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Assay of plasma dehydroepiandrosterone and its sulphate by competitive protein binding
Clinica Chimica Acta, 43 (1973) 295-303 © Elsevier Scientific Publishing Company, A m s t e r d a m - P r i n t e d in The N e t h e r l a n d s
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ASSAY OF PLASMA D E H Y D R O E P I A N D R O S T E R O N E AND ITS SULPHATE BY COMPETITIVE P R O T E I N BINDING
C. M. A N D R E AND V. H. T. J A M E S
Steroid Research Unit, Department of Chemical Pathology, St. Mary's Hospital Medical School, London, W.2. (U.K.) (Received J u l y 26, I972)
SUMMARY
A competitive protein-binding method for the assay of dehydroepiandrosterone and its sulphate in plasma is described. After extraction from plasma, and solvolysis in the case of dehydroepiandrosterone sulphate, dehydroepiandrosterone is purified by paper chromatography and transformed to 5-androstene-3fl, I7fl-diol by reduction with sodium borohydride. The 5-androstene-3fl, I7fl-diol so formed is purified by paper chromatography prior to assay. The assay end-point uses the binding property of testosterone-binding globulin, which binds with high affinity those steroids with an unhindered I7fl-hydroxyl group. Tile reliability criteria of the method in terms of precision, accuracy, sensitivity and specificity have been evaluated.
INTRODUCTION
During the last few years the principle of competitive protein binding has been successfully applied to the assay of plasma androgens 1. All the methods developed for the assay of androgens by this technique use, as an assay end-point, the binding property of testosterone-binding globulin which binds with high affinity steroids with an unhindered I7fl-hydroxyl group S. In order to assay androgens like androstenedione or dehydroepiandrosterone, which are i7-oxosteroids, it is necessary firstly to separate the I7-Oxosteroid from the I7fl-hydroxysteroids in the sample by chromatography and then transform the I7-Oxosteroid to the corresponding I7fl-hydroxysteroid by reduction. Dehydroepiandrosterone can be readily transformed to 5-androstene-3fl, I7fldiol which is known to bind with high affinity to testosterone-binding globulin 3. Advantage of these facts was taken to develop the present method. Reliable methods, e.g. gas-liquid chromatography or the double isotope dilution derivative technique, have been developed for the assay of dehydroepiandrosterone*, 5 or its sulphate 6. However, the present study was undertaken to investigate the possibility of using the simpler technique of competitive protein binding as a useful alter-
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native to the existing methods. Recently two competitive proteii, binding methods for the assay of plasma dehydroepiandrosterone 7 and its sulphateT, ~ have been published. The present method is similar to the method of Halnpl and Starka ~ but differs in several practical aspects, summarised below, which are incorporated in order to improve the reliability and specificity of the assay. Sufficient tritiated dehydroepiandrosterone is added to tile plasma extracts to enable accurate localisation by scanning the radioactivity on each strip of the paper chroniatogram. This is desirable not only because it results in more reproducible recoveries but also reduces the likelihood of contaminating the eluate from the chromatogram with adjacent substances which may also interfere in the protein-binding part of the assay. The specificity may thus be improved 9. A second chromatogram is essential after the reduction of dehydroepiandrosterone to 5-androstene-3/3, I7/5-diol with sodium borohydride as it is a common experience that the reduction of nanogram quantities of steroids with this technique can be erratic. It is essential therefore to separate the reduction products by chromatography to isolate the generated 5-androstene-3/3, I7/3-diol otherwise it is impossible to assess methodological losses accurately. In the protein-binding part of the assay the present method makes use of a homogeneous system, i.e. the tracer is chemically identical to the test compound apart from the radioactive isotope incorporated in the tracer molecule. In a heterogeneous system as used by Hampl and Starka a tracer, namely tritiated testosterone, of a different reaction energy to the test compound is reacted with the binding protein. In this type of system the interaction between tracer, unlabelled hormone and binding protein is more complex and the effect on the final equilibrium state of the reactants for a given assay condition is unpredictable ~° even more so should other steroidal or non-steroidal substances which partially interfere with the binding reactions be present in the assay mixture. For these reasons the simpler homogeneous s?stem is preferable as errors are least likely to arise. A further point to be considered is the different techniques used in the separation of bound from unbound hormone after equilibration with the binding protein. None of the methods currently available absolutely separate bound from unbound hormone. Hence the method of choice is the most precise and there i~; evidence to indicate that ammonium sulphate protein precipitation, as used here, is more precise than the dextran-coated charcoal methodt'. In addition the present method has also been modified for the assay of unconjugated dehydroepiandrosterone. MATERIALS AND REAGENTS
Dichloromethane, methanol and ethyl acetate are redistilled before use. Ethanol, benzene and petroleum ether (IOO°--I20 °) a r e purified as described by Townsend and James '~ except that in addition, benzene and petroleum ether are prewashed twice with 3o ml of concentrated sulphuric acid per litre. Aluminium oxide "CAMAG'" MFC lOO-24o mesh alkaline Brockmann activity = I (Hopkin and Williams Ltd., Essex, England) is purified according to Mayes and Nugent a and activated by heating at IiO°-i2o ° for 3 h and stored in a sealed container.
