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Development and application of a direct radioimmunoassay for aldosterone in saliva
845 DEVELOPMENTAND APPLICATIONOF A DIRECTRADIOIMMUNOASSAY FOR ALDOSTERONEIN SALIVA Shelia M. Atherden,John E.T.Carrie*,David 3, Jones, Emad A.S. Al-Dujalli and ChristopherR.N.Edwards Departmentof Medicine and *MRC Clinical and PopulationCytogenetics Unit, Western General Hospital,EdinburghEH4 2XU, U.K. Received g-26-85 (Revised 11-13-851 ABSTRACT A previously describeddirect radioimmunoassay for plasma aldosterone has been modified to enable direct measurementof the steroidin saliva. The specificityof the method has been demonstratedby assay after high pressureliquid chromatographicpurificationof saliva extracts. Assay of matched plasma and saliva samples taken from normal subjectsduring unrestrictedand controlledsodium intakes,either under basal conditionsor while undergoingACTH stimulationor dexamethasone suppression,confirmsthat salivary aldosteronevalues provide a good reflectionof levels in plasma. Mean salivary aldosteronevalues are approximatelyone-thirdof those in plasma. Sampling immediatelyupon waking appearsto provide reliable values for salivary aldosteroneand the potentialapplicationof this techniqueto the screeningof hypertensive patients is discussed. INTRODUCTION Measurementsof steroidsin saliva and their clinical relevance have received considerableattentionin recent years (e.g., I>, but aldosteronehas been relatively neglected. The first paper in the field describedan assay which requiredlarge sample volumes and involved extractionand chromatography(21,and a later enzyme i~unoassay
alSO
requiredsolvent extraction(3). Recently,Few and co-workershave describeda direct assay using a radioiodinatedtracer (4),andwe have made a preliminaryreport of a similar system (51. The present paper details the developmentand validationof our assay and comparesplasma and salivary aldosteroneresponsesin normal subjectsto varying dietary sodium intakes,ACTH stimulationand dexamethasonesuppression. We hoped by establishingthis assay to provide a simple, non-invasive means for outpatientscreeningof hypertensivepatientsto detect those October,November 1985
Steroids
Volume 46,Numben 4,5
with possible primary hyperaldosteronism (Corm's syndrome). Because of the effects of time and posture on aldosterone levels, hospital admission is normally required in order to obtain controlled plasma samples. If patients could collect saliva samples at home upon waking, both time and posture could be controlled and the procedure could be readily repeated on successive days. Data on the still unknown prevalence (6) of Corm's syndrome could become available, with consequent benefit to patients with this readily treatable condition.
MATERIALS AND METHODS Shee anti-aldosterone antiserum, aldosterone-3-(@carboxymethyl)oxime- fs5 II-iodohistamine cl25I-aldosterone) and commercial reagents were obtained as previously described (7). Donkey anti-sheep antiserum was from the Scottish Antibody Production Unit, Carluke, Lanarkshire, U.K., and was coupled to Sephacryl S300 (Pharmacia) as described (8). Methanol and water for high pressure liquid chromatography (HPLC) were from Rathburn Chemicals Ltd., Walkerburn, Peebleshire, U.K., and HPLC was carried out on equipment from Waters Associates, Cheshire, U.K., using an M45 pump, U6K injector, 441 UV detector with 254 nm filter and a 3.9 mm x 30 cm uBondapak Cl8 column. The mobile phase was methanol/ water (I:1 by vol) and flow rate was 0.7 mL/min. Dichloromethane (BDH Chemicals) was redistilled immediately prior to use. Saliva samples were collected into polystyrene tubes, 5-10 min after rinsing the mouth with water, and were stored at -2O'C for 24 h, then centrifuged to remove debris and refrozen until required for assay. Assay diluent was 0.05 mol/L sodium phosphate pH 7.4, containing sodium azide (1 g/L) and bovine serum albumin (I g/L). Aldosterone standards were prepared in this diluent over the range 6.25-250 pg/mL, equivalent to 17.5-700 pmol/L in saliva. '251-Aldosterone was diluted to give 40,000 cpm/mL (11.2 fmol/mL) in 0.5 mol/L sodium phosphate pH 7.4, containing sodium aside (1 g/L) and bovine serum albumin (IO g/L). Polystyrene tubes (55 x 12 mm) for the direct assay were from Sarstedt, Leicester, U.K.,and disposable glass tubes (75 x 12 mm) for the postHPLC assay were from Corning Medical, Essex, U.K. Plasma aldosterone values were determined by the direct assay previously described (7). Direct assay Duplicate aliquots (400 p.L)of saliva or aldosterone standards were and aldosterone dispensed into assay tubes and 1251-aldosterone (50 I.~L) antiserum at 50,000-fold dilution (50 pL) added. Tubes were incubated at room temperature for 4 h and an aliquot (50 PL) of Sephacryl-coupled donkey anti-sheep serum at 20-fold dilution of the initial antiserum was added. The tubes were agitated on an orbital shaker for 30 min and the
