Clinical Science (1981) 61,201-206
20 1
Evaluation of the mineralocorticoid activity of
18-hydroxycorticosterone
G. H U S T O N * , V . I. M A R T I N , E. A. S. A L - D U J A I L I A N D C. R . W. E D W A R D S
Departments of Clinical Pharmacology and Endocrinology, St BartholomewS Hospital, London
(Received 17 November 197814 July 1980; accepted 25 February 1981)
Summary
1. The mineralocorticoid activity of 18hydroxycorticosterone (18-OH-B) has been compared with that of aldosterone by using human
bioassay in vivo with measurement of rectal
potential difference and urinary log 10 Na+/K+
ratio.
2. A log-linear relationship was found between maximum change in rectal potential difference and increasing doses of aldosterone.
3. No mineralocorticoid activity could be
demonstrated after an intravenous bolus and
infusion of 18-OH-B.
4. The half-life of clearance of 18-OH-B was
measured in three subjects and found to be 28,48
and 24 min.
Key words:
sterone.
aldosterone,
18-hydroxycortico-
Introduction
18-Hydroxycorticosterone (18-OH-B) is thought
to be an intermediate in aldosterone Piosynthesis
[ l ] and is produced by the 18-hydroxylation of
corticosterone [21. In man the secretion rate of
18-OH-B parallels that of aldosterone [31. However, despite this association and the fact that the
secretion rate of 18-OH-B is approximately twice
Abbreviations: 18-OH-B, 18-hydroxycorticosterone; 18-OH-DOC, 18-hydroxydeoxycorticosterone.
Correspondence: Professor C. R. W. Edwards,
Department of Medicine, Western General Hospital,
Edinburgh EH4 2XU, Scotland, U.K.
* Present address: Department of Rheumatology,
Guy’s Hospital, London.
0143-5221/81/080201-06$01.50/1
that of aldosterone, very little is known of its
biological activity.
Feldman & Funder [41 and Baxter, Schambelan, Mattulich, Spindler, Addison & Bartter 151
demonstrated that 18-OH-B has a low affinity for
mineralocorticoid or glucocorticoid receptors. In
man the low plasma concentrations of 18-OH-B
in conjunction with this low affinity would
suggest that 18-OH-B has no significant
mineralocorticoid or glucocorticoid effects [3, 61.
It has, however, been suggested that the constant
hypersecretion of even a weak mineralocorticoid
may lead to hypertension [71. The mineralocorticoid activity of 18-OH-B has not been
evaluated in man. These studies were therefore
undertaken to determine the effect of infusions of
18-OH-B on transmural rectal potential difference and urinary sodium and potassium
excretion and to compare the effect of 18-OH-B
with that of aldosterone. In addition, studies were
also carried out to determine the half-life of
clearance of 18-OH-B from the circulation.
Materials and methods
Rectal potential difference was measured with
silver, silver chloride, saline/agar electrodes [81
used in conjunction with an Orion 701 millivoltmeter. An EEL flame photometer was used
for urinary electrolyte determination and a Sage
(Orion) constant-infusion pump for infusion
studies.
Experiments were carried out with five normal
male volunteer subjects between the ages of 29
and 55 years to whom the protocol was fully
explained and who gave their informed consent.
Quantitative mineralocorticoid bioassays were
established in subjects nos. 1, 2 and 3; subjects
nos. 4 and 5 took part in qualitative studies.
0 1981 The Biochemical Society and the Medical Research Society
G. Huston et al.
202
TABLE1. Experimentalprotocolfor aIdosterone and 18-OH-B bioassays
Bolus dose
(pg/kg)
(pg min-’ kg-’)
Infusions
14.284
7.142
3.571
1.785
0.892
0.446
0.223
0.111
Control
0.494
0.247
0.1235
0.0617
0.0308
0.0154
0.0077
0.0038
Control
18-OH-B
Aldosterone
Expt .... I
***
.
.
1
***
* 0.
-
***
1
.
1
**I
***
2
3
1
2
3
4
5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
...
***
-
*I*
t”*
* *I
* *I
01.
**I
***
1
.
