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In the overnight dexamethasone suppression test, 1.0 mg loading is superior

to 0.5 mg loading for diagnosing subclinical adrenal Cushing’s syndrome
based on plasma dexamethasone le...

Article  in  Endocrine Journal · June 2017

DOI: 10.1507/endocrj.EJ17-0083

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2017, 64 (1), 1-10
Advance Publication
doi: 10.1507/endocrj.EJ17-0083
Original

In the overnight dexamethasone suppression test, 1.0

mg loading is superior to 0.5 mg loading for diagnosing
subclinical adrenal Cushing’s syndrome based on
plasma dexamethasone levels determined using liquid
chromatography-tandem mass spectrometry
Yosuke Sasaki1), 2), Takuyuki Katabami1), Shiko Asai1), Hisashi Fukuda1) and Yasushi Tanaka2)
1)
Division of Metabolism and Endocrinology, Department of Internal Medicine, St. Marianna University School of Medicine
Yokohama City Seibu Hospital, Yokohama, Japan
2)
Division of Metabolism and Endocrinology, Department of Internal Medicine, St. Marianna University School of Medicine,
Kawasaki, Japan

Abstract. The low-dose dexamethasone suppression test (DST) is one of the commonly used initial tests for endogenous
Cushing’s syndrome (CS). However, there are two loading dose regimens (0.5-mg and 1-mg), which may cause some
confusion in daily practice in Japan; furthermore, there are no reports regarding whether 0.5-mg DST is a better loading
dose for detecting adrenal subclinical CS (SCS) based on the plasma dexamethasone (DEX) levels. Therefore, the aims of
this study were (a) to develop a novel assay to measure DEX by using liquid chromatography tandem-mass spectrometry
(LC-MS/MS) and (b) to compare between the 0.5-mg and 1-mg DST for SCS diagnosis based on the DEX levels. The
study retrospectively analyzed 52 consecutive subjects hospitalized for diagnosis of adrenal incidentaloma but who did not
exhibit an overt CS phenotype; eight (15.4%) patients were affected with adrenal SCS. Inter-individual variability of DEX
levels after the DST was high, but intra-individual variability was low. DEX levels after 1-mg loading in each patient was
around two times higher than those after 0.5-mg loading (ρ = 0.853 and p < 0.001). There were 45 (86.5%) and 17 (32.7%)
subjects with DEX levels ≤2.2 ng/mL after the 0.5-mg and 1-mg DST, respectively (p < 0.001). Twenty-eight (93.3%) of
30 subjects and four (21.1%) of 19 subjects with detectable ACTH levels after the 0.5-mg and 1.0-mg DST, respectively,
did not exhibit DEX levels >2.2 ng/mL. These results clearly indicate that the 1-mg DST is superior to 0.5-mg loading for
the diagnosis of adrenal SCS.

Key words: Dexamethasone suppression test, Plasma dexamethasone concentration, Liquid chromatography tandem-mass
spectrometry, Adrenal subclinical Cushing’s syndrome