antibody-bound and free fractions separated by two passes of the previously described sucrose separation technique (9). The remaining (antibody-bound) fraction was counted in a Wallac gamma counter and data were processed using a modified four-parameter logistic fit (IO). Assay validation using HPLC The HPLC system was calibrated using authentic steroids (Fig. 1). Subsequently, a mixture of unlabeled and tritiated aldosterone was injected and fractions were collected for B-counting. The fraction which co-eluted with aldosterone (11.75-13.25min) contained 97.5% of the radioactivity applied, and this figure was checked at the beginning and end of each working day. Saliva specimens (1 mL) were dispensed into glass tubes and extracted with dichloromethane (5 mL). The extracts were washed in turn with 0.1 mol/L sodium hydroxide (1 mL) and water (1 mL), and duplicate aliquots (2 mL) of each extract were evaporated at 45'C under a nitrogen stream. Residues were dissolved in HPLC mobile phase (100 uL),and aliquots (75 pL) were injected onto the column. The appropriate fraction of eluate was collected, the methanol was evaporated under nitrogen and the aqueous residue extracted with dichloromethane (1.5 mL). An aliquot (1 mL) of this extract was evaporated under nitrogen and the residue was dissolved in assay diluent (400 uL) and assayed as above. The overall recovery was determined using [SH]-aldosterone and values determined by radioimmunoassay were corrected by this factor. Physiological studies Full details of the experimental protocol have been described elsewhere (II). Briefly, each of six normal male volunteers was studied on three separate occasions, with an interval of at least one week between each study. During the first study, subjects ate their usual food,but on the second and third occasions they consumed diets containing respectively 20 and 240 mm01 sodium per day. Diets were started at 0800 h on Day 1 and subjects provided matched plasma and saliva samples on Day 4 (0900, 1300, 1800 and 2400 h), on Day 5 before and after corticotropin (ACTH) stimulation (Synacthen 250 ug given intravenously) and on Days 6, 7 and 8 while taking dexamethasone (0.5 mg 6-hourly from 0900 h on Day 6). Subjects were recumbent throughout the ACTH stimulation test and for 30 min before all other blood/saliva samples were taken, but otherwise maintained their normal activities. Validity of aldosterone values in saliva obtained on waking Normal volunteers collected saliva at home immediately upon waking after overnight sleep, then remained in bed for a further hour and collected a second sample. Before each collection subjects rinsed their mouths with water as described. Aldosterone values were measured in all samples by the direct assay.
RESULTS Effect of antibody dilution and tracer mass Standard curves were set up concurrently under the conditions of the previously described (7) plasma assay (final antibody dilution 1 in 300,000; tracer 5000 cpm (1.4 fmol)/tube) and as described above (final antibody dilution 1 in 500,000; tracer 2000 cpm (0.56 fmol)/tube). The masses of analyte required to give a decrease in the percentage binding (B) of 50% of the percentage tracer bound in the absence of unlabeled aldosterone (B,) were 59 and 48 pg/tube respectively. Further decreases in tracer mass caused only minor improvement in sensitivity at the expense of prolonged counting times. Sensitivity and working range The formal sensitivity of the assay , corresponding to the mass of analyte required to give a decrease in the %B of tracer of 2.5 SD of (B,) was 2.8 pg/tube (19.4 pmol/L in saliva).
In practice, we have
taken the lower limit of the working range as 60 pmol/L (8.6 pg/tube), which is the concentration of aldosterone required to cause a 20% depression of B/B,. Precision and accuracy Figure 2 shows a typical standard curve for the direct salivary assay and within-assay component of the variation of duplicate estimates of concentration for saliva samples measured in six routine assays. The curve represents the averaged precision profiles (mean + 1 SD) for the individual assays and shows that the mean within-assay CV was less than 10% for all levels of salivary aldosterone above 60 pmol/L. Table 1 shows data for between-assay variability and analytical recovery of aldosterone added to pooled saliva.
S
%?EIEOSDrn
Parallelism Twenty-nine saliva specimens assayed as described, and at serial 2fold dil.utionsover an 8-fold range gave 73 results within the working range of the assay.
