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1
Laboratoire de Biochimie Hormonale, Hôpital Saint-Louis, 75010 Paris, France.
2
Laboratoire de Chimie Organique
3
Service de Néphrologie, Hôpital Foch, 92151
Suresnes, France.
4
Laboratoire d'Explorations Endocriniennes,
Hôpital Trousseau, 75012 Paris, France.
5
Service des Molécules Marquées, CEA/Saclay,
91191 Gif-sur-Yvette, France.
6
Department of Biochemistry and National Diagnostics
Centre, University College, Galway, Ireland.
7
Biochimie, Faculté de Pharmacie, 75006 Paris,
France.
a Address correspondence to this author at: Laboratoire de Biochimie Hormonale, Hôpital Saint-Louis, 1 ave. Claude-Vellefaux, 75475 Paris cédex 10, France. Fax + 33 1 42 49 42 80; e-mail bio.horm.fiet{at}chu-stlouis.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: indexing terms: scintillation proximity assay • apparent mineralocorticoid excess • 11ß-hydroxysteroid dehydrogenase • Cushing syndrome
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inversely, conversion of E to F takes place mainly in the liver (6), under the action of 11-HSD-1. This enzyme probably regulates access of glucocorticoids to their receptors (7).
In the adult, plasma F exceeds E 10-fold, whereas in the fetus, in which 11-HSD-2 is present in many tissues (3), the E concentration is threefold higher than that of F (11-HSD-2 is also abundant in the placenta [8, 9]). In the fetus, 11-HSD-2 is thought to prevent the deleterious effects of high F concentrations and to help adrenal development (3). Patients with mutations in the 11-HSD-2 gene (10)(11)(12)(13)(14) have been reported to have a severe and sometimes fatal (15)(16)(17) hypertensive syndrome called apparent mineralocorticoid excess type 1 (AME-1) (18) and to have a low birth weight (19). Thus, the F–E shuttle appears to be implicated in fetal development and arterial hypertension. Methods that aim to evaluate its status may be clinically relevant.
Ulick et al. used gas chromatography to establish a diagnosis of AME on
the basis of urinary assays of the tetrahydrometabolites of F (THF and
aTHF) and E (THE) (18). While useful, this method is
lengthy and time-consuming and the THF + aTHF/THE ratio obtained
reflects not only the altered activities of 11-HSD-2, but also those of
the 5
- and 5ß-reductases encountered in AME-1 (20).
We describe a new, sensitive, specific, and simple RIA for E that, when used in combination with a F RIA, provides an accurate and unambiguous assessment of F/E equilibrium.
After validating the new E RIA, we studied variations in basal and
post-Cosyntropin {Synacthen® [-1-24
corticotropin (ACTH)]} and dexamethasone (DXM) concentrations of E
and F over 24 h in simultaneous samples of serum, saliva, and
urine.
We also evaluated the clinical applications of this new E assay in Cushing syndrome and in chronic renal insufficiency (CRI), as well as its diagnostic potential in AME.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Healthy volunteers.
Fifty healthy Caucasian
nonsmoking volunteers (27 women and 23 men) ranging in age from 21 to
46 years (mean 28) taking no medication or contraceptives and not
eating licorice were included in the study to establish normal values.
Thirty-one of them (17 women and 14 men) participated in a circadian
study. Sixteen underwent dynamic testing with short-acting Synacthen
(Novartis, Rueil-Malmaison, France) (0.25 mg intramuscularly), five
with long-acting Synacthen (1 mg), and three were subjected to standard
DXM suppression tests (3 mg x 5 days).
Patients.
We also studied a selected population of
patients in university teaching hospital endocrinology and nephrology
services in the Paris area. Two of the patients had primary adrenal
insufficiency (Addison disease), two had secondary adrenal
insufficiency (hypopituitarism), 10 had adrenal adenoma, two had
Cushing disease, five had ectopic ACTH secretion, 10 were hypertensive
patients with CRI, and two were cases of 11ß-OH steroid dehydrogenase
deficiency (AME-1).
