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A rapid method for the radioimmunoassay of urinary aldosterone
Clin. Biochem. 6, 285-294 (1973)
A R A P I D METHOD FOR THE R A D I O I M M U N O A S S A Y OF U R I N A R Y A L D O S T E R O N E
J. S T U A R T , P. M. K E A N E , G. VIOL, and R. N. G U P T A
Department of Laboratory Medicine and Medicine, St. Joseph's Hospital, Hamilton, Ontario. (Received February 1~, 1973)
CLBIA, 6 (4): 285-294 (1973)
Clin. Biochem. Stuart, J., Keane, P. M., Viol, G. and Gupta, R. N.
Departments of Labarato~ T Medicine and Medicine, St. Joseph's Hospital, Hamilton 20, Ontario. A R A P I D M E T H O D F O R T H E R A D I O I M M U N O A S S A Y OF U R I N A R Y ALDOSTERONE 1. The availability of an antiserum to aldosterone 7-1actone allows the rapid assay of aldosterone in urine (1). 2. Following acid hydrolysis of the 18 oxo-conjugate, aldosterone is oxidized to its 7-1actone. 3. The extremely low cross reactivity of the antiserum with structurally related steroids obviates the need f o r chromatographic purification before radio° immunoassay. 4. This procedure is sensitive enough to allow the assay of aldosterone in the equivalent of 25 UL of hydrolyzed urine.
A NUMBER OF COLORIMETRIC AND FLUORIMETRIC PROCEDURES have been described for the a s s a y of urinary aldosterone (2-4). Double isotope derivative (5) and gas liquid chromatographic (6-8) techniques have also been used. In order to achieve the desired degree of specificity all of these methods have utilised multiple chromatographic steps in the isolation of the compound. These m e t h o d s are time consuming and necessitate the extraction of large volumes of urine; and as a result, the handling of large volumes of organic solvents.
Correspondence: Dr. P. M. Keane, Department of Laboratory Medicine, St. Joseph's Hospital, Hamilton 20, Ontario.
286
S T U A R T et al.
The procedure described here is basically that described by Farmer et al (i) and utilizes a commercially available anti-serum raised against aldosterone 7-1actone. Purification is achieved by differential solvent extraction. MATERIALS
AND
EQUIPMENT
D-Aldosterone (&~-Pregnen-18-Al-llb, 21-Diol-3,20-Dione) Aldosterone ~-Lactone (3-oxo-11~, 18 Epoxy-androst-4-ene-17~. 1 8 - C a r b o l a c t o n e ) . S i g m a Cat. No.'s A 6628 a n d A 1385 respectively. A l d o s t e r o n e - 1 , 2-'~H, 53 C l / m m o l e , A m e r s h a m Searle. All of the above w e r e used w i t h out further purification. A l d o s t e r o n e "~-lactone a n t i s e r u m ( L o t 185-5-71). A n t i b o d i e s Inc. Davis, C a l i f o r n i a . H u m a n "~-globulin - - C o n n a u g h t Medical R e s e a r c h Lab., T o r o n t o ChloroformFisher, Reagent Grade Benzene -- Analar BDH Periodic Acid - - F r e d r i c k S m i t h Chem. Co., Columbus, Ohio Dioxane -- Fisher, Reagent Grade P y r i d i n e - - A n a l a r , B D H , redistilled a n d stored in a b r o w n bottle A q u a s o l S c i n t i l l a t i o n Cocktail - - N e w E n g l a n d N u c l e a r B u f f e r (1) 1 g g e l a t i n w a s dissolved in 100 ml h o t w a t e r . 3.09 g boric acid w a s dissolved in 300 ml w a t e r a n d t h e p H a d j u s t e d to 8.5 w i t h sodium h y d r o x i d e . T h e g e l a t i n solution w a s a d d e d a n d t h e volume m a d e up to 1 1. B u f f e r (2) - - Add h u m a n ~-globulin to b u f f e r (1) to o b t a i n 1 g / 1 0 0 m l v - g l o b u l i n solution. Periodic acid solution - - 100 m g periodic acid w a s dissolved in 5.0 ml w a t e r . 5.0 ml dioxa n e a n d 0.1 m l p y r i d e w a s added a n d the solution mixed. The acid solution w a s m a d e u p f r e s h f o r each oxidation. S a t u r a t e d a m m o n i u m s u l p h a t e - - A s a t u r a t e d solution w a s p r e p a r e d in deionized w a t e r . T h i s solution w a s f i l t e r e d t h r o u g h W h a t m a n ~:2 p a p e r p r i o r to use. D e x t r a n coated c h a r c o a l - - 100 m g d e x t r a n 70 ( P h a r m a c i a ) a n d 1 g N o r i t A C h a r c o a l w a s added to 100 ml of 0.1 M p h o s p h a t e b u f f e r p H 7.4. F l a s h e v a p o r a t o r - - Bfichi R o t o v a p o r Rinco I n s t r u m e n t Co. Inc., Greenville, Ill. (Canlab). Liquid scintillation system - - N u c l e a r Chicago U n i l u x I I - - A S o r v a l G e n e r a l P u r p o s e RC*3 a u t o m a t i c r e f r i g e r a t e d C e n t r i f u g e , Allied Scientific Co., Toronto.
