Determination of dehydroepiandrosterone sulfate in human plasma by competitive protein binding

CLINICA

CHIMICA

77

ACTA

DETERMINATION

OF DEHYDROEPIANDROSTERONE

HUMAN PLASMA BY COMPETITIVE

R. HAMPL Research

AND

I.,.

March

SULFATE

IN

BINDING

STARKA

Institute of Endocrinology,

(Received

PROTEIN

Prague (Czechoslovakia)

XI, 1971)

SUMMARY

A competitive protein-binding method for the determination of dehydroepiandrosterone sulfate in human plasma is described. After deproteinisation and solvolysis the plasma extract is chromatographed on paper and dehydroepiandrosterone is reduced with sodium borohydride. The resulting 5-androstene-3@,17/3-diol is determined by competitive protein-binding, using displacement of [3H]testosterone from a complex with sexual hormone-binding globulin from late pregnancy human plasma. Free and bound fractions are separated on dextran-coated charcoal. The criteria of accuracy, sensitivity, precision and specificity are evaluated and the results are compared with gas-chromatographic determinations. The mean level of peripheral dehydroepiandrosterone was found to be 92.9 & 34.5 ,ug/roo ml of male plasma and 74.4 f 31.6 ,ug/roo ml of female plasma.

INTRODUCTION

For the estimation of dehydroepiandrosterone (3&hydroxy-5-androsten-r7one, DHA) levels in normal human plasma several methods were elaborated based either on photometry after suitable reaction (e.g., l.c. in ref. I) or on gas chromatographyz-*_ Gas-chromatographic methods are reliable and except for very low levels of steroids, are very practical. However, the recent deveIopment of saturation analysis5 and the excellent results of testosterone and 4-androstene-3,r7-dione estimations on sub-nanogram scale by competitive protein-binding methods suggested the working out of a sensitive method for dehydroepiandrosterone determination in plasma that would require no gas-chromatographic equipment. The method utilizes the selective binding of C,,- and &-steroids containing r7@-hydroxygroup by sexual hormone-binding @-globulin (SHBG) from human pregnancy plasmas. It was established in competition studies with this protein that 5-androstene-3#I,r7&diol as well as its gee-epimer approximate in binding affinity to SHBG to that of testosterone and that they can displace testosterone from the steroidprotein complex6-7. A method based on displacement of [3H]testosterone from the CZi=.CkiPvs. Acta,

34 (1971)

77-83

HAMPL, STARKA

78 protein binding by 5-androstene-3/?,17,%diol formed by sodium borohydride reduction is described.

from dehydroepiandrosterone

EXPERIMENTAL Solvents

of analytical

grade were redistilled

before use.

Steroids. Radioactive steroids were purchased from Radiochemical Centre, Amersham, England. [1,2-~Hl testosterone (spec. activity 41.4 C/mmole) was purified by chromatography on Whatman No. I paper in the system cyclohexane-dioxanemethanol-water IOO: 25 : IOO: IO (v/v) for 20 h. [7a-3H]3/?-hydroxy-5-androsten-r7one 3-sulfate (spec. activity 605 mC/mmole) was purified by thin-layer chromatography on silica gel (Kieselgel G, Merck A.G.) in the system 1% ammonia-ethanol ~:g. The purity of non-radioactive steroids (Koch and Light Laboratories Ltd.) was checked by chromatography. Paper chvomatogvaphy was carried out on Whatman No. I paper pre-washed with hot methanol in Soxhlet extractor. Glassware was rinsed with acetone and ether before use. Radioactivity was determined in Nuclear Chicago 6860 Mark I liquid scintillation spectrometer. Scintillation fluid contained 5 g PPO and 0.3 g dimethyl-POPOP in I 1 of toluene. Aqueous solutions were measured in mixture containing additional 2% of methanol or in scintillation fluid consisting of IO ml of NE,,, (Nuclear Enterprises, Edinburgh) to which 4% Cab-o-sil (Packard Instr.) was added to form a thixotropic gel. Preparation of the complex of [3H]testosterone with SHBGfrompregnancy plasma. Late human pregnancy plasma from third trimester was diluted I : g with 0.05 M borate buffer pH 7.8. To I ml of the diluted plasma 0.1 ng of purified [3H]testosterone (approx. 8000 counts/min) were addede. De&an-coated charcoal was prepared tran (Dextran 70, Roth OHG, Karlsruhe) 0.125-0.40 mesh) IO min before use.