Samples.
For the circadian study, in addition to 24-h
urines, saliva and serum samples were collected at 8, 12, 16, and
20 h, and at 0 and 4 h the next day. Urine samples were also
obtained at approximately the same times from 20 of the healthy
subjects. Aliquots were stored at -20 °C until assay. We tested the
sera and saliva of the CRI patients at 8 h, before they began
dialysis. A neutral (flavorless) chewing-gum was chewed by both healthy
subjects and patients, who had nothing by mouth for at least 1 h
before obtaining 8-mL saliva samples from them.
Reagents.
Steroids were purchased from Sigma
(Saint-Quentin-Fallavier, France) and Steraloids (Wilton, NH), and
tritiated F 2.40 TBq/mmol from Amersham (Les Ulis, France). Solvents
were of analytical or HPLC grade (Merck, Nogent/Marne, France).
Phosphate gelatin buffer (PGB) was prepared from 0.04 mol/L phosphate
buffer, pH 7.4, + 1 g/L gelatin. BCS scintillation liquid was purchased
from Amersham and RPN140 antirabbit scintillation proximity assay
reagent (SPA) from Amersham.
125I-labeled ACTH IRMA kit.
A Nichols
Allegro HS-ACTH kit (ref. CA2194) purchased from
Mallinckrodt-Diagnostica-France (Evry, France) was used to assay ACTH.
125I-labeled aldosterone RIA kit.
Aldosterone was assayed with DPC Coat-a-count® kit
(TKAL20) obtained from SAPB Höechst Behring (Rueil-Malmaison,
France).
Angiotensin I, RIA
125I kit.
RENCTK (P2721) bought from Sorin-Biomedica (Antony, France) was used
for plasma renin activity assay.
125I-labeled F RIA kit.
An Incstar kit
(ref. CA1549) obtained from Sorin-Biomedica was used for F
determination, after extraction and Celite chromatography separation
(see below).
Preparation of [1,2-
3H]E.
Prednisone (10 mg) in ethanol (3 mL) solution was reduced by tritium
gas with tristri-phenylphosphine rhodium chloride (Wilkinson
catalyst) (20 mg) under constant stirring at ambient temperature and
pressure for 18 h. Separation of prednisone and E was carried out
by HPLC and a Zorbax C18 column with a mobile phase consisting of
methanol:water (60:40 by vol) at a flow rate of 2 mL/min. The specific
activity of tritiated E was found to be 1.04 TBq/mmol (28 Ci/mmol).
Preparation of the cortisone-3-(
O-carboxymethyl)
oxime hapten (cortisone-3-CMO). To a cooled (0–5 °C) solution
of E (0.180 g, 0.5 mmol) in 20 mL of methanol, anhydrous pyrrolidine
(0.071 g, 1 mmol) in 20 mL of methanol was added. After stirring for 5
min, the same quantity of pyrrolidine was added, followed by
O-(carboxymethyl) hydroxylamine hemihydrochloride (0.055 g,
0.5 mmol). The mixture was brought to and kept at 50 °C for 5 min,
then cooled to room temperature. The methanol was evaporated under
vacuum and the residue redissolved in 20 mL of distilled water. This
aqueous solution was adjusted to pH 2 with HCl and extracted with
methylene chloride (3 x 20 mL). The combined organic extracts
were washed with water (1 x 10 mL), then dried over sodium
sulfate and concentrated to dryness, yielding 0.190 g (86%) of
cortisone-3-CMO. The product was a mixture of two geometric isomers: E
(57%) and Z (43%) (Mp = 86–91 °C), 1H-NMR
(CDC13, TMS, Bruker 270 MHz): 0.63 (s, 18-CH3); 1.28 (s,
19-CH3); 4.21 and 4.61 (dd, Jgem = 19 Hz, J21H-OH
= 4 Hz, 21-CH2OH); 4.60 (s, CH2CO); 5.80 (s,
4-H, E-isomer); 6.50 (s, 4-H, Z-isomer).
