Department
of
Pharmacology, University Heidelberg, Germany
of
Heidelberg
RADIOIMMUNOASSAYS OF TETRAHYDROALDOSTERONE (TH-ALDO) IN HUMAN URINE
By K.-H. Kohl, P. Vecsei and S. Abdelhamid ABSTRACT
Specific
antisera
against tetrahydroaldosterone (TH-Aldo)
two white New Zealand rabbits.
were
raised in
3\g=a\,5\g=b\-TH-aldo-20-oxime-bovine-serum
albumin complex was used as antigen. The resulting titers were 1:18 000 and 1:16 000. Except tetrahydrocortisol (THF) (0.23%) and tetrahydro\x=req-\ 18-hydroxy-11-dehydrocorticosterone (18-OH-THA) (3.2 %), all steroids and steroid metabolites gave negligible cross-reactions. Immunograms of the paper chromatograms made from the n-butanol-extract of the urines, as well as after \g=b\-glucuronidase treatment and dichlormethane extraction, were studied to further define the specificity of the antiserum. Antibody H1 (used in this study) reacted with aldosterone-18-gluc., a TH-aldosterone-glucuronide (probably the 21-glucuronide) and an unidentified less polar material. Two methods were developed: a) TH-Aldo-glucuronidc(s) estimation after ethylacetate pre-extraction as a rapid screening test of endogenous aldosterone production. b) estimation of TH-aldosterone using one chromatographic system. The results of method a) showed a significant correlation with the values obtained by technique b). Normal values (method b) were 25.88 \m=+-\16.50 \g=m\g/24h(range 9.5\p=n-\64.8\g=m\g/24h). A significant correlation was also shown between the TH-aldo (technique b) and 18-gluc. values.
TH-Aldo is the major metabolite of aldosterone isolated from human urine (Ulick 8c Liebermann 1957; Ulick 8c Vetter 1962). It is formed in the liver and excreted as glucuronide. 15-40 °/o of the secreted aldosterone is excreted in the
Partly presented at the V International Congress of Endocrinology, Hamburg, July 1976. ') Deutsche Klinik für Diagnostik, Wiesbaden, GFR. Requests for reprints should be addressed to Dr. P. Vecsei, Pharmakologisches der Universität, Im Neuenheimer Feld 366, Heidelberg. 596
Institut
form of this metabolite, mann
mostly (80-90 %)
as
the 3a,
5/?-isomer (Kelly
8c Lieber¬
1964).
Measurement of TH-Aldo in urine can be expected to be more representative of the aldosterone production than measurement of the aldosterone 18-gluc, which represents only 5-15 °/o of the endogenous aldosterone. Preparation of a TH-Aldo antibody and a radioimmunoassay of TH-Aldo with TH-Aldo-antiserum have not been reported in the literature.
MATERIAL AND METHODS
(Aldo), Cortisol (F), corticosterone (B), compound S, tetrahydrocortisone (THE), tetrahydrocortisol (THF) and pregnantriol were obtained from Merck, Darm¬ stadt, GFR. The 3- and 21-glucuronides of the tetrahydrocorticosteroid metabolites were generously provided by V. R. Mattox, Rochester, Minn., and tetrahydro-18hydroxy-11-dehydrocorticosterone (18-OH-THA) was provided by S. Ulick, New York, Aldosterone
N.Y. All other corticosteroids and corticosteroid metabolites,
List
as
listed in the
of abbreviations: Aldo ll/?,21-dihydroxy-4-pregnene-3,20-dion-18-al F ll/?,17a,21-trihydroxy-4-pregnene-3,20-dione B 11/?,21 -dihydroxy-4-pregnene-3,20-dione S 17a,21-dihydroxy-4-pregnene-3,20-dione THE 3a, 17a,21 -trihydroxy-5/?-pregnane-l 1,20-dione THF 3a, 11/?, 17 a,21-tetrahydroxy-5/?-pregnane-20-dione Pregnanetriol 3a,17a,20a-trihydroxy-5/?-pregnane 3a,5yS-TH-Aldo 3 0% 0.18 "la 0"la 0.5 °/o 0.23 "la 0"la 0"la 0"la 0"la 2 "la 0»/o 0.22 "la 0»/o 0°/o 0°/o 0.3 «Vo
pg 600 500
500
¿00
400
300
300
200
200
100
100
Fig. la. Immunogram after chromatography in system I, A aldosterone-18-oxo-glucuronide.
n-butanol extract of
a
normal urine.
