The Third Department of Internal Medicine, University of Tokyo Faculty of Medicine, Hongo, Tokyo 113

Endocrinol. Japon. 1974, 21 (2), 115 ` 119 A Radioimmunoassay for Serum Dehydroepiandrosterone HISAHIKO SEKIHARA, TOHRU YAMAJI, NAKAAKI OHSAWA AND HIROSHI IBAYASHI * The Third Department of Internal Medicine, University of Tokyo Faculty of Medicine, Hongo, Tokyo 113 Synopsis A radioimmunoassay for serum dehydroepiandrosterone has been developed by using the anti-dhea antiserum. After DHEA-7ƒ -3H was added to 0.2 to 1.0ml of serum and 1ml of water for recovery, DHEA was extracted with methylene chloride, and the extract was evaporated to dryness. The residue was applied to paper chromatography. The eluates and DHEA-7ƒ -3H which was added to determine the % free of DHEA were evaporated to dryness, and the residues were incubated with the antiserum containing pepsin treated human immune serum globulin and bovine serum albumin at 4 Ž overnight. Ammonium sulfate was used to separate free from bound DHEA. The accuracy, precision and specificity were satisfactory. The sensitivity was 300pg per sample. The blank value could not be differentiated from zero. Although the antiserum reacted with the other 3ƒÀ-OH-ƒ 5-steroids as well as with DHEA, complete separation of DHEA from the other 3ƒÀ-OH-ƒ 5-steroids was achieved chromatographically. Serum DHEA levels in normal subjects and their responses to ACTH stimulation and dexamethasone suppression were evaluated by using the radioimmunoassay. Dehydroepiandrosterone (DHEA) and its sulfate ester, dehydroepiandrosterone sulfate (DHEAS) are the major adrenal C19 steroids in man. Concerning DHEAS many extensive studies have been reported using Zimmerman reaction and gas-liquid chromatography. Only a few papers, however, have appeared concerning DHEA. One of the causes of minute amount of information about DHEA was the complicated assay procedure such as double isotope derivative technique. Recently the advent of a sensitive radioimmunoassay has made it possible to measure steroid hormones easily. Nieschlag et al. (1972) published a radioimmunoassay for DHEA and DHEAS and we described a radioimmunoassay for serum DHEAS in a Received for publication December 15, 1973. *Present address: The Third Department of Internal Medicine, University of Kyushu Faculty of Medicine, Fukuoka. previous paper (Sekihara et al. 1972). We further developed a radioimmunoassay for serum DHEA by using the same antiserum. The present paper describes the results. Materials Isotope, chemicals and paper and Methods DHEA-7ƒ 3H with a specific activity of 20.8 Ci/mM was obtained from the New England Nuclear Corp. The isotope was purified by thin layer chromatography with the system, benzene: ethyl acetate (2: 1). DHEA was obtained from Sigma Chemical Co. The pepsin treated human immune serum globulin (Gamma-Venin) was obtained from Hoechst Japan Co. Other materials and their sources were: bovine serum albumin (BSA) (Armour Pharmaceutical Co.), methylene chloride, methanol and other solvents (Wako Pure Chemical Industries) and paper (Toyo, No.51). Anti DHEA-antiserum DHEA-17-oxime-BSA conjugate was prepared by

SEKIHARA et al. Endocrinol.Japon. April 1974 the method of Erlanger et al. (1957). Details of the procedures of immunization were described in a previous paper (Sekihara et al. 1972). Table 1. Accuracy and precision of the assay Extraction and purification procedure After the addition of 3000 dpm of DHEA-7ƒ -3H for recovery, 0.2 to 1.0ml of serum was added to 1ml of water, and extracted with 10ml of methylene chloride. The extract was evaporated to dryness under a stream of nitrogen. The residue was subjected to paper chromatography using the system, petroleum ether: methanol: water (500: 350: 150). A part of DHEA was eluted with 2ml of methanol. 0.2ml of the eluate was used for assay, and 1ml to calculate recovery. Inter-assay precision Radioimmunoassay 10000 dpm of DHEA-7ƒ -3H was added to 0.2ml of the eluate, and 0, 10, 25, 50, 100, 200, 300, 500, 750 and 1000pg of DHEA standard in 0.2ml of methanol. The respective solutions were evaporated to dryness under a stream of nitrogen. 0.3ml of the antiserum diluted 1: 6000 with a borate buffer (0.05 M, ph 7.8) and containing 0.025% pepsin treated human immune serum globulin and 0.075%BSA were added, and the mixture was incubated at 4 Ž overnight. Then, 0.3ml of saturated ammonium sulfate was added, mixed on a vortex mixer, allowed to stand for 10min, and centrifuged for 15min at 3000 rpm. 0.3ml of the supernatant was transferred to a counting vial. 10ml of toluene scintillator (4g PPO and 100mg POPOP in 1000ml toluene) was added. The radioactivity was counted in the Aloka model LSC-601 liquid scintillation spectrometer. Standard Results and Discussion curve The antiserum gave the optimal standard curve between 0 and 1000pg of DHEA in a dilution of 1: 6000. Recovery The mean recovery of DHEA-7ƒ 3H added to samples was 74.1 }7.5%(N=100). Accuracy (Table 1) Accuracy was evaluated by adding 1.0 and 2.0ng of DHEA respectively to 0.2ml of serum. The recoveries of DHEA added were 104 }5 and 101 }6%, respectively. Sample 1. 0.2ml of serum from a 24 year old woman with Addison's disease Sample 2. 0.2ml of serum from a 32 year old, normal man CV Coefficient of variation Precision (Table 1) Intra-assay precision was examined. The coefficients of variation for analysis of 1.0 and 2.0ng of DHEA added to 0.2ml of serum were 4.8 and 5.4%. The coefficient of variation for serum sample from a normal male was 4.3%. The serum sample was measured on separate days, and variations between assays were examined. The coefficient of variation was 5.6%. Sensitivity The minimal detectable amount of DHEA was 300pg per sample. (N= 5, P < 0.05) Specificity The cross-reactivity of the antiserum was described in a previous paper (Sekihara et al. 1972). 3ƒÀ-OH-ƒ 5-steroids such as pregnenolone, 17-OH-pregnenolone and androstenediol showed marked interference. Paper chromatography gave complete separation of DHEA from the other 3ƒÀ-OH-ƒ 5-steroids. The blank values could not be differentiated from zero.