Preparation of the cortisone-3-CMO/bovine serum albumin (BSA)
immunogen.
BSA was coupled to cortisone-3-CMO with the mixed
anhydride method described by Erlanger et al. (21).
Preparation of anti-E antibodies.
Three New Zealand
White male rabbits were immunized according to the method of
Vaitukaitis et al. (22).
Preparation of
125I-labeled E.
The coupling of histamine to cortisone-3-CMO, radioactive labeling with
125I by the chloramine-T method of Greenwood and Hunter,
modified, and purification of radiolabeled E by HPLC were carried out
as previously described (23). The specific activity of
iodinated E was found to be 25 TBq/mmol (450 mCi/mmol).
Celite and columns.
Celite obtained from Touzard et
Matignon (Vitry/Seine, France) was washed in cyclohexane and heated for
16 to 18 h at 800 °C, then kept dry at 100 °C until use.
Five mL x 5 mm (i.d.) Kimble pipettes obtained from STP (Paris, France)
were siliconized and stoppered at the bottom with a glass bead.
Measuring instruments.
The following measuring
instruments and equipment were used: a Wallac 1409 beta-ray counter
(Pharmacia-LKB, Saint-Quentin-en-Yvelines, France), a model 400 HPLC
solvent delivery system (Applied Biosystems, Foster City, CA), and a
Zorbax ODS HPLC column, 5 µ, 250 x 9.4 mm from Interchim
(Montluçon, France).
RIA of F and E.
These assays were carried out after
extraction and Celite chromatography (24), with slight
changes. Two thousand cpm each of [3H]F and
[3H]E in PGB (50 µL) were added to the serum samples
(0.5 mL), as well as to the saliva (2 mL) and urine (0.5 mL) samples,
both of which had been supplemented with 0.5 mL of steroid-free serum.
Two milliliters of dichloromethane was added, the samples vortex-mixed
for 60 s, and then centrifuged for 10 min at 2500g. The
aqueous phase was decanted and the organic phase first evaporated, then
redissolved in 1.5 mL of isooctane + 20 mL/L dichloromethane. The
extract obtained was introduced into the top of a 5 mL x 5 mm (i.d.)
pipette packed with 0.75 g of Celite/ethylene glycol (1 g/0.5 mL)
to a depth of 6 cm and eluted under positive air pressure. The column
was flushed first with 5 mL of 35:65, then with 5 mL of 50:50
dichloromethane:isooctane. One 5-mL faction of 80:20
dichloromethane:isooctane (E) and one 5-mL fraction of 60:40 ethyl
acetate:isooctane (F) were collected. The eluates were evaporated in an
air current and the eluted steroids redissolved in 1 mL of PGB.
One-hundred microliters were counted in 4 mL of scintillation liquid
for 300 s to estimate recovery. F RIA was carried out in duplicate
in various aliquot volumes: 300 µL for saliva, 100 µL for urine,
and 30 µL for serum, to each of which 10 µL of zero calibrator was
added, with the Incstar kit. E RIA was also carried out in duplicate on
100 µL (saliva and serum) and 40 µL (urine). Calibrators consisted
of duplicates containing 0, 10, 20, 50, 100, 200, 500, 1000, and 2000
pg of steroid/tube, in 100 µL of PGB solution. To these samples were
added 100 µL of 125I-labeled E (10 000 cpm), 100 µL of
rabbit anti-E antibody, and 100 µL of antirabbit SPA reagent
(25). The samples were vortex-mixed, then incubated at
room temperature for 15 to 20 h under rotating agitation (2000
rpm) before being counted for 60 s with a ß-ray counter.
Calibration curves were plotted by using RIAcalc Sofware (Pharmacia
LKB, Bromma, Sweden) and smooth-spline function fitting. We also
performed this same E assay using [3H]E to compare the
sensitivity obtained with the tritiated and the iodinated tracers.
