| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
1
Clinical Chemistry Laboratory, Kobe Pharmaceutical University, 4-19-1, Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan.
2
Department of Laboratory Medicine, Osaka University
Medical School, Osaka 565-0871, Japan.
3
Kuma Hospital, Kobe 650-0011, Japan.
a Author for correspondence. Fax 81-78-441-7559; e-mail t-noriko{at}kobepharma-u.ac.jp
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Methods: Steroids extracted with methanol from serum sample were separated into an unconjugated fraction (DHEA) and a monosulfate fraction (DHEA-S and PREG-S), using a solid-phase extraction and an ion-exchange column. After separation of unconjugated steroids by HPLC, the DHEA concentration was measured by enzyme immunoassay. The monosulfate fraction was treated with arylsulfatase, and the freed steroids were separated by HPLC. The DHEA and PREG fractions were determined by gas chromatography–mass spectrometry, and the concentrations were converted into those of DHEA-S and PREG-S.
Results: Serum concentrations of DHEA, DHEA-S, and PREG-S were all significantly lower in patients with hypothyroidism (n = 24) than in age- and sex-matched healthy controls (n = 43). By contrast, in patients with hyperthyroidism (n = 22), serum DHEA-S and PREG-S concentrations were significantly higher, but the serum DHEA concentration was within the reference interval. Serum concentrations of these three steroids correlated with serum concentrations of thyroid hormones in these patients. Serum albumin and sex hormone-binding globulin concentrations were not related to these changes in the concentration of steroids.
Conclusions: Serum concentrations of DHEA, DHEA-S, and PREG-S were decreased in hypothyroidism, whereas serum DHEA-S and PREG-S concentrations were increased but DHEA was normal in hyperthyroidism. Thyroid hormone may stimulate the synthesis of these steroids, and DHEA sulfotransferase might be increased in hyperthyroidism.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The serum concentration of cholesterol is known to change markedly in patients with thyroid dysfunction (11), i.e., hypercholesterolemia is associated with hypothyroidism and hypocholesterolemia is associated with hyperthyroidism (12). Less is known, however, about the serum concentrations of DHEA and DHEA-S in patients with thyroid dysfunction. In this study, we examine serum DHEA, DHEA-S, and pregnenolone sulfate (PREG-S) concentrations in patients with hyperthyroidism and hypothyroidism and discuss the mechanism of change in the concentration of these steroids in thyroid dysfunction.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). Patients with Graves hyperthyroidism had thyrotoxic
symptoms, and the diagnosis was confirmed by increased concentrations
of free thyroxine (FT4) and free triiodothyronine
(FT3), decreased thyrotropin (TSH), and positive
TSH receptor antibody and/or high radioiodine thyroid uptake. Patients
with hypothyroid autoimmune thyroiditis had hypometabolic symptoms, and
the diagnosis was confirmed by decreased FT4 and
FT3 concentrations, increased TSH, and a positive
test for anti-thyroid microsomal and/or thyroglobulin antibodies.
|
calibrator and sample preparation
Stock solutions containing 1.20 mmol/L DHEA, DHEA-S, and PREG-S
(Steraloids) in methanol were serially diluted with methanol; these
were the methanol calibrators. To prepare serum steroid calibrators
containing DHEA (0–521 nmol/L), DHEA-S (0–18.8 µmol/L), or PREG-S
(0–8.54 µmol/L), the methanol calibrators were added into the tubes
(0–0.10 nmol DHEA, 0–3.75 nmol DHEA-S, 0–1.71 nmol PREG-S) followed
by 0.2 mL of steroid-free serum (13).
Separation of DHEA, DHEA-S, and PREG-S from serum samples was carried out as reported previously (14). Briefly, 0.2 mL of serum sample or serum calibrator was extracted three times with 1 mL of methanol. The obtained solvent was combined and evaporated under reduced pressure. The residue was applied to a solid-phase extraction column, Sep-Pak Vac tC18® (Waters), and an anion-exchange column, Accell Plus QMA® (Waters). Because the DHEA serum calibrators did not contain DHEA-S and PREG-S, the Accell Plus QMA was not used for the treatment of DHEA serum calibrators. After these treatments, the steroid glucuronides were excluded, and the unconjugated fraction (DHEA) and monosulfate fraction (DHEA-S and PREG-S) were obtained.
dhea assay
The unconjugated fraction was evaporated under reduced pressure.