=
B X
=
=
TH-Aldo-glucuronide(s). unidentified substance. =
-
=
radioactivity in 24 h urine after administration of [^H]TH-Aldo intravenously. immunoactivity with Hj, TH-Aldo antibody.
-----
600
TH-Aldo
P9
2500
-s-
Fig. lb. Immunograms
after
chromatography
in
system III. Dichlormethane
yS-glucuronidase
= -
-=
extract after
treatment.
radioactivity of f3H]TH-Aldo added to the extract graphy. immunoactivity with Hj, TH-Aldo antibody.
before chromato¬
Immunologie analysis of a thin layer chromatogram (system V) is shown in Fig. lc. After intravenous administration of [1,2-3H] 18-OH-corticosterone, urine was collected for 24 h from a healthy volunteer. After addition of ßglucuronidase and [l,2-3H]TH-Aldo (15 000 cpm) to the urine sample, and extraction with
dichlormethane, the first immuno- and radioactive
area corres-
Fig. le.
Immunogram =
-
-=
after
chromatography
in
system V (thin layer system). Dichlormethane
extract after /?-glucuronidase treatment. radioactivity in 24 h urine after administration of
immunoactivity
with
Hj,
TH-Aldo
601
antibody.
[3H]18-OH-B
per
os.
to 18-OH-THA, the second to TH-Aldo. The immunoactive area x contained an unidentified material which may be identical with the unidenti¬ fied material shown in Fig. la.
ponded
///. Determination of "TH-Aldo-glucuronide" and TH-Aldo Two methods were developed: a) the estimation of "TH-Aldo-glucuronide" in diluted urine as a potential test for the screening of the aldosterone pro¬ duction and b) the determination of the TH-Aldo in the urine after treatment with /?-glucuronidase, extraction, and chromatography. Flow sheets of the two methods are shown in Table 2. When "TH-Aldo-glucuronide" was determined in preliminary experiments in diluted unpurified urine, no significant correlation was found between these values and the results after chromatography (Kohl 8c Vecsei, unpublished). The correlation was significant, however, when the urine samples were extracted with ethylacetate prior to the dilution of the "TH-Aldo-glucuronide" assay (Fig. 2). For the "TH-Aldo-glucuronide" assay, the intra-assay and the interassay variations were 5.82 °/o and 8.82 °/o, respectively (n 35). =
Table 2. Flow sheet of the estimation of "TH-Aldo-glucuronide" and determination of TH-Aldo in human urine. For details of radioimmunoassay technique see the section "Materials and Methods".
"TH-Aldo-glucuronide" 0.5 ml urine
TH-Aldo 5-20 ml urine, pH 5, 0.5-2 ml Ketodase®, 37°C, 24 h
extraction by 5 ml ethyl
+
acetate
dilution of the extracted urine
15 000 CPM [3H]TH-Aldo extraction by 30-50 ml
(1/50-1/100)
+
dichlormethane
radioimmunoassay: (0.1 ml)
Chromatography: system
III
elution: 2 ml ethanol 0.1 ml recovery
dilution 1/30-1/50
(0.1 ml)
radioimmunoassay (0.1 ml) 602
TH-Aldo-glue" /jg/24 h
r»0,84S p< 0,001 n
500
=
66
1000 TH-Aldo chrom. ¿jg/24 h
Fig. 2. Correlation between the TH-Aldo values determined after chromatography and the "TH-Aldo-gluc." values estimated after extraction with ethyl acetate.