Direct RIAs of F and E (without extraction and chromatography) were
also carried out in serum, saliva, and urine and the results of these
analyses compared with those obtained after extraction and Celite
chromatography.
Statistics.
The results of the F and E concentrations
obtained by the two methods were compared with the Wilcoxon test.
Comparisons between group mean values were carried out with Student's
t-test or the Mann–Whitney U-test. Variations in
F and E and in the F/E ratio over a 24-h period, as well as before and
after Synacthen or DXM administration, were evaluated by ANOVA for
repeated measurements. P values >0.05 were considered
nonsignificant.
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Results |
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Affinity constant.
The resulting affinity constant,
calculated according to Scatchard, was K = 2.33 x
109 L mol-1.
Specificity
(Table 1
). Anti-E antiserum cross-reactions corresponding to 50%
displacement of the iodinated tracer were determined according to
Abraham (26) and with SPA. Antiserum 3101 was much more
highly specific for E than for F, with only 0.42% cross-reactivity.
Most of the interference was from the following compounds: prednisone
(52%), 5-THE (9.75%), 6ß-OH-cortisone (0.71%), 11-deoxycortisol
(0.70%), 5ß-THE (0.53%), and prednisolone (0.46%).
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assay reliability
Separation of F and E by Celite chromatography.
Less
than 3% of each steroid eluate was found in the other fraction,
resulting in negligible errors (<1% in the worst case, which was a
serum E assay in which the F concentration was 10- to 20-fold that of
E). In a preliminary study, we compared the direct F and E assays with
the new assay, which includes extraction and Celite chromatography
steps. We obtained significantly higher concentrations of F and E with
the direct assays (Wilcoxon test, P <0.001). For serum
(n = 28), saliva (n = 20), and urine (n = 39), they were
respectively 1.20-, 1.6-, and 1.5-fold higher for F and 1.2-, 1.4-, and
1.8-fold higher for E. Therefore we decided to carry out the assays
after extraction and Celite chromatography steps.
Recovery of tritiated markers.
The recovery rates after
extraction and chromatography for F were (mean ± SD, n =
28): 70.1 ± 9.1, 64.5 ± 4.2, and 65.8 ± 9.3
respectively for serum, urine, and saliva; for E they were 75.7 ±
10.5, 68.4 ± 6.5, and 63.2 ± 7.2.
Blank values.
Blank assay values were found to be <30
pg/tube for F and <4 pg/tube for E.
Accuracy.
Two serum, two urine, and two saliva samples
were supplemented with known quantities of F and E to the following
concentrations (nmol/L): S1: F = 310, E = 72;
S2: F = 560, E = 165; U1: F =
22, E = 72; U2: F = 300, E = 360;
Sa1: F = 10, E = 20; Sa2: F =
40, E = 80, and assayed five times. The recovery rates were
respectively (mean ± SD): S1: F = 91 ±
6.4, E = 86 ± 6.5%; S2: F = 93 ±
6.3, E = 97 ± 5.7%; U1: F = 110 ±
7.1, E = 104 ± 5.6%; U2: F = 93 ±
6.1, E = 95 ± 6.1%; Sa1: F = 98 ±
6.2, E = 99 ± 7.3%; Sa2: F = 101 ±
7.5, E = 94 ± 5.9%. One serum, one urine, and one saliva
with high concentrations of F and E (S3: F = 1186,
E = 266; U3: F = 728, E = 794;
Sa3: F = 43, E = 78 nmol/L) were progressively
diluted (2- to 10-fold) with steroid-free serum and water
(respectively) and assayed in triplicate. Mean results ranged from 89%
to 112% of the expected values in serum, 92% to 109% in urine, and
94% to 108% in saliva.
Reproducibility.
Intra- (n = 10) and interassay
reproducibility (n = 20) were respectively: S1: 6.6%,
10.3%; S2: 7.8%, 10.4%; U1: 6.2%, 11.8%;
U2: 4.7%, 7.9%; Sa1: 8.2%, 10.8;
Sa2: 6.4%, 8.8% for F and S1: 8.5%, 11.6%;
S2: 7.7%, 11%; U1: 5.7%, 11%;
U2: 7.0%, 10.2%; Sa1: 5.9%, 8.1%;
Sa2: 5.3%, 6.7% for E.