The residue was dissolved in 0.3 mL of the HPLC mobile phase solvent,
water-acetonitrile (70:30, by volume), and 0.25 mL of the sample was
subjected to HPLC using a system (Waters) of two pumps (model 510), an
ultraviolet detector (model 481) operating at 210 nm, an
autosampler (model 710B), and a Wakosil-II 5C18-HG column [150 x
4.6 mm (i.d.); Wako Pure Chemical Industries]. The separations were
performed at 40 °C and at a flow rate of 1 mL/min with a linear
gradient of acetonitrile (2%/min) from 30% to 70% in water. The
eluate between 15.2 and 17.7 min (DHEA retention time, 16.2 min) was
collected as the DHEA fraction. The solvent was evaporated under
reduced pressure, and the residue was dissolved in 0.25 mL of methanol.
Aliquots of 0.1 mL of this solution were transferred into two tubes for
duplication. After evaporation of the solution, the enzyme
immunoassay was performed.
Anti-DHEA antiserum [raised in rabbits against DHEA 7-(O-carboxymethyl)oxime-bovine serum albumin; UCB-Bioproducts S.A.] was diluted (1:3200) with the assay buffer (0.07 mol/L phosphate buffer solution containing 2.5 g/L bovine serum albumin, pH 7.4). Alkaline phosphatase (ALP)-labeled DHEA was prepared by conjugation of DHEA 7-(O-carboxymethyl)oxime (Sigma) and ALP (EC 3.1.3.1, from calf intestine; Boehringer Mannheim) according to the carbodiimide method (15), and was diluted with the assay buffer containing 6 mL/L normal rabbit serum. To the tubes prepared as above, 0.5 mL of anti-DHEA antiserum and 0.1 mL of ALP-labeled DHEA were added. After the tubes stood at room temperature for 1 h, 0.1 mL of 30 mL/L second antibody was added, and the mixture was allowed to stand at 4 °C overnight. After bound/free separation, the enzyme activity of the resulting precipitate was measured using the Alkaline Phospha K-Test® kit (Wako) (15). The measurable range was 0.13–521 nmol/L. The overall recovery was 97.2% ± 4.8%. The intra- and interassay CVs for DHEA (mean, 4.24 nmol/L; n = 5) were 5.6% and 8.9%, respectively.
assays for dhea-s and preg-s
The monosulfate fraction was hydrolyzed with arylsulfatase (EC
3.1.6.1, from Helix pomatia; Boehringer Mannheim)
(14). The resulting samples were subjected to HPLC in the
same manner as the above DHEA assay. The eluate was collected from 15.2
to 17.7 min (DHEA) and 20.5 to 23.0 min (PREG retention time, 21.5 min)
and was combined in a tube. Androstenediol diacetate in methanol (0.11
nmol/sample) was added to each sample as an internal standard. After
evaporation, derivatization was performed with 0.1 mL of
heptafluorobutyric anhydride (GL Sciences) at 60 °C for 30 min.
Under these conditions, no dehydrated or decomposed compounds were
observed. The reaction mixture was evaporated to dryness. The residue
was dissolved in 0.4 mL of dichloromethane, and 2 µL of the
sample was analyzed by gas chromatography–mass spectrometry
(14). Selective-ion monitoring was carried out at
m/z 270 [M - 214]+ for DHEA,
m/z 298 [M - 214]+ for PREG,
and m/z 314 [M - 60]+ for the
internal standard. The concentrations obtained by gas
chromatography–mass spectrometry were expressed as the values of serum
steroid sulfates. The overall recoveries were 91.3% ± 4.4% for
DHEA-S and 89.2% ± 4.3% for PREG-S, respectively. The measurable
range for each steroid was as follows: DHEA-S, 0.01–18.8 µmol/L;
PREG-S, 0.01–8.54 µmol/L. The intra- and interassay CVs for DHEA-S
(mean, 1.36 µmol/L; n = 5) or PREG-S (mean, 0.63 µmol/L;
n = 5) were 2.3% and 5.4%, and 2.1% and 3.1%, respectively.
other assays
Sex hormone-binding globulin (SHBG).
SHBG was measured by an
ELISA sandwich assay according to the reported method (16)
with minor modifications. Diluted serum samples (1:500 or 1:1000) and
SHBG calibrators (0–0.53 nmol/L, purified from human serum;
Calbiochem-Novabiochem) were diluted with 0.07 mol/L phosphate-buffered
saline (PBS, pH 7.4) containing 5 g/L gelatin. The plates were
incubated at room temperature for 3 h and then washed six times
with 0.07 mol/L PBS containing 0.5 g/L Tween 20 (Bio-Rad). To
each well, 0.1 mL of ALP-labeled anti-SHBG antibody diluted 1:50 with
0.07 mol/L PBS was added. The plates were left to stand at room
temperature for 2 h, and then were washed six times with 0.3 mL of
25 mmol/L Tris-HCl, pH 7.4. The ALP activity in each well was measured
using an Alkaline Phospha K-Test. The measurable range was 0.002–0.53
nmol/L. The recovery was 101.0% ± 3.1%. The intra- and interassay
CVs (129 nmol/L; n = 5) were 4.3% and 5.4%, respectively.