ASSAY OF PLASMA D H A
AND ITS SULPHATE
297
Paper for chromatography, W h a t m a n No. 2 is prepared by cutting lanes 2-cm wide and 4o-cm long with a o.5-cm gap between lanes and washed by descending chromatography in methanol-dichloromethane 3 :I for at least 48 h. Dehydroepiandrosterone sodiunl sulphate and 5-androstene-3/5, I7/%dioI are used as supplied by Steraloids, Inc. E7-3H~ Dehydroepiandrosterone specific activity 25.1 C/mmole (New England Nuclear, Boston, U.S.A.) is purified by paper chromatography on pre-washed W h a t m a n No. 2 chromatography paper in the B3 system of Bush 13 (petroleum ether-benzene-methanol-water 33:17:4o:1o ) for 3 h at 24 ° and stored in benzene at 4 ° . The working standard is prepared from this stock solution by dilution with ethanol. E7-~Hl5-Androstene-3/~, i7/~-diol is prepared b y reducing E7-3HJdehydro epiandrosterone with sodium borohydride, as described under borohydride reduction, and purified by chromatography on pre-washed W h a t m a n No. 2 paper in the B~ system of Bush for 4 h at 24 °. The ~7-~Hl5-androstene-3/~, I7/~-diol is located with a strip scanner, eluted with dichloromethane-methanol 3:2 and stored in 5 % ethanol in benzene at 4 ° . The working standard is prepared from this stock solution by dilution with ethanol. Third trimester pregnancy plasma is pooled from normal donors and stored in o.5-ml aliquots at --20 °. Under these conditions it is stable for at least 18 months. The scintillation fluid is prepared by adding 2,5-bis-E2-(5-t-butylbenzoxaloyl)lthiophene (BBOT), 6 g/litre (Ciba), naphthalene 80 g/litre to a mixture of 2-methoxyethanol and toluene 2 : 3. Disposable glass specimen tubes, glass capillaries and disposable Pasteur pipettes are washed in distilled water, ethanol and dried. All other glassware is washed in a detergent, RBS 25 (Chemical Concentrates Limited, London), in an ultrasonic cleaning bath, thoroughly rinsed with tap water and distilled water and dried in an oven at 12o ° . Glass wool, for plugging columns, is washed in methanol and dichloromethane and dried. Blood samples are collected in plastic tubes containing lithium heparin. The plasma is separated immediately and stored at --20 ° until analysed. METHOD
Extraction and solvolysis To io-ml glass-stoppered tubes containing 2 ml of o.I N sulphuric acid saturated with sodium chloride 14, o.i ml plasma samples are added. After mixing, 5 ml of ethyl acetate is added to each tube. The tubes are then shaken in a mechanical shaker for 15 min and afterwards centrifuged at 800 g for 5 rain at room temperature. The supernatants are transferred with Pasteur pipettes to I5-ml glass-stoppered tubes containing 25000 disint/min of E7-3Hldehydroepiandrosterone in o.I ml ethanol. The same amount of radioactive tracer is added in triplicate to counting vials to calculate the recovery through the method. The samples are again extracted with 5 ml ethyl acetate as before. The appropriate extracts are pooled, mixed and incubated at 37 ° for 22-24 h. The cooled extracts are transferred to 5o-ml separating funnels containing 2 ml of 1%, (w/v) sodium carbonate. The tubes are rinsed with i ml of ethyl acetate and the washings transferred to the appropriate separating funnel. After shaking for I rain the phases are allowed to separate and the aqueous layer is discarded. The ex-
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tracts are then washed with 2-ml volumes of distilled water until the washings are neutral. The extracts are transferred to conical glass tubes and evaporated to dryness under a stream of nitrogen, in a water b a t h at 4 o°.
First chromatography The residues are quantitatively transferred to pre-washed W h a t m a n No. 2 c h r o m a t o g r a p h y paper with diehloromethane-methanol 3:2. After equilibration the c h r o m a t o g r a m is developed in the B~ system of BuslO a for 3 h at 24 ~. The radioactive dehydroepiandrosterone areas are located with a strip scanner and the appropriate sections cut out. Microcolumns of alumina, 3-cm long, are prepared in disposable Pasteur pipettes (0.6 × 15 cm) plugged with glass wool% and the sections of paper to be eluted (approx. 4.5 × 2 cm) are folded longitudinally and placed on top. The columns and sections of paper are washed twice with dichloromethane by filling the pipettes and allowing them to drain to waste, and eluted with approximately 5 ml of dichloromethanol 3 : 2. The eluates are collected in 6-ml glass-stoppered tubes and evaporated to dryness under nitrogen.
Borohydride reduction To each tube 0.2 ml of a freshly prepared 0.25% (w/v) methanolic solution of sodium borohydride is added and mixed. After 5 min at room temperature o. 5 ml of distilled water is added and the reduction products extracted once by adding 4.5 ml of dichloromethane to the tube and shaking for 3 min. The extracts are washed once with o.5 ml of distilled water and evaporated to dryness under nitrogen.
Second chromatography ]'be residues are chromatographed in the B3 system as before except that the c h r o m a t o g r a p h y time is increased to 4 tl. The radioactive 5-androstene-3fl, I7fl-diol areas are located with a strip scanner and the appropriate sections (approx 4 >: 2 cm) are cut out and eluted by soaking twice in 2 ml of dichloromethane-methanol 3 : 2 for IO rain each time in I2 7. IOO-inm test tubes. The eluates are collected in io x 5 om m glass specimen tubes and evaporated to dryness under nitrogen. Microeolumns of alumina are prepared as described abow~ and the columns washed twice with dichloromethane immediately before use by filling the pipettes and allowing t h e m to drain to waste. The residues are transferred to the alumina colunms, with glass capillary tubes, in small volumes of dichloromethane (6 drops from a Pasteur pipette) three times and once with 2. 5 % methanol in dichloronmthane (4 drops from a Pasteur pipette). The colunms are washed twice with dichloromethane by filling the Pasteur pipettes and allowing them to drain to waste, and eluted with 2.5 nd of 2.5% methanol in dichloromethane which is collected in IO x 5o-mm glass specimen tubes. The eluates are evaporated to dryness under nitrogen and redissolved in I ml of ethanol.
Competitive protein binding Standards are prepared in triplicate by adding o, 0.25, o.5, i.o, 1.5 and 2.5 ng of 5-androstene-3fl, I7fl-diol in ethanol to IOX5o-nlm glass specimen tubes and
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I6OOO disint./min of [7-"H]5-androstene-3fl, I7fl-diol in o.I ml ethanol to each tube. After mixing the ethanol is evaporated to dryness under nitrogen. The same amount of radioactive 5-androstene-3fl, I7fl-diol is added in triplicate to counting vials to calculate the proportion of free radioactive 5-androstene-3fl, I7fl-diol after the ammonium sulphate precipitation of testosterone-binding globulin. From each test sample an aliquot (0.2 ml) is taken and added to a counting vial to calculate the recovery. Duplicate aliquots expected to contain not more than 2.0 ng of 5-androstene-3fl, I7fl-diol (usually between IO and 5 °/~1) are then taken with a Hamilton syringe and added to IO × 5o-mm glass specimen tubes containing i6ooo disint./min of [7-aHl5-androstene-3fl, ITfl-diol in o.I inl ethanol. After mixing the ethanol is evaporated to dryness under nitrogen. To each standard and test sample is added 0.3 ml of a freshly prepared 2% solution of pregnancy plasma in distilled water. After mixing thoroughly but gently to avoid frothing, the tubes are covered with parafilm and left overnight at 4 °.
Ammonium sulphate protein precipitation To each tube o. 3 ml of a cold saturated solution of ammonium sulphate, p H 6, is added, mixing immediately and thoroughly but avoiding frothing. The tubes are incubated at 4 ° for 5 rain and centrifuged at IOOOg for IO rain at 4 °. From each supernatant 0.2 ml is taken and added to a counting vial.
