~IOIM~OASSAYS Received: y/14/76
IN PLASMA AND URINE
A.B.KURTZ* and F.C.D~TTER, The National Heart and Lung Institute, The National Institutes of Health, Bethesda, Maryland. ABSTRACT:Radioimmunoassays for aldosterone and deoxycorticosterone (DOC)e described in which a simple separation procedure using a~onium sulfate stabilization of bound steroid and extraction of free steroid into toluene scintilla& allows an "in vial" assay without mechanical separation of the two phase system. Extraction and thin layer chrsmatographic methods for purification of aldosterone and DOG are free of solvent and plate blank effects. Normal values are given for unconjugated aldosterone and DOC in urine, for aldosterone and DOC in plasma and for aldosterone 18-glucuronide in urine. Research into the endocrinological aspects of hypertension requires the measurement of the salt
retaining steroids aldosterone
and deoxycorticosterone (DOG): both are present in very low concentrations in plasma and urine in comparison with many other steroids. Radioimmunoassay of steroid haptens is complicated by steroid cross-reactions and by non-specific interference from substances in solvents, chromatography materials, plasma and urine. We use readily available and only moderately specific antisera with extraction and chromatography systems which do not have solvent or plate blank 3ffects: if, however, antisera of particularily high specificity are available a chromatographic or other separation step can be omitted (1,2). Cross-reaction problems increase at low hormone levels and are most troublesome in assays designed for maximum sensitivity (3). The singular characteristic of the 18-glucuronide of aldosterone, its hydrolysis at pH 1, allows easy separation of this metabolite from other steroids so that chromatography is not needed (4). * Current address: Central Middlesex Hospital, London NWlO, England.
Plasma steroid levels fluctuate a great deal so that their measurement is of most value when many samples are taken at close intervals and short term changes are followed. The free, or unconjugated, urinary steroid excretion represents the integrated concentration of unbound steroid in plasma: as there is less variability and problems of protein binding in plasma are avoided it is theoretically a most satisfactory measurement. Urinary free cortisol has been shown to be superior to other direct measurements in assessing oortisol over-production in Gushing's syndrome (5). Conn and his coworkers have described an assay for urinary free aldosterone which they did not find superior to measurement of the 18-glucuronide (6). We report assay systems for both aldosterone and DOC in plasma and, as free steroid, in urine. The excretion of free aldosterone and the 18-gluouronide are compared. MATERIALS AND mTHODS: Aldosterone (Sigma Chem. Corp.), DOC (Schwartz Plan), l-2 3H aldosterone and l-2 3H DOG (New England Nuclear) are checked for purity by paper and thin layer chromatography and used without further purification. All solvents are analytical grade: chloroform (with 1% ethanol) cyclohexane, methanol, methylene chloride (all from the Bodman Chem. Corp.) and t-butanol (Fisher Scientific Corp.) are checked to make sure there are no substances causing interference in the assay and are then used without purification. TLC is on silica gel (Silplate 60-F-254, Merck): the plates are prewashed with hot methanol (65'~) by ascending chromatography. The TLC tanks are lined with methanol washed glass paper. Elution of steroids from the silica gel is through Pasteur pipettes with 1 cm of silioic acid (Sil-b-325, Sigma Chem. Corp.) over a glass wool Plug. The buffer used throughout is TRIS 0.05 mMol/f, adjusted to pH 8 with hydrochloric acid, with sodium chloride 0.34 mMol/f and bovine gamma globulin (Sigma Chem. Corp.) 2g/l. The assays are carried out in 25 ml low potassium glass counting vials (Kimble). The antiserum to aldosterone was raised to the 18,21-dihemisuocinate conjugated to bovine serum albumin (supplied by the Hormone Distribution Committee of the Rational Institutes of Health). The antiserum 30 DOG was raised to the 3-oarboxymethyloxime conjugated to bovine serum albumin by Arnold and James ('i'), (available from the Medical Research Council's Division of