SUBCLINICAL CUSHING’S SYNDROME (SCS)
is characterized by subtle autonomous cortisol secre-
tion from an adrenal incidentaloma (AI) without the
phenotype of overt Cushing’s syndrome (CS). This
low-grade cortisol excess may worsen cardiovascu-
lar (CV) risk factors, increase CV events, and result
in a higher mortality rate [1]. However, there is no
consensus concerning establishment of a biochemical
diagnosis for SCS [2]. The low-dose dexamethasone
Submitted Feb. 20, 2017; Accepted May 4, 2017 as EJ17-0083
Released online in J-STAGE as advance publication Jun. 21, 2017
Correspondence to: Takuyuki Katabami, M.D., Ph.D., Division of
Metabolism and Endocrinology, Department of Internal Medicine,
St. Marianna University School of Medicine Yokohama City Seibu
Hospital, 1197-1, Yasashicho, Asahi-ku, Yokohama, Kanagawa 241-
0811, Japan. E-mail: t2kataba@marianna-u.ac.jp
©The Japan Endocrine Society
suppression test (DST), originally described by Liddle
in 1960 [3], is one of the commonly used initial tests
for diagnosing endogenous CS. Administration of
the potent synthetic glucocorticoid dexamethasone
(DEX) suppresses the hypothalamic-pituitary-adrenal
(HPA) axis in normal individuals, but SCS patients are
at least partially resistant to this negative feedback.
The simpler overnight low-dose DST was advocated
by Nugent in 1965 [4]; doses ranging between 0.5 and
2 mg have subsequently been proposed for the DST,
and various diagnostic cut-offs have been applied.
Because of its greater simplicity, lower cost, and high
sensitivity, recent published guidelines agree with the
use of the 1-mg overnight DST for hypercortisolism
screening [5].
2 Sasaki et al.
According to diagnostic criteria for adrenal SCS
set forth by the Intractable Disease Research Grant of
the Ministry of Health, Labour, and Welfare (MHLW),
Japan [6], cortisol levels >3 µg/dL following the 1-mg
DST are used to diagnose adrenal SCS. Clinical prac-
tice guidelines from the Endocrine Society recommend
use of the test, with a cortisol cut-off of <1.8 µg/dL,
which is lower than previous criteria, to exclude auton-
omous cortisol secretion from AI [5].
In 2009, Oki et al. [7] described that a 0.5-mg DST
with a serum cortisol cut-off level of 3 µg/dL may be
more sensitive and specific for the diagnosis of ACTH-
dependent CS compared to the 1-mg DST, with a
cut-off of 1.8 µg/dL. Accordingly, the two loading dose
regimens may cause some confusion in daily practice,
at least for general physicians. Furthermore, to avoid
false positive and negative results, some experts have
advocated simultaneous measurement of both cortisol
and DEX for DST to ensure adequate plasma DEX lev-
els (2.2 ng/mL) [8]. Although many studies report that
DEX dose concentrations increase plasma DEX levels
in a dose-dependent manner [9, 10], Oki et al. [7] did
not report any data related to this.
Due to cross-reactivity with possible metabolites of
DEX, radioimmunoassays (RIAs) and enzyme-linked
immunosorbent assays (ELISAs) require a highly
selective antibody for accurate quantification of DEX,
which is a serious drawback of these methods [11]. In
contrast, structurally based assays such as liquid chro-
matography-tandem mass spectrometry (LC-MS/MS)
do not present this problem [12]. Currently, there is no
information regarding whether 0.5 mg is a better load-
ing dose for detecting adrenal SCS based on plasma
DEX levels. Therefore, the aim of this study was: (a)
to develop a novel assay for DEX by using LC-MS/MS
and (b) to compare between the 0.5-mg and 1-mg DST
for SCS based on plasma levels of DEX in Japanese
subjects with AI.
Materials and Methods
Subjects
We retrospectively collected 52 consecutive sub-
jects hospitalized for diagnosis of AI without any spe-
cific clinical features of CS between October 2013
and August 2015 at St. Marianna University School of
Medicine Yokohama City Seibu Hospital (Yokohama,
Japan). Patients taking steroids, oral estrogen, or
antiepileptic agents were excluded, because these
Endocrine Journal Advance Publication
medications can influence the HPA axis [13]. Those
with a history of alcohol abuse and psychiatric disor-
ders were also excluded from this study. Hypertension
was defined as the need for drug therapy, systolic
blood pressure ≥140 mmHg, or diastolic pressure ≥90
mmHg. Diabetes mellitus (DM) was diagnosed as a
need for antidiabetic drug therapy or a diabetic pattern
on a 75-g oral glucose tolerance test. Hyperlipidemia
was defined as the need for antilipidemic drug therapy,
low-density lipoprotein cholesterol levels ≥140 mg/dL
as calculated using Friedewald’s formula [14], or tri-
glyceride levels ≥150 mg/dL [15]. Patients with body
mass index (BMI) ≥25 kg/m2 were defined as obese.
This study was approved by the Human Ethical
Committee of St. Marianna University School of
Medicine (No. 3324) and was performed according to
the principles of the Helsinki Declaration.
Dexamethasone suppression test
The standard overnight 0.5-mg and 1-mg DST was
performed in all subjects. Serum cortisol and plasma
ACTH were measured at 0800 h after an overnight fast,
and 0.5 mg DEX was administered at 2300 h on the
same day. The next morning at 0800 h, serum cortisol,
plasma ACTH, and plasma DEX measurements were
obtained, and 1 mg DEX was administered at 2300 h.
On day 3, a blood sample was collected again at 0800 h