Note The ammonium sulphate protein precipitation step is carried out on batches of eighteen tubes to avoid prolonged contact with ammonium sulphate 3.
Radioactive counting To avoid large differences in quenching between the various counting vials in the batch, ammonium sulphate is added to the standard and recovery vials so that finally each vial contains 0.2 inl of 50% (w/v) solution of ammonium sulphate. To each counting vial io ml of the BBOT scintillation fluid is added and the vial contents are thoroughly mixed before counting in a liquid scintillation spectrometer to a theoretical error of 20/0.
Calculation The percentage of free radioactivity in the standards and test samples and the percentage recovery of the radioactive tracer through the method are calculated. A graph is drawn plotting the percentage free radioactivity for the standard curve samples as a function of the added unlabelled 5-androstene-3/~, I7/%diol (Fig. I). The mass of 5-androstene-3fl, I7fl-diol in each test sample is read from the graph and corrected for methodological losses. The concentration of total dehydroepiandrosterone (D), i.e. the sum of dehydroepiandrosterone and its sulphate fraction, in # g / I o o ml plasma is calculated from the formula: I00
D=A
IOO
I
x T- xT-- x
where A = ng 5-androstene-3fl, I7fl-diol from graph, R = % Recovery, V = Volume of plasma assayed and S ~ aliquot in/~1 of purified extract assayed.
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70
6O
5O
g b-
40
30
20
0.5 1.0 1; 2D ' ' ' ng 5 - A m d r o s t e n e - 3p, 17/9-diol
2 i5
Fig. I. S t a n d a r d c u r v e . T h e p e r c e n t a g e of r a d i o a c t i v i t y in t h e s u p e r n a t a n t f u n c t i o n of t h e u n l a b e l l e d 5 - a n d r o s t e n e - 3 f l , i T f l - d i o l c o n t e n t of e a c h t u b e .
(free) is p l o t t e d as a
Assav of uuconjugated dehydroepiaudrosterone It is possible to assay only unconjugated dehydroepiandrosterone by the same method with certain slight modifications as follows: T() z5-ml glass-stoppered tubes containing z ml of distilled water, z 4 ooo disint./ lnin of [7-aHjdehydroepiandrosterone in o.I ml of ethanol and o.z ml of plasma samples are added. After mixing, Iz ml of dichloromethane is added to each tube. The tubes are then shaken in a mechanical shaker for 5 min and afterwards centrifuged at 8oo g for 5 rain at room temperature. The extracts are washed once with z ml of distilled water and then evaporated to dryness under nitrogen in a water bath at 4 o°. The subsequent procedures, as from First chromatography, are as described before except that the whole of the final purified extract is used in the competitive protein-binding part of the assay. An aliquot (o.o5 ml) is taken from each test sample, to calculate recovery, after the overnight incubation with the z% dilution of pregnancy plasma. Then o.z5 ml of the cold saturated solution of ammonium sulphate is added to each test sample tube. The procedures are otherwise identical to those already described. The formula used to calculate the concentration ()f unconjugated dehydroepiandrosterone (D '~) in ng/Ioo ml plasma is : IOO I) ~'-
- A
X
- -
R
>(
iOO
V
The symbols are the same as those used above.
Assessment of method The mean recovery of '7-3H]dehydroepiandrosterone in the assay of total dehydroepiandrosterone was 63.9 ~ 6.9 (S.D.)g,o, ~ = 44 and for the assay of the unconjugated fraction 67. 9 ~ 5.7 (S.D.)%, n 5o.
ASSAY OF PLASMA
DHA
AND ITS SULPHATE
301
Precision Precision was examined by assaying samples in duplicate within batch and b y carrying out replicate assays of a plasma pool. The results are shown in Table I.
Accuracy and sensitivity Accuracy was evaluated by adding known amounts of dehydroepiandrosterone within the range 0.6 to 2.0 ng and dehydroepiandrosterone sulphate within the range 30 to 12o ng to distilled water and plasma. The mean recovery of dehydroepiandroTABLE
I
PRECISION STUDIES
Total DHA
Unconjugated DHA
R e p l i c a t e a s s a y of a p l a s m a pool
76.8 ± 4.4 n = I2
350 ± 34.5 n = 8
D u p l i c a t e a s s a y s of plasma samples Range
54.8 4- 2.2 n = i8 18-118
679 4- 69.6 n = i8 2oo-15oo
N o t e s : F i g u r e s g i v e n are m e a n ± s t a n d a r d d e v i a t i o n in /~g of d e h y d r o e p i a n d r o s t e r o n e (DHA) per IOO m l of p l a s m a in t h e a s s a y of t o t a l D H A a n d n g / i o o ml p l a s m a in t h e a s s a y of u n c o n j u g a t e d DHA. The s t a n d a r d d e v i a t i o n from d u p l i c a t e a s s a y s is c a l c u l a t e d f r o m :
S . D . = , ~nn W h e r e d is t h e difference b e t w e e n d u p l i c a t e a s s a y s a n d n is t h e n u m b e r of d u p l i c a t e s .
sterone was 97% (range 87-1o5), n = 4 from distilled water and 99% (range 83-113), n ~ 12 from plasma. Similarly, the mean recovery of dehydroepiandrosterone sulphate was 91% (range 84-98), n = 12 and 96% (range 84-1o9) , n ---- 12 respectively. The sensitivity of the method can be calculated theoretically from the precision of the assay at low levels 15. The precision was determined from replicate analyses of a water blank, which gave a S.D. of 4- 50 pg. On this basis, the theoretical sensitivity for the method is approximately IOO pg (p = 0.05) per incubation mixture.
Specificity Non-specific interference was assessed by processing water blanks. The processed residue had no significant effect on the distribution of E7-3Hl5-androstene-3fl, I7fl-diol between the bound and free fraction in the incubation mixture as compared to the zero point of the standard curve. Similarly, plasma from two adrenalectomised oophorectomised subiects had levels of dehydroepiandrosterone and dehydroepiandrosterone sulphate of less than IOO pg per incubation mixture. The specificity of the method for the assay of total plasma dehydroepiandrosterone was further studied b y comparing the results with those obtained by a gasliquid chromatographic (GLC) method described for the assay of plasma dehydroepiandrosterone sulphate e. The result are shown in Table II. The samples were from patients with malignant or benign breast disease. The overall correlation coefficient r equals 0.96 and the regression line equation is y ---- 0.98 x + 7.2. y ~ GLC, x = competitive protein binding.