There was a significant correlation between the TH-Aldo values determined after chromatography in system III, and the aldosterone values measured after pH 1 hydrolysis of aldosterone-18-gluc. followed by extraction and chromato¬
graphy (Fig. 3). Aliquots of the eluates from system III were re-chromatographed in system II, followed by TH-Aldo radioimmunoassay. TH-Aldo was determined after chromatography in system II and III. A significant correlation was found. The
Aldo
pHI /jg/24h
30J r
=
0,8729
P« 0,001
1000
TH-Aldo chrom.
pqllU h
Fig. 3. Correlation between the TH-Aldo values and the aldosterone-18-oxo-glucuronide values. Both determinations were carried out after chromatography. 603
values determined after separation in system II and III correlated significantly (r 0.9528, P < 0.001, n 18). For the TH-Aldo determination after chromato¬ graphy the intra-assay variation was 6.23 % and the inter-assay variation 9.67% (n 35), respectively. When water was used instead of urine the values did not differ from 0 in the calibration curve. Known amounts (30-300 ng) of TH-Aldo dissolved in 3 ml water had a recovery rate of 99 %> ± 4.3. The overall recovery of [3H] TH-Aldo after Chromatographie separation was 35-45 °/o. =
=
=
IV. Clinical results
TH-Aldo and
aldosterone-18-gluc.
were
measured in urines of normal sub¬
jects, and in urines of essential hypertensive patients with normal, and with low plasma renin activity. The plasma renin activities (ng angiotensin I/ml/h) were: normal subjects, supine: 0.18-1.8 (mean 0.50), upright: 0.4-3.6 (1.32); hypertension and low plasma renin activity, supine: 0.05-0.3 (0.18), upright: 0.1-0,45 (0.3); hyper¬ tension and normal plasma renin activity, supine: 0.21-1.4 (0.44), upright: 0.55-3.7 (1.25). The resulting TH-Aldo values after chromatography are given in Table 3.
Aldo-18-gluc.
Table 3a. and TH-Aldo values determined in normal
1
2 3 4 5 6 7 8 9 10 11 12
Mean
Range
14.6 4.5 13.9 9.5 8.8 7.0 8.6 4.3 8.0 6.4 11.6 6.0 8.6 4.3-14.6
604
subjects.
33.7 9.5 39.6 35.8 64.8 12.0 16.4 9.7 13.3 27.4 33.0 15.4
25.9 9.5-64.8
Table 3b.
Aldo-18-gluc.
and TH-Aldo in
No.
patients with essential hypertension renin activity.
and normal
Aldo-18-gluc. «g/24 h
TH-Aldo fig/24 h
1.2 19.0 11.9 18.7
24.2 78.2 18.0 27.1 50.1 58.7
1
2 3 4 5 6 7 8 9 10
6.3 11.0 8.5 11.8 5.8 13.6 17.1 4.4 8.1
11
12 13 14
17.0 19.7 64.2 8.9 35.5 25.3 44.3 64.5
12.0
Table 3c.
Aldo-18-gluc.
and TH-Aldo in
with low
plasma
7.6 4.7 9.1 5.4 7.1 9.5 10.5 10.1 9.2 8.8
1
2 3 4 5 6
7 8 9 10
Mean
Range *not
patients
605
activity.
19.8 11.3 95.0 17.4 26.0 42.0 51.1 74.4 52.4 40.4
8.2 4.7-10.5
significantly different
renin
from normal
43.0* 11.3-95.0
subjects
plasma
Aldo-18-gluc.
Table 3d. and TH-Aldo levels in patients with
No.
Aldo-18-gluc. ,«g/24 h 49.4 23.8
primary hyperaldosteronism. TH-Aldo ,«g/24 h 322.5 125.0
Two out of 10
patients with low renin hypertension had a normal excretion of aldosterone-18-gluc. but moderate elevation of TH-Aldo (Table 3c, cases No. 3 and 8). In case 2 of the normal plasma renin activity group a similarly elevated excretion of TH-Aldo was found with the 18-gluc. excretion level close to the upper normal border (Table 3b). Values of two patients with sus¬ pected primary hyperaldosteronism, not yet however confirmed by surgery, are given in Table 3d. These patients showed both 18-oxo-glucuronide and TH-Aldo elevated values. DISCUSSION