to complete the study. Nurses in our institute con-
firmed ingestion of DEX. According to Endocrine
Society guidelines, false-positive results for DST were
defined as 1) plasma DEX levels ≤2.2 ng/mL and 2)
serum cortisol levels after DST above the cut-off value.
False negative results were defined as 1) plasma DEX
levels >2.2 ng/mL and 2) normal results for all endocri-
nological tests related to HPA axis function other than
the DST.
In the preliminary examination, we assayed plasma
DEX levels at 24 h after 0.5-mg DEX administration in
several patients with chronic adrenal insufficiency or
non-functioning AI. Glucocorticoids were not detected
in any sample, indicating that 0.5-mg DEX dose on day
2 is less likely to affect its plasma levels on day 3.
Diagnostic method for adrenal SCS
A diagnosis of SCS was defined as the presence of
an AI, absence of specific clinical features of OCS,
normal morning serum cortisol levels on more than
two different evaluations, the presence of autono-
mous cortisol secretion confirmed by overnight 1-mg
 1-mg DST is superior to 0.5-mg DST
DST (≥3.0 μg/dL serum cortisol following the DST),
and at least one endocrine test including low plasma
ACTH levels in the early morning (ACTH <10 pg/mL
at 0800 h) or poor ACTH response to CRH (i.e., peak
ACTH <30 pg/mL or less than 1.5 times greater than
baseline), no diurnal changes in serum cortisol levels
(serum cortisol levels ≥5.0 μg/dL at 2300 h), and low
serum dehydroepiandrosterone sulfate (DHEA-S) lev-
els (lower than age- and sex-matched reference lev-
els). This diagnostic method followed the criteria set
forth by the MHLW, Japan [6]. The Endocrine Society
guidelines [5] and a recent report from Japan [16] pro-
posed an alternative cut-off of serum cortisol >1.8 μg/
dL after the 1-mg DST for the diagnosis of adrenal
SCS. Therefore, we also used this value for determi-
nation of DST results.
Hormone assays
Blood samples were immediately centrifuged at 4°C
and then stored at −80°C until assayed. DHEA-S RIA
kits (Mitsubishi Chemical Co., Tokyo, Japan), ACTH
immunoradiometric assay kits (Mitsubishi Chemical
Co.), and serum cortisol chemiluminescence enzyme
immunoassay kits (Beckman Coulter, CA, USA) were
used for hormone measurements.
Measurements of other parameters
To evaluate hepatorenal function, we also measured
aspartate aminotransferase (AST), alanine aminotrans-
ferase (ALT), γ-glutamyltranspeptidase (γ-GTP), cre-
atinine, and blood urea nitrogen. A sex-matched ref-
erence was used for the determination of γ-GTP levels
(women: 9–36 IU/L and men: 10–72 IU/L).
LC-MS/MS
DEX was obtained from Sigma-Aldrich (St. Louis,
MO, USA). The deuterated internal standard, 4,6a,21,
21-d4-DEX (DEX-d4) was obtained from C/D/N iso-
topes (Quebec, Canada). LC-MS grade solvents (meth-
anol and water) and other reagents were purchased from
Wako Pure Chemical Industries (Osaka, Japan).
The sample was prepared as follows to measure
dexamethasone. As an internal standard, DEX-d4 dis-
solved in methanol was added to sample tubes, and
methanol was evaporated to dryness. Human plasma
(0.1 mL) was added to the sample tubes containing the
internal standard. DEX was then extracted with 4 mL
t-butyl methyl ether. After the organic layer was evap-
orated to dryness, the residue was dissolved in 0.5 mL
Endocrine Journal Advance Publication
3
methanol and diluted with 1 mL distilled water. The
sample was applied to an Oasis MAX cartridge (60 mg,
3 mL, Waters Corporation, Milford, MA, USA), which
had been successively conditioned with 3 mL of meth-
anol and 3 mL of distilled water. The cartridge was
successively washed with 1 mL distilled water, 1 mL
methanol/distilled water/acetic acid (50:50:1, v/v/v),
and 1 mL 1% pyridine. DEX was then eluted with
1 mL of methanol/distilled water/pyridine (80:20:1,
v/v/v). After the solvent was evaporated, the residue
was dissolved in 0.1 mL methanol/distilled water (2:3,
v/v), and 10 µL of the solution was subjected to analy-
sis in the LC-MS/MS instrument as described below.
Each analytical standard (50 µL) of DEX (0, 50, 100,
500, 1,000, 5,000, 20,000 pg/μL) and internal standard
(50 μL; 2,000 pg/50 μL) were added and evaporated.
Distilled water/acetic acid (1 mL; 100:1, v/v) was then
added to each solution to determine the calibration
curves for DEX.
As quality control (QC) samples, 50 μL of analytical
standards of DEX (low QC: 75 pg/50 μL, middle QC:
2,000 pg/50 μL, high QC: 16,000 pg/50 μL) and 50 μL
of internal standard (2,000 pg/50 μL) were added and
evaporated. Distilled water/acetic acid (1 mL; 100:1,
v/v) was then added to each solution. As a plasma
matrix sample, 50 µL of an internal standard (2,000
pg/50 μL) was added and evaporated. The collected
blood plasma (0.1 mL) and distilled water/acetic acid
(0.9 mL; 100:1, v/v) were then added to the solution.
DEX was analyzed using a technique similar to
that in a previous report [17] using an API-4000 tri-
ple stage quadrupole mass spectrometer equipped with
a positive electrospray ionization source (AB SCIEX,
Framingham, MA, USA) and Nexera UHPLC systems
(Shimadzu Co. Ltd., Kyoto, Japan). Regarding LC-MS/
MS analysis of DEX, 10 μL of the reconstituted sam-
ples were applied to a Kinetex C18 column (2.1 × 150
mm, 1.7 μm, Shimadzu Co. Ltd., Kyoto, Japan). The
mobile phase consisted of acetonitrile (Solvent A), and