METHODS
A 2 ml aliquot of a 24 h. urine sample was adjusted to pH 1 with 2 N HCI, and 3,000 cpm :'H aldosterone was added. Hydrolysis was allowed to proceed 20-24 h. at room temperature. The urine was saturated with sodium sulphate, transferred to a clean test tube, and extracted once with 20 ml of chloroform. The aqueous phase was removed and the organic phase washed with 1 ml 0.1 N sodium hydroxide and then I ml water. The organic phase was reduced in volume to approximately 0.2 ml using a flash evaporator (water bath temperature 40 °, transferred with 2 x 0.3 ml rinses of chloroform into a clean test tube, and evaporated to dryness under high purity nitrogen. B ml water and 1 ml benzene were added to the tube and the contents
R A D I O I M M U N O A S S A Y OF A L D O S T E R O N E
287
well mixed. The tube was c e n t r i f u g e d and the aqueous phase t r a n s f e r r e d to a clean t e s t tube. This was e x t r a c t e d w i th 6 ml chloroform, t he organic phase t r a n s f e r r e d to a clean tube, and dried under n i t r o g e n ( w a t e r b a t h temp. 40°). To th e dried e x t r a c t was added 0.1 ml of the periodic acid solution. T he t u be was r o t a t e d so t h a t t he acid solution came into contact with all of the dried residue, stoppered, and kept at room t e m p e r a t u r e , in t he dark, f o r 90 min. 0.5 ml of w a t e r was added, and a f u r t h e r e x t r a c t i o n carried out with 4 ml chloroform. The aqueous phase was discarded and the organic phase w as h ed with 1 ml h a l f - s a t u r a t e d sodium bicarbonate followed by 1 ml w ater . Th e organic phase was t r a n s f e r r e d to a clean tube, and e v a p o r a t e d to d r y n e s s u n d er nitrogen. T h e tubes c o n t a i n i n g dried e x t r a c t s were stored at -20 ° until assayed. R a d i o i m m u n o a s s a y - - The dried residue was r e c o n s t i t u t e d in 2.0 ml of B u f f e r (1) and a 0.5 ml aliquot t r a n s f e r r e d to a scintillation vial f o r r e co v er y . 1:4 and 1:8 dilutions of t he r e c o n s t i t u t e d e x t r a c t were made in b u f f e r (1) and duplicate 0.1 ml aliquots of each t r a n s f e r r e d to disposable 10 x 75 m m glass t e s t tubes. 2 mg of the t a n d a r d aldosterone ~-lactone was dissolved in 10 ml c h l o r o f o r m / m e t h a n o l 50/50 v-v. This was f u r t h e r diluted 1 in 10 with m e t h a n o l and the n with b u f f e r (1) to give final concentrations of 0.1. 0.2, 0.4, 0.8, and 1.6 ng/0.1 ml. 0.1 ml volumes of t hese s t a n d a r d s were added in triplicate to 10 x 75 m m glass t e s t tubes. 25 ul of ~'H aldosterone was dried under ni t rogen and oxidized following the above p r o c e d u r e ; however, t h e sodium bicarbonate wash was omitted. T h e dried residue was r e c o n s t i t u t e d in 2.0 ml b u f f e r (1) and a 0.1 ml aliquot tak en f o r r a d i o a c t i v i t y counting. T he r e m a i n i n g :~H aldosterone ~/-lactone was diluted with b u f f e r (1) to give a p p r o x i m a t e l y 20,000 c p m / 0.- ml. 0.1 ml of this solution was added to each of t he s t a n d a r d and sample tubes. 0.3 ml of a 1/1,800 aldosterone ~-lactone a n t i s e r u m dilution in b u f f e r (2) was added to each tube giving a final a n t i s e r u m dillution of 1/3,000 and a f in al v-globulin c o n c e n t r a t i o n of 0.6 g/ 100 ml. Th e tubes were v o r t e x e d and incubated at 4: for 30 min. 0.5 ml of cold s a t u r a t e d a m m o n i u m sulphate was added to each tube, and th e tu b es were v o r t e x e d t h e n c e n t r i f u g e d at 3,000 rpm f o r 15 min at 4 °. T h e tubes were r e t u r n e d to a 4 ° ice b a t h and 0.5 ml of t h e s u p e r n a t a n t was p ip etted into counting vials containing 10 ml of "aquasol". T he vials were shaken and counted f or 10 min in a liquid scintillation counter.
288
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C a l c u l a t i o n s - - T he s t a n d a r d curve was c o n s t r u c t e d by plotting t he CPM in t h e a m m o n i u m sulphate s u p e r n a t a n t s agai nst t h e mass of unlabelled aldosterone T-lactone added to t he s t a n d a r d tubes. No correction was m ade f o r th e "H aldosterone added to t he samples f o r r e c o v e r y : since, f o r 609;, recovery, t h e additional r a d i o a c t i v i t y in t h e assay tubes was calculated t o be 15 cpm and could t h e r e f o r e be ignored.
Th e equation below was used to calculate t he aldosterone excretion per 24 h, using th e value of the unknown read o f f t he s t a n d a r d curve. F o r a 1:4 e x t r a c t dilution : Y u g / d a y -- 0.025 where Y
100 X % recovery
× 0.911 X urine volume in litres
---- the value read off the standard curve in ng.
0.025
= t hat factor which allows for the volume of the reconstituted extract taken for radioimmunoassay.
0.911
= ratio of the molecular weights of aldosterone to aldosterone -r-lactone.
% r e c o v e r y --- r e c o v e r y of ~H aldosterone added to t he original uri ne sample. T h e p e r c e n t a g e binding of '~H aldosterone ~-lactone to ant i body was calculated f o r t h e a m m o n i u m sulphate ant i body t i t r a t i o n curve as follows: % bound --
T'--S T'
X 100
where T ' = the radioactivity in 0.5 ml of supernatant in the absence of antiserum.
S
-- the radioactivity in 0.5 ml of supernatant of each antibody dilution.
Plas ma R e n i n A c t i v i t y (P.R.A.) - - P l a s m a renin a c t i v i t y was m e a s u r e d using a modification of t he Schwarz-Mann r a d i o i m m u n o a s s a y m e t h o d (9). Urine Sodium Concentration -F l a me P h o t o m e t e r .
This was m e a s u r e d using an I. L. 143
RESULTS AND DISCUSSION
R e c o v e r y o f 3H A l d o s t e r o n e A d d e d to U r i n e S a m p l e s - - T h e m e a n r e c o v e r y o f :'H a l d o s t e r o n e added to 102 u r i n e s a m p l e s w a s 61.1'/; w i t h a s t a n d a r d d e v i a t i o n of 8.5%. R a t e o f A t t a i n m e n t o f E q u i l i b r i u m m :'H A l d o s t e r o n e T-lactone w a s added to a 1 / 6 , 0 0 0 d i l u t i o n o f t h e a l d o s t e r o n e T-lactone a n t i s e r u m . A t
RADIOIMMUNOASSAY
OF ALDOSTERONE
289
100-
90"
• ammonium sulphate sepn. o charcoal sepn.