Linear regression analysis of these results gave
the equation y = 0.99x - 1.38 (r=0.994; p
Levels of endogenous steroids in saliva
were too low to detect by UV absorptionand collection of the fractions containing aldosterone was by carefully monitored timing as described. The reagent blank following extraction of assay diluent and HPLC fractionation of the extract was (7 pmol/L, which was not significantly different from zero. Overall recovery of [3H]-aldosterone was 92.7 _c 4.4% (mean 2 SD),and this correction was applied to values measured following HPLC fractionation. Thirty-seven saliva samples with aldosterone values in the range 70-425 pmol/L (direct assay) were assayed following HPLC fractionation. Linear regression analysis of aldosterone values measured directly (x) and after HPLC (y) gave the equation y
q
0.95x + 7.43 (r = 0.979; p
Stability of aldosterone in saliva Aliquots of the pools shown in Table 1 allowed to stand at room temperature for up to 14 days showed no significant change in aldosterone values measured by direct assay. Effects of clock time and sodium intake Table 2 shows mean plasma and salivary aldosterone levels throughout
S
850
!EPBROfDb
the day on each different diet. As expected, aldosterone levels rose in response to sodium restriction, while on all diets the lowest concentrations of aldosterone
were found at 2400 h.
The patterns of aldo-
sterone levels in saliva followed closely those in plasma.
For the 53
data pairs where salivary aldosterone values lay within the range of the assay, plasma (x) and salivary (y) aldosterone concentrations were related by the equation y
q
0.26~ + 20 (r = 0.938; p
TABLE 1. Analytical recovery and between-assay variability (n=20) for aldosterone added to pooled saliva.
Aldosterone, pmol/L Added Measured
% Recovery
Between-assay CV, %
68 108~ 105 315 595
218 427 687
8.9 8.8 7.7 6.4 7.2
104.7 101.3 96.6
aThis pool was used for preparation of recovery pools.
cortlcosterone Cortisal-
t
ABSORBENCE
FIGURE 1. Elution pattern of steroids from HPLC. The trace is that of an extract of saliva spiked with the steroids shown.
Response to ACTH stimulation Table 3 shows for each diet the plasma and salivary levels of aldosterone before and after administration of ACTH.
Significant increases
in aldosterone were observed in all cases,and the percentage increases
S
fl?DROID=
851
above basal levels were essentially identical in plasma and saliva.
FOP
the 52 data pairs where salivary aldosterone concentrations lay within the range of the assay, plasma (x) and salivary (y) aldosterone levels were related by the equation y = 0.28~
175
35
70
ALDOSTERONE
+ 27.5
175
(r = 0.958; pC:O.OOl).
350
700
(pmol/L)
and within-assay precision profile FIGURE 2. Standard curve (*-$ (mean + SD;) for direct salivary aldosterone assay.
Effect of dexamethasone administration Table 4 shows for each diet the plasma and salivary aldosterone concentrations before and after 24 and 48 h on dexamethasone. The percentage decreases below basal levels were similar in plasma and saliva, although this was obscured in saliva on the high sodium diet, where most values fell below the lower limit of the assay. Validity of aldosterone measurements in saliva taken on waking Aldosterone levels in saliva specimens taken on waking (107.6+ 36 pmol/L; mean_eSD) and 1 h thereafter (101.3 + 38) were not significantly different (n=20; p)O.lO; Student's paired 4 test).
TABLE 2. Plasma (P) and salivary (S) aldosterone concentrations (pmol/L) in response to clock time and sodium intake. Values are shown as mean + SD for 6 normal male subjects.
Sodium intake Clock Time
Ad libitum P
0900 1300 1800 2400
348+101 313+127a 210+95c 152273'
Low S
113237 (93~20a (74~22~ C66_c13b
High
P
S
608+246 965;228c 609:133a 444z255a
167268 278~55~ 158~32~ 6143~87~
P
S
201+62 280561' 167+87a 139Z50b
478220 91+19a ~$70~24~ 4.63~6~
Probabilities (Student'spaired t test) that values differ significantly from 0900 h levels: anot significant; bp\t0.05;cp
TABLE 3. Plasma (P) and salivary (S) aldosterone concentrations (pmol/L) during different sodium intakes in response to ACTH administration. Synacthen (250 pg iv) was given at 0900 h. Values are shown as mean 2 SD for 6 normal male subjects.
Sodium intake Clock Time 0855 0930 1000
P
Ad libitum S
3142182 6222237' 608y38'
112-1_39 210z8ga 209267'
High
Low P 5792341 1419+411c 14422373'
S
P
S
176276 422+87b 452538'
213259 459+118c 530+137b
474219 148~31~ 172~46~
Probabilities (Student's paired & test) that values differ from 0855 h levels: ap(0.025; bp(O.Ol; 'p
TABLE 4. Plasma (P) and salivary (S) aldosterone concentrations (pmol/L) at 0900 h during different sodium intakes in response to dexamethasone (Dex) administration. Values are shown as mean + SD for 6 normal male subjects.