0.1% formic acid (Solvent B) was used with a gradi-
ent elution of A:B = 25:75 (0–1.5 min), 35:65 (1.5–4
min), 100:0 (4–4.9 min), and 25:75 (4.9–5.5 min) at a
flow rate of 0.5 mL/min. The column temperature was
maintained at 50°C. The column eluent was subjected
to electron spray ionization (positive mode, ES+).
DEX was determined using selective reaction
monitoring of the transitions m/z 393.1→147.2, and
397.27→341.2 were selected for DEX and DEX-d4,
respectively.
4 Sasaki et al.
Statistical analysis
Results are presented as the median (interquar-
tile range [IQR]). Statistical analysis was performed
using the Mann-Whitney U or Fisher’s exact tests.
Correlations among plasma DEX after DST, anthropo-
metric measurements, and laboratory data were evalu-
ated using Spearman’s rank correlation analysis. The
clinical factors were predicted plasma DEX levels
by the univariate regression analysis. After identify-
ing factors that influenced the plasma DEX levels by
univariate analysis, multiple linear regression analysis
was performed with the factors showing p <0.05 to find
independent determinants of the plasma DEX levels.
When data were skewed, logarithmic transformation
was performed before multiple analysis. All statistical
analyses were performed using EZR (Saitama Medical
Center, Jichi Medical University, Saitama, Japan) and
graphical user interface for R (The R Foundation for
Statistical Computing, Vienna, Austria), a modified
version of R commander that includes statistical func-
tions frequently used in biostatistics. Statistical signifi-
cance was defined as a p value < 0.05.
Results
Validation of LC-MS/MS analysis
As an additional recovery test, three concentrations
of DEX (0, 500, and 1,000 pg) were spiked into human
plasma samples to obtain samples with three different
DEX levels. Subsequently, the spiked samples were
analyzed in parallel with the calibration curve sam-
ples and QC samples by LC-MS/MS. The QC samples
(three of each level, for a total of six samples) were ana-
lyzed before and after the human samples. Measured
values were only adopted if the accuracy of measur-
ing ≥4 QC samples was in the range of 85–115% and
the two QC samples outside this reference range were
not both samples with the same concentration. The
acceptable range of the recovery rate was defined as
80–120%. In this test, the accuracy of all QC samples
was in the range of 97.4–111%. The addition recovery
rate was 99.2–107.9%.
As a dilution test, three doses (0.01, 0.1, or 0.2 mL)
of human plasma samples were spiked with DEX and
analyzed in parallel with the calibration curve samples
and QC samples using LC-MS/MS. The QC samples
(three of each dilution level, for a total of six samples)
were analyzed before and after the human plasma.
Measured values were only adopted if the accuracy
Endocrine Journal Advance Publication
of measuring ≥4 QC samples was in the range of
85–115% and the two QC samples outside this refer-
ence range were not both samples with the same con-
centration. The acceptable error was defined as being
within ±20% of the error calculated with an analytical
standard of 0.2 mL. In the dilution test, the accuracy
of measuring all QC samples was in the range of 90.4–
106%, and the dilution level error was 3.0–12.4%.
As a test of intra-assay reproducibility (between-
run), five human plasma samples were spiked with
DEX (50 pg) and were analyzed in parallel with the
samples for the calibration curve and the QC samples
by LC-MS/MS. The specified accuracy of measure-
ment at 50 pg, the lower limit quantification (LLQ),
was 80–120% [18], and the specified precision (the
precisions below were shown coefficient validation)
for human plasma samples was ±15%. The QC sam-
ples (three of each level, for a total of six samples) were
analyzed before and after the human plasma. Measured
values were only adopted if the accuracy of measuring
≥4 QC samples was in the range of 85–115% and the
two QC samples outside this reference range were not
both samples with the same concentration. In this test,
the accuracy of measuring all QC samples was in the
range of 86.4–101%, and the precision was 2.4–5.5%.
Regarding inter-assay reproducibility (between-day),
the accuracy and precision were 97.5–97.8% and 3.3–
9.1%, respectively. The LLQ and quantitation range of
DEX were 0.5 ng/mL and 0.5–200 ng/mL, respectively,
using 0.1 mL of plasma or 0.25 ng/mL and 0.25–100
ng/mL, respectively, using 0.2 mL of plasma. Using 1
mL of plasma, the LLQ of this assay was 0.74 ng/mL,
and the quantitation range was 1.2–23.5 ng/mL.
Patient characteristics
Clinical and laboratory characteristics of the sub-
jects at baseline are shown in Table 1. The median
(IQR) age of 52 subjects was 65 (53, 73) years. The
prevalence of obese, impaired glucose tolerance,
hypertension, and dyslipidemia was 48.1%, 59.6%,
71.2%, and 46.2%, respectively. The median maxi-
mum diameter of adrenal tumor was 17.5 (12.8, 25.0)
mm. Six of the 52 (11.5%) subjects were taking cyto-
chrome P-450 (CYP) 3A4 inhibitors [19], such as
clarithromycin (n = 1), cimetidine (n = 4), and diltia-
zem (n = 1), which could increase plasma DEX levels
[5], while none of them were taking CYP3A4 induc-
ers such as antiepileptic agents or rifampin [20]. No
subjects were infected with hepatitis B or C virus or
 1-mg DST is superior to 0.5-mg DST 5