80-
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60-
0
c 0
L.
¢~ 50"O a
z
40-
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=
° =
n
30-
20-
10-
1/500
I
i
i
I
I
1/1000
1/2000
1/4000
1/8000
1/16000
~
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2000
1/64000
antibody d i l u t i o n Fig. 1. Antibody titration curves using dextran coated charcoal and ammonium sulphate separation procedures.
timed intervals 0.5 ml aliquots were removed and the bound and free radioactivity separated using ammonium sulphate. The percentage binding of the ~H compound was estimated as in the radioimmunoassay procedure.
S T U A R T et al.
290
/ sample
1600"
A
1400-
~sample B
1200"
O
"-a 1 0 0 0 " a @
o"-
8 00'
0
a
600-
& 400-
200-
1'o
2'o
3'o
!
40
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~.Ls extract assayed Fig. 2. Relationship between the volumes of extract used in the radioimmunoassay procedure and their aldosterone content.
It was found t h a t equilibrium, i.e., maximal binding of the added ~H aldosterone ~-lactone, was arrived at in less t h a n 10 min. at 4 °. A n t i b o d y T i t r a t i o n C u r v e - - Serial dilutions up to 1/128,000 were made of the a n t i s e r u m in b u f f e r s (1) and (2). 1.0 ml volumes were added to tubes containing dried ~H aldosterone ~/-lactone. A f t e r 30 min. incubation
RADIOIMMUNOASSAY OF ALDOSTERONE TABLE 2
TABLE 1 WITHIN BATCH VARIATION
BETWEEN BATCH VARIATION
Where N = The number of assaysper sample.
ALDOSTERONE CONCENTRATION
Urine A 21.8 ng/ml 23.3 ng/ml 23.6 ng/ml
Urine B 50.0 ng/ml 50.5 ng/ml 50.6 ng/ml
291
Sample l 2 3
Mean Aldosterone Concentration N ng/ml C.V. % 8 22.6 8 13 11.4 15 13 22.4 9
at 4 ° , the antibody bound and free radioactivity were separated by adding 1 ml of ice-cold s a t u r a t e d a m m o n i u m sulphate to the tubes containing b u f f e r (2) and 0.1 ml of Dextran and coated charcoal suspension to t h e others. Following centrifugation at 4 °, aliquots of the s u p e r n a t a n t were removed for counting. The t i t r a t i o n curves are shown in Fig. 1. As m a y be seen, the degree of binding at low antibody dilution is g r e a t e r when ammonium sulphate is used for the separation procedure t h a n when charcoal is used. The apparent failure of the antibody to bind more t h a n 90% of the added aldosterone ~-lactone even in the presence of gross antibody excess, when dextran-coated charcoal was used as the separating agent, is possibly due to non-specific absorption of about 10% of the antigen-antibody complex to the dextran-coated charcoal. This effect has been previously described in the r a d i o i m m u n o a s s a y of insulin ( 1 0 ) . As the a m m o n i u m sulphate procedure is routinely used in this laborat o r y for other steroid radioimmunoassays, it was decided to use it in this assay. The dextran-coated charcoal procedure, however, is equally efficient. Blank - - When distilled w a t e r was carried t h r o u g h the entire procedure, the resulting blank values ranged f r o m 0 t o 1 n g / m l . This represents a f u r t h e r advantage of the technique described here. The problem of non-specific blanks due to solvent e x t r a c t s and materials used in chromotographic separations has recently been discussed ( 1 1 ) .
Linearity - - The linearity of the procedure was checked by a s s a y i n g increasing volumes of the e x t r a c t s of 2 urines taken t h r o u g h the method. The results are shown in Fig. 2. A linear relationship held up to values of 1.4 ng on the s t a n d a r d curve. F r o m this, it m a y be assumed t h a t the material assayed in the urine samples and t h a t used in t h e s t a n d a r d curve are identical in t h e i r cross-reactivity with the antibody.
292
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et al.
TABLE 3 REPRODUCIBILITY OF THE STANDARD
CUnVE Aldosterone v-lactone ng
F/Fo
0.1 0.2 0.4 O.8 1.6
1.43 1.63 1.89 2.10 2.30
C.V. % 4.2 4.2 5.0 5.0 5.0
Where F = No. of cpm in the INH4)._,SO,t supernatant of the standards. Fo = No. of cpm in the INH4),.,EO.~ s u p e r n a t a n t in the absence of unlabelled aldosterone -/-lactone.
Reproducibility of Results - - Within batch variation was evaluated as follows: 3 aliquots of 2 urine samples were extracted, oxidized, and assayed as 6 separate samples. The results are shown in Table 1, while between batch is shown in Table 2. Oxidation of Aldosterone to Aldosterone y-Lactone m The completeness of oxidation was checked by treated 5 /zg of adosterone with 0.1 ml of t h e periodic acid mixture. The oxidized material was t h e n c h r o m a t o g r a p h e d on thin layer plates of silica gel GF 254 (Solvent System, ethyl acetate cyelohexane 60/40 v-v), along with s t a n d a r d s of aldosterone and aldosterone y-lactone. The degree of oxidation was determined by visualization under UV light. Oxidation was found to be complete a f t e r 90 min. Reproducibility of the Standard Curve ~ This was examined by comparing s t a n d a r d curves run on 12 separate occasions; the results shown in Table 3. The between batch variation of the s t a n d a r d curve is much lower t h a n the between batch variation of the urine samples. This difference would appear to be due to variation introduced by the oxidation and extraction procedures which m a y not be fully compensated for by the use of 3H aldosterone as an index of the overall recovery. Stability of 3H Aldosterone y-Lactone ~ The ~H aldosterone y-lactone was routinely used within 4 days of oxidation, as it was found t h a t the sensitivity of the s t a n d a r d curve decreased if the ~H compound was used beyond this time. Stability of Oxidized Urine Extracts - - Following oxidation and purification, t h e urine e x t r a c t s have been shown to be stable when stored at 4 ° for at least 2 weeks, or when kept overnight at room t e m p e r a t u r e . This is advantageous in t h a t a large n u m b e r of samples m a y be prepared over a period of days and then measured concurrently by radioimmuoassay.