Sodium Intake Time on Dex
0
+24 h +48 h
P
Ad libitum S
4062333 175+106a 181~177~
122252 ,(73+2aa S63;6b
Low P
7772337 557_?301b 339+193C
High S
P
S
219+90 165+4gb 11125b
225277 139+51a 126263'
76217 d61+2a <62z4a
Probabilities (Student'spairbadt test) that valxes differ from basal p~O.001. values: anot significant; p40.05; cpso.ol;
DISCUSSION Modification of the previously described direct assay for aldosterone in plasma (71, by the simple expedient of using a higher dilution of antiserum and reduced mass of tracer, readily yielded a method with a small increase in sensitivity which was sufficient to enable quantitation of aldosterone concentrations in salivary specimens. The accuracy of the assay was confirmed by the excellent agreement of results obtained by direct assay with those determined following chromatographic fractionation of saliva extracts.
Assay of serial
dilutions of saliva showed excellent parallelism of the aldosterone values measured. Plasma aldosterone levels showed the expected changes in response to time of day, sodium status, ACTH stimulation and dexamethasone suppression (121, and salivary aldosterone concentrations reflected all these changes. Although the ratio of salivary to plasma aldosterone concentrations showed considerable intra- and inter-individual variability (range 0.17-0.511,the mean ratio (0.32 + 0.06) was remarkably stable.
The only significant differences were in the ratios for the basal circadian levels on the low and high sodium intakes, which were 0.29 t_ 0.06 and 0.34 + 0.07 respectively (~(0.025). These minor differences are probably artificial, since they were not apparent across the different diets during ACTH stimulation or dexamethasone suppression, nor did the ratios during these tests differ from those under basal conditions. This is in contrast to the more limited data of McVie et al
(131,who
reported relative increases and decreases in saliva/plasma aldosterone ratios during ACTH and dexamethasone administration respectively, but little change with changes in sodium intake. Resolution of this discrepancy must await further study, but it would be surprising if the saliva/plasma aldosterone ratio truly responds in different ways to changes (either up or down) in plasma aldosterone concentration wrought by changes in dietary sodium compared with changes of similar magnitude wrought by pharmacological agents. As discussed above, a major reason for establishing this assay was the potential for outpatient screening of hypertensive subjects, with saliva samples being collected after overnight sleep. However, it has been reported (14) for at least one other steroid (testosterone) that samples taken on waking contain unrepresentatively high concentrations, and it has been suggested that this phenomenon may be in some way related to the very low salivary flow rate during sleep. However, in our hands salivary aldosterone levels showed no significant change in the first hour after waking (provided that subjects remained supine),and the sampling technique therefore appears to be valid for this steroid. In conclusion, it has been shown that salivary aldosterone concentrations measured by the direct assay described herein show a high
degree of correlation with plasma aldosterone concentrations in normal subjects during a variety of procedures designed to manipulate aldosterone levels.
These data and those of other workers who described
similarly good correlation during exercise or postural stimuli (4) suggest that measurement of salivary aldosterone will provide a useful clinical tool. TRIVIAL AND IUPAC NAMES Aldosterone: 3,18,20-Trioxo-4-pregnene-llE,21-diol Dexamethasone: 9a-fluoro-1~-methyl-11t3,17d,21-trihydroxy-l,4pregnadiene-3,20-dione REFERENCES 1.
2. 3.
4.
Immunoassay of Steroids in Saliva (Read, G.F., Riad-Fahmy, D., Walker, R.F. and Griffiths, K., Editors), Alpha Omega, Cardiff (1984). McVie, R., Levine, L. and New, M.I., PEDIATR. RES. 13, 755 (1979). Hubl, W., Taubert, H., Freymann, E., Hofmann, F., Meissner, D., Garten, C.D., Schmidt, P.H.K., Thiele, H.J. and Neef, B., EXP. CLIN. ENDOCRINOL. 82, 188 (1983). Few, J.D., Chaudry, S. and James, V.H.T., J. STEROID BIOCHEM.21, 87 (1984).
5.
6. 7. 8. 9.
Atherden, S.M., Al-Dujaili,E.A.S., Corrie, J.E.T.and Edwards, C.R.W.,Abstract 75, 3rd Joint Meeting of British Endocrine Societies, Edinburgh, March 1984. Swales, J.D., BR. MED. J. 287, 702 (1983). Al-Dujaili, E.A.S.and Edwards, C.R.W.,J.STEROID BIOCHEM.,l4, 481 (1981).