The cross-reactions of TH-Aldo antiserum with other steroids and steroid me¬ tabolites are low (Table 1) with the exception of 18-OH-THA (3.2%). The cross-reaction of THF (0.23 %) should also be regarded, due to the high urinary THF concentration. Both materials are, however, easily separated by chromatography from TH-Aldo. The TH-Aldo antiserum binds with the glucuronide (s) of TH-Aldo (Fig. la); however, the 3-glucuronide conjugate of the 3a,5jÔ-TH-Aldo did not crossreact. On the basis of analogies with other steroid antibodies, we assume that a high cross-reactivity exists with the 21-glucuronide but this could not be assessed because the 21-glucuronide and the 3,21-diglucuronide of TH-Aldo were not available. Antisera raised by other TH-corticosteroid antigens have indeed shown high cross-reactivity with the corresponding 21-glucuronides {Vecsei et al. 1976). The immunograms (Fig. la and c) revealed that the TH-Aldo antiserum reacts with an unidentified less polar material. This compound may be a free steroid alcohol, since it could be extracted by benzene from the unprocessed urine. It may have structural similarities to TH-Aldo in view of its binding with the TH-Aldo antibody. The amount of unidentified compound in dif¬ ferent urine samples was unrelated to the amounts of TH-Aldo and aldo¬ sterone-18-gluc. Currently no further information is available regarding the identity of this material. 606
Fig. la shows a high 18-gluc. radioactive peak in comparison to the THAldo peak. The cause of this unexpected event is not known. It is possible that the excretion of the TH-Aldo-gluc. is delayed. Unfortunately we have not analyzed the urine sample of the second 24 h. The "TH-Aldo-glucuronide" estimation in the ethylacetate extracted residue of urine has proven to be of practical use as a simple screening technique to exclude elevated aldosterone production. The unidentified cross-reacting ma¬ terial mentioned above, is removed by ethylacetate extraction of the urine. In addition to TH-Aldo-glucuronide(s), also aldosterone-18-gluc. reacts with the TH-Aldo antibody (Fig. la). This does not, however, limit the usefulness of this method for screening purposes. The radioimmunoassay of TH-Aldo after hydrolysis, extraction, and one paper chromatography, is more practical than the methods described previously (Coppage et al. 1962; Pasqualini et al. 1963; New et al. 1966; Melby et al. 1967; Nielsen et al. 1972). Re-chromatography and repeated determinations have confirmed that substances interfering with the radioimmunoassay of THAldo have been separated from the TH-Aldo by chromatography in system III. Very recently, a radioimmunoassay of TH-Aldo was reported that employs chromatography and aldosterone antibodies which bind to TH-Aldo (Delasalle, personal communication). The normal range of TH-Aldo found by our method agrees with the lowest ranges estimated by other investigators, Ulick (1976) found 34-93 pig/24 h, New et al. (1966) 15-80 /¿g/24 h, Melby et al. (1967) 10-50 pig/24 h, Nicolis et al. (1968) 4-31 jig/24 h, Nielsen et al. (1972) 18-77 /¿g/24 h. The TH-Aldo values obtained in hypertensive patients of different groups and in primary hyperaldosteronism also indicate that the radioimmunoassay of TH-Aldo is a suitable parameter to assess endogenous aldosterone production.
ACKNOWLEDGMENTS The authors wish to thank Thomas
Connelly
for his
help
in
preparing the manuscript.
REFERENCES
Abraham G. E.: J. clin. Endocr. 29 (1969) 866. Brunner H. R., Laragh J. H., Baer L., Newton M. A., Goodwin F. T., Krockoff L. R., Bard R. H. 8c Bühler F. R.: New Engl. J. Med. 286 (1972) 441. Bush I. E.: Biochem. J. 50 (1951) 370. Coppage W. S. Jr., Island D. P., Cooner A. E. Se Liddle G. W.: J. clin. Invest. 41
(1962)
1672.
Erlanger B. F., Borek F., Beiser S. M. & Liebermann S.: J. biol. Grose J. H., Nowaczynski W'., Kachel O. Se Genest J.: J. Steroid 607
Chem. 228 Biochem. 4
(1957) (1973)
713. 551.
Haber E., Koerner T.,
Page
L. B., Kliman B. Se Pamode A.:
J.
clin. Endocr. 29
(1969)
1349.
Kelly
W. G. Se Liebermann S. In: Baulieu E. E. and Robel P., Eds. Aldosterone. Blackwell, Oxford (1964) p. 103. Laragh J. H., Baer L., Brunner H. R., Bühler F. R.. Sealey J. E. Se Vaughan E. D.:
Amer. J. Med. 52 (1972) 633. Mattox V. R. Se Lewbart M. L.: Arch. Biochem. 76 (1958) 362. Melby J. G, Spark R. F., Dale S. L., Egdahl R. E. Se Kahn P. G: New Engl. J. Med. 277
1050.