Sensitivity.
For Incstar F RIA, the least detectable
dose (zero + 3SD) was 38 pg/tube, corresponding to the usual lower
limit of F detection (loss-corrected) of 55 pg/mL (0.15 nmol/L) in
saliva, 2.5 ng/mL (7 nmol/L) in serum, and 0.7 ng/mL (2 nmol/L) in
urine. For E RIA, the least detectable dose was 4.5 pg/tube,
corresponding to a pratical E detection limit of 21 pg/mL (0.06 nmol/L)
in saliva, 90 pg/mL (0.25 nmol/L) in serum, and 210 pg/mL (0.58 nmol/L)
in urine. When the sensitivity of the 125I-labeled E RIA
was compared with the same RIA carried out with [3H]E, it
was found to be lower (4.5 ± 1.6 vs 10 ± 2.3 pg/tube,
P <0.001, n = 6).
normal results in healthy subjects
Normal values and circadian fluctuations.
Usual values
(n = 50) (Table 2
, upper part): Whereas in our healthy adult subjects the serum E
concentration was found to be around one-tenth the F concentration, its
concentrations in saliva and urine were twice those of F. At 0800,
there was a positive correlation between F/E and F [F/E = 0.057F
(nmol/L) + 0.248; r = 0.610; P =
0.003] and a negative correlation between F/E and E [F/E =
-0.437E (nmol/L) + 18.239; r = 0.688;
P = 0.0001], but no significant correlation between E
and F (r = 0.063; P = 0.0921).
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Circadian variations (Figs. 1
and
2, Table 3
) in the serum E concentrations among our healthy subjects were
seen to parallel those classically described for F; however, their
individual E concentrations were practically identical at 0800 and
noon. Circadian variations were also found in their saliva and urine.
The highest E concentrations were observed in these subjects' serum
and saliva at 0800 and the lowest at midnight. In urine this occured at
noon and at 0400. The F/E ratio fluctuated significantly over the
course of the day (Fig. 2
and Table 3
) (ANOVA P <0.001 for
serum and saliva, P = 0.045 for urine). In serum and
saliva, the highest F/E ratio was found at 0800 and in urine at 1600
(P <0.001). This ratio was significantly lower for urine
during the day (0800–1600) than at night (2000–0400)
(P = 0.0042).
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Dynamic tests (Table 4
).
Synacthen stimulation tests: While a quite classically
significant increase in the serum F concentration was observed in our
healthy subjects 1 h after injection of short-acting Synacthen, a
significant decrease in E was found. In contrast, serum E was found to
be increased 4 h after administration of long-acting Synacthen.
However, both these stimulation tests resulted in a significant
increase in the F/E ratio (P <0.001).
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DXM suppression test: Serum F and E significantly decreased (P <0.001) in our healthy subjects during the DXM suppression test. However, the decrease in F was greater than that of E, resulting in a nonsignificant decrease in the F/E ratio.
pathological results in patients
Adrenal diseases (Table 2
).
Primary adrenal
insufficiency: The two Addisonians had nearly indetectable serum F
(
12 nmol/L) and E (<2 nmol/L) that failed to respond to
short-acting Synacthen infusion.
Secondary adrenal insufficiency: The two hypopituitary patients had depressed serum F and E concentrations that responded to short-acting Synacthen stimulation 1 h later. The responses of these two steroids to 1-24 ACTH were parallel, resulting in a constant F/E ratio.
