A radioimmunoassay for plasma androstenedione using 125I tracer

165

Clinica Chimica Acta, 103 (1980) 165-168 @ Elsevier/North-Holland Biomedical Press

CCA 1330

A RADIOIMMUNOASSAY 12sI TRACER

FOR PLASMA ANDROSTENEDIONE

USING

B. PELC * and A.L. HOLMES Medical Research Council, Mineral Metabolism Unit, The General Infirmary, Street, Leeds LS13EX (U.K.) (Received

September

Great George

3rd, 1979)

Summary A radioimmunoassay for plasma androst-4-en-3,17-dione is described using ether extracts of plasma and 12’ I radioligand. The reagent is easily prepared by an established radiochemical method from androst-4-en-3,17-dione-Go(carboxymethyl) thioether and 12SI-histamine. Introduction Previously described methods for radioimmunoassay (RIA) of androstenedione (AD) [l-7] use tritiated AD and antibodies raised against androst-C en-3,17dione, substituted in positions 3, 6 or 11 by homologous [1,2] or heterologous [3--51 systems. Most use chromatographic separation of AD from other steroid hormones; these can interfere with the assay, but assays without chromatographic purification also have been published [8-lo]. Some recent communications describe RIA of steroid hormones using “‘1 tracers e.g. progesterone [ 11,141, oestrone [ 111, oestradiol [ 121, cortisol [ 13,15,16] and aldosterone [ 151. We have prepared a suitable lzsI radioligand (II) from androstenedione-6/3(carboxymethyl) thioether (I) for RIA of AD, an important intermediate in the synthesis of male and female sex hormones.

SCHzCOOH (I) * To whom correspondence should be addressed.

SCH2 CONH( CH2 )2 H

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Materials Androst-4-en-3, 17dione-6/3-(carboxymethyl) thioether (I) was prepared from 6P-bromoandrost-4-en-3,17-dione [ 181 and thioglycolic acid by the method of Lindner et al. [17]. The crude acid was purified on a thin layer chromatography (TLC) plate (silica gel 60 F254, E. Merck) developed in ethyl acetate and 10% ethanol. It was about 93% pure, calculated from X,,, 243 nm (~10,500) [17] but resisted crystallisation. [IR: 1680, 1735, 2500-2800 and 3580 cm-’ - NMR: G(CDCl,), 0.92 (s, 18-H3), 1.25 (s, 19-H3), 2.18 (s, CHz), 3.4 (s, 4-H or 6-H). MS: methyl ester: m/e 390, 359 (-0CH3), 331 (-CO), 285 (-S.CH*); m/e 462 for TMS -enolether methyl ester]. The iodinated derivative, G&thiomethylenecarbonyl- [ ‘*‘I] iodohistamine (II) was prepared by the mixed anhydride method [ 111 and finally purified on a TLC silica gel plate developed in a mixture of benzene and ethanol 3 : 1, R, c.a. 0.6. Incorporation of “‘1 in the histamine derivative was about 7%; the product was stable for at least four months when stored in ethanol at 0°C. 1,2,6,7-[3H4]androstenedione, (specific activity about 90 Ci/mmol) and Na12SI were obtained from the Radiochemical Centre, Amersham, Buckinghamshire (U.K.). Sodium phosphate buffer (0.05 mol/l, pH 7.0) containing 0.15 mol/l saline solution, 0.1% gelatine and 0.1% NaN3 as preservative was used as the assay medium. 0.9% saline solution, containing 0.1% gelatine and 0.1% NaN3 was used as the solvent for the standards and to redissolve plasma extracts. Crystalline AD was obtained from Sigma Ltd. and recrystallised from methanol. It was dissolved in A.R. ethanol and serially diluted in saline solution to give a range of 125-6000 pmol/l. The antiserum, raised in rabbits against AD-7a-carboxyethylthioether-BSA, was supplied by Miles Laboratories, Slough, U.K. It was stored at 4°C as freezedried powder and reconstituted as required in buffer and when stored at 4°C it was stable for 2-3 weeks. It was used at the recommended dilution. Dextran-coated charcoal was prepared from 2.5 g charcoal and 0.25 g dextran T 70 in 1 1 phosphate buffer. Methods Extraction of plasma [lo0 ~1 plasma were extracted with 1.5 ml diethyl ether, in duplicate, on a Baird and Tatlock Multi-Vortex shaker, for 5 min. After standing a short time, the plasma was frozen in a dry ice/ethanol bath and the ether extract poured into an assay tube and the solvent was removed in a vacuum oven at 37°C. Assay To the extracts were added 400 ~1 of saline solution and the tubes were vortexed for 10 s to redissolve the residue. 100 ~1 were taken for assay, and 100 ~1 of saline solution added. 100 ~1 of standard solutions of AD were added to the standard tubes. Balancing the standard curve was achieved by placing 3 ml of diethyl ether in several tubes and evaporating down in the vacuum oven. 800 ~1 saline solution were added to each tube, vortexed and 100 ~1 of the