Wright, J.F. and Hunter,W.M., J. IMMUNOL. METHODS 48, 311 (1982). Corrie, J.E.T.,Ratcliffe, W.A.and Macpherson,J.S., STEROIDS 3, 709 (1981). 10. Raab, G.M. and McKenzie, I.G.M.,in: Quality Control in Clinical Endocrinology (Wilson, D., Gaskell, S.J. and Kemp, K., Editors), Alpha Omega, Cardiff (19811, p 225. 11. Corrie, J.E.T.,Edwards, C.R.W., Jones, D.B., Padfield, P.L. and Budd, P.S., CLIN. ENDOCRINOL.(in press). 12. Bravo, E.L., Tarazi, R.C., Dustan, H.P., Fouad, F.M., Textor, S-C., Gifford, R.W. and Vidt, D.G., AM. J. MED. 74, 641 (1983). 13. McVie, R., Levine, L.S. and New, M.I., in: Immunoassay of Steroids in Saliva (Read, G.F., Riad-Fahmy, D., Walker, R.F. and Giffiths, K., Editors), Alpha Omega, Cardiff (19841, p 295. 14. Baxendale, P.M., Jacobs, H.S. and James, V.H.T.,CLIN. ENDOCRINOL. &, 595 (1982).
alSO
requiredsolvent extraction(3). Recently,Few and co-workershave describeda direct assay using a radioiodinatedtracer (4),andwe have made a preliminaryreport of a similar system (51. The present paper details the developmentand validationof our assay and comparesplasma and salivary aldosteroneresponsesin normal subjectsto varying dietary sodium intakes,ACTH stimulationand dexamethasonesuppression. We hoped by establishingthis assay to provide a simple, non-invasive means for outpatientscreeningof hypertensivepatientsto detect those October,November 1985
Steroids
Volume 46,Numben 4,5
with possible primary hyperaldosteronism (Corm's syndrome). Because of the effects of time and posture on aldosterone levels, hospital admission is normally required in order to obtain controlled plasma samples. If patients could collect saliva samples at home upon waking, both time and posture could be controlled and the procedure could be readily repeated on successive days. Data on the still unknown prevalence (6) of Corm's syndrome could become available, with consequent benefit to patients with this readily treatable condition.
MATERIALS AND METHODS Shee anti-aldosterone antiserum, aldosterone-3-(@carboxymethyl)oxime- fs5 II-iodohistamine cl25I-aldosterone) and commercial reagents were obtained as previously described (7). Donkey anti-sheep antiserum was from the Scottish Antibody Production Unit, Carluke, Lanarkshire, U.K., and was coupled to Sephacryl S300 (Pharmacia) as described (8). Methanol and water for high pressure liquid chromatography (HPLC) were from Rathburn Chemicals Ltd., Walkerburn, Peebleshire, U.K., and HPLC was carried out on equipment from Waters Associates, Cheshire, U.K., using an M45 pump, U6K injector, 441 UV detector with 254 nm filter and a 3.9 mm x 30 cm uBondapak Cl8 column. The mobile phase was methanol/ water (I:1 by vol) and flow rate was 0.7 mL/min. Dichloromethane (BDH Chemicals) was redistilled immediately prior to use. Saliva samples were collected into polystyrene tubes, 5-10 min after rinsing the mouth with water, and were stored at -2O'C for 24 h, then centrifuged to remove debris and refrozen until required for assay. Assay diluent was 0.05 mol/L sodium phosphate pH 7.4, containing sodium azide (1 g/L) and bovine serum albumin (I g/L). Aldosterone standards were prepared in this diluent over the range 6.25-250 pg/mL, equivalent to 17.5-700 pmol/L in saliva. '251-Aldosterone was diluted to give 40,000 cpm/mL (11.2 fmol/mL) in 0.5 mol/L sodium phosphate pH 7.4, containing sodium aside (1 g/L) and bovine serum albumin (IO g/L). Polystyrene tubes (55 x 12 mm) for the direct assay were from Sarstedt, Leicester, U.K.,and disposable glass tubes (75 x 12 mm) for the postHPLC assay were from Corning Medical, Essex, U.K. Plasma aldosterone values were determined by the direct assay previously described (7). Direct assay Duplicate aliquots (400 p.L)of saliva or aldosterone standards were and aldosterone dispensed into assay tubes and 1251-aldosterone (50 I.~L) antiserum at 50,000-fold dilution (50 pL) added. Tubes were incubated at room temperature for 4 h and an aliquot (50 PL) of Sephacryl-coupled donkey anti-sheep serum at 20-fold dilution of the initial antiserum was added. The tubes were agitated on an orbital shaker for 30 min and the
antibody-bound and free fractions separated by two passes of the previously described sucrose separation technique (9). The remaining (antibody-bound) fraction was counted in a Wallac gamma counter and data were processed using a modified four-parameter logistic fit (IO). Assay validation using HPLC The HPLC system was calibrated using authentic steroids (Fig. 1). Subsequently, a mixture of unlabeled and tritiated aldosterone was injected and fractions were collected for B-counting. The fraction which co-eluted with aldosterone (11.75-13.25min) contained 97.5% of the radioactivity applied, and this figure was checked at the beginning and end of each working day. Saliva specimens (1 mL) were dispensed into glass tubes and extracted with dichloromethane (5 mL). The extracts were washed in turn with 0.1 mol/L sodium hydroxide (1 mL) and water (1 mL), and duplicate aliquots (2 