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ANI)Rt,2, J A M E S
TABLE
l I
COMPARISON B E T W E E N A G L C METHOD AND THE C P B
METHOD
P l a s m a level r a n g e p e r 2 oo m l 50--zoo #g
loo
GLC
- 50 #g CPB
GLC
CPB
GLC
CI-'B
GLC
CPB
25 30
19 33 15 13 !7 32
72 03
119 124
212 284 ]54 239 !72 ~67 197 182
255 299
44 45 43
76 81 9o 66 52 90 79 97 58 6i
27. 5 ~ 13. 3
75.o k I5.2
2oo.8 [ 43
191. 5 t 50
I2
II 2I 32 i0 4~ 34 ti
M e a n 27.o H S . I ) . ": 12. 3
14
Note. The results plasma.
are expressed
.15o p g
> 15o I~g
]o 3
]oo
61 52 7° .54 97 55 46
103 I29 13.5 lO 5 141 137 149
124 lOO 94 94 i 11 122 1t 5 ]5 ° 12.5 T 13
7o.3 20.6
124.2 z!: 17. I
]]4.8 ]0.9
i n /*g of u n c o n j u g a t e d
dehydroepiandrosterone
14 {)
t 85 t.53 171} 17.5
i ~3
per
Ioo
m l of
Stability or frozen plasma The concentration of total dehydroepiandrosterone in a plasma pool kept at --2(}" was determined on two occasions one year apart. The mean level found, ex-
pressed in Fg/Ioo ml plasma, was 74 and 76 respectively. I)ISCUSSION
A method is described for the assay of dehydroepiandrosterone and its sulphate. After extraction and solvolysis dehydroepiandrosterone is purified by paper chromatography and transformed to 5-androstene-3fl, I7fl-diol by reduction with sodium borohydride. The 5-androstene-3fl, I7fl-diol formed is purified by paper chromatography prior to assay. Eighteen samples can normally be processed in 4½ days. Unconjugated dehydroepiandrosterone may also be assayed by the same method using a modified extraction procedure as described. The assay end-point is sensitive enough to measure IOO pg of dehydroepiandrosterone as 5-androstene-3fl, I7fl-diol in the final incubation mixture. The precision studies reveal a coefficient of variation of lO% for the assay of unconjugated dehydroepiandrosterone and 6% for the assay of dehydroepiandrosterone and its sulphate, in the range of values usually encountered in peripheral plasma. The accuracy studies also reveal that there is no large systematic error. However, to obtain an accuracy better than 8o% of the expected value in the assay of dehydroepiandrosterone sulphate it was found necessary to extract the sample twice with ethyl acetate and to extend the extraction time to 15 min. In this context, it should be noted that losses occurring during extraction and solvolysis are not corrected for because the radioactive tracer is only added subsequent to these stages. To avoid this potential source of error it would be preferable to add radioactive dehydroepiandrosterone sulphate to the sample before proceeding with the extraction. The specificity of the method for the assay of total dehydroepiandrosterone
ASSAY OF PLASMA
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was examined b y comparing the results obtained with those of a GLC method. The overall correlation is good but there are some discrepancies, the reasons for which were not investigated. It should be noted that the GLC method assays dehydroepiandrosterone sulphate whereas the present method assays the unconjugated and sulphate fraction together. As the reported levels of unconjugated dehydroepiandrosterone in peripheral plasma is less than 2 #g/Ioo mP, it is unlikely that this will contribute to any extent to differences between the methods, because, taking into account the precision of the present method, a difference in the total dehydroepiandrosterone content of the sample of 2/~g/Ioo ml would not be statistically significant. The GLC method tends to give results which are on the whole slightly higher than the present method. This m a y be due partly to losses, which are not corrected for in this method, during the extraction and solvolysis procedures. Non-specific interference b y plasma constituents other than gonadal and adrenocortical steroids was assessed by analysing plasma samples from two patients who had been oophorectomised and adrenalectomised. The levels of dehydroepiandrosterone or its sulphate found in these plasmas were below the detection limit ot the method, i00 pg per incubation mixture, therefore indicating that non-specific interference from plasma extracts is minimal. It would appear from the results obtained by the present method that competitive protein binding offers a useful alternative to existing methods for the assay of plasma dehydroepiandrosterone and its sulphate. ACKNOWLEDGEMENTS
We thank Dr. D. Y. Wang, Imperial Cancer Research Fund, London, W.C. 2. for providing the results of the dehydroepiandrosterone sulphate assays by his method. This work was carried out with the aid of a grant from the Medical Research Council. REFERENCES I 2 3 4 5 6 7 8 9 io
ii 12 13 14 15
E. PEARSON MURPHY, Acta Endocrinol., Suppl. 147 (197 o) 37. KATO AND R. HORTON, J. Clin. Endocrinol., 28 (1968) 116o. MAYES AND C. A. NUGENT, J. Clin. Endocrinol., 28 (1968) 1169. GOLDFIEN, J. JONES, M. E. YANNONE AND B. WHITE, i n M. B. L1PSETT (Ed.), Gas Chromatography of Steroids in Biological Fluids, P l e n u m P r e s s , N e w Y o r k , 1965, p. 35. H. M. GANDY AND R. E . PETERSON, J. Clin. Endocrinol., 28 (1968) 949D. Y. WANG, R . D. BULBROOK, B. S. THOMAS AND M. FRIEDMAN, J. Endocrinol., 42 (1968) 567. R. L. ROSENFIELD, Steroids, 17 (1971) 689. R. HAMPL AND L. STARKA, Clin. Chim. Acta, 34 (1971) 77R. L. ROSENFIELD, J. Clin. Endocrinol., 32 (1971) 717 • R. P. EKINS, G. B. NEWMAN AND J. L. H . O'RIORDAN, i n R. I. HAYES, F. A. GOSWlTZ AND B. E . P. MURPHY (Eds.), Radioisotopes in Medicine: In Vitro Studies, U S A E C , O a k R i d g e , T e n n e s s e e , 1968, p. 59. A. DE LA PENA AND J. W . GOLDZIEHER, Steroids, 18 (1971) 195. J. TOWNSEND AND V. H . T. JAMES, Steroids, I I (1968) 497. I. E . BUSH, Biochem. J., 5 ° (1952) 37 ° • S. BORSTEIN AND S. LIEBERMANN, J. Biol. Chem., 233 (1958) 331J. B. BROWN, R. D. BOLBROOK AND F. C. GREENWOOD, J. Endocrinol., 16 (1957) 41. B. T. D. A.