Albumin, FT4, FT3, and TSH.
Serum
albumin was measured by the "Dry Chemistry" method using DRI-CHEM
3000 (FUJIFILM). The slide used was ALB-P®. The
serum concentrations of FT4,
FT3, and TSH were measured with the commercial
kits, AxSYM Free T4
Dainapack® (Dainabott),
Mab-FreeT3® (Amerlex), and
AxSYM TSH Dainapack (Dainabott), respectively. Anti-TSH receptor
antibody was measured by radioreceptor assay using a TRAb-II
kit® (Cosmic). Anti-thyroid microsomal and
thyroglobulin antibodies were measured with passive particle
agglutination kits (Fuji Rebio).
statistical analysis
Differences between two groups were analyzed statistically by the
Mann–Whitney U-test. To examine the relationship between
two hormones, the log-normal distributions of the hormone
concentrations were confirmed by Kolmogorov–Smirnov tests, the
regression lines were calculated by standard least-squares methods, the
ellipses of constant distance that represent 95% bivariate tolerance
regions were calculated based on assumption of an underlying bivariate
parametric distribution, and Pearson correlation coefficients were
determined. P <0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). In hyperthyroidism, the serum DHEA-S concentration was
significantly increased, but DHEA was normal. In hypothyroidism, the
serum concentrations of both DHEA and DHEA-S were significantly
decreased. Individual values of DHEA and DHEA-S are shown in Fig. 1
. DHEA values were widely distributed even in the controls.
Interestingly, the serum concentration of PREG-S, the precursor of DHEA
and DHEA-S, was increased in hyperthyroidism and decreased in
hypothyroidism.
|
We examined serum concentrations of albumin, a binding protein of DHEA
and DHEA-S, and SHBG, a weak binding protein of DHEA. Serum albumin was
decreased in hyperthyroidism, and serum SHBG was increased in
hyperthyroidism (Table 1
).
Because DHEA and DHEA-S concentrations are gender-dependent, we analyzed these data using only female or male subjects, but the results were the same as above.
thyroid hormone and dhea, dhea-s, or preg-s
To elucidate the mechanism of change in the steroid hormone
concentrations, the relationships between the serum concentrations of
thyroid hormones and those of DHEA, DHEA-S, and PREG-S were studied.
The distributions of FT4, DHEA, DHEA-S, and
PREG-S in total (including hyperthyroidism, hypothyroidism, and
controls) were confirmed to fit the log-normal distribution by
Kolmogorov–Smirnov tests. Therefore, they were transformed to
logarithmic scale, and linear correlations of log (DHEA), log (DHEA-S),
and log (PREG-S) with log (FT4) were analyzed
(Fig. 2
). The serum concentrations of these steroids correlated with
that of serum FT4. The correlation was strongest
between FT4 and PREG-S (Fig. 2C
). Similar results
were obtained between serum FT3 and these
steroids (data are not shown).
|
Values of DHEA, DHEA-S, or PREG-S were not related to titers of anti-thyroid microsomal or thyroglobulin antibodies, or anti-TSH receptor antibodies.
relationship between dhea and dhea-s
Because DHEA and DHEA-S are interconvertible and DHEA was not
increased in hyperthyroidism, we examined the relationship between DHEA
and DHEA-S in patients and controls. The distributions of DHEA and
DHEA-S for each (hyperthyroid, hypothyroid, and control) group were
confirmed to fit the log-normal distribution by Kolmogorov–Smirnov
tests. Therefore, they were transformed to logarithmic scale, and
linear correlations of log (DHEA) and log (DHEA-S) were analyzed (Fig. 3
). The two steroids were correlated in controls and patients
with hypothyroidism. However, there was no correlation between the two
in hyperthyroidism. The ratio (nmol/nmol) of DHEA-S to DHEA in controls
was 181, and that in hyperthyroidism was increased to 480, indicating
that DHEA-S is more predominant in hyperthyroidism.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the present study, we confirmed the decrease of serum DHEA and DHEA-S in hypothyroid patients. In the patients with Graves hyperthyroidism, serum DHEA-S was increased but serum DHEA was normal, similar to the earlier report by Földes at al. (18). We also found that serum PREG-S was increased in hyperthyroidism and decreased in hypothyroidism.