1
0.0
-
***
-
-
-
-
-
..t
011
***
*I*
&*b
111
I**
I*
*I
.*
-
**
* Measurement of rectal potential difference.
** Measurement of urinary Na+/K+ excretion.
t Subject no. 1 was given two infusions of 18-OH-Bat this dose level.
The infused doses of steroid and the parameters measured are shown in Table 1. All
subjects were allowed a sodium intake ad libitum.
18-OH-B was bought from Fluka Fluorochem
Ltd, Switzerland. Ampoule concentrations and
plasma levels of 18-OH-B and 18-hydroxydeoxycorticosterone (18-OH-DOC) were assayed as
described previously [6, 91. Plasma aldosterone
was measured by a highly specific direct radioimmunoassay [ 101. Plasma cortisol levels were
assayed with a commercial kit (Cortipac; The
Radiochemical Centre, Amersham, Bucks.,
U.K.).
The infusion rate required to maintain a
constant plasma concentration of 18-OH-B or
aldosterone after a bolus dose was calculated
from the equation: infusion rate = D0(0.693/t+),
where tt is the biological half-life of the infused
steroid and Do is the bolus dose of administered
steroid [ 111. In establishing dose-response curves
for aldosterone, bolus doses of between 0.11 1
and 14.25 pg/kg were used.
The infusion calculation was performed with
an approximated half-life for aldosterone of
20 min [ 121. In the absence of information on the
half-life of 18-OH-B the same figure was used in
the calculation of the infusion dose for this
steroid.
Experimental protocol
All experiments were performed on fasting
subjects who were hydrated by being given
200 ml of water every 2 h. An interval of at least 1
week was allowed to elapse between infusion
experiments. All experiments were commenced at
09.00 hours, the subjects remaining recumbent
throughout the experimental period apart from
111 I
I
1
0
I
I
2
I
I
4
I
I
6
I
J
8
Time (h)
FIG. 1. Flow chart of infusion experiment; urine
samples were obtained and rectal potential differences
(p.d.) measured together at the times indicated.
rising to pass urine. The design of the infusion
experiments is shown in Fig. 1. Measurements of
transmural rectal potential difference were made
7 cm from the anal margin avoiding pressure
artifacts; the reference site was an area of skin
injected intradermally with sodium chloride
solution (150 mmol/l) [13]. Urinary sodium
and potassium concentrations were measured by
flame photometry.
The bolus injection of either aldosterone or
18-OH-B was given over 1 min in 10 ml of 5%
(wh) glucose solution and was followed by a
constant-rate infusion in 45 ml of 5% (w/v)
glucose solution over 60 min; a control bolus and
infusion of 5% (w/v) glucose solution alone was
also given. 18-OH-B or glucose solutions were
administered in a non-blind cross-over fashion to
subjects nos. 1 , 2 and 3.
To assess the half-life of clearance of 18-OH-B,
blood samples were taken at intervals up to 150
203
18-Hydroxycorticosteronebioactivity
rnin after the infusion of 18-OH-B was stopped in
subjects nos. 1, 2 and 3. In addition to 18-OH-B,
plasma levels of aldosterone, cortisol and 18OH-DOC were also measured. In all subjects
urine samples were collected immediately before
the bolus injection and at 2 h intervals for 8 h.
Further samples were obtained at 12 and 24 h.
Blood pressure measurements were made at
15 min intervals for 30 min before delivery of the
bolus, at 15 rnin intervals for 1 h after delivery of
the bolus, at 30 min intervals for 2 h and then
hourly until 7 h after the end of the infusion. A
London School of Hygiene and Tropical Medicine sphygmomanometer [141 was used to
measure blood pressure with Korotkoff's first and
fifth sound for each measurement.
log 10 Na+/K+ found in subjects nos. 1, 2 and 3
after aldosterone and Table 3 the maximum
changes in these parameters in response to
18-OH-B infusion.
In subjects nos. 4 and 5 changes in only the log
10 Na+/K+ ratio were measured. Neither of the
parameters demonstrated any evidence of
mineralocorticoid activity after 18-OH-B bolus
and infusion in any subject.