(1967)
New M. J., Miller B. Se Peterson R. E.: J. clin. Invest. 45 (1966) 412. Nicolis G. L., Wotis H. H. Se Gabrilove J. L.: J. clin. Endocr. 28 (1968) 547. Nielsen M. D., Lund J. O. Se Munck O.: Acta endocr. (Kbh.) 71 (1972) 198. Pasqualini J. R., Legrand J. C. Se Jayle M. F.: Acta endocr. (Kbh.) 43 (1963) 67. Ulick S.: J. clin. Endocr. 43 (1976) 92. Ulick S. Se Liebermann S.: J. Amer. chem. Soc. 79 (1957) 6567. Ulick S. Se Vetter K. K.: J. biol. Chem. 237 (1962) 3364. Vecsei P. In: Jaffe B. M. and Behrmann H. R., Eds. Methods of Hormone Radioimmuno¬ assay. Academic Press, New York, London (1974) p. 393. Vecsei P. Se Gless K. H.: Aldosteron-Radioimmunoassay. Ferdinand Enke Verlag, Stutt¬
gart (1975).
Vecsei P., Kohi K. H., Mok M., Pallai P., Penke B., Vielhauer W. Se Will H.: V. Int. Congr. Endocr., Hamburg (1976) Abstract 373. Vecsei P., Onyechi R., Hornung J., Dietz R., Mast G. Se Hobler H.: J. Steroid Biochem. 6
(1975)
383.
Weber G.: Biochem. Received
on
J.
July 8th,
51
(1952)
155.
1977.
608
of
Pharmacology, University Heidelberg, Germany
of
Heidelberg
RADIOIMMUNOASSAYS OF TETRAHYDROALDOSTERONE (TH-ALDO) IN HUMAN URINE
By K.-H. Kohl, P. Vecsei and S. Abdelhamid ABSTRACT
Specific
antisera
against tetrahydroaldosterone (TH-Aldo)
two white New Zealand rabbits.
were
raised in
3\g=a\,5\g=b\-TH-aldo-20-oxime-bovine-serum
albumin complex was used as antigen. The resulting titers were 1:18 000 and 1:16 000. Except tetrahydrocortisol (THF) (0.23%) and tetrahydro\x=req-\ 18-hydroxy-11-dehydrocorticosterone (18-OH-THA) (3.2 %), all steroids and steroid metabolites gave negligible cross-reactions. Immunograms of the paper chromatograms made from the n-butanol-extract of the urines, as well as after \g=b\-glucuronidase treatment and dichlormethane extraction, were studied to further define the specificity of the antiserum. Antibody H1 (used in this study) reacted with aldosterone-18-gluc., a TH-aldosterone-glucuronide (probably the 21-glucuronide) and an unidentified less polar material. Two methods were developed: a) TH-Aldo-glucuronidc(s) estimation after ethylacetate pre-extraction as a rapid screening test of endogenous aldosterone production. b) estimation of TH-aldosterone using one chromatographic system. The results of method a) showed a significant correlation with the values obtained by technique b). Normal values (method b) were 25.88 \m=+-\16.50 \g=m\g/24h(range 9.5\p=n-\64.8\g=m\g/24h). A significant correlation was also shown between the TH-aldo (technique b) and 18-gluc. values.
TH-Aldo is the major metabolite of aldosterone isolated from human urine (Ulick 8c Liebermann 1957; Ulick 8c Vetter 1962). It is formed in the liver and excreted as glucuronide. 15-40 °/o of the secreted aldosterone is excreted in the
Partly presented at the V International Congress of Endocrinology, Hamburg, July 1976. ') Deutsche Klinik für Diagnostik, Wiesbaden, GFR. Requests for reprints should be addressed to Dr. P. Vecsei, Pharmakologisches der Universität, Im Neuenheimer Feld 366, Heidelberg. 596
Institut
form of this metabolite, mann
mostly (80-90 %)
as
the 3a,
5/?-isomer (Kelly
8c Lieber¬
1964).
Measurement of TH-Aldo in urine can be expected to be more representative of the aldosterone production than measurement of the aldosterone 18-gluc, which represents only 5-15 °/o of the endogenous aldosterone. Preparation of a TH-Aldo antibody and a radioimmunoassay of TH-Aldo with TH-Aldo-antiserum have not been reported in the literature.
MATERIAL AND METHODS
(Aldo), Cortisol (F), corticosterone (B), compound S, tetrahydrocortisone (THE), tetrahydrocortisol (THF) and pregnantriol were obtained from Merck, Darm¬ stadt, GFR. The 3- and 21-glucuronides of the tetrahydrocorticosteroid metabolites were generously provided by V. R. Mattox, Rochester, Minn., and tetrahydro-18hydroxy-11-dehydrocorticosterone (18-OH-THA) was provided by S. Ulick, New York, Aldosterone
N.Y. All other corticosteroids and corticosteroid metabolites,
List
as
listed in the
of abbreviations: Aldo ll/?,21-dihydroxy-4-pregnene-3,20-dion-18-al F ll/?,17a,21-trihydroxy-4-pregnene-3,20-dione B 11/?,21 -dihydroxy-4-pregnene-3,20-dione S 17a,21-dihydroxy-4-pregnene-3,20-dione THE 3a, 17a,21 -trihydroxy-5/?-pregnane-l 1,20-dione THF 3a, 11/?, 17 a,21-tetrahydroxy-5/?-pregnane-20-dione Pregnanetriol 3a,17a,20a-trihydroxy-5/?-pregnane 3a,5yS-TH-Aldo 3 0% 0.18 "la 0"la 0.5 °/o 0.23 "la 0"la 0"la 0"la 0"la 2 "la 0»/o 0.22 "la 0»/o 0°/o 0°/o 0.3 «Vo
pg 600 500
500
¿00
400
300
300
200
200
100
100
Fig. la. Immunogram after chromatography in system I, A aldosterone-18-oxo-glucuronide.
n-butanol extract of
a
normal urine.
=
B X
=
=
TH-Aldo-glucuronide(s). unidentified substance. =
-
=
radioactivity in 24 h urine after administration of [^H]TH-Aldo intravenously. immunoactivity with Hj, TH-Aldo antibody.
-----
600
TH-Aldo
P9
2500
-s-
Fig. lb. Immunograms
after
chromatography
in
system III. Dichlormethane
yS-glucuronidase
= -
-=
extract after
treatment.
radioactivity of f3H]TH-Aldo added to the extract graphy. immunoactivity with Hj, TH-Aldo antibody.
before chromato¬
Immunologie analysis of a thin layer chromatogram (system V) is shown in Fig. lc. After intravenous administration of [1,2-3H] 18-OH-corticosterone, urine was collected for 24 h from a healthy volunteer. After addition of ßglucuronidase and [l,2-3H]TH-Aldo (15 000 cpm) to the urine sample, and extraction with
dichlormethane, the first immuno- and radioactive
area corres-
Fig. le.
Immunogram =
-
-=
after
chromatography
in
system V (thin layer system). Dichlormethane
extract after /?-glucuronidase treatment. radioactivity in 24 h urine after administration of
immunoactivity
with
Hj,
TH-Aldo
601
antibody.
[3H]18-OH-B
per
os.
to 18-OH-THA, the second to TH-Aldo. The immunoactive area x contained an unidentified material which may be identical with the unidenti¬ fied material shown in Fig. la.
ponded
///. Determination of "TH-Aldo-glucuronide" and TH-Aldo Two methods were developed: a) the estimation of "TH-Aldo-glucuronide" in diluted urine as a potential test for the screening of the aldosterone pro¬ duction and b) the determination of the TH-Aldo in the urine after treatment with /?-glucuronidase, extraction, and chromatography. Flow sheets of the two methods are shown in Table 2. When "TH-Aldo-glucuronide" was determined in preliminary experiments in diluted unpurified urine, no significant correlation was found between these values and the results after chromatography (Kohl 8c Vecsei, unpublished). The correlation was significant, however, when the urine samples were extracted with ethylacetate prior to the dilution of the "TH-Aldo-glucuronide" assay (Fig. 2). For the "TH-Aldo-glucuronide" assay, the intra-assay and the interassay variations were 5.82 °/o and 8.82 °/o, respectively (n 35). =
Table 2. Flow sheet of the estimation of "TH-Aldo-glucuronide" and determination of TH-Aldo in human urine. For details of radioimmunoassay technique see the section "Materials and Methods".