Cushing syndrome: (a) Adrenal adenoma (10 cases). Both serum F and E concentrations were high and failed to increase after ACTH in the two patients who underwent the short-acting Synacthen test. Nonetheless in these two patients, as well as in the eight others who did not undergo the Synacthen test, serum F/E ratios were normal. However, all adrenal adenoma patients were found to have abnormally high urinary F/E ratios (in the 1.1 to 2.0 range). (b) Cushing disease (two cases). The explosive increase in serum F following short-acting Synacthen was accompanied by a smaller increase in serum E, and the increase in their serum F/E ratio was similar to that observed in healthy subjects (2.78 ± 0.91). However, their urinary F/E ratios were higher than those found in healthy subjects. (c) Ectopic ACTH secretion (five cases). Excessively high serum F concentrations were associated in these patients with a much lower excess of serum E, and consequently with a greatly increased F/E ratio in both serum and urine (P <0.001). (d) OP'DDD therapy. The return of serum F to normal concentrations was accompanied by a return to normal E concentrations and to a normal F/E ratio.
CRI patients (10 patients) (Table 2
).
All 10 patients
had been on hemodialysis (4 h, 3 times a week) for more than a year and
all were hypertensive (mean ± SD, 141.7 ± 10.9/86.2 ±
9.5 mmHg). Hypertension was not the cause of their nephropathy, but
occurred secondarily; all had normal liver function. Their serum F was
slightly reduced (386 vs 475 nmol/L for the controls, P
= 0.04), but E was dramatically reduced (18.4 vs 51.4 nmol/L,
P = 0.0001), and the F/E ratio frankly increased
compared with controls (23.5 vs 9.8, P = 0.0001), but
less increased than in 11-HSD-2 deficiency (23.5 vs 40.5,
P = 0.03). Saliva F was normal (9.9 vs 9.3 for the
controls) and E slightly but not significantly increased (25.1 vs 17.9,
P = 0.054). Nevertheless, the F/E ratio was frankly
reduced (0.38 vs 0.50, P = 0.0075).
11ß-OH-Steroid dehydrogenase-2 deficiency (two cases).
Two young brothers (A and JC, respectively 21/2 and 6 years of
age) who had high blood pressure, hypokalemia, undetectable plasma
aldosterone (<14 pmol/L), and undetectable plasma renin activity (PRA
<0.05 nmol/L s) (27) presented low normal concentrations
of serum F associated with very low concentrations of E and an F/E
ratio three to five times the mean normal ratio. Urinary free F was
normal and free urinary E depressed below the lower limit of the normal
range with, as in plasma, an increased F/E ratio. The two brothers were
successfully treated with DXM alone (1 mg x 10 days), resulting
in transient normalization of aldosterone and PRA and correction of
arterial hypertension. JC has since ceased to follow his therapeutic
regimen, but A remains under combined DXM (0.5 mg/day) + Nifedipine (20
mg x 2/day) treatment. Since he has been under DXM treatment,
A's serum and urine F and E concentrations have decreased. However, in
spite of the clinical effectiveness of his therapy, the F/E ratios in
these two media remain unchanged, which accords with the absence of
decrease in the ratio of the hydrogenated metabolites of F and E (THF +
aTHF/THE) previously described (27). Moreover, A's PRA
and aldosterone have stabilized at low concentrations [upright PRA
0.048 nmol/L (normal range 0.53–1.79), aldosterone <28 pmol/L (normal
range 196–826)] (unpublished data). In addition, A's saliva F/E
ratio before and during DXM treatment, as well as JC's before DXM
treatment, were at the upper limit of normal values.
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Discussion |
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emission, emits Auger electrons, well-suited for SPA. The use of SPA
(35) has greatly improved RIA in general. This new E RIA +
SPA assay is carried out in a homogeneous (or pseudohomogenous) phase
and does not require time-consuming and laborious methods for
separating the bound and free fractions, such as the use of
charcoal–dextran or precipitation with a second antibody. The
imperfect separation by Celite chromatography of prednisone from E and
of prednisolone and methylprednisolone from F requires 24-h abstention
from these therapeutic agents in patients who are taking them, to
obtain true E and F results (data not shown).
physiological variations in f and e concentrations
The usual values of F and E that we found accord well with
previously published results for serum
(4)(31)(32), saliva
(29)(33), and urine
(28)(36). E varies in these three media over
the course of the day, with timing similar to those for F and ACTH. One
previous study (6) found no significant variation in the
serum E concentration (probably because it included only a small number
of healthy subjects, n = 7), but other authors have reported
cyclic variations of E in serum (32) and saliva
(29), which agrees with our results.
In our fractionated urine samples, maximum and minimum E concentrations occur respectively at noon and 0400, slightly later than in serum and saliva (respectively at 0800 and midnight), reflecting the additional time it takes for urine to be produced and stored. E concentrations in saliva and urine are higher than those of F because of the oxidase activity of 11-HSD-2 (present in parotid gland and kidney) (2)(3). The F/E fluctuation in serum is slightly attenuated compared with those encountered in saliva and urine, probably because serum reflects the antagonistic oxidation of F to E (by NAD-dependent 11-HSD-2 in kidney) and the reduction of E to F (by NADP-dependent 11-HSD-1 isoenzyme in liver).
cushing syndrome
Only our ectopic ACTH patients had increased serum F/E ratios, not
those with Cushing disease or the adrenal adenoma patients, as
previously found by Walker et al. (6). In contrast, we
found that all Cushing syndrome patients had increased urinary F/E
ratios, which corroborates previous results for the THF+aTHF/THE ratio
found by Ulick et al. (37) and more recently by Stewart et
al. (38).
role of acth
At present, the interaction between ACTH and 11-HSD-2 is still in
dispute, some authors arguing in favor of an inhibitory effect of ACTH
on 11-HSD-2 activity, others against. As Walker et al. (6)
have previously shown, although they did not find any significant
variations in plasma E, we observed that circadian fluctuations in E
and F in both serum and saliva displayed an increased F/E ratio at
0800, when ACTH is maximum. Katz and Shannon (29) reported
that 2 h after administration of 40 IU of ACTH there was an
increase in the F/E ratio in parotid fluid, as we found in serum after
short- and long-acting Synacthen stimulation. Kornel (39)
also demonstrated that ACTH prolongs the half-life of F and shortens
the half-life of E and found an increased F/E ratio in urine
(40). All this suggests a possible inhibitory effect of
ACTH on 11-HSD-2, as first proposed by Walker et al. (6).
In contrast, Ulick et al. (37) explained the high urinary F/E ratio encountered in Cushing syndromes (that we also observed) as being due to an overload of the capacity of 11-HSD-2 to oxidize F to E in these hypercortisolic patients. Such overload has been demonstrated in vitro by Diederich et al. (41). Our observation of the absence of increase in the F/E ratio after ACTH in patients with secondary adrenal insufficiency also argues against the inhibitory effect of ACTH on 11-HSD-2 activity, but in hypocortisolic patients.
A comprehensive explanation would also take into account the retroinhibition of 11-HSD-2 activity by increased serum E that Rusvai and Fejs-Toth (42) and Stewart et al. (3) have demonstrated in vitro.
At this time, no single hypothesis can explain all the observations reported. Some combination of the three phenomena might explain (for example) the data we observed in the sera of the healthy subjects. Progressive 11-HSD-2 overload could account for the positive correlation between the F/E ratio and the F concentration, whereas retroinhibition of 11-HSD-2 by E might play a role in the negative correlation between F/E and E. Finally, ACTH inhibition of 11-HSD-2 could explain these two simultaneous correlations.
cri patients on hemodialysis
Whitworth et al. (4) found plasma creatinine and E to
be inversely correlated in renal disease, due to progressive
destruction of kidney tissue (and 11-HSD-2). Because some functional
11-HSD-2 activity did remain in our CRI patients, their serum E
concentrations were less decreased than in AME. In saliva, the increase
in E and the low F/E ratio suggest stimulation of parotid 11-HSD-2 in
response to the impaired renal 11-HSD-2 activity encountered in CRI
patients.
In our study, all the CRI patients were hypertensive and had excessive serum F/E ratios. Vierhapper et al. (43), studying 22 CRI patients not on hemodialysis, found that the ratio of the urinary metabolites (THF/THE) increased from control subjects to normotensive CRI patients to hypertensive CRI patients. This matter deserves further investigation to determine whether 11-HSD-2 deficiency plays a causal role in the onset of hypertension among CRI patients.
ame-1
We used our E assay to study the blood and urine of two young
brothers in whom a diagnosis of 11ß-HSD-2 deficiency (AME-1) had been
made by capillary chromatography assay of the hydrogenated urinary
metabolites of F (THF, aTHF) and of E (THE). According to Ulick et
al.'s criteria (18), the ratio of the urinary metabolites
(r = THF + aTHF/THE) was very high in both brothers
(for JC, r = 42; for A, r = 21; normal
range 0.58–2.65) (27). It is therefore not surprising
that their urinary F/E ratios were >4, whereas this ratio is normally
<1 (normal range 0.12–0.91). The serum F/E ratio in these two
patients also was higher than normal. In addition, during effective DXM
treatment of A, the unmodified F/E ratios found in both his plasma and
urine corroborated our previously published results on the urine
THF/THE ratio (27). In spite of decreased serum and urine
F and E concentrations and low PRA and aldosterone concentrations under
long-term DXM therapy, the increased F/E ratios we found suggest that
the effectiveness of DXM treatment is potentially mediated partly by
the formation of a DXM-MR complex (44). Indeed, the
transcriptional efficiency of this complex is 100-fold lower than those
of F-MR and aldosterone-MR (45)(46).
The fact that salivary F and E concentrations and the F/E ratio were normal in these two AME-1 patients demonstrates that saliva is not appropriate for carrying out AME-1 diagnosis.
The F–E shuttle has become a subject of investigation in the study of hypertension, both clinically and experimentally. Numerous factors may potentially interfere with the interpretation of results: sample timing; licorice ingestion by the subject (47); and liver (48), thyroid (49), renal, and adrenal status. These must all be carefully taken into account. The E RIA we describe is simple, selective, sensitive, and well suited for E assay in serum, saliva, and urine for diagnostic purposes, as well as for experimental and therapeutic applications. The easy-to-carry out parallel RIAs of E and F are an alternative or complement to gas chromatography assay of the urinary metabolites of these two steroids.
Using these parallel RIAs, we obtained abundant physiological and pathological data concerning the F–E shuttle. We demonstrated that the circadian variation in the F/E ratio is similar to those of F and E themselves, suggesting that the activity of 11-HSD-2 varies over the course of 24 h.
We showed that the urinary F/E ratio is increased in Cushing syndrome patients, but that only patients with ectopic ACTH secretion had an increased F/E ratio in serum.
We found the serum F/E ratio to be increased in anuric CRI patients under hemodialysis, which we attribute to the destruction of renal 11-HSD-2. In contrast, we found the salivary F/E ratio to be decreased in these patients, which could be due to a compensatory increase in parotid 11-HSD-2 activity.
In addition, in the two cases of AME we studied, the serum and urinary F/E ratios were found to be highly increased. These data suggest that parallel serum and urinary RIA of F and E may be a useful indicator in the detection of AME in patients who consult for hypertension associated with hypoaldosteronemia and hyporeninemia.
Although the data we present appear to be of interest for the study of AME as well as for the differential diagnosis of Cushing syndrome, the small number of patients included did not enable us to determine F/E ratio cutoff values usable as diagnostic criteria. To do so would require additional study and larger numbers of subjects.
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Acknowledgments |
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Footnotes |
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-tetrahydrocortisol; aTHF, 5ß-tetrahydrocortisol; THE, 5-tetrahydrocortisone; ACTH, corticotropin; DXM, dexamethasone; CRI, chronic renal insufficiency; PGB, phosphate gelatin buffer; SPA, scintillation proximity assay; cortisone-3-CMO, cortisone-3-(O-carboxymethyl) oxime; BSA, bovine serum albumin; and PRA, plasma renin activity. 
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