had chronic hepatitis or liver cirrhosis. According to
the MHLW criteria in Japan, 30 of 52 (57.7%) sub-
jects were finally diagnosed as non-functional adre-
nal tumor. Seven subjects (13.5%) were diagnosed
as SCS without primary aldosteronism (PA), one sub-
ject (1.9%) was diagnosed as SCS with PA, and 14
(26.9%) were diagnosed with PA. Thus, eight of 52
(15.4%) patients were affected with SCS.
Plasma dexamethasone levels after the 0.5-mg or 1-mg
DST
Box-and-whisker plots of the morning plasma DEX
levels after the 0.5- and 1-mg DST are shown in Fig.
1A. No patient had undetectable circulating plasma
DEX levels, showing that all subjects had taken the
drug. Irrespective of administration dose, plasma
DEX values varied considerably, ranging from 0.2 to

Table 1 Subject characteristics A 10

Plasma DEX level (ng/mL)

Total (n = 52) 8
Female, n (%) 22 (42.3)
Age, years 65 (53, 73) 6
Height, cm 162 (157, 168)
4
Body weight, kg 61 (56, 75)
2.2
Body mass index, kg/m2 23.8 (21.9, 27.2) 2
Body surface area, m2 1.63 (1.55, 1.82)
0
Obesity, n (%) 25 (48.1)
0.5-mