between 200 and 5000 pg of F and between 200 and 2000 pg of E were prepared in duplicate. 12,000d.p.rn. of the respective tritiated steroid and 500~1 of antiserum were added to standards and unknowns. The same antiserum. containing a rabbit cortisone-21-hemisuccinate-BSA antibody, was used for E, F and S, in dilutions of 1:60,000. 1: 10,000 and 1: 30,000. respectively. Corticosterone, on the other hand, was assayed using a rabbit anti-l l-desoxycorticosterone-3-CMO-BSA antibody in a dilution of 1:5.000. Incubations, separation of bound from unbound fractions, radioactivity counting and calculations were performed exactly as described for aldosterone. RIA of prqesterone (P), 17-h!,dro.u~-proyesrerorle (170HP) UK.! I I -neso.u~~orticoste~one (DOC). The P fraction obtained by the second chromatography was divided (after evaporating and redissolving it in 2.0 ml of ethanol) into 750~1 (=37.5”,,) for recovery and 2 x 500pl (=25”;J for RIA, whereas the 2.0 ml ethanolic 17-OHP- and DOC-fractions were split each into 400pl (=20”,,) for recovery and 2 x 750~1 (= 37.5”,,) for RIA. Duplicate standards were prepared covering the range between 50 and 6000 pg of P and between 50 and 2000 pg of l7-OH-P and DOC. respectively. To every RIA-tube. 12.000 d.p.m. of tritiated steroid and 500 pI of antiserum diluted in gamma globulin buffer, were added. For assaying P and l7-OH-P. a rabbit anti I I x-hydroxy-progesterone- I I x-hemisuccinate-BSA antibody and a rabbit anti l7-hydroxy-progesterone-3-CMO-BSA antibody, respectively. were used. both having a titre of I : 15.000. In the DOC assay, a sheep I I-deoxycorticosterone-21-hemisuccinate-BSA antibody in a dilution of I : 10,000 was used (kindly supplied by Prof. P. Vecsei, Ph. D.. Heidelberg). Incubations were carried out with gentle shaking at 37-C’ for one h and subsequent standing at rest at 4 C for another two h. Separation of bound from free fractions. liquid scintillation counting and calculations were done as in the aldosterone RIA. A flowsheet of the entire procedure (extraction, chromatography and radioimmunoassays) is outlined in Fig. 3. RESULTS

(1) Extruction

and recoreries

(LI) Extraction. Two sequential methylene chloride extractions removed X7.5 to 95.X”; of the radioactive steroids from the plasma samples. Since a third

extraction mcrcascd thcsc Ircco\crIcs b\ oiil~ less than I”,, on the average. 1~0 cstractions \F.\rc used. (b) Rcco~~c~~.. The a\cragc reco~ct-J t>f radioacti>c steroids added to plasma varied from 52.0 to 6X.2”,, after extraction and one (B. S. A. E. F) or t\+o (P, DOC. 17-OH-P) Scphadex LH-20 chrom~ltographies (Table I). Mean coclticients of variation of percent recoveries between sets of 8 columns elutcd in parallel were ranging between IO.9 and lY.2”,, after one and between I I .Y and 24.Y”0 after two chromatographics. (2) E&uurioif

of f/I~~Hlc’t/?orl

(a) Studrrrd ~IIIY~Y.~. In Table 2, the moan displaccments of the labelled steroid from the antibody by known amounts of added steroid is expressed as the percentage of radioactivity of the 7ero samples in which no unlabelled steroid was present. For each of the eight radioimmunoassays, these figures art‘ listed as mean percentage of bound radioactivit) + I S.D. together Mith the mc;m dispersion of the duplicate standards from IX typical standard curves. (b) Specjficiry. Percent cross-reactions. calculated according to Abraham[??] between the antisera used for each radioimmunoassay and other major plasma steroids are listed in Table 3. Steroid cross-reactions within chromatographic fractions were observed onl) in the case of 4-enc-andro-stenedione which is eluted (fractions I9- 23 in Fig. 2) to almost IOO”,, togcthrr with the progesterone fraction. and in the cast of testosterone. IO 15”,, of which are elutcd with the corticosterone fraction (Fig. I ). In both instances. these cross reactions were vcrc MelI tolerahlc: 0.02 and 4.3”,,. rcspcctively. On the 60-cm columns. pregnenolone and 17-hydroxypregnenolone arc eluted from 33 to 3X ml and from 35 to 40 ml, rcspcctively. resulting in a crosscontamination of prcgncnolonr with I7-hydroxy-progestcrone of no more than IO”,, which was further reduced by the subsequent chromatography on the 40-cm columns. Moreover. the cross-reaction of tho 17-hydroxyprogestcrone antiserum with pregnenolonc any significant interferwas only 0.3”,,. thus excluding encc of pregncnolone in the l7-hydroayprogesteronc assay. 17-hydroxypregnenolone did not overlap with any of the steroids isolated. I X-hydroxy-I I-dco\!corticostcronc (I X-OH-DOC) and I X-hydroxycorticostcrone ( I X-OH-B) were eluted on the 60-cm columns from 30 to 54 ml and from 56 to 62 ml. respectively. This resulted in an overlapping of the I I-deoxycortisol peak of about W,, bq IX-OH-DOC and of 5 IO”,, by IX-OH-B. However. the cross reactions of the antiserum used in the I X-OH-DOC and I I-deowycortisol RIA with 1X-OH-B were only 0.X?,, and 0.3 I”,, respectively. The aldostcrone antiserum showed a cross-reaction of only 0.12”,, with IX-OH-B. which would contaminate the aldosterone peak to about 35”,,. T~LIS. suticiently specific measurement of both 11-deoxycortisol and aldosterone seems possible even under pathological conditions.

67

SimultaneousRIA of 8 plasma steroids of Plasma Extract: 3 to 1,O ml plasma + 8x 1500 cpm H-steroid (internal standard)

Preparation 1.

0,s

2.

Extract

3.

Wash with

4.

Evaporate

5.

Redissolve

Separation

(ice-cold, anal. grade)

twice with 5 ml CH2C12

3 ml sterile distilled

water

to dryness under gentle N2-stream in 250 ~1 CH2C12

: MeOH (98:2, v/v)

of Corticosteroids:

6.Mechanized multi-column chromatoqraphy on Sephadex LH-20 (60 x 1 cm) with constant elution (40 ml/h)

'P + DOC + 17-OH-P‘

B,

S,

A,

E,

F

7.

2 nd LH-20 chromatography (40 x 1 cm ; solvent: n-heptane : CHCl : EtOH 50:50.0.25, wager to sat.) l-11=: P , DOC , 17-OH-P

8.

Divide each of the 8 steroid fractions

(redissolved in 2.0 ml EtOH)

2 x aliquot for radioimmunoassay (in RIA test tubes)

2 x aliquot for recovery counting (in counting vials)

Radioimmunoassaocedure: --_-Aliquot of unknown samples or 9. }(evaporate known samples for standard curve + 100 ~1 3H-steroid + 500 ~1 antibody

containing

to dryness)

5000 cpm

solution

10.

Shake all tubes 20 min at 37' C Let tubes stand for 2 h in ice bath

11.

Add 100 ~1 dextran-coated charcoal to each tube Shake briefly, return all tubes for 15 min to ice bath

12.

Centrifuqe

13.

Decant supernatant

at 4" C (swinq-out head, 1500 q x 20 min) in countinq vials containinq 10 ml Bray's solution

14.

Liquid scintillation

15.

Computerized calculation of nq steroid per ml plasma by comparison with automatically analyzed standard curve using the individual recovery of each sample Fig. 3. Flowsheet

Table

1. Recoveries

the

tritiated

(to 10,000 cts per vial)

multi-steroid

Progesterone* 11-Deoxycorticosterone* 17-Hydroxyprogesterone* Corticosterone 11-Deoxycortisol Aldosterone Cortisone Cortisol obtained

determination

plasma steroids chromatography Radioactivity recovered + S.D. (%)

Steroid

* Steroids

of

of

counting

68.2 55.4 52.0 57.5 58.3 65.1 59.7 56.4 by a subsequent

+ _t k + + k + _t

8.1 13.8 10.8 10.9 12.4 11.6 9.9 8.7

elution

in

procedure

mechanized

multi-column

Number of columns eluted

Mean intercol. C.V. (%) (col. 1-8)

200 200 200 200 48 200 200 200

11.9 24.9 20.7 10.9 19.2 12.8 11.6 12.1

on 40 cm Sephadex

LH-20

columns.

w.

G.

SIPWLL

C’f c/1.

Table 2. Standard curves for progesterone. deoxycorticosterone. corticosterone. I I-deoxycortisol, aldosterone, cortisone and cortisol; mean dispersion

of duplicates:

Amount of steroid added (pg)

Progesterone

0 SO 100 250 500 1000 3000 6000

kS.D.

Mean dispersion of duplicates

i_ 1.36 +0.x4 k 0.63 1-0.57 iO.61 *0.34

100.00 73.37 62.62 52.08 37.17 20.19 12.62

+9.24 k9.1 I f 10.86 k8.29 + 8.74 k6.10 + 3.94

* 1.40 * 1.31 * I.17 f I .07 * 0.80 &OS2 * 0.42

100.00

+9.3x k4.68 + 5.93 i_ 3.07 +4.55 +3.57 & I .93

i I.18 jy 1.33 kO.95 io.77 +0.64 + 0.39 io.47

+ 6.26 i_ 5.0’) + 6.07 f 6.59 k5.14 k4.19 +3.53

& 2.52 f 1.83 i_ 1.81

3000

I00.00 88.88 69.6 1 55.15 39.92 26.28 22.26 100.00 89.25 81.66 58.68 40.95 25.97 10.42

+11.‘)2 * 2.50 is.12 * 5.39 * 3.08 12.56 + I .05

+ I.26 f 0.99 il.99 + 1.74 kO.86 iO.67 kO.31

50 100 200 400 800

100.00 66.24 48.43 34.13 23.48 14.55

k6.19 * 4.05 * 4.09 k5.21 k4.64 + 3.72

i I.68 20.76 _t 1.12 +0.59 + 0.66 + 0.42

I00.00 40.94 26.78 15.12 7.63

* 5.72 k4.32 * 2.79 k3.01 +0.54

2 I .30 iO.61 +0X i 0.62 iO.15

100.00 53.20 36.27 18.90 8.15 5.90

k6.28 +5.35 i4.56 * 2.66 + 1.35 * I.10

f 1.14 i_ I.10 ,0.x3 * 0.93 io.23 + 0.22

2000 0

50 100 250 500 1000 2000 Corticosterone 50 200 400 800 1600 2000 0 50 100

250 500 1000 Aldosterone

Cortisone 200 400 1000

Cortisol

“,, of bound radioactivity

*7.X k4.7’) f 4.08 k4.73 + 2.95 + 2.45 f 1.30 f I.35

50 100 200 400 IO00

1 1-Deoxycortisol

II = 18

84.04 71.62 48.57 31.54 23.13 11.4’) 7.84

Deoxycorticosterone

17-Hydroxyprogesterone

17-hydroxyprogesterone. mean values & S.D. and

0 200

400 1000 3000 5000

Similarly, other steroids (1 I-dehydrocorticosterone, dehydroepiandrosterone, 20~ and 20/Ghydroxypregnenolone) either showed no chromatographical overlapping with collected steroid fractions or did not exhibit significant cross-reactions with overlapping steroids in the radioimmunoassays chosen. Overall in-

100.00

72.45 57.16 33.46 22.45 14.03 x.1 I

&I.51 & 1.72

* I .09 *0.x7 kO.95 F0.57

terference, therefore, in no instance exceeded 0.6%. Table 3 also includes (at the top) the site of conjugation of the steroid antigen used for immunization and (at the bottom) the antiserum titer used in radioimmunoassay at which approximately SOS< of the labelled steroid are bound.

Simultaneous RIA of 8 plasma steroids Table 3. Steroid-cross-reactions

(in %) with antisera used in radioimmunoassays

c-11

Anti-DOC c-21

Anti-17-OHP c-3

Anti-B c-3

Anti-S c-21

Anti-A c-3

Anti-E .___ c-21

Anti-F ~_~ c-21

100.00 1.36

54.30 100.00

12.10 0.11

100.00 100.00

41.60 32.10

0.15 0.13

35.90 29.20

10.60 32.10

0.07 0.92 1.22