Biological Standards, London). Tritium is counted in toluene scintillant; 42 ml of Liquafluor (New England Nuclear) per liter of toluene (Scintanalyzed grade, Fisher Scientific Corp.). Samples of urine, without preservative, and heparinized plasma are stored at -2O'C before assay. Assay Procedure: To each two to three ml plasma sample 2000 dpm each of tritiated aldosterone and DOC are added for recovery estimation. The samples are extracted with 25 ml of cold methylene chloride and the extracts washed with 2 ml of sodium hydroxide 0.1 Mel/l, then with 2 ml of acetic acid 0.1 Mel/l and lastly with 2 ml of water. Urine samples of volume 5-10 ml are treated similarily. Thg methylene chloride extracts are evaporated to dryness in air at 40 C and the residues partitioned between 4 ml cyclohexane and 4 ml water. The cyclohexane fraction, which contains the DOC, is evaporated to dryness and the residues run on TLC plates in a chloroform: methanol:water system (gg.6:0.25:0.15), with progesterone, 17-hydroxy progesterone, testerone and DOC markers. The plates are run twice to 15 cm. The area containing the DOC is eluted through silicic acid columns in Pasteur pipettes with 3 ml of methanol, the eluate is evaporated to dryness in air at 40°C and the residue dissolved in one ml of buffer. The aqueous fraction containing the aldosterone is extracted with 25 ml of methyisne chloride and after evaporation the residue is transferred to TLC plates with methanol. The plates are run twice to 15 cm in a system of chloroform:methanol:t-butanol (95:2.5:2.5) with markers of coricosterone, cortisone, cortisol, prednisone and aldosterone. Elution is as described for DOG and the residue is dissolved in one ml of buffer. Of each extract 0.3 ml is counted for recovery and two 0.3 ml aliquots assayed. For urinary aldosterone only 0.1 ml is needed in the assay. For the assay of urinary aldosterone 18-glucuronide the free steroids are first extracted from 2 ml of urine with 25 ml of methylene chloride and the urine acidified to pH 1 with concentrated hydrochloric acid. After 24 hours at room temperature one ml of urine is extracted with 25 ml of methylene chloride and the extract washed as described for plasma. After evaporation the residues are dissolved in 5 ml of buffer and 0.05 ml aliquots assayed. The assay standards are made up in buffer. 0.3 ml of standard or unknown is added to each vial in duplicate: with smaller volumes the volume is brought to 0.3 ml with buffer. Standards for aldosterone range from 5 to 300 pg per vial and for DOC from 9 to 600 pg. Twenty to 30 pg of labelled aldosterone or DOC is then added in 0.1 ml of buffer followed by antiserum in 0.1 ml of buffer: the aldosterone antiserum is used at a final dilution of 1:700,000 and the DOC antiserum at 1:4O,c)OO.The total volume incubated is 0.5 ml; the incubation being either at room temperature for two hours or overnight (16h) at 4'C. Then 0.5 ml of a cold saturated solution of ammonium sulfate , pH 8, is added to each vial and the vials shaken. Ten ml of cold scintillant is then added to each vial, the vials are capped, shaken for one minute and then counted, after one hour's
equilbration, in a cooled counter at 4'C. The assay results are calculated by log-logit transformation of the data using a computer program (8). RESULTS: Chromatography: Aldosterone is satisfactorily separated from both cortisol and cortisone. Separation from prednisone is not however possible. The Rf values for the different steroids chromatographed are shown in table I as ratios of the Rf of corticosterone. For DOC separation is also satisfactory and the Rf values are shown as ratios of the Rf of progesterone. Cross-reactivity is also shown in table I at 5Oj%displacement of bound hormone. Table I. Rf ratios for TLC systems for aldosterone and DOG. The per cent cross-reaction is at B/So = 0.5. Rf,/Rf corticosterone cortisol aldosterone prednisone cortisone corticosterone
cross-reaction with aldosterone 0.006
0.47 0.53 0.63 1.00
Rf/Rf progesterone testosterone l'j'-OH-progesterone deoxycorticosterone progesterone
cross-reaction with DOC
0.58 0.63 0.76 1.00
Recovery: After extraction and chromatography the recovery aldosterone from urine averaged 6%
and from plasma 75%. For DOC the
recovery from urine averaged 55y and from plasma 48%. Blank:
The solvents are entirely free of effect on the assay.