by mixing and 0.5%

equal volumes of 0.05% dexsuspension of charcoal (Hiag,

PROCEDURE Deproteinisation and solvolysis To 0.5 ml of heparinised plasma in 5 ml glass-stoppered centrifuge tube approx. IOOO countsjmin of [7cr-3H]dehydroepiandrosterone sulfate in 0.05 ml of redistilled water and 0.03 ml of 0.5 N sodium hydroxide were added. The mixture was pre-extracted with 2 ml of diethyl ether and the organic phase was removed by suction. To the plasma I ml of acetone was added, the mixture was stirred thoroughly and left overnight at o-2’. Precipitated proteins were removed by centrifugation at 2000 Y g, the supernatant was decanted into another glass-stoppered 15 ml tube. Precipitate was then resuspended in I ml of mixture of acetone-water 4: I (v/v), stirred and centrifuged again and the supernatant was added to the first portion. Acetone was removed by a gentle stream of nitrogen. To the aqueous residue I ml of 0.5% sulphuric acid in saturated NaCl solution was added and the mixture was vigorously extracted with ethyl acetate (6 ml). After 24 h standing at 37O, ethyl acetate extract was washedonce with 0.5 ml of 5 N sodium hydroxide and twice with water. The aqueous phases were removed by suction and the solvent was evaporated to dryness in vacua. Clin. Chim.

Acta,

34 (1971)

77-83

DHA SULFATE

79

IN PLASMA

Chromatography

The samples were then chromatographed on paper in the system cyclohexanetoluene-methanol-water 270 : 30: 240 : 60 (v/v) after overnight equilibration. Chromatography was carried out on 3.5-cm wide paper strips, separated one from other by cutting a strip of I mm between them. Dehydroepiandrosterone as a reference substance was chromatographed simultaneously on one strip, another strip was left free for the determination of blank value. After chromatography the 4-cm area corresponding to the reference spot of DHA, localised by the Zimmermann reaction, was cut off and eluted with the mixture of chloroform-methanol g5 : 5 (v/v) (twice 1.5 ml and I ml) during 2 h. The collected eluate was filtered through small bunches of cottonwool (prewashed with hot methanol) into r5-ml glass-stoppered tubes and evaporated to dryness in zlaczto.

Borohydride

reductiom

The samples were resolved in 0.2 ml of methanolic sodium borohydride (IO mg NaBH, in IO ml of methanol, prepared immediately before use), the tubes were stoppered and after IO min standing in an ice box at oD were left overnight at room temperature. Then 0.1 ml of 10% acetic acid and 0.5 ml of water were added and the mixture was extracted with 7 ml of diethyl ether. The aqueous phase was removed by suction and ether solution was washed once with I ml of 0.05 N sodium hydroxide and twice with 0.5 ml of water. The extract was dried up by a pinch of anhydrous sodium sulfate and filtered quantitatively through cottonwool into calibrated tubes. An aliquot (115) was taken for competitive protein-binding assay and the remaining portion (4/5) was put into a counting vial, where it was evaporated to dryness for recovery determination.

Competitive

protein-binding

assay

Ether extract (I/5) of each sample and the extract of the blank were evaporated in 5-ml glass tubes. Simultaneously, a standard series for calibration curve, consisting of o, IO, 30, 50, 70 and IOOng of 5-androstene-3j3,17/?-diol evaporated in 5-ml tubes, was prepared. To each sample, blank and standards, I ml of diluted plasma-[3H]testosterone complex was added and the mixture was incubated for 15 min at room temperature under stirring every 5 min. Then, I ml of dextran-charcoal mixture was pipetted into each tube and the samples were incubated in the same way for additional 15 min. The content of the tubes was then placed into conical tubes and centrifuged for 6 min at 2000 x g. One ml of the supernatant was then transferred into a counting vial and II ml of scintillation fluid containing 2% of methanol was added. The radioactivity was measured after 8 h.

Calibration

cwve

and calculation

The radioactivity found in standard samples was plotted against the amount of 5-androstene-3/?,r7/Ldiol added and from the resulting curve the amount of DHA assessed as 5-androstene-3o,r7B-diol was obtained. From each value the blank was substracted and results were corrected for methodical losses.