mL) of each extract were evaporated at 45'C under a nitrogen stream. Residues were dissolved in HPLC mobile phase (100 uL),and aliquots (75 pL) were injected onto the column. The appropriate fraction of eluate was collected, the methanol was evaporated under nitrogen and the aqueous residue extracted with dichloromethane (1.5 mL). An aliquot (1 mL) of this extract was evaporated under nitrogen and the residue was dissolved in assay diluent (400 uL) and assayed as above. The overall recovery was determined using [SH]-aldosterone and values determined by radioimmunoassay were corrected by this factor. Physiological studies Full details of the experimental protocol have been described elsewhere (II). Briefly, each of six normal male volunteers was studied on three separate occasions, with an interval of at least one week between each study. During the first study, subjects ate their usual food,but on the second and third occasions they consumed diets containing respectively 20 and 240 mm01 sodium per day. Diets were started at 0800 h on Day 1 and subjects provided matched plasma and saliva samples on Day 4 (0900, 1300, 1800 and 2400 h), on Day 5 before and after corticotropin (ACTH) stimulation (Synacthen 250 ug given intravenously) and on Days 6, 7 and 8 while taking dexamethasone (0.5 mg 6-hourly from 0900 h on Day 6). Subjects were recumbent throughout the ACTH stimulation test and for 30 min before all other blood/saliva samples were taken, but otherwise maintained their normal activities. Validity of aldosterone values in saliva obtained on waking Normal volunteers collected saliva at home immediately upon waking after overnight sleep, then remained in bed for a further hour and collected a second sample. Before each collection subjects rinsed their mouths with water as described. Aldosterone values were measured in all samples by the direct assay.
RESULTS Effect of antibody dilution and tracer mass Standard curves were set up concurrently under the conditions of the previously described (7) plasma assay (final antibody dilution 1 in 300,000; tracer 5000 cpm (1.4 fmol)/tube) and as described above (final antibody dilution 1 in 500,000; tracer 2000 cpm (0.56 fmol)/tube). The masses of analyte required to give a decrease in the percentage binding (B) of 50% of the percentage tracer bound in the absence of unlabeled aldosterone (B,) were 59 and 48 pg/tube respectively. Further decreases in tracer mass caused only minor improvement in sensitivity at the expense of prolonged counting times. Sensitivity and working range The formal sensitivity of the assay , corresponding to the mass of analyte required to give a decrease in the %B of tracer of 2.5 SD of (B,) was 2.8 pg/tube (19.4 pmol/L in saliva).
In practice, we have
taken the lower limit of the working range as 60 pmol/L (8.6 pg/tube), which is the concentration of aldosterone required to cause a 20% depression of B/B,. Precision and accuracy Figure 2 shows a typical standard curve for the direct salivary assay and within-assay component of the variation of duplicate estimates of concentration for saliva samples measured in six routine assays. The curve represents the averaged precision profiles (mean + 1 SD) for the individual assays and shows that the mean within-assay CV was less than 10% for all levels of salivary aldosterone above 60 pmol/L. Table 1 shows data for between-assay variability and analytical recovery of aldosterone added to pooled saliva.
S
%?EIEOSDrn
Parallelism Twenty-nine saliva specimens assayed as described, and at serial 2fold dil.utionsover an 8-fold range gave 73 results within the working range of the assay.
Linear regression analysis of these results gave
the equation y = 0.99x - 1.38 (r=0.994; p
Levels of endogenous steroids in saliva
were too low to detect by UV absorptionand collection of the fractions containing aldosterone was by carefully monitored timing as described. The reagent blank following extraction of assay diluent and HPLC fractionation of the extract was (7 pmol/L, which was not significantly different from zero. Overall recovery of [3H]-aldosterone was 92.7 _c 4.4% (mean 2 SD),and this correction was applied to values measured following HPLC fractionation. Thirty-seven saliva samples with aldosterone values in the range 70-425 pmol/L (direct assay) were assayed following HPLC fractionation. Linear regression analysis of aldosterone values measured directly (x) and after HPLC (y) gave the equation y
q
0.95x + 7.43 (r = 0.979; p
Stability of aldosterone in saliva Aliquots of the pools shown in Table 1 allowed to stand at room temperature for up to 14 days showed no significant change in aldosterone values measured by direct assay. Effects of clock time and sodium intake Table 2 shows mean plasma and salivary aldosterone levels throughout
S
850
!EPBROfDb
the day on each different diet. As expected, aldosterone levels rose in response to sodium restriction, while on all diets the lowest concentrations of aldosterone
were found at 2400 h.