RADIOIMMUNOASSAY
293
OF ALDOSTERONE
TABLE 4 RESULTS OF CLINICAL STUDIES
Na +
Exrcetion
PRA
meq/24 hrs
ng/ml/hr
Subject
Dayl
Day5
1 2 3 4 5 6 7 8 9 10 11 12 Range
268 155 136 152 140 219 164 252 167 271 156 ll2
21 20 14 19 10 19 27 34 18 15 9 24
Dayl 1.8 1.2 1.9 0.5 2.2 1.0 3.8* 2.2 1.6 1.5 2.0 3.0
Day5 3.2 2.7 6.2 3.0 4.4 5.6 14.3" 8.6 3.9 5.2 6.8 3.5
Aldosterone Excretion ug/24 hrs Dayl
Day5
4 33 8 18 9 30 7 48 14 56 8 16 5 14 14 28 7 23 14 63 8 30 8 lO 4 - 14 10- 63 ( 4 - 17) (20-67)
*Taken at 1200 hrs. Figures in brackets from ref. 12.
Clinical Studies - - 24-hour urine samples were collected from 12 normal subjects on an unrestricted sodium diet. A 10 m e q / d a y sodium diet w a s then instituted for f o u r days. Following this, f u r t h e r 24-hour urine samples were collected from the subjects. U r i n a r y sodium and aldosterone p H 1 labile fraction excretion were estimated on both samples. Plasma samples for the a s s a y of P R A were taken on both days at 0800 hours. The results are shown in Table 4. The range of values for u r i n a r y aldosterone excretion agrees well with t h a t of Schambelan et al (12) who used a m o r e complex procedure. The expected rise in aldosterone excretion following a low sodium diet is evident in 11 cases. In case n u m b e r 10, t h e r e w a s a m a r k e d aldosterone response with a poor renin response; and in case 12, the opposite effect occurred.
The m e t h o d described here is basically the same as t h a t of F a r m e r et al (1) with slight modifications. It is much less time consuming t h a n those previously described. Small volumes of urine only are required and this obviates the necessity for using large volumes of organic solvents. A urine volume of 2 ml is used compared to 25 ml used in t h e above mentioned procedure. A single extraction using 20 ml of chloroform w a s found to be adequate and the acetic acid w a s h of chloroform e x t r a c t h a s been found to be unnecessary. The r a d i o i m m u n o a s s a y procedure has been
294
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altered, in t h a t s a t u r a t e d a m m o n i u m s u l p h a t e h a s been used to s e p a r a t e t h e a n t i b o d y b o u n d and f r e e r a d i o a c t i v i t y r a t h e r t h a n charcoal. T h e r e p r o d u c i b i l i t y of t h e m e t h o d a p p e a r s a d e q u a t e f o r clinical p u r p o s e s . H o w e v e r , t h e b e t w e e n b a t c h v a r i a t i o n is q u i t e m a r k e d a n d we a r e u n a b l e to a c c o u n t f o r this. T h e c o m m e r c i a l a v a i l a b i l i t y of t h e a l d o s t e r o n e a - l a c t o n e a n t i s e r u m a n d t h e lack of r e q u i r e m e n t f o r c o m p l e x c h r o m a t o g r a p h i c p u r i f i c a t i o n would allow t h i s a s s a y to be c a r r i e d o u t in a n y r o u t i n e l a b o r a t o r y w i t h facilities f o r liquid scintillation c o u n t i n g .
REFERENCES 1. FARMER, R. W., ROUP, W. G. Jr., PELLIZZARI, E. D., and FABRE, L. F. Jr. A rapid aldosterone radioimmunoassay. J. Clin. Endocr. 34, 1'8-22 (1972). 2. NOWACZYNSKI, W., KOIW, E., and GENEST, J. Chemical method for the de~ termination of urinary aldosterone. Can. J. Biochem. and Physiology 35, 425-443 (1957). 3. UNDERWOOD, R. H., FLOOD, C. A., TAIT, S. A. S., and TAIT, J. F. A comparison of methods for the acid hydrolysis of a urinary conjugate of aldosterone. J. Clin. Endocr. and Metab. 21, 1092-1098 (1961). ~. SOBEL, C., HENRY, R. J., GOLUB, O. J., and RUDY, M. Chemical determination of aldosterone in urine. J. Clin. Endocr. and Metab. 19, 1302-1311 (1959). 5. KLIMAN, B., and PETERSON, R. E. Double isotope derivative assay of aldosterone in bioloegical extracts. J. Biol. Chem. 235, 1639-1648 (1960). 6. RAPP, J. P., and EIK-NES, K. B. Determination of desoxycorticosterone and aldosterone in biological samples by gas chromatography with electron capture detection. Analytical Biochem. 15, 386-408 (1966). 7. BRAVO, E. L., and TRAVIS, R. H. A quantitative gas-liquid chromatograph|c method for aldosterone in human urine. J. Lab. Clin. Med. 70, 831-840 (1967). 8. LEUNG, F. Y., and GRIFFITHS, J. A chromatographic method for aldosterone determination in human urine. Clin. Chim. Acta. 37, 423-432 (1972). 9. VIOL, G. W., KEANE, P. M., SPEED, J., and SMITH, E. K. The assay of plasma renin activity using commercially available reagents. Clin. Biochem. 5, 251265 (1972). 10. KEANE, P. M., PEARSON, J., and WALKER, ~V. H. C. Dextran coated charcoal
radioimmunoassay of insulin. Diabetelogia 4, 339-344 (1968). 11. LEYENDICKER, G., WARDLAW, S., and NOCKE, W. Gamma globulin pro-
tection of radioimmunoassay and competitive protein binding saturation of steroids. J. Clin. Endocr. 34, 430-433 (1972). 12. SCHAMBELAN, M., STOCKIGT, J. R., and BIGLIERI, E. G. Isolated hypoaldosteronism in adults. N. E. J. Med. 287, 573-578 (1972).
A R A P I D METHOD FOR THE R A D I O I M M U N O A S S A Y OF U R I N A R Y A L D O S T E R O N E
J. S T U A R T , P. M. K E A N E , G. VIOL, and R. N. G U P T A
Department of Laboratory Medicine and Medicine, St. Joseph's Hospital, Hamilton, Ontario. (Received February 1~, 1973)
CLBIA, 6 (4): 285-294 (1973)
Clin. Biochem. Stuart, J., Keane, P. M., Viol, G. and Gupta, R. N.
Departments of Labarato~ T Medicine and Medicine, St. Joseph's Hospital, Hamilton 20, Ontario. A R A P I D M E T H O D F O R T H E R A D I O I M M U N O A S S A Y OF U R I N A R Y ALDOSTERONE 1. The availability of an antiserum to aldosterone 7-1actone allows the rapid assay of aldosterone in urine (1). 2. Following acid hydrolysis of the 18 oxo-conjugate, aldosterone is oxidized to its 7-1actone. 3. The extremely low cross reactivity of the antiserum with structurally related steroids obviates the need f o r chromatographic purification before radio° immunoassay. 4. This procedure is sensitive enough to allow the assay of aldosterone in the equivalent of 25 UL of hydrolyzed urine.
A NUMBER OF COLORIMETRIC AND FLUORIMETRIC PROCEDURES have been described for the a s s a y of urinary aldosterone (2-4). Double isotope derivative (5) and gas liquid chromatographic (6-8) techniques have also been used. In order to achieve the desired degree of specificity all of these methods have utilised multiple chromatographic steps in the isolation of the compound. These m e t h o d s are time consuming and necessitate the extraction of large volumes of urine; and as a result, the handling of large volumes of organic solvents.
Correspondence: Dr. P. M. Keane, Department of Laboratory Medicine, St. Joseph's Hospital, Hamilton 20, Ontario.
286
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The procedure described here is basically that described by Farmer et al (i) and utilizes a commercially available anti-serum raised against aldosterone 7-1actone. Purification is achieved by differential solvent extraction. MATERIALS
AND
EQUIPMENT
D-Aldosterone (&~-Pregnen-18-Al-llb, 21-Diol-3,20-Dione) Aldosterone ~-Lactone (3-oxo-11~, 18 Epoxy-androst-4-ene-17~. 1 8 - C a r b o l a c t o n e ) . S i g m a Cat. No.'s A 6628 a n d A 1385 respectively. A l d o s t e r o n e - 1 , 2-'~H, 53 C l / m m o l e , A m e r s h a m Searle. All of the above w e r e used w i t h out further purification. A l d o s t e r o n e "~-lactone a n t i s e r u m ( L o t 185-5-71). A n t i b o d i e s Inc. Davis, C a l i f o r n i a . H u m a n "~-globulin - - C o n n a u g h t Medical R e s e a r c h Lab., T o r o n t o ChloroformFisher, Reagent Grade Benzene -- Analar BDH Periodic Acid - - F r e d r i c k S m i t h Chem. Co., Columbus, Ohio Dioxane -- Fisher, Reagent Grade P y r i d i n e - - A n a l a r , B D H , redistilled a n d stored in a b r o w n bottle A q u a s o l S c i n t i l l a t i o n Cocktail - - N e w E n g l a n d N u c l e a r B u f f e r (1) 1 g g e l a t i n w a s dissolved in 100 ml h o t w a t e r . 3.09 g boric acid w a s dissolved in 300 ml w a t e r a n d t h e p H a d j u s t e d to 8.5 w i t h sodium h y d r o x i d e . T h e g e l a t i n solution w a s a d d e d a n d t h e volume m a d e up to 1 1. B u f f e r (2) - - Add h u m a n ~-globulin to b u f f e r (1) to o b t a i n 1 g / 1 0 0 m l v - g l o b u l i n solution. Periodic acid solution - - 100 m g periodic acid w a s dissolved in 5.0 ml w a t e r . 5.0 ml dioxa n e a n d 0.1 m l p y r i d e w a s added a n d the solution mixed. The acid solution w a s m a d e u p f r e s h f o r each oxidation. S a t u r a t e d a m m o n i u m s u l p h a t e - - A s a t u r a t e d solution w a s p r e p a r e d in deionized w a t e r . T h i s solution w a s f i l t e r e d t h r o u g h W h a t m a n ~:2 p a p e r p r i o r to use. D e x t r a n coated c h a r c o a l - - 100 m g d e x t r a n 70 ( P h a r m a c i a ) a n d 1 g N o r i t A C h a r c o a l w a s added to 100 ml of 0.1 M p h o s p h a t e b u f f e r p H 7.4. F l a s h e v a p o r a t o r - - Bfichi R o t o v a p o r Rinco I n s t r u m e n t Co. Inc., Greenville, Ill. (Canlab). Liquid scintillation system - - N u c l e a r Chicago U n i l u x I I - - A S o r v a l G e n e r a l P u r p o s e RC*3 a u t o m a t i c r e f r i g e r a t e d C e n t r i f u g e , Allied Scientific Co., Toronto.
METHODS
A 2 ml aliquot of a 24 h. urine sample was adjusted to pH 1 with 2 N HCI, and 3,000 cpm :'H aldosterone was added. Hydrolysis was allowed to proceed 20-24 h. at room temperature. The urine was saturated with sodium sulphate, transferred to a clean test tube, and extracted once with 20 ml of chloroform. The aqueous phase was removed and the organic phase washed with 1 ml 0.1 N sodium hydroxide and then I ml water. The organic phase was reduced in volume to approximately 0.2 ml using a flash evaporator (water bath temperature 40 °, transferred with 2 x 0.3 ml rinses of chloroform into a clean test tube, and evaporated to dryness under high purity nitrogen. B ml water and 1 ml benzene were added to the tube and the contents
R A D I O I M M U N O A S S A Y OF A L D O S T E R O N E
287
well mixed. The tube was c e n t r i f u g e d and the aqueous phase t r a n s f e r r e d to a clean t e s t tube. This was e x t r a c t e d w i th 6 ml chloroform, t he organic phase t r a n s f e r r e d to a clean tube, and dried under n i t r o g e n ( w a t e r b a t h temp. 40°). To th e dried e x t r a c t was added 0.1 ml of the periodic acid solution. T he t u be was r o t a t e d so t h a t t he acid solution came into contact with all of the dried residue, stoppered, and kept at room t e m p e r a t u r e , in t he dark, f o r 90 min. 0.5 ml of w a t e r was added, and a f u r t h e r e x t r a c t i o n carried out with 4 ml chloroform. The aqueous phase was discarded and the organic phase w as h ed with 1 ml h a l f - s a t u r a t e d sodium bicarbonate followed by 1 ml w ater . Th e organic phase was t r a n s f e r r e d to a clean tube, and e v a p o r a t e d to d r y n e s s u n d er nitrogen. T h e tubes c o n t a i n i n g dried e x t r a c t s were stored at -20 ° until assayed. R a d i o i m m u n o a s s a y - - The dried residue was r e c o n s t i t u t e d in 2.0 ml of B u f f e r (1) and a 0.5 ml aliquot t r a n s f e r r e d to a scintillation vial f o r r e co v er y . 1:4 and 1:8 dilutions of t he r e c o n s t i t u t e d e x t r a c t were made in b u f f e r (1) and duplicate 0.1 ml aliquots of each t r a n s f e r r e d to disposable 10 x 75 m m glass t e s t tubes. 2 mg of the t a n d a r d aldosterone ~-lactone was dissolved in 10 ml c h l o r o f o r m / m e t h a n o l 50/50 v-v. This was f u r t h e r diluted 1 in 10 with m e t h a n o l and the n with b u f f e r (1) to give final concentrations of 0.1. 0.2, 0.4, 0.8, and 1.6 ng/0.1 ml. 0.1 ml volumes of t hese s t a n d a r d s were added in triplicate to 10 x 75 m m glass t e s t tubes. 25 ul of ~'H aldosterone was dried under ni t rogen and oxidized following the above p r o c e d u r e ; however, t h e sodium bicarbonate wash was omitted. T h e dried residue was r e c o n s t i t u t e d in 2.0 ml b u f f e r (1) and a 0.1 ml aliquot tak en f o r r a d i o a c t i v i t y counting. T he r e m a i n i n g :~H aldosterone ~/-lactone was diluted with b u f f e r (1) to give a p p r o x i m a t e l y 20,000 c p m / 0.- ml. 0.1 ml of this solution was added to each of t he s t a n d a r d and sample tubes. 0.3 ml of a 1/1,800 aldosterone ~-lactone a n t i s e r u m dilution in b u f f e r (2) was added to each tube giving a final a n t i s e r u m dillution of 1/3,000 and a f in al v-globulin c o n c e n t r a t i o n of 0.6 g/ 100 ml. Th e tubes were v o r t e x e d and incubated at 4: for 30 min. 0.5 ml of cold s a t u r a t e d a m m o n i u m sulphate was added to each tube, and th e tu b es were v o r t e x e d t h e n c e n t r i f u g e d at 3,000 rpm f o r 15 min at 4 °. T h e tubes were r e t u r n e d to a 4 ° ice b a t h and 0.5 ml of t h e s u p e r n a t a n t was p ip etted into counting vials containing 10 ml of "aquasol". T he vials were shaken and counted f or 10 min in a liquid scintillation counter.
288
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C a l c u l a t i o n s - - T he s t a n d a r d curve was c o n s t r u c t e d by plotting t he CPM in t h e a m m o n i u m sulphate s u p e r n a t a n t s agai nst t h e mass of unlabelled aldosterone T-lactone added to t he s t a n d a r d tubes. No correction was m ade f o r th e "H aldosterone added to t he samples f o r r e c o v e r y : since, f o r 609;, recovery, t h e additional r a d i o a c t i v i t y in t h e assay tubes was calculated t o be 15 cpm and could t h e r e f o r e be ignored.
Th e equation below was used to calculate t he aldosterone excretion per 24 h, using th e value of the unknown read o f f t he s t a n d a r d curve. F o r a 1:4 e x t r a c t dilution : Y u g / d a y -- 0.025 where Y
100 X % recovery
× 0.911 X urine volume in litres
---- the value read off the standard curve in ng.
0.025
= t hat factor which allows for the volume of the reconstituted extract taken for radioimmunoassay.
0.911
= ratio of the molecular weights of aldosterone to aldosterone -r-lactone.
% r e c o v e r y --- r e c o v e r y of ~H aldosterone added to t he original uri ne sample. T h e p e r c e n t a g e binding of '~H aldosterone ~-lactone to ant i body was calculated f o r t h e a m m o n i u m sulphate ant i body t i t r a t i o n curve as follows: % bound --
T'--S T'
X 100
where T ' = the radioactivity in 0.5 ml of supernatant in the absence of antiserum.
S
-- the radioactivity in 0.5 ml of supernatant of each antibody dilution.
Plas ma R e n i n A c t i v i t y (P.R.A.) - - P l a s m a renin a c t i v i t y was m e a s u r e d using a modification of t he Schwarz-Mann r a d i o i m m u n o a s s a y m e t h o d (9). Urine Sodium Concentration -F l a me P h o t o m e t e r .
This was m e a s u r e d using an I. L. 143
RESULTS AND DISCUSSION
R e c o v e r y o f 3H A l d o s t e r o n e A d d e d to U r i n e S a m p l e s - - T h e m e a n r e c o v e r y o f :'H a l d o s t e r o n e added to 102 u r i n e s a m p l e s w a s 61.1'/; w i t h a s t a n d a r d d e v i a t i o n of 8.5%. R a t e o f A t t a i n m e n t o f E q u i l i b r i u m m :'H A l d o s t e r o n e T-lactone w a s added to a 1 / 6 , 0 0 0 d i l u t i o n o f t h e a l d o s t e r o n e T-lactone a n t i s e r u m . A t
RADIOIMMUNOASSAY
OF ALDOSTERONE
289
100-
90"
• ammonium sulphate sepn. o charcoal sepn.