Clin.Chim. Acta, 34 (1971) 77-83

HAMPL, STARKA

80

RESULTS

Thecriteriaof accuracy,sensitivity,precisionandspecificityaswellasthe methodical losses during the whole procedure and each purification step were determined. In order to decrease the blank, different solvents were tested for their suitability for extraction and elution. The values of the DHA sulfate level were compared with those obtained by independent gas-chromatographic method. Methodical losses of individual analytical steps were assessed in IO analyses. Final recovery of the tracer was 45.6 + 5.2%. Losses during solvolysis and extraction averaged 34.4%, during the chromatographic step 10.2% and during borohydride treatment 9.8%. Accuracy and sensitivity. To the duplicate samples of normal pooled plasma (0.3 ml each) containing 137.6 ,ug of DHA sulfate per IOO ml, increasing amounts of DHA sulfate were added. The recovery ranged from 92-118% (see Table I). The sensitivity of the method was about 0.5-1 ,ug/roo ml. TABLE I RECOVERY

OF

DHA

ADDED

Amount added to the samfile analysed (~cg DHA imll

SULFATE

TO

THE

NORMAL

POOLED

Dehydroepiandrostevone

0 0.10 0.25 0.50

expected (pslml)

found (pglml)

1.48 1.63 1.88

1.38 1.49 1.65 1.84

PLASMA

Recovery iuglml

%

0.12

118

0.27

108

92

0.46

Presicion. In IO analyses of pooled normal plasma the mean content of DHA sulfate was found to be 66.0 f 6.8 ,ug/roo ml. The coefficient of variation was 10.3%. Specijkity. As demonstrated elsewhere St&,the specificity of CPB methodsdepends upon the selectivity of the B-globulin used, the binding affinity to which depends on characteristic functional groups of the related steroids. Since several steroids occurring in human plasma are reduced by borohydride to r7/3-hydroxysteroids, which are bound to SHBG, their interference in the method was examined. Addition of steroids in concentrations near physiological levels did not practically interfere in the determination of DHA (Table II). The accompanying steroids are separated by paper chromatography, as shown by the RF values listed in Table III. TABLE INFLUENCE OF

II OF

ADDITION

OF

FIVE

c,,

STERODIS

TO

THE

PLASMA

DHA

Steroid added

,wlml

No

Androsterone sulfate Etiocholanolone sulfate 5-Androstene-3/?,17,%diol Testosterone 5a-Androstane-3.17.dione Clin. Chim. Acta. 34

(1971)77-83

2.00 2.00

0.10 0.01 0.01

Found DHA (pglml) 1.37 I.39 I.35 1.38 I.39 1.36

SAMPLE

ON

THE

DETERMINATION

DHA SULFATE IN PLASMA TABLE RF

81

III

VALUES

OF

SOME

C,,

Paper chromatography

STEROIDS

OCCURRING

IN HUMAN

PLASMA

in the system cyclohexane-toluene-methanol-water,

Steroid

RF

Dehydroepiandrosterone Androsterone Etiocholanolone 5-Androstene-3B,I7/?-diol Testosterone 4-Androstene-3,r7-dione 5u-Androstane-3,17-dione

0.43 0.64 0.56 0.14 0.29 0.60 0.78

270: 30: 240: 60 (v/v).

The specificity of the method was ascertained further by identical results of DHA analysis obtained after additional paper chromatography in the system cyclohexanedioxane-methanol-water IOO: 25 : IOO : IO after borohydride reduction and correction for losses during the procedure by recoveries of tracer [aH]DHA. Blank. The average blank value of 28 analyses was 3.3 ng, range 1.2 to 5.6 ng per sample. When different solvents were tested for their suitability for extraction and elution procedures, it was found that the blank increased in the sequence diethyl ether-chloroform-methanol-benzene-dichloromethane. The position of dichloromethane differs from that reported by Murphy9, perhaps because of different provenience of the solvent. When thin-layer chromatography or column chromatography on prewashed alumina were employed instead of paper chromatography, the blank values rose beyond the analytical range. Thus, diethyl ether was used for the extraction procedure and less volatile chloroform with methanol for elution. Com$arison of the CPB method with gas chromatography. The values of peripheral plasma levels of DHA sulfate in 8 healthy subjects measured by CPB method were compared with those obtained by the gas-chromatographic method of DeMoor and Heynslo. The results are shown in Table IV. Normal levels of dehydroepiandrosterone sadfate reported in the literature and those found by means of the present method in plasma of 14 men and 14 women are listed in Table V. TABLE THE

IV

ESTIMATION

BINDING

OF

TECHNIQUE

PLASMA AND

BY

DEHYDROEPIANDROSTERONE GAS

SULFATE

Subject

Sex

Competitive protein binding (,ug DHA /roe ml)

Gas chromatografihy (,ug DHA 1x00 ml)

M.T. M.K. I.P. J.C. V.P. O.B. R.S. L.N. Mean

F F F F F M M M -

43 76 98 143 157 I35 67

46 72 94 I35 90 166 139 71

101

IO2

82

BY

COMPETITIVE

PROTEIN-

CHROMATOGRAPHY

Clin. Chim. Acta, 34

(1971)

77-83

HAMPL, STARKA

82 TABLE NORMAL

V LEVELS

OF PLASMATIC

DEHYDROEPIANDROSTERONE

Author

Method

Hudson and Oertell

spectrometry

Wang et a1.$ Kumari et aZ.* Present paper * Expressed

Group (n)

men women men gas chromatography women gas chromatography men women gas chromat. (elect. Capt.) women competitive men protein binding women

Vihko2

SULFATE

(26)

(I 8) (62) (54) (41)

(51) (IO)

(14) (14)

DHA sulfihate* Mean & S.D.