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ASSAY OF PLASMA D E H Y D R O E P I A N D R O S T E R O N E AND ITS SULPHATE BY COMPETITIVE P R O T E I N BINDING
C. M. A N D R E AND V. H. T. J A M E S
Steroid Research Unit, Department of Chemical Pathology, St. Mary's Hospital Medical School, London, W.2. (U.K.) (Received J u l y 26, I972)
SUMMARY
A competitive protein-binding method for the assay of dehydroepiandrosterone and its sulphate in plasma is described. After extraction from plasma, and solvolysis in the case of dehydroepiandrosterone sulphate, dehydroepiandrosterone is purified by paper chromatography and transformed to 5-androstene-3fl, I7fl-diol by reduction with sodium borohydride. The 5-androstene-3fl, I7fl-diol so formed is purified by paper chromatography prior to assay. The assay end-point uses the binding property of testosterone-binding globulin, which binds with high affinity those steroids with an unhindered I7fl-hydroxyl group. Tile reliability criteria of the method in terms of precision, accuracy, sensitivity and specificity have been evaluated.
INTRODUCTION
During the last few years the principle of competitive protein binding has been successfully applied to the assay of plasma androgens 1. All the methods developed for the assay of androgens by this technique use, as an assay end-point, the binding property of testosterone-binding globulin which binds with high affinity steroids with an unhindered I7fl-hydroxyl group S. In order to assay androgens like androstenedione or dehydroepiandrosterone, which are i7-oxosteroids, it is necessary firstly to separate the I7-Oxosteroid from the I7fl-hydroxysteroids in the sample by chromatography and then transform the I7-Oxosteroid to the corresponding I7fl-hydroxysteroid by reduction. Dehydroepiandrosterone can be readily transformed to 5-androstene-3fl, I7fldiol which is known to bind with high affinity to testosterone-binding globulin 3. Advantage of these facts was taken to develop the present method. Reliable methods, e.g. gas-liquid chromatography or the double isotope dilution derivative technique, have been developed for the assay of dehydroepiandrosterone*, 5 or its sulphate 6. However, the present study was undertaken to investigate the possibility of using the simpler technique of competitive protein binding as a useful alter-
296
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native to the existing methods. Recently two competitive proteii, binding methods for the assay of plasma dehydroepiandrosterone 7 and its sulphateT, ~ have been published. The present method is similar to the method of Halnpl and Starka ~ but differs in several practical aspects, summarised below, which are incorporated in order to improve the reliability and specificity of the assay. Sufficient tritiated dehydroepiandrosterone is added to tile plasma extracts to enable accurate localisation by scanning the radioactivity on each strip of the paper chroniatogram. This is desirable not only because it results in more reproducible recoveries but also reduces the likelihood of contaminating the eluate from the chromatogram with adjacent substances which may also interfere in the protein-binding part of the assay. The specificity may thus be improved 9. A second chromatogram is essential after the reduction of dehydroepiandrosterone to 5-androstene-3/3, I7/5-diol with sodium borohydride as it is a common experience that the reduction of nanogram quantities of steroids with this technique can be erratic. It is essential therefore to separate the reduction products by chromatography to isolate the generated 5-androstene-3/3, I7/3-diol otherwise it is impossible to assess methodological losses accurately. In the protein-binding part of the assay the present method makes use of a homogeneous system, i.e. the tracer is chemically identical to the test compound apart from the radioactive isotope incorporated in the tracer molecule. In a heterogeneous system as used by Hampl and Starka a tracer, namely tritiated testosterone, of a different reaction energy to the test compound is reacted with the binding protein. In this type of system the interaction between tracer, unlabelled hormone and binding protein is more complex and the effect on the final equilibrium state of the reactants for a given assay condition is unpredictable ~° even more so should other steroidal or non-steroidal substances which partially interfere with the binding reactions be present in the assay mixture. For these reasons the simpler homogeneous s?stem is preferable as errors are least likely to arise. A further point to be considered is the different techniques used in the separation of bound from unbound hormone after equilibration with the binding protein. None of the methods currently available absolutely separate bound from unbound hormone. Hence the method of choice is the most precise and there i~; evidence to indicate that ammonium sulphate protein precipitation, as used here, is more precise than the dextran-coated charcoal methodt'. In addition the present method has also been modified for the assay of unconjugated dehydroepiandrosterone. MATERIALS AND REAGENTS
Dichloromethane, methanol and ethyl acetate are redistilled before use. Ethanol, benzene and petroleum ether (IOO°--I20 °) a r e purified as described by Townsend and James '~ except that in addition, benzene and petroleum ether are prewashed twice with 3o ml of concentrated sulphuric acid per litre. Aluminium oxide "CAMAG'" MFC lOO-24o mesh alkaline Brockmann activity = I (Hopkin and Williams Ltd., Essex, England) is purified according to Mayes and Nugent a and activated by heating at IiO°-i2o ° for 3 h and stored in a sealed container.