Results
Rectal potential direrence and urinary log
1 ONa+IK+ ratio
The maximum increase in rectal potential
difference in response to intravenous aldosterone
occurred 4 to 5 h after the administration of the
bolus. A typical time course of response in
subject no. 1 is shown in Fig. 2. The relationship
between the log-bolus dose of aldosterone and
maximum change in rectal potential difference in
the same subject is shown in Fig. 3. Plasma
aldosterone levels achieved by the bolus and
infusion technique in subjects nos. 1 and 2 are
shown in Fig. 4. Table 2 includes the maximum
changes in rectal potential difference and urinary
0
100
10
1
Aldosterone bolus (gg/kg)
FIG.3. Dose-response relationship between maximal
change in rectal potential difference (p.d.) and bolus
dose of aldosterone in subject no. 1.
100
h
r
-20
2
4 10
0
e
-30
8
s
5
2
-
0
P
2
-40
3
a
1
8
3
X
c
n
-50
2
-60
0.1
#
-10
-70
0
2
I
I
I
4
Time (h)
6
8
FIG. 2. Time course of response of rectal potential
difference (p.d.) to intravenous aldosterone in subject
no. 1 (bolus dose 7-142 yg/kg, infusion dose 0.247 pg
min-' kg-').
1
0
1
1
20
1
1
1
40
1
60
1
1
80
1
1
100
Time (min)
FIG. 4. Plasma aldosterone levels attained by bolus
and infusion techniques in subjects nos. 1 and 2.
Subject no. 1: 0, bolus 3.571 yg/kg and infusion
0.1235 yg min-' kg-'; 0, bolus 0.446 yg/kg and
infusion 0.0154 pg min-' kg-'. Subject no. 2:W, bolus
0.892yglkg; infusion 0-0308yg min-' kg-'.
G. Huston et al.
204
TABLE
2. Maximum changes in rectal potential difference and urinary log 10 N a + f K +responses to aldosterone bolus and
infusion
Bolus
dose
Subject no. I
Infusion
Max.
change in p.d.
(pglkg)
(mV)
14.284
7.142
3.571
1.785
0.892
0.446
0.223
0.111
Control
Subject no. 2
Subject no. 3
(pg min-' kg-I)
0.494
0.247
0. I235
0.06 I7
0.0308
0.0154
0.0077
0.0038
Control
Max.
change in
log 10 Na+/K+
of urine
-36
-35
-3 1
-27
Max.
change in p.d.
(mV)
-
-
-
-0.5 1
-0.3 1
-0.39
-0.37
-0.32
-
-
-49
-39
-35
-3 1
-19
-0.6
-0.35
-0.42
-0.36
-0.43
-39
-39
-37
-27
-I2
-
-0.36
-0.30
-0.18
+0.04
-I
Max.
change in p.d.
-
-0.56
-0.87
-0.58
-0.70
-22
-19
-9
Max.
change in
log 10 Na+/K+
of urine
-
(mV)
-
-3
-
-
-
-0.08
Max.
change in
log 10 Na+/K+
of urine
-3
-0.05
TABLE3. Maximum changes in rectal potential direrence and log 10 Na+/K+ ratio in response to bolus and infusion of
18-OH-B
Bolus
dose
Infusion
(pg/kg)
(pdkg)
Subject no. 2
Subject no. 1
Subject no. 3
Max.
change in
p.d. (mv)
Max.
change in
log
10 Na+/K+
Max.
change in
p.d. (mV)
Max.
change in
log
10 Na+/K+
+0.37
+0.13
-
-
3.571
0.1235
(1)+3
(2) -1
1.785
0.892
Control
0.0617
0.0308
-
-2
-
-
+0.08
-1
-
-3
Blood pressure responses
In subject no. 1 (experiment 1) a blood
pressure rise of 40 mmHg systolic and 30 mmHg
diastolic was observed 2 h after the bolus
injection of 18-OH-B; this was not reproduced
when the experiment was repeated after an
interval of 3 months or after the infusion of
18-OH-B into any other subject. No subject
showed a significant change in pulse rate or other
side effects during the infusion. None of the
aldosterone infusions produced a change in blood
pressure.