The patterns of aldo-
sterone levels in saliva followed closely those in plasma.
For the 53
data pairs where salivary aldosterone values lay within the range of the assay, plasma (x) and salivary (y) aldosterone concentrations were related by the equation y
q
0.26~ + 20 (r = 0.938; p
TABLE 1. Analytical recovery and between-assay variability (n=20) for aldosterone added to pooled saliva.
Aldosterone, pmol/L Added Measured
% Recovery
Between-assay CV, %
68 108~ 105 315 595
218 427 687
8.9 8.8 7.7 6.4 7.2
104.7 101.3 96.6
aThis pool was used for preparation of recovery pools.
cortlcosterone Cortisal-
t
ABSORBENCE
FIGURE 1. Elution pattern of steroids from HPLC. The trace is that of an extract of saliva spiked with the steroids shown.
Response to ACTH stimulation Table 3 shows for each diet the plasma and salivary levels of aldosterone before and after administration of ACTH.
Significant increases
in aldosterone were observed in all cases,and the percentage increases
S
fl?DROID=
851
above basal levels were essentially identical in plasma and saliva.
FOP
the 52 data pairs where salivary aldosterone concentrations lay within the range of the assay, plasma (x) and salivary (y) aldosterone levels were related by the equation y = 0.28~
175
35
70
ALDOSTERONE
+ 27.5
175
(r = 0.958; pC:O.OOl).
350
700
(pmol/L)
and within-assay precision profile FIGURE 2. Standard curve (*-$ (mean + SD;) for direct salivary aldosterone assay.
Effect of dexamethasone administration Table 4 shows for each diet the plasma and salivary aldosterone concentrations before and after 24 and 48 h on dexamethasone. The percentage decreases below basal levels were similar in plasma and saliva, although this was obscured in saliva on the high sodium diet, where most values fell below the lower limit of the assay. Validity of aldosterone measurements in saliva taken on waking Aldosterone levels in saliva specimens taken on waking (107.6+ 36 pmol/L; mean_eSD) and 1 h thereafter (101.3 + 38) were not significantly different (n=20; p)O.lO; Student's paired 4 test).
TABLE 2. Plasma (P) and salivary (S) aldosterone concentrations (pmol/L) in response to clock time and sodium intake. Values are shown as mean + SD for 6 normal male subjects.
Sodium intake Clock Time
Ad libitum P
0900 1300 1800 2400
348+101 313+127a 210+95c 152273'
Low S
113237 (93~20a (74~22~ C66_c13b
High
P
S
608+246 965;228c 609:133a 444z255a
167268 278~55~ 158~32~ 6143~87~
P
S
201+62 280561' 167+87a 139Z50b
478220 91+19a ~$70~24~ 4.63~6~
Probabilities (Student'spaired t test) that values differ significantly from 0900 h levels: anot significant; bp\t0.05;cp
TABLE 3. Plasma (P) and salivary (S) aldosterone concentrations (pmol/L) during different sodium intakes in response to ACTH administration. Synacthen (250 pg iv) was given at 0900 h. Values are shown as mean 2 SD for 6 normal male subjects.
Sodium intake Clock Time 0855 0930 1000
P
Ad libitum S
3142182 6222237' 608y38'
112-1_39 210z8ga 209267'
High
Low P 5792341 1419+411c 14422373'
S
P
S
176276 422+87b 452538'
213259 459+118c 530+137b
474219 148~31~ 172~46~
Probabilities (Student's paired & test) that values differ from 0855 h levels: ap(0.025; bp(O.Ol; 'p
TABLE 4. Plasma (P) and salivary (S) aldosterone concentrations (pmol/L) at 0900 h during different sodium intakes in response to dexamethasone (Dex) administration. Values are shown as mean + SD for 6 normal male subjects.