DHEA has been reported to enhance immune function, and some patients with autoimmune disease have decreased serum DHEA and DHEA-S concentrations (2). Reported autoimmune diseases include rheumatoid arthritis (20) and systemic lupus erythematosus (21), but these are generalized, not organ specific. Patients with pemphigoid/pemphigus, one of the organ-specific autoimmune diseases, are also reported to have decreased serum DHEA-S concentrations (22). In animal experiments, DHEA-S retarded the onset of autoimmune disease (23). It is speculated that abnormalities of DHEA or DHEA-S might have some pathological relation to autoimmune disease. Both Graves disease and autoimmune thyroiditis are representative of organ-specific autoimmune disease, but serum DHEA or DHEA-S concentrations in these patients were not consistently decreased. Thus, DHEA and DHEA-S may not have important roles in autoimmune thyroid disease.
We found a significant correlation between the serum thyroid hormone concentration and DHEA or DHEA-S, suggesting that thyroid hormone regulates serum concentration of DHEA and DHEA-S. Steroid hormones are synthesized from cholesterol in the adrenal glands and other organs. The side chain of cholesterol at C-17/C-20 is cleaved by cholesterol side-chain cleavage enzyme (cytochrome P-450SCC), producing PREG. This rate-determining step precedes the biosynthesis of all steroid hormones, including DHEA and DHEA-S. A significant reduction in the activity of P-450SCC in rat adrenal cortex-mix was detected by thyroidectomy (24). Therefore, the significantly lower concentrations of DHEA, DHEA-S, and PREG-S in patients with hypothyroidism could be explained by the decreased adrenal steroidogenesis induced by the low concentration of thyroid hormone. Additionally, this mechanism would contribute to the hypercholesterolemia in patients with hypothyroidism, although the metabolic rate of cholesterol principally decreased in those patients (25)(26).
Inversely, the steroidogenesis in hyperthyroidism could be activated by
thyroid hormone. This possibility is supported by reports that
treatment with T3 in some patients caused an
increase of androsterone production (27)(28)(29). Although
different enzymes participate in the biosynthesis of androsterone and
it is unknown which step in the metabolic pathway from cholesterol to
androsterone is activated by thyroid hormone, the increased DHEA-S and
PREG-S concentrations in the patients with hyperthyroidism are
explained by the hypothesis that P-450SCC,
17
-hydroxylase, C17,20-lyase, and
sulfotransferase are activated in hyperthyroidism. However, that there
was no difference in the DHEA concentrations between patients with
hyperthyroidism and controls is harder to explain. It is possible that
the rate of conversion from DHEA to DHEA-S is increased in
hyperthyroidism because of activated sulfotransferase.
As for the evaluation of serum concentrations of steroid hormones,
their binding proteins may influence them when extreme changes in
binding proteins are observed. Therefore, we measured albumin and SHBG.
Only a small portion of serum DHEA binds to SHBG, and 
90% of this
steroid binds weakly to albumin (30). Likewise, >90% of
serum DHEA-S binds weakly to albumin, but DHEA-S does not bind to SHBG
(9)(31)(32). In this study, we found
that only slight perturbation occurs as a result of thyroid
function. Small changes in these binding proteins may not
affect the serum concentration of DHEA and DHEA-S.
DHEA is rapidly cleared from the blood at a rate of 2000 L/day,
whereas DHEA-S has a clearance of 13 L/day (33). Thus,
DHEA has a short half-life of 1–3 h, whereas the half-life of DHEA-S
is 10–20 h (34). In the hyperthyroid condition, the rate of
clearance of blood constituents would be increased more than in the
euthyroid condition. These differences might partly explain the normal
DHEA and increased DHEA-S in hyperthyroidism.
In conclusion, considering these results, one should be careful when interpreting the serum concentrations of DHEA and DHEA-S in patients with thyroid dysfunction.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  Y.-H. Huang, C.-Y. Lee, P.-J. Tai, C.-C. Yen, C.-Y. Liao, W.-J. Chen, C.-J. Liao, W.-L. Cheng, R.-N. Chen, S.-M. Wu, et al. Indirect Regulation of Human Dehydroepiandrosterone Sulfotransferase Family 1A Member 2 by Thyroid Hormones Endocrinology, May 1, 2006; 147(5): 2481 - 2489. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), patients with hyperthyroidism (•), and patients with
hypothyroidism (