Vol.21, No.2 RIA OF DHEA WITH PAPER CHROMATOGRAPHY Table 2. Serum DHEA and DHEAS levels in normal men and women (M }SD)* at different ages in both sexes are shown in Table 2. The serum DHEA levels are low before puberty, reach the highest levels between the ages of high teens and twenties and decline progressively with the advancing age. The serum DHEAS levels obtained by radioimmunoassay are also presented for comparison. *Blood samples were obtained between 9 and 10a.m. **Values are expressed as Đg of free DHEA Serum DHEA levels in normal subjects (Table 2). The serum DHEA levels in normal subjects. Correlation between serum DHEA and DHEAS The correlation between serum DHEA and DHEAS in normal subjects was evaluated. DHEA (Male)= 0.132+0.00311 EDHEAS (r=0.69) (N=67) DHEA (Female)= 0.205+0.00280 EDHEAS (r= 0.52) (N=70) The coefficients of correlation are 0.69 for males and 0.52 for females. Response of serum DHEA to ACTH stimulation (Fig. 1) ACTH stimulation was performed by injecting 0.25mg of Ĉ1-24-acth intravenously every hour, 5 times in total to maintain the maximum stimulation for 5hr.The response Fig. 1 The response of serum DHEA, DHEAS and cortisol to ACTH stimulation for 5hr.

SEKIHARA et al. Endocrinel. April 1974 Japon. Fig. 2 The response of serum DHEA, DHEAS and cortisol to dexamethasone suppression during 4 days. of serum DHEA is shown in Figure 1. The serum DHEA was elevated within 30min by the initial stimulation and reached the maximum level by the second or the third stimulation. The maximum level could be maintained by the following stimulations. The responses of the serum DHEAS and cortisol are also presented for comparison. The serum DHEAS did not vary 30min and 1hr after the first stimulation, started to increase by the second or the third stimulation, and showed gradual increases by the following stimulations. The response of the serum cortisol showed the same response pattern as the DHEA. Response of serum DHEA to dexamethasone suppression (Fig. 2) Dexamethasone suppression was performed by giving 0.5mg of dexamethasone orally every six hr during 4 days. Samples were collected at 9 a.m. on each of the rst, second, third, fourth and fifth day. As is shown in Figure 2, the serum DHEA declined gradually. The responses of the serum DHEAS and cortisol to dexamethasone suppression are presented for comparison. The serum DHEAS also declined gradually. A good correlation was found between serum DHEA and DHEAS during dexamethasone suppression. DHEA= 0.017+0.00375 EDHEAS (r=0.82) (N= 30) In contrast to DHEA and DHEAS, the serum cortisol declined rapidly colse to the zero level on the second day of suppression and remained at the level by the following suppressions. Conclusion From the above-mentioned results, the radioimmunoassay for serum DHEA described in the present paper seems to be accurate, precise, sensitive and specific. The present method is suitable for routine use. For further simplification of the procedure, the omission of paper chromatography is a problem to be dissolved. Acknowledgements We are grateful to Prof. Kinori Kosaka, Director of the Third Department of Internal Medicine, University of Tokyo Faculty of Medicine, for his advice

Vol. 21, No. 2 RIA OF DHEA WITH PAPER CHROMATOGRAPHY and encouragement throughout these studies. References Erlanger, B. F., F. Borek, S. H. Bieser and S. Lieberman (1957). J. Biol. Chem. 228, 713. Nieschlag, E., D. L. Loriaux and M. B. Lipsett (1972). Steroids 19, 669. Sekihara, H., N. Ohsawa and H. Ibayashi (1972). Ibid. 20, 813.