They are tested by evaporating 20ml of the solvent, taking up the residue in one ml of buffer and using 0.3 ml of this extract as an unknown in the assay.
Material with considerable effect is eluted
from unwashed TLC plates but this material is removed by washing the plates in hot methanol.
Paper also contains interfering
using methanol washed glass paper to line the TLC
tanks results in no detectable solvent or plate blank effect in either assay.
Plasma and urine from Addisonian patients on full
replaoement therapy oontains small amounts of cross reacting material.
This effect is at or below the level of assay
sensitivity and for both steroids is less than 2 ngf100 ml in plasma and less than 4 r.g/lof urine.
Serial dilution of
aldosterone and DOC in Addisonian plasma or urine give plots which are not statistically different from the standard curve.
correcting for procedural losses the recovery of aldosterone was
95s (95s confidence limits 87-104s) and of DOC 103$, (95% confidence limits 95-113s). The unchromatographed aldosterone from pH 1 hydrolysis of the 18-glucuronidc dilui.ed~~arallelto -i??e ctanJ.r.rd cu.rvc :
~0 crooo rc;;cti. 1~ I ~~~tc~~‘.~hwas detected in urir.e I^rom
addisonian patients on full replacement. Assay Separation Procedure:
Ammonium sulfate reduces the
solubility of aldosterone in water and favours partition to toluene. At equilibrium at 4'C the ratio of aldosterone concentration in toluene to water is 0.24. When 2 Mel/l ammonium sulfate, pH 8, is substituted for the water the ratio is 1.8. There is little further improvement if saturated ammonium sulfate (4 Mel/l) is used;
ratio being 2.1. Ammonium sulfate at 2 Mel/l precipitates IgG and with it bound steroid.
The precipitated complexes are fairly stable:
the rate of
release of steroid was calculated by serial counts of a two phase system of 3H steroid-antiserum complex in 2 Mel/l ammonium sulfate, PW 8, and toluene scintillant.
As dissociation occurs free steroid
enters the toluene phase and the count rate increases. The dissociation rate is markedly temperature dependant and at 6’~
the rate is sufficiently low to allow an assay to 'cc counted without separation of the phases. Dissociation rate values are given in table II. Table II. The dissociation rates and half lives fox -ntibody bound steroid in a 2 Mel/l ammonium sulfate : toluene scintillant system. Steroid
Aldosterone ,t It
0 6 24 6
Q.00063 0.0049 0.013
t; h 1lOcf 143 54
The ammonium sulfate stabilizes the bound steroid. In its absence the dissociation rate is about ten times greater: for aldosterone at b°C it is 0.048 h
. with a half life of 14.6 h.
This separation procedure was compared with conventional ammonium sulfate separation. The precipitate was removed by centrifugation and the supernatant counted in a dispersed scintillant. The assay plots are not significantly different so it appears that counting a two phase system does not introduce any obvious misclassification errors. Assay Data: For both aldosterone and DOC the log-lo&t
formation gives linear plots. The data from a series of assays is summarised in table III. Clinical results: The aldosterone measurements in normals are shown in table Iv. The correlation of the excretion of free aldosterone with the 18-glucuronide is good (r=O.88): the ratio of the excretion of free aldosterone to the 18-glucuronide averaged 0.014.
Table III. Data for the Aldosterone and DOC assays. The results are given with the standard errors of the mean. Aldosterone Number of assays 7 Bound/Total (Be/T) 0.63 Slope -1.09 Correlation coefficient (r) -0.997 Concentration at B/Bo=0.5, pg/ml 117 Minimum detectable dose, pg/vial 2.6 Coefficient of variation: within assay, $ 4.4 between assay, $ 12.2
+/- 0.02 +/- 0.015 +/- 0.0005
0.68 +/- 0.02 -1.03 +/- 0.016 -0.995 +/- 0.0014
Table IV. Aldosterone results in normals. llr"indicates recumbency and W*l upright for four hours. Sodium intake mMol/day
w/day Urinary Free Aldosterone
9 110 240
Urinary Aldosterone 18-glucuronide
9 110 240
9 ,1 110 11
275 120 82
170-370 69-134 37-106
12-35 2.8-21 0.5-9.3
08:3Or 12:3Ou 08r3Or 12:3Ou 08:3Or 12:3Ou
:; 8.9 Es
8-48 40-100 4.5-18 11-40 2-14 3.5-19
Above normal values were found in two patients one with an aldosterone producing adenoma and the other with idiopathic hyperaldosteronism: free aldosterone excretion ranged from 540 to 2,700 ng/day on a daily intake of 110 mMo1 of sodium, with a range for the 18-glucuronide of 64 to 285 pg/day.
Plasma and urinary free DOC were measured in normal subjects: at O8:30 the mean plasma DOC was 6.8 ng/lOOml (range 3-19), and the urinary DOC averaged 33 ng per day (range 16-54). There was no detectable variation between high (240 mMol/day) and low (9 mMol/day) sodium intakes. A patient with ad&sons
disease, on replacement
therapy, had a plasnz 3OC of 1.2 ng/lOOml and a urinary DOC of 4.5 ng/day: a patient with an aldosterone producing adenoma had moderate elevation of plasma DOC with recumbent 08:30 levels of 55 and 60 ng/lOOml and upright l2:3O levels of 32 and 37 ng/lOOml: three patients with adrenal carcinomas had elevated levels of 30, 90 and 390 ng/lOOml; the subject with the highest level having hypertension and hypokalaemic alkalosis. Metyrapone increased a normal subject's plasma DOC to 325 ng/lOOml. DISCUSSION: The extraction, chromatography and assay, based on the method described by Ito et al (9), which we have used for alaosterone and DOC, are reasonably routine. The assay separation technique is new and has been the subject of a preliminary communication (10). It is a simple method which combines features of ammonium sulfate precipitation and toluene partition techniques: Jowett et al (11) have described the use of toluene scintillant to extract free steroid'in an aldosterone radioimmunoassay. The method we describe makes use of the salting out effect of ammonium sulfate which decreases the solubility of steroid in the aqueous phase. Using this separation technique an assay can be incubated and counted in the same vial without mechanical separation of free from bound steroid. There is the problem of the slow increase of counts in the toluene
phase as steroid dissociates from the precipitated complexes. This dissociation is greatly reduoed at lower temperatures so that counting should be carried out as near O'C as is practicable. In order to cancel out minimal count changes the vials can be counted twice, the second time in reverse order, and the two counts added, Cope and Loisou (12) report an assay for urinary free DOC. Using two paper chromatography systems and the same antiserum which we use they found a similar normal level (a mean excretion of 41 ng per day). Our plasma DOC levels are in good agreement with previous reports (7,13,14,15). In plasma the concentration of DOG is about half that of aldosterone, (on 110 mMo1 of sodium per day), while the urinary free DOG excretion is only a quarter of the free aldosterone excretionr the relatively lower amount of DOC excreted probably reflects its greater binding to plasma proteins. The correlation of urinary free aldosterone excretion with the excretion of the 18-glucuronide is so close that measurement of free aldosterone does not seem to offer any advantage as it is technically more difficult; our findings agreeing with those of Deck et al (6). However in situations of altered metabolism or protein binding, suoh as pregnancy or renal failure, (the 18-glucuronide is formed in the kidney), there could well be an advantage as the free hormone would give a more accurate indication of biological activity (5). With ACTH stimulation in pregnancy a reciprocal relationship between DOC and aldosterone was found by Ehrlich et al (16)~ they measurea the excretion of tetrahydro-DOG and aldosterone 18-glucuronide. It is not known whether the high levels of DOC which occur in pregnancy, and which Brown et al suggest are of placental origin (17),
exert a physiologically significant salt retaining effect: studies using urinary free steroid excretion could help to provide the answer. ACKNOWLEDGEMENTS; A.K. was the recipient of a United States Public Health International Postdoctoral Fellowship. The aldosterone antiserum was provided by Dr. Robert W. Bates of the Pituitary Hormone Distribution Program, NIA~D, The National Institutes of Health, Bethesda, Maryland. The DOC antiserum was given by Dr. C.R.W. Edwards, St. Bartholomews Hospital, London. ~FE~NCES: The following trivial names are used in this paper: Aldosterone= the 18,11-hemiacetal of 11,8,21-aihydroxy-3,20-.dioxo4-pregnen-18-al; Corticosterone= ll,6,21-dihydroxy-4-pregnene-3,20dione; Cortisol= 1$5,170(,2l-trihydroxy-4-pregnene-3,20-dione; Cortisone= 17,2l-dihydroxy-4-pregnene-3,lI,2O-trione; Deoxycorticosterone (DOG)= Zl-hydroxy-4-pregnene-3,20-dione; Prednisone= 17,21dihydroxy-1,4-pregnadiene-3,11,20-trione; Progesterone= 4-pregnene3,20-dione; Testosterone= 17d-hydroxy-4-androsten-3-one. 587 (1974) 1. Pham-Hun-Trung M.T. and Corral P., Steroids&, 2. McKenzie J.K. and elements J.A., J Clin Endocrinol Me-tab2, 622 (1974) 3. Ekins R.P., Newman G.B., Piyasena R., Banks P. and Slater J.D.H., J Steroid Bioohem 2, 289 (1972) 4. Langan J., Jackson R., Adlin E.V. and Channick B.J., J Cfin Endocrinol Metab 38, 189 (1974) 5. cope C.L. and Black E.G., Br Med. J 2, 1117 (1959) 6. Deck K.A., Champion P.K. and Conn J.W., J Clin Endocrinol Metab 3, 756 (1973) Arnold M.L. and James V.H.T., Steroids 18, 789 (1972) ;5: Rodbard D. and Frazier G.R., Second Edition PB 217366 and PB 217367, National Technical Information Service, Springfield, Virginia (1973) 9. Ito T., Woo J., Haning R. and Horton R., 3 Clin Endocrinol Metab 2, 106 (1972) _ lfl Edwards C.R.W., Tay1or A.A., Baum C.K. and Kurtz A.B., Tenobus Workshops on Steroid Immunoassays, Alpha and Omega Publishers, Cardiff 229 (1975) 11. Jowett T.P., Slater J.D.H., Piyasena R.D. and Ekins R.P., Clin Sci Mol Neda, 607 (1973) 12. cope C.L. ana Loizou s., Clin Sci Mo1 Med @, 97 (1975) 13. Brown R.D. and Strott C.A., J Clin Endocrinol Metab 32, 744 (1971) 14. Castro A., Bartos D., Jelen B. and Kutas M., Steroids 22, 851 (1973) 15. Tan S.Y. and Mulrow P.J., Steroids a, 1 (1975) 16. Ehrlich E.N., Biglieri E.G. and Lindheimer M.D., 3 Clin Endocrinol Mefab 2, 701 (1974) 17. Brown R.D., Strott C.A. and Liddle G.W., J Clin Endocrinol Metab x, 736 (1972) A”.