"TH-Aldo-glucuronide" 0.5 ml urine
TH-Aldo 5-20 ml urine, pH 5, 0.5-2 ml Ketodase®, 37°C, 24 h
extraction by 5 ml ethyl
+
acetate
dilution of the extracted urine
15 000 CPM [3H]TH-Aldo extraction by 30-50 ml
(1/50-1/100)
+
dichlormethane
radioimmunoassay: (0.1 ml)
Chromatography: system
III
elution: 2 ml ethanol 0.1 ml recovery
dilution 1/30-1/50
(0.1 ml)
radioimmunoassay (0.1 ml) 602
TH-Aldo-glue" /jg/24 h
r»0,84S p< 0,001 n
500
=
66
1000 TH-Aldo chrom. ¿jg/24 h
Fig. 2. Correlation between the TH-Aldo values determined after chromatography and the "TH-Aldo-gluc." values estimated after extraction with ethyl acetate.
There was a significant correlation between the TH-Aldo values determined after chromatography in system III, and the aldosterone values measured after pH 1 hydrolysis of aldosterone-18-gluc. followed by extraction and chromato¬
graphy (Fig. 3). Aliquots of the eluates from system III were re-chromatographed in system II, followed by TH-Aldo radioimmunoassay. TH-Aldo was determined after chromatography in system II and III. A significant correlation was found. The
Aldo
pHI /jg/24h
30J r
=
0,8729
P« 0,001
1000
TH-Aldo chrom.
pqllU h
Fig. 3. Correlation between the TH-Aldo values and the aldosterone-18-oxo-glucuronide values. Both determinations were carried out after chromatography. 603
values determined after separation in system II and III correlated significantly (r 0.9528, P < 0.001, n 18). For the TH-Aldo determination after chromato¬ graphy the intra-assay variation was 6.23 % and the inter-assay variation 9.67% (n 35), respectively. When water was used instead of urine the values did not differ from 0 in the calibration curve. Known amounts (30-300 ng) of TH-Aldo dissolved in 3 ml water had a recovery rate of 99 %> ± 4.3. The overall recovery of [3H] TH-Aldo after Chromatographie separation was 35-45 °/o. =
=
=
IV. Clinical results
TH-Aldo and
aldosterone-18-gluc.
were
measured in urines of normal sub¬
jects, and in urines of essential hypertensive patients with normal, and with low plasma renin activity. The plasma renin activities (ng angiotensin I/ml/h) were: normal subjects, supine: 0.18-1.8 (mean 0.50), upright: 0.4-3.6 (1.32); hypertension and low plasma renin activity, supine: 0.05-0.3 (0.18), upright: 0.1-0,45 (0.3); hyper¬ tension and normal plasma renin activity, supine: 0.21-1.4 (0.44), upright: 0.55-3.7 (1.25). The resulting TH-Aldo values after chromatography are given in Table 3.
Aldo-18-gluc.
Table 3a. and TH-Aldo values determined in normal
1
2 3 4 5 6 7 8 9 10 11 12
Mean
Range
14.6 4.5 13.9 9.5 8.8 7.0 8.6 4.3 8.0 6.4 11.6 6.0 8.6 4.3-14.6
604
subjects.
33.7 9.5 39.6 35.8 64.8 12.0 16.4 9.7 13.3 27.4 33.0 15.4
25.9 9.5-64.8
Table 3b.
Aldo-18-gluc.
and TH-Aldo in
No.
patients with essential hypertension renin activity.
and normal
Aldo-18-gluc. «g/24 h
TH-Aldo fig/24 h
1.2 19.0 11.9 18.7
24.2 78.2 18.0 27.1 50.1 58.7
1
2 3 4 5 6 7 8 9 10
6.3 11.0 8.5 11.8 5.8 13.6 17.1 4.4 8.1
11
12 13 14
17.0 19.7 64.2 8.9 35.5 25.3 44.3 64.5
12.0
Table 3c.
Aldo-18-gluc.
and TH-Aldo in
with low
plasma
7.6 4.7 9.1 5.4 7.1 9.5 10.5 10.1 9.2 8.8
1
2 3 4 5 6
7 8 9 10
Mean
Range *not
patients
605
activity.
19.8 11.3 95.0 17.4 26.0 42.0 51.1 74.4 52.4 40.4
8.2 4.7-10.5
significantly different
renin
from normal
43.0* 11.3-95.0
subjects
plasma
Aldo-18-gluc.