Sodium Intake Time on Dex
0
+24 h +48 h
P
Ad libitum S
4062333 175+106a 181~177~
122252 ,(73+2aa S63;6b
Low P
7772337 557_?301b 339+193C
High S
P
S
219+90 165+4gb 11125b
225277 139+51a 126263'
76217 d61+2a <62z4a
Probabilities (Student'spairbadt test) that valxes differ from basal p~O.001. values: anot significant; p40.05; cpso.ol;
DISCUSSION Modification of the previously described direct assay for aldosterone in plasma (71, by the simple expedient of using a higher dilution of antiserum and reduced mass of tracer, readily yielded a method with a small increase in sensitivity which was sufficient to enable quantitation of aldosterone concentrations in salivary specimens. The accuracy of the assay was confirmed by the excellent agreement of results obtained by direct assay with those determined following chromatographic fractionation of saliva extracts.
Assay of serial
dilutions of saliva showed excellent parallelism of the aldosterone values measured. Plasma aldosterone levels showed the expected changes in response to time of day, sodium status, ACTH stimulation and dexamethasone suppression (121, and salivary aldosterone concentrations reflected all these changes. Although the ratio of salivary to plasma aldosterone concentrations showed considerable intra- and inter-individual variability (range 0.17-0.511,the mean ratio (0.32 + 0.06) was remarkably stable.
The only significant differences were in the ratios for the basal circadian levels on the low and high sodium intakes, which were 0.29 t_ 0.06 and 0.34 + 0.07 respectively (~(0.025). These minor differences are probably artificial, since they were not apparent across the different diets during ACTH stimulation or dexamethasone suppression, nor did the ratios during these tests differ from those under basal conditions. This is in contrast to the more limited data of McVie et al
(131,who
reported relative increases and decreases in saliva/plasma aldosterone ratios during ACTH and dexamethasone administration respectively, but little change with changes in sodium intake. Resolution of this discrepancy must await further study, but it would be surprising if the saliva/plasma aldosterone ratio truly responds in different ways to changes (either up or down) in plasma aldosterone concentration wrought by changes in dietary sodium compared with changes of similar magnitude wrought by pharmacological agents. As discussed above, a major reason for establishing this assay was the potential for outpatient screening of hypertensive subjects, with saliva samples being collected after overnight sleep. However, it has been reported (14) for at least one other steroid (testosterone) that samples taken on waking contain unrepresentatively high concentrations, and it has been suggested that this phenomenon may be in some way related to the very low salivary flow rate during sleep. However, in our hands salivary aldosterone levels showed no significant change in the first hour after waking (provided that subjects remained supine),and the sampling technique therefore appears to be valid for this steroid. In conclusion, it has been shown that salivary aldosterone concentrations measured by the direct assay described herein show a high
degree of correlation with plasma aldosterone concentrations in normal subjects during a variety of procedures designed to manipulate aldosterone levels.
These data and those of other workers who described
similarly good correlation during exercise or postural stimuli (4) suggest that measurement of salivary aldosterone will provide a useful clinical tool. TRIVIAL AND IUPAC NAMES Aldosterone: 3,18,20-Trioxo-4-pregnene-llE,21-diol Dexamethasone: 9a-fluoro-1~-methyl-11t3,17d,21-trihydroxy-l,4pregnadiene-3,20-dione REFERENCES 1.
2. 3.
4.
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6. 7. 8. 9.
Atherden, S.M., Al-Dujaili,E.A.S., Corrie, J.E.T.and Edwards, C.R.W.,Abstract 75, 3rd Joint Meeting of British Endocrine Societies, Edinburgh, March 1984. Swales, J.D., BR. MED. J. 287, 702 (1983). Al-Dujaili, E.A.S.and Edwards, C.R.W.,J.STEROID BIOCHEM.,l4, 481 (1981).
Wright, J.F. and Hunter,W.M., J. IMMUNOL. METHODS 48, 311 (1982). Corrie, J.E.T.,Ratcliffe, W.A.and Macpherson,J.S., STEROIDS 3, 709 (1981). 10. Raab, G.M. and McKenzie, I.G.M.,in: Quality Control in Clinical Endocrinology (Wilson, D., Gaskell, S.J. and Kemp, K., Editors), Alpha Omega, Cardiff (19811, p 225. 11. Corrie, J.E.T.,Edwards, C.R.W., Jones, D.B., Padfield, P.L. and Budd, P.S., CLIN. ENDOCRINOL.(in press). 12. Bravo, E.L., Tarazi, R.C., Dustan, H.P., Fouad, F.M., Textor, S-C., Gifford, R.W. and Vidt, D.G., AM. J. MED. 74, 641 (1983). 13. McVie, R., Levine, L.S. and New, M.I., in: Immunoassay of Steroids in Saliva (Read, G.F., Riad-Fahmy, D., Walker, R.F. and Giffiths, K., Editors), Alpha Omega, Cardiff (19841, p 295. 14. Baxendale, P.M., Jacobs, H.S. and James, V.H.T.,CLIN. ENDOCRINOL. &, 595 (1982).