(figlIo0 Range

130 97 100 60

(20-253) (26-197) (19-323)

122

(12-392) (10-248) (22-68)

107 33 zt 13.8 93 i 34.5 74 * 31.6

ml)

(0-188)

(56-157) (33-143)

-

as free DHA.

DISCUSSION

The relative binding capacity of 5-androstene-$?,r7!-diol was shown to be very high6~7+‘~11, however, this fact has not been utilized for DHA determination by CPB analysis until now. The present method uses the experiences for sample preparation, especially deproteinisation, solvolysis and extraction, described by Aakvaag and Fylling12 for the gas-chromatographic determination of DHA. For separation of DHA from other interfering steroids, paper chromatography is used followed by sodium borohydride reduction. It was found, that no additional purification of the resulting 5-androstene-$?,rT/?-diol is necessary. 5-Androstene-@,r7p-diol is assessed by modification of the CPB method for testosterone determinations. [SH]Testosterone is displaced from the complex with SHBG of human late pregnancy plasma and the separation of the free and bound [3H]testosterone is achieved by adsorption on dextran-coated charcoal. The criteria of accuracy, precision, sensitivity and specificity are satisfactory and comparable to those of the best gas-chromatographic methods (refs. 2, 4, 12, 13).Its practicability makes the method suitable to be employed for routine use also in laboratories without gas-chromatographic equipment. The method has been worked out for normal human plasma, but its sensitivity allows the estimation in plasma of very low DHA content. It should be emphasized that the specificity of the method has been established for human plasma samples. When plasma of experimental animals is to be analysed, the specificity is to be reevaluated with respect to the steroid spectra in the plasma of the animal. Sodium borohydride reduction has proved to be a simple tool for transforming a steroid with low affinity to SHBG to a compound with a very high one. Similarly, other r7-ketosteroids important for steroid diagnostic-as 4-androstene-x,17-dione, androsterone, adrenosterone, 7-ketodehydroepiandrosterone-could be treated with borohydride to obtain derivatives suitable for protein-binding analyses. ACKNOWLEDGEMENT

We are indebted to Mrs. I. Gregorova, Dipl. Ing., CSc., Laboratory of Endocrinology and Metabolism, Medical Faculty of Charles University, for gas-chromatographic analyses. Cl&..Chim. Acta, 34

(1971)

77-83

DHA SULFATE IN PLASMA

83

REFERENCES I 2 3 4

5 6 7 8 g IO II

12 13

B. HUDSON AND G. W. OERTEL, Anal. Biochem., z (1961) 248 R. VIHKO, Acta Endocrinol., Suppl. Iog (1966). D.Y. WANG,R.D.BULBROOK,B.S.THOMASANDM.FRIEDMAN, J.Endocrinol.,42(1g68)567. G. L. KUMARI, W. P. COLLINSAND I.F. SOMMERVILLE,J.Chromatog., 4~ (1969) 22. B. E. P. MURPHY, Recent Pvogr. Hormone Res., 25 (1969)563. T. KATO AND R. HORTON, J.CZin. Endocrinol., 28 (1968) 1160. W. HEYNS AND P. DEMOOR, Ann. Endocvinol. (Paris), 31 (1970)811. R. HORTON, T. KATO AND R. SHERINS,Steroids, IO (1967)245. B. E. P. MURPHY, Acta Endocrinol., Suppl. 147 (1970)37. P. DEMOOR AND W. HEYNS, Excerpta Med., Intern. Congr. Ser., IOI (1965)54. A. VERMEULEN AND L. VERDONCK, ActaEndocrinol., Suppl. 147(1g7o)239. A. AAKVAAG AND P. FYLLING,Acta Endocrinol., 57 (1968) 447. 0. JKNNE, R.VIHKO, J. SJGVALL AND K.SJ~VALL,C~&Z.Chim. Acta, 23(196g) 405. C&z. Chim. Acta, 34 (1971) 77-83