ASSAY OF PLASMA D H A
AND ITS SULPHATE
297
Paper for chromatography, W h a t m a n No. 2 is prepared by cutting lanes 2-cm wide and 4o-cm long with a o.5-cm gap between lanes and washed by descending chromatography in methanol-dichloromethane 3 :I for at least 48 h. Dehydroepiandrosterone sodiunl sulphate and 5-androstene-3/5, I7/%dioI are used as supplied by Steraloids, Inc. E7-3H~ Dehydroepiandrosterone specific activity 25.1 C/mmole (New England Nuclear, Boston, U.S.A.) is purified by paper chromatography on pre-washed W h a t m a n No. 2 chromatography paper in the B3 system of Bush 13 (petroleum ether-benzene-methanol-water 33:17:4o:1o ) for 3 h at 24 ° and stored in benzene at 4 ° . The working standard is prepared from this stock solution by dilution with ethanol. E7-~Hl5-Androstene-3/~, i7/~-diol is prepared b y reducing E7-3HJdehydro epiandrosterone with sodium borohydride, as described under borohydride reduction, and purified by chromatography on pre-washed W h a t m a n No. 2 paper in the B~ system of Bush for 4 h at 24 °. The ~7-~Hl5-androstene-3/~, I7/~-diol is located with a strip scanner, eluted with dichloromethane-methanol 3:2 and stored in 5 % ethanol in benzene at 4 ° . The working standard is prepared from this stock solution by dilution with ethanol. Third trimester pregnancy plasma is pooled from normal donors and stored in o.5-ml aliquots at --20 °. Under these conditions it is stable for at least 18 months. The scintillation fluid is prepared by adding 2,5-bis-E2-(5-t-butylbenzoxaloyl)lthiophene (BBOT), 6 g/litre (Ciba), naphthalene 80 g/litre to a mixture of 2-methoxyethanol and toluene 2 : 3. Disposable glass specimen tubes, glass capillaries and disposable Pasteur pipettes are washed in distilled water, ethanol and dried. All other glassware is washed in a detergent, RBS 25 (Chemical Concentrates Limited, London), in an ultrasonic cleaning bath, thoroughly rinsed with tap water and distilled water and dried in an oven at 12o ° . Glass wool, for plugging columns, is washed in methanol and dichloromethane and dried. Blood samples are collected in plastic tubes containing lithium heparin. The plasma is separated immediately and stored at --20 ° until analysed. METHOD
Extraction and solvolysis To io-ml glass-stoppered tubes containing 2 ml of o.I N sulphuric acid saturated with sodium chloride 14, o.i ml plasma samples are added. After mixing, 5 ml of ethyl acetate is added to each tube. The tubes are then shaken in a mechanical shaker for 15 min and afterwards centrifuged at 800 g for 5 rain at room temperature. The supernatants are transferred with Pasteur pipettes to I5-ml glass-stoppered tubes containing 25000 disint/min of E7-3Hldehydroepiandrosterone in o.I ml ethanol. The same amount of radioactive tracer is added in triplicate to counting vials to calculate the recovery through the method. The samples are again extracted with 5 ml ethyl acetate as before. The appropriate extracts are pooled, mixed and incubated at 37 ° for 22-24 h. The cooled extracts are transferred to 5o-ml separating funnels containing 2 ml of 1%, (w/v) sodium carbonate. The tubes are rinsed with i ml of ethyl acetate and the washings transferred to the appropriate separating funnel. After shaking for I rain the phases are allowed to separate and the aqueous layer is discarded. The ex-
298
ANDRE, JAMES
tracts are then washed with 2-ml volumes of distilled water until the washings are neutral. The extracts are transferred to conical glass tubes and evaporated to dryness under a stream of nitrogen, in a water b a t h at 4 o°.
First chromatography The residues are quantitatively transferred to pre-washed W h a t m a n No. 2 c h r o m a t o g r a p h y paper with diehloromethane-methanol 3:2. After equilibration the c h r o m a t o g r a m is developed in the B~ system of BuslO a for 3 h at 24 ~. The radioactive dehydroepiandrosterone areas are located with a strip scanner and the appropriate sections cut out. Microcolumns of alumina, 3-cm long, are prepared in disposable Pasteur pipettes (0.6 × 15 cm) plugged with glass wool% and the sections of paper to be eluted (approx. 4.5 × 2 cm) are folded longitudinally and placed on top. The columns and sections of paper are washed twice with dichloromethane by filling the pipettes and allowing them to drain to waste, and eluted with approximately 5 ml of dichloromethanol 3 : 2. The eluates are collected in 6-ml glass-stoppered tubes and evaporated to dryness under nitrogen.
Borohydride reduction To each tube 0.2 ml of a freshly prepared 0.25% (w/v) methanolic solution of sodium borohydride is added and mixed. After 5 min at room temperature o. 5 ml of distilled water is added and the reduction products extracted once by adding 4.5 ml of dichloromethane to the tube and shaking for 3 min. The extracts are washed once with o.5 ml of distilled water and evaporated to dryness under nitrogen.
Second chromatography ]'be residues are chromatographed in the B3 system as before except that the c h r o m a t o g r a p h y time is increased to 4 tl. The radioactive 5-androstene-3fl, I7fl-diol areas are located with a strip scanner and the appropriate sections (approx 4 >: 2 cm) are cut out and eluted by soaking twice in 2 ml of dichloromethane-methanol 3 : 2 for IO rain each time in I2 7. IOO-inm test tubes. The eluates are collected in io x 5 om m glass specimen tubes and evaporated to dryness under nitrogen. Microeolumns of alumina are prepared as described abow~ and the columns washed twice with dichloromethane immediately before use by filling the pipettes and allowing t h e m to drain to waste. The residues are transferred to the alumina colunms, with glass capillary tubes, in small volumes of dichloromethane (6 drops from a Pasteur pipette) three times and once with 2. 5 % methanol in dichloronmthane (4 drops from a Pasteur pipette). The colunms are washed twice with dichloromethane by filling the Pasteur pipettes and allowing them to drain to waste, and eluted with 2.5 nd of 2.5% methanol in dichloromethane which is collected in IO x 5o-mm glass specimen tubes. The eluates are evaporated to dryness under nitrogen and redissolved in I ml of ethanol.
Competitive protein binding Standards are prepared in triplicate by adding o, 0.25, o.5, i.o, 1.5 and 2.5 ng of 5-androstene-3fl, I7fl-diol in ethanol to IOX5o-nlm glass specimen tubes and
A S S A Y OF P L A S M A
DHA
AND ITS SULPHATE
299
I6OOO disint./min of [7-"H]5-androstene-3fl, I7fl-diol in o.I ml ethanol to each tube. After mixing the ethanol is evaporated to dryness under nitrogen. The same amount of radioactive 5-androstene-3fl, I7fl-diol is added in triplicate to counting vials to calculate the proportion of free radioactive 5-androstene-3fl, I7fl-diol after the ammonium sulphate precipitation of testosterone-binding globulin. From each test sample an aliquot (0.2 ml) is taken and added to a counting vial to calculate the recovery. Duplicate aliquots expected to contain not more than 2.0 ng of 5-androstene-3fl, I7fl-diol (usually between IO and 5 °/~1) are then taken with a Hamilton syringe and added to IO × 5o-mm glass specimen tubes containing i6ooo disint./min of [7-aHl5-androstene-3fl, ITfl-diol in o.I inl ethanol. After mixing the ethanol is evaporated to dryness under nitrogen. To each standard and test sample is added 0.3 ml of a freshly prepared 2% solution of pregnancy plasma in distilled water. After mixing thoroughly but gently to avoid frothing, the tubes are covered with parafilm and left overnight at 4 °.
Ammonium sulphate protein precipitation To each tube o. 3 ml of a cold saturated solution of ammonium sulphate, p H 6, is added, mixing immediately and thoroughly but avoiding frothing. The tubes are incubated at 4 ° for 5 rain and centrifuged at IOOOg for IO rain at 4 °. From each supernatant 0.2 ml is taken and added to a counting vial.