Plasma levels of 18-OH-B
Plasma concentrations of 1 8-OH-B, 18-OHDOC, aldosterone and cortisol during infusion of
18-OH-B in subjects nos. 1, 2 and 3 are shown in
Table 4. Assuming that the distribution of
+0.25
-
-0.08
Max.
change in
p.d. (mV)
-5
Max.
change in
log
10 Na+/K+
-0.21
Subject no. 4 Subject no. 5
Max.
Max.
change in
change in
log
log
10 Na+/K+ 10 Na+/K+
+0.19
-
-
-
-
-
-0.10
+O.ll
-3
-
+0.04
-0.03
18-OH-B throughout the tissue was complete
during the infusion and that the exponential decay
of plasma concentration of 18-OH-B after cessation of the infusion represents only metabolism
and excretion, the half-lives of elimination of
18-OH-B from the plasma of subjects nos. 1, 2
and 3 were 28,48 and 24 min respectively.
Plasma levels of 18-OH-DOC showed only a
minimal rise after infusion of 18-OH-B in subjects
nos. 1 and 2; a fivefold rise in plasma 18OH-DOC concentration was seen at 60 min in
subject no. 3. In this subject, in comparison with
the other two, plasma cortisol levels rose during
the 18-OH-B infusion and it would seem likely
that the rise in 18-OH-DOC was secondary to
adrenocorticotropic hormone release in response
to the experimental stress.
Plasma aldosterone levels before and after
18-OH-B infusions are shown in Table 4. There
was no consistent change of plasma aldosterone
during or after the 18-OH-B infusion.
18-Hydroxycorticosteronebioactivity
205
Discussion
oolnoooo
wr-m"i]m
A log-linear relationship between maximum
change in rectal potential difference and increasing doses of intravenously administered aldosterone has previously been demonstrated [ 151.
This relationship was confirmed in the present
study as was the threshold nature of the urinary
electrolyte response to increasing mineralocorticoid activity.
The synchronous use of two mineralocorticoid
assay systems was thought appropriate as
circumstantial evidence has been obtained for a
dissociation of renal and large gut mineralocorticoid response to an unidentified adrenal
steroid in a hypertensive patient [161. A similar
dissociation appeared to be present in the
responses of normal subjects to carbenoxolone
treatment, after which rectal effects predominated [17, 181. It seemed important therefore to measure both renal and gastrointestinal
responses.
It has been demonstrated that supine plasma
aldosterone levels and electrical asymmetry
across sodium transporting epithelia are almost
completely suppressed by a sodium intake of
100-300 mmol/day [191, this being the range of
sodium intake encountered in developed countries
[20, 211. In this study, in which no alterations to
the subjects' normal diets were made, there was
no consistent depression of plasma aldosterone
by the 18-OH-B infusion which might have
masked a minor mineralocorticoid activity of
18-OH-B.
No mineralocorticoid activity could be demonstrated in response to an intravenous bolus and
infusion of 18-OH-B in any subject. As might
have been predicted from the findings of Vecsei,
Purjesz & Wolff [221 conversion of 18-OH-DOC
was minimal and did not produce a measurable
mineralocorticoid response. Log 10 Na+/K+ ratio
was greater after 18-OH-B bolus and infusion
than after control infusion in four of the five
subjects. It has been shown that sodium excretion in adrenalectomized rats is increased by the
infusion of 18-OH-B [23]. These results, therefore, raise the possibility of a natriuretic effect in
man. This requires further investigation and also
measurement of titratable H+ and NH: excretion
which are increased by 18-OH-B in the rat [231.
The hypertensive response observed after the
first infusion of 18-OH-B in subject no. 1 was not
reproducible and remains unexplained. If 18OH-B does play a role in the genesis of
hypertension in man, the present study would
suggest that it is unlikely to be mediated through
mineralocorticoid activity.
206
G.Huston et al.
Acknowledgments
We are indebted to the Medical Research
Council, the British Heart Foundation and the
North East Thames Regional Health Authority
for financial support.
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