Table 3d. and TH-Aldo levels in patients with
No.
Aldo-18-gluc. ,«g/24 h 49.4 23.8
primary hyperaldosteronism. TH-Aldo ,«g/24 h 322.5 125.0
Two out of 10
patients with low renin hypertension had a normal excretion of aldosterone-18-gluc. but moderate elevation of TH-Aldo (Table 3c, cases No. 3 and 8). In case 2 of the normal plasma renin activity group a similarly elevated excretion of TH-Aldo was found with the 18-gluc. excretion level close to the upper normal border (Table 3b). Values of two patients with sus¬ pected primary hyperaldosteronism, not yet however confirmed by surgery, are given in Table 3d. These patients showed both 18-oxo-glucuronide and TH-Aldo elevated values. DISCUSSION
The cross-reactions of TH-Aldo antiserum with other steroids and steroid me¬ tabolites are low (Table 1) with the exception of 18-OH-THA (3.2%). The cross-reaction of THF (0.23 %) should also be regarded, due to the high urinary THF concentration. Both materials are, however, easily separated by chromatography from TH-Aldo. The TH-Aldo antiserum binds with the glucuronide (s) of TH-Aldo (Fig. la); however, the 3-glucuronide conjugate of the 3a,5jÔ-TH-Aldo did not crossreact. On the basis of analogies with other steroid antibodies, we assume that a high cross-reactivity exists with the 21-glucuronide but this could not be assessed because the 21-glucuronide and the 3,21-diglucuronide of TH-Aldo were not available. Antisera raised by other TH-corticosteroid antigens have indeed shown high cross-reactivity with the corresponding 21-glucuronides {Vecsei et al. 1976). The immunograms (Fig. la and c) revealed that the TH-Aldo antiserum reacts with an unidentified less polar material. This compound may be a free steroid alcohol, since it could be extracted by benzene from the unprocessed urine. It may have structural similarities to TH-Aldo in view of its binding with the TH-Aldo antibody. The amount of unidentified compound in dif¬ ferent urine samples was unrelated to the amounts of TH-Aldo and aldo¬ sterone-18-gluc. Currently no further information is available regarding the identity of this material. 606
Fig. la shows a high 18-gluc. radioactive peak in comparison to the THAldo peak. The cause of this unexpected event is not known. It is possible that the excretion of the TH-Aldo-gluc. is delayed. Unfortunately we have not analyzed the urine sample of the second 24 h. The "TH-Aldo-glucuronide" estimation in the ethylacetate extracted residue of urine has proven to be of practical use as a simple screening technique to exclude elevated aldosterone production. The unidentified cross-reacting ma¬ terial mentioned above, is removed by ethylacetate extraction of the urine. In addition to TH-Aldo-glucuronide(s), also aldosterone-18-gluc. reacts with the TH-Aldo antibody (Fig. la). This does not, however, limit the usefulness of this method for screening purposes. The radioimmunoassay of TH-Aldo after hydrolysis, extraction, and one paper chromatography, is more practical than the methods described previously (Coppage et al. 1962; Pasqualini et al. 1963; New et al. 1966; Melby et al. 1967; Nielsen et al. 1972). Re-chromatography and repeated determinations have confirmed that substances interfering with the radioimmunoassay of THAldo have been separated from the TH-Aldo by chromatography in system III. Very recently, a radioimmunoassay of TH-Aldo was reported that employs chromatography and aldosterone antibodies which bind to TH-Aldo (Delasalle, personal communication). The normal range of TH-Aldo found by our method agrees with the lowest ranges estimated by other investigators, Ulick (1976) found 34-93 pig/24 h, New et al. (1966) 15-80 /¿g/24 h, Melby et al. (1967) 10-50 pig/24 h, Nicolis et al. (1968) 4-31 jig/24 h, Nielsen et al. (1972) 18-77 /¿g/24 h. The TH-Aldo values obtained in hypertensive patients of different groups and in primary hyperaldosteronism also indicate that the radioimmunoassay of TH-Aldo is a suitable parameter to assess endogenous aldosterone production.
ACKNOWLEDGMENTS The authors wish to thank Thomas
Connelly
for his
help
in
preparing the manuscript.
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