Note The ammonium sulphate protein precipitation step is carried out on batches of eighteen tubes to avoid prolonged contact with ammonium sulphate 3.
Radioactive counting To avoid large differences in quenching between the various counting vials in the batch, ammonium sulphate is added to the standard and recovery vials so that finally each vial contains 0.2 inl of 50% (w/v) solution of ammonium sulphate. To each counting vial io ml of the BBOT scintillation fluid is added and the vial contents are thoroughly mixed before counting in a liquid scintillation spectrometer to a theoretical error of 20/0.
Calculation The percentage of free radioactivity in the standards and test samples and the percentage recovery of the radioactive tracer through the method are calculated. A graph is drawn plotting the percentage free radioactivity for the standard curve samples as a function of the added unlabelled 5-androstene-3/~, I7/%diol (Fig. I). The mass of 5-androstene-3fl, I7fl-diol in each test sample is read from the graph and corrected for methodological losses. The concentration of total dehydroepiandrosterone (D), i.e. the sum of dehydroepiandrosterone and its sulphate fraction, in # g / I o o ml plasma is calculated from the formula: I00
D=A
IOO
I
x T- xT-- x
where A = ng 5-androstene-3fl, I7fl-diol from graph, R = % Recovery, V = Volume of plasma assayed and S ~ aliquot in/~1 of purified extract assayed.
300
A N D R E , JAMES
70
6O
5O
g b-
40
30
20
0.5 1.0 1; 2D ' ' ' ng 5 - A m d r o s t e n e - 3p, 17/9-diol
2 i5
Fig. I. S t a n d a r d c u r v e . T h e p e r c e n t a g e of r a d i o a c t i v i t y in t h e s u p e r n a t a n t f u n c t i o n of t h e u n l a b e l l e d 5 - a n d r o s t e n e - 3 f l , i T f l - d i o l c o n t e n t of e a c h t u b e .
(free) is p l o t t e d as a
Assav of uuconjugated dehydroepiaudrosterone It is possible to assay only unconjugated dehydroepiandrosterone by the same method with certain slight modifications as follows: T() z5-ml glass-stoppered tubes containing z ml of distilled water, z 4 ooo disint./ lnin of [7-aHjdehydroepiandrosterone in o.I ml of ethanol and o.z ml of plasma samples are added. After mixing, Iz ml of dichloromethane is added to each tube. The tubes are then shaken in a mechanical shaker for 5 min and afterwards centrifuged at 8oo g for 5 rain at room temperature. The extracts are washed once with z ml of distilled water and then evaporated to dryness under nitrogen in a water bath at 4 o°. The subsequent procedures, as from First chromatography, are as described before except that the whole of the final purified extract is used in the competitive protein-binding part of the assay. An aliquot (o.o5 ml) is taken from each test sample, to calculate recovery, after the overnight incubation with the z% dilution of pregnancy plasma. Then o.z5 ml of the cold saturated solution of ammonium sulphate is added to each test sample tube. The procedures are otherwise identical to those already described. The formula used to calculate the concentration ()f unconjugated dehydroepiandrosterone (D '~) in ng/Ioo ml plasma is : IOO I) ~'-
- A
X
- -
R
>(
iOO
V
The symbols are the same as those used above.
Assessment of method The mean recovery of '7-3H]dehydroepiandrosterone in the assay of total dehydroepiandrosterone was 63.9 ~ 6.9 (S.D.)g,o, ~ = 44 and for the assay of the unconjugated fraction 67. 9 ~ 5.7 (S.D.)%, n 5o.
ASSAY OF PLASMA
DHA
AND ITS SULPHATE
301
Precision Precision was examined by assaying samples in duplicate within batch and b y carrying out replicate assays of a plasma pool. The results are shown in Table I.
Accuracy and sensitivity Accuracy was evaluated by adding known amounts of dehydroepiandrosterone within the range 0.6 to 2.0 ng and dehydroepiandrosterone sulphate within the range 30 to 12o ng to distilled water and plasma. The mean recovery of dehydroepiandroTABLE
I
PRECISION STUDIES
Total DHA
Unconjugated DHA
R e p l i c a t e a s s a y of a p l a s m a pool
76.8 ± 4.4 n = I2
350 ± 34.5 n = 8
D u p l i c a t e a s s a y s of plasma samples Range
54.8 4- 2.2 n = i8 18-118
679 4- 69.6 n = i8 2oo-15oo
N o t e s : F i g u r e s g i v e n are m e a n ± s t a n d a r d d e v i a t i o n in /~g of d e h y d r o e p i a n d r o s t e r o n e (DHA) per IOO m l of p l a s m a in t h e a s s a y of t o t a l D H A a n d n g / i o o ml p l a s m a in t h e a s s a y of u n c o n j u g a t e d DHA. The s t a n d a r d d e v i a t i o n from d u p l i c a t e a s s a y s is c a l c u l a t e d f r o m :
S . D . = , ~nn W h e r e d is t h e difference b e t w e e n d u p l i c a t e a s s a y s a n d n is t h e n u m b e r of d u p l i c a t e s .
sterone was 97% (range 87-1o5), n = 4 from distilled water and 99% (range 83-113), n ~ 12 from plasma. Similarly, the mean recovery of dehydroepiandrosterone sulphate was 91% (range 84-98), n = 12 and 96% (range 84-1o9) , n ---- 12 respectively. The sensitivity of the method can be calculated theoretically from the precision of the assay at low levels 15. The precision was determined from replicate analyses of a water blank, which gave a S.D. of 4- 50 pg. On this basis, the theoretical sensitivity for the method is approximately IOO pg (p = 0.05) per incubation mixture.
Specificity Non-specific interference was assessed by processing water blanks. The processed residue had no significant effect on the distribution of E7-3Hl5-androstene-3fl, I7fl-diol between the bound and free fraction in the incubation mixture as compared to the zero point of the standard curve. Similarly, plasma from two adrenalectomised oophorectomised subiects had levels of dehydroepiandrosterone and dehydroepiandrosterone sulphate of less than IOO pg per incubation mixture. The specificity of the method for the assay of total plasma dehydroepiandrosterone was further studied b y comparing the results with those obtained by a gasliquid chromatographic (GLC) method described for the assay of plasma dehydroepiandrosterone sulphate e. The result are shown in Table II. The samples were from patients with malignant or benign breast disease. The overall correlation coefficient r equals 0.96 and the regression line equation is y ---- 0.98 x + 7.2. y ~ GLC, x = competitive protein binding.