167

resulting solution were added to each standard tube. 200 ~1 of antibody in phosphate buffer were added and the contents mixed, followed by 100 1.11of tracer (3H or ‘*‘I) in buffer. The tubes were vortexed for 5 s and incubated overnight at 4°C. 1 ml of dextran-coated charcoal was added and the tubes centrifuged at 1500 X g for 15 min. The supernatant was counted on a Beckman LS-233 liquid scintillation counter or on a L.K.B. Rack-gamma counter with RIA evaluation program giving automatic printout of the standard curve and assay values. Results The assay using “‘1 tracer was compared with the 3H assay, which has been in use in this laboratory for three years. A comparison of the standard curves obtained with the same highly specific antiserum is shown in Fig. 1. They are of similar shape, except that the lzsI curve shows a higher zero-point binding and a more sensitive curve for a given number of counts. This comparison held good whether the incubation time was 3 h at room temperature, or overnight at 4°C. The curves cover the same range of concentration so no changes in the methodology were required. A comparison of results obtained by both methods is shown in Fig. 2. The results were obtained for the ‘?‘I method from the printout of a L.K.B. Rackgamma counter. This produced a standard curve plot, from which results are obtained by a linear interpolation between standard curve points. The 3H results were obtained using a logit/log concentration method in which the best fit to the points and subsequent calculation of values was by a least squares routine on an Olivetti P652 microcomputer. The regression line (eq. Y = 1.05X - 0.307) did not differ significantly from

L

nMol/l

ANDROSTENEDIONE

5b

ebo 4km 2bD ANDROSTENEDIONE fMol/tvbo

Fig. 1. Standard curves 3,17-dione m-----m.

using androst-4-en-3.17-dione-6C1

Fig. 2. Comparison of androstenedione 4-en-3,17-dione-6[12sIl.

levels measured

3H

2s11

l

-

and

TRACER

[1,2.6,7-JHlandrost-4.en-

by C3Hlandrost-4-en-3,17-dione

and by androst-

168

the 45°C line, and a high correlation coefficient was obtained, r = 0.968, (Y, lz51] ; X, value with [‘Hlandrost-4-envalue with androst-4-en-3,17-dione-6-[ 3,17dione). Repeated measurements of a plasma pool gave an intra-assay precision of 7.03% and inter-assay precision of 9.20% over six assays. (Mean 3.05, S.D. 0.28, n = 39, inter-assay; mean 3.03, S.D. 0.13, n = 10, intra-assay). Discussion ‘*‘I compared very favourably with the The new AD tracer incorporating existing 3H material. The tracer can be substituted without any alterations in procedure, except the elimination of preparation for liquid scintillation (L.S.) counting. Additionally the expense of L.S. materials is saved. Furthermore the ‘*‘I assay had better precision than the 3H assay. The tracer proved to be stable for a least four months, with no sign of deterioration other than radioactive decay. The Rack-gamma counter provides automatic calculation of the standard curve and assay values. This facility is not provided on simple L.S. counters, which require external and sometimes expensive computing equipment. Possible alternative methods include counting both bound and free fractions to improve precision and the development of a solid-phase antibody. This would combine antibody and separating agent into a single reagent, thereby simplifying the procedure and reducing the assay time. Acknowledgement We are grateful to the Chemistry Department ing out N.M.R. and mass spectrum measurements.

of Leeds University

for cany-

References 1 2 3 4 5 6 7 8 9 10 11

12 13 14 15 16 17 18

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