80-
70e)
c a u
o
60-
0
c 0
L.
¢~ 50"O a
z
40-
°-
=
° =
n
30-
20-
10-
1/500
I
i
i
I
I
1/1000
1/2000
1/4000
1/8000
1/16000
~
1/
i
2000
1/64000
antibody d i l u t i o n Fig. 1. Antibody titration curves using dextran coated charcoal and ammonium sulphate separation procedures.
timed intervals 0.5 ml aliquots were removed and the bound and free radioactivity separated using ammonium sulphate. The percentage binding of the ~H compound was estimated as in the radioimmunoassay procedure.
S T U A R T et al.
290
/ sample
1600"
A
1400-
~sample B
1200"
O
"-a 1 0 0 0 " a @
o"-
8 00'
0
a
600-
& 400-
200-
1'o
2'o
3'o
!
40
i
50
~.Ls extract assayed Fig. 2. Relationship between the volumes of extract used in the radioimmunoassay procedure and their aldosterone content.
It was found t h a t equilibrium, i.e., maximal binding of the added ~H aldosterone ~-lactone, was arrived at in less t h a n 10 min. at 4 °. A n t i b o d y T i t r a t i o n C u r v e - - Serial dilutions up to 1/128,000 were made of the a n t i s e r u m in b u f f e r s (1) and (2). 1.0 ml volumes were added to tubes containing dried ~H aldosterone ~/-lactone. A f t e r 30 min. incubation
RADIOIMMUNOASSAY OF ALDOSTERONE TABLE 2
TABLE 1 WITHIN BATCH VARIATION
BETWEEN BATCH VARIATION
Where N = The number of assaysper sample.
ALDOSTERONE CONCENTRATION
Urine A 21.8 ng/ml 23.3 ng/ml 23.6 ng/ml
Urine B 50.0 ng/ml 50.5 ng/ml 50.6 ng/ml
291
Sample l 2 3
Mean Aldosterone Concentration N ng/ml C.V. % 8 22.6 8 13 11.4 15 13 22.4 9
at 4 ° , the antibody bound and free radioactivity were separated by adding 1 ml of ice-cold s a t u r a t e d a m m o n i u m sulphate to the tubes containing b u f f e r (2) and 0.1 ml of Dextran and coated charcoal suspension to t h e others. Following centrifugation at 4 °, aliquots of the s u p e r n a t a n t were removed for counting. The t i t r a t i o n curves are shown in Fig. 1. As m a y be seen, the degree of binding at low antibody dilution is g r e a t e r when ammonium sulphate is used for the separation procedure t h a n when charcoal is used. The apparent failure of the antibody to bind more t h a n 90% of the added aldosterone ~-lactone even in the presence of gross antibody excess, when dextran-coated charcoal was used as the separating agent, is possibly due to non-specific absorption of about 10% of the antigen-antibody complex to the dextran-coated charcoal. This effect has been previously described in the r a d i o i m m u n o a s s a y of insulin ( 1 0 ) . As the a m m o n i u m sulphate procedure is routinely used in this laborat o r y for other steroid radioimmunoassays, it was decided to use it in this assay. The dextran-coated charcoal procedure, however, is equally efficient. Blank - - When distilled w a t e r was carried t h r o u g h the entire procedure, the resulting blank values ranged f r o m 0 t o 1 n g / m l . This represents a f u r t h e r advantage of the technique described here. The problem of non-specific blanks due to solvent e x t r a c t s and materials used in chromotographic separations has recently been discussed ( 1 1 ) .
Linearity - - The linearity of the procedure was checked by a s s a y i n g increasing volumes of the e x t r a c t s of 2 urines taken t h r o u g h the method. The results are shown in Fig. 2. A linear relationship held up to values of 1.4 ng on the s t a n d a r d curve. F r o m this, it m a y be assumed t h a t the material assayed in the urine samples and t h a t used in t h e s t a n d a r d curve are identical in t h e i r cross-reactivity with the antibody.
292
STUART
et al.
TABLE 3 REPRODUCIBILITY OF THE STANDARD
CUnVE Aldosterone v-lactone ng
F/Fo
0.1 0.2 0.4 O.8 1.6
1.43 1.63 1.89 2.10 2.30
C.V. % 4.2 4.2 5.0 5.0 5.0
Where F = No. of cpm in the INH4)._,SO,t supernatant of the standards. Fo = No. of cpm in the INH4),.,EO.~ s u p e r n a t a n t in the absence of unlabelled aldosterone -/-lactone.
Reproducibility of Results - - Within batch variation was evaluated as follows: 3 aliquots of 2 urine samples were extracted, oxidized, and assayed as 6 separate samples. The results are shown in Table 1, while between batch is shown in Table 2. Oxidation of Aldosterone to Aldosterone y-Lactone m The completeness of oxidation was checked by treated 5 /zg of adosterone with 0.1 ml of t h e periodic acid mixture. The oxidized material was t h e n c h r o m a t o g r a p h e d on thin layer plates of silica gel GF 254 (Solvent System, ethyl acetate cyelohexane 60/40 v-v), along with s t a n d a r d s of aldosterone and aldosterone y-lactone. The degree of oxidation was determined by visualization under UV light. Oxidation was found to be complete a f t e r 90 min. Reproducibility of the Standard Curve ~ This was examined by comparing s t a n d a r d curves run on 12 separate occasions; the results shown in Table 3. The between batch variation of the s t a n d a r d curve is much lower t h a n the between batch variation of the urine samples. This difference would appear to be due to variation introduced by the oxidation and extraction procedures which m a y not be fully compensated for by the use of 3H aldosterone as an index of the overall recovery. Stability of 3H Aldosterone y-Lactone ~ The ~H aldosterone y-lactone was routinely used within 4 days of oxidation, as it was found t h a t the sensitivity of the s t a n d a r d curve decreased if the ~H compound was used beyond this time. Stability of Oxidized Urine Extracts - - Following oxidation and purification, t h e urine e x t r a c t s have been shown to be stable when stored at 4 ° for at least 2 weeks, or when kept overnight at room t e m p e r a t u r e . This is advantageous in t h a t a large n u m b e r of samples m a y be prepared over a period of days and then measured concurrently by radioimmuoassay.
RADIOIMMUNOASSAY
293
OF ALDOSTERONE
TABLE 4 RESULTS OF CLINICAL STUDIES
Na +
Exrcetion
PRA
meq/24 hrs
ng/ml/hr
Subject
Dayl
Day5
1 2 3 4 5 6 7 8 9 10 11 12 Range
268 155 136 152 140 219 164 252 167 271 156 ll2
21 20 14 19 10 19 27 34 18 15 9 24
Dayl 1.8 1.2 1.9 0.5 2.2 1.0 3.8* 2.2 1.6 1.5 2.0 3.0
Day5 3.2 2.7 6.2 3.0 4.4 5.6 14.3" 8.6 3.9 5.2 6.8 3.5
Aldosterone Excretion ug/24 hrs Dayl
Day5
4 33 8 18 9 30 7 48 14 56 8 16 5 14 14 28 7 23 14 63 8 30 8 lO 4 - 14 10- 63 ( 4 - 17) (20-67)
*Taken at 1200 hrs. Figures in brackets from ref. 12.
Clinical Studies - - 24-hour urine samples were collected from 12 normal subjects on an unrestricted sodium diet. A 10 m e q / d a y sodium diet w a s then instituted for f o u r days. Following this, f u r t h e r 24-hour urine samples were collected from the subjects. U r i n a r y sodium and aldosterone p H 1 labile fraction excretion were estimated on both samples. Plasma samples for the a s s a y of P R A were taken on both days at 0800 hours. The results are shown in Table 4. The range of values for u r i n a r y aldosterone excretion agrees well with t h a t of Schambelan et al (12) who used a m o r e complex procedure. The expected rise in aldosterone excretion following a low sodium diet is evident in 11 cases. In case n u m b e r 10, t h e r e w a s a m a r k e d aldosterone response with a poor renin response; and in case 12, the opposite effect occurred.
The m e t h o d described here is basically the same as t h a t of F a r m e r et al (1) with slight modifications. It is much less time consuming t h a n those previously described. Small volumes of urine only are required and this obviates the necessity for using large volumes of organic solvents. A urine volume of 2 ml is used compared to 25 ml used in t h e above mentioned procedure. A single extraction using 20 ml of chloroform w a s found to be adequate and the acetic acid w a s h of chloroform e x t r a c t h a s been found to be unnecessary. The r a d i o i m m u n o a s s a y procedure has been
294
STUART et al.
altered, in t h a t s a t u r a t e d a m m o n i u m s u l p h a t e h a s been used to s e p a r a t e t h e a n t i b o d y b o u n d and f r e e r a d i o a c t i v i t y r a t h e r t h a n charcoal. T h e r e p r o d u c i b i l i t y of t h e m e t h o d a p p e a r s a d e q u a t e f o r clinical p u r p o s e s . H o w e v e r , t h e b e t w e e n b a t c h v a r i a t i o n is q u i t e m a r k e d a n d we a r e u n a b l e to a c c o u n t f o r this. T h e c o m m e r c i a l a v a i l a b i l i t y of t h e a l d o s t e r o n e a - l a c t o n e a n t i s e r u m a n d t h e lack of r e q u i r e m e n t f o r c o m p l e x c h r o m a t o g r a p h i c p u r i f i c a t i o n would allow t h i s a s s a y to be c a r r i e d o u t in a n y r o u t i n e l a b o r a t o r y w i t h facilities f o r liquid scintillation c o u n t i n g .
REFERENCES 1. FARMER, R. W., ROUP, W. G. Jr., PELLIZZARI, E. D., and FABRE, L. F. Jr. A rapid aldosterone radioimmunoassay. J. Clin. Endocr. 34, 1'8-22 (1972). 2. NOWACZYNSKI, W., KOIW, E., and GENEST, J. Chemical method for the de~ termination of urinary aldosterone. Can. J. Biochem. and Physiology 35, 425-443 (1957). 3. UNDERWOOD, R. H., FLOOD, C. A., TAIT, S. A. S., and TAIT, J. F. A comparison of methods for the acid hydrolysis of a urinary conjugate of aldosterone. J. Clin. Endocr. and Metab. 21, 1092-1098 (1961). ~. SOBEL, C., HENRY, R. J., GOLUB, O. J., and RUDY, M. Chemical determination of aldosterone in urine. J. Clin. Endocr. and Metab. 19, 1302-1311 (1959). 5. KLIMAN, B., and PETERSON, R. E. Double isotope derivative assay of aldosterone in bioloegical extracts. J. Biol. Chem. 235, 1639-1648 (1960). 6. RAPP, J. P., and EIK-NES, K. B. Determination of desoxycorticosterone and aldosterone in biological samples by gas chromatography with electron capture detection. Analytical Biochem. 15, 386-408 (1966). 7. BRAVO, E. L., and TRAVIS, R. H. A quantitative gas-liquid chromatograph|c method for aldosterone in human urine. J. Lab. Clin. Med. 70, 831-840 (1967). 8. LEUNG, F. Y., and GRIFFITHS, J. A chromatographic method for aldosterone determination in human urine. Clin. Chim. Acta. 37, 423-432 (1972). 9. VIOL, G. W., KEANE, P. M., SPEED, J., and SMITH, E. K. The assay of plasma renin activity using commercially available reagents. Clin. Biochem. 5, 251265 (1972). 10. KEANE, P. M., PEARSON, J., and WALKER, ~V. H. C. Dextran coated charcoal
radioimmunoassay of insulin. Diabetelogia 4, 339-344 (1968). 11. LEYENDICKER, G., WARDLAW, S., and NOCKE, W. Gamma globulin pro-
tection of radioimmunoassay and competitive protein binding saturation of steroids. J. Clin. Endocr. 34, 430-433 (1972). 12. SCHAMBELAN, M., STOCKIGT, J. R., and BIGLIERI, E. G. Isolated hypoaldosteronism in adults. N. E. J. Med. 287, 573-578 (1972).