302
ANI)Rt,2, J A M E S
TABLE
l I
COMPARISON B E T W E E N A G L C METHOD AND THE C P B
METHOD
P l a s m a level r a n g e p e r 2 oo m l 50--zoo #g
loo
GLC
- 50 #g CPB
GLC
CPB
GLC
CI-'B
GLC
CPB
25 30
19 33 15 13 !7 32
72 03
119 124
212 284 ]54 239 !72 ~67 197 182
255 299
44 45 43
76 81 9o 66 52 90 79 97 58 6i
27. 5 ~ 13. 3
75.o k I5.2
2oo.8 [ 43
191. 5 t 50
I2
II 2I 32 i0 4~ 34 ti
M e a n 27.o H S . I ) . ": 12. 3
14
Note. The results plasma.
are expressed
.15o p g
> 15o I~g
]o 3
]oo
61 52 7° .54 97 55 46
103 I29 13.5 lO 5 141 137 149
124 lOO 94 94 i 11 122 1t 5 ]5 ° 12.5 T 13
7o.3 20.6
124.2 z!: 17. I
]]4.8 ]0.9
i n /*g of u n c o n j u g a t e d
dehydroepiandrosterone
14 {)
t 85 t.53 171} 17.5
i ~3
per
Ioo
m l of
Stability or frozen plasma The concentration of total dehydroepiandrosterone in a plasma pool kept at --2(}" was determined on two occasions one year apart. The mean level found, ex-
pressed in Fg/Ioo ml plasma, was 74 and 76 respectively. I)ISCUSSION
A method is described for the assay of dehydroepiandrosterone and its sulphate. After extraction and solvolysis dehydroepiandrosterone is purified by paper chromatography and transformed to 5-androstene-3fl, I7fl-diol by reduction with sodium borohydride. The 5-androstene-3fl, I7fl-diol formed is purified by paper chromatography prior to assay. Eighteen samples can normally be processed in 4½ days. Unconjugated dehydroepiandrosterone may also be assayed by the same method using a modified extraction procedure as described. The assay end-point is sensitive enough to measure IOO pg of dehydroepiandrosterone as 5-androstene-3fl, I7fl-diol in the final incubation mixture. The precision studies reveal a coefficient of variation of lO% for the assay of unconjugated dehydroepiandrosterone and 6% for the assay of dehydroepiandrosterone and its sulphate, in the range of values usually encountered in peripheral plasma. The accuracy studies also reveal that there is no large systematic error. However, to obtain an accuracy better than 8o% of the expected value in the assay of dehydroepiandrosterone sulphate it was found necessary to extract the sample twice with ethyl acetate and to extend the extraction time to 15 min. In this context, it should be noted that losses occurring during extraction and solvolysis are not corrected for because the radioactive tracer is only added subsequent to these stages. To avoid this potential source of error it would be preferable to add radioactive dehydroepiandrosterone sulphate to the sample before proceeding with the extraction. The specificity of the method for the assay of total dehydroepiandrosterone
ASSAY OF PLASMA
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AND ITS S U L P H A T E
303
was examined b y comparing the results obtained with those of a GLC method. The overall correlation is good but there are some discrepancies, the reasons for which were not investigated. It should be noted that the GLC method assays dehydroepiandrosterone sulphate whereas the present method assays the unconjugated and sulphate fraction together. As the reported levels of unconjugated dehydroepiandrosterone in peripheral plasma is less than 2 #g/Ioo mP, it is unlikely that this will contribute to any extent to differences between the methods, because, taking into account the precision of the present method, a difference in the total dehydroepiandrosterone content of the sample of 2/~g/Ioo ml would not be statistically significant. The GLC method tends to give results which are on the whole slightly higher than the present method. This m a y be due partly to losses, which are not corrected for in this method, during the extraction and solvolysis procedures. Non-specific interference b y plasma constituents other than gonadal and adrenocortical steroids was assessed by analysing plasma samples from two patients who had been oophorectomised and adrenalectomised. The levels of dehydroepiandrosterone or its sulphate found in these plasmas were below the detection limit ot the method, i00 pg per incubation mixture, therefore indicating that non-specific interference from plasma extracts is minimal. It would appear from the results obtained by the present method that competitive protein binding offers a useful alternative to existing methods for the assay of plasma dehydroepiandrosterone and its sulphate. ACKNOWLEDGEMENTS
We thank Dr. D. Y. Wang, Imperial Cancer Research Fund, London, W.C. 2. for providing the results of the dehydroepiandrosterone sulphate assays by his method. This work was carried out with the aid of a grant from the Medical Research Council. REFERENCES I 2 3 4 5 6 7 8 9 io
ii 12 13 14 15
E. PEARSON MURPHY, Acta Endocrinol., Suppl. 147 (197 o) 37. KATO AND R. HORTON, J. Clin. Endocrinol., 28 (1968) 116o. MAYES AND C. A. NUGENT, J. Clin. Endocrinol., 28 (1968) 1169. GOLDFIEN, J. JONES, M. E. YANNONE AND B. WHITE, i n M. B. L1PSETT (Ed.), Gas Chromatography of Steroids in Biological Fluids, P l e n u m P r e s s , N e w Y o r k , 1965, p. 35. H. M. GANDY AND R. E . PETERSON, J. Clin. Endocrinol., 28 (1968) 949D. Y. WANG, R . D. BULBROOK, B. S. THOMAS AND M. FRIEDMAN, J. Endocrinol., 42 (1968) 567. R. L. ROSENFIELD, Steroids, 17 (1971) 689. R. HAMPL AND L. STARKA, Clin. Chim. Acta, 34 (1971) 77R. L. ROSENFIELD, J. Clin. Endocrinol., 32 (1971) 717 • R. P. EKINS, G. B. NEWMAN AND J. L. H . O'RIORDAN, i n R. I. HAYES, F. A. GOSWlTZ AND B. E . P. MURPHY (Eds.), Radioisotopes in Medicine: In Vitro Studies, U S A E C , O a k R i d g e , T e n n e s s e e , 1968, p. 59. A. DE LA PENA AND J. W . GOLDZIEHER, Steroids, 18 (1971) 195. J. TOWNSEND AND V. H . T. JAMES, Steroids, I I (1968) 497. I. E . BUSH, Biochem. J., 5 ° (1952) 37 ° • S. BORSTEIN AND S. LIEBERMANN, J. Biol. Chem., 233 (1958) 331J. B. BROWN, R. D. BOLBROOK AND F. C. GREENWOOD, J. Endocrinol., 16 (1957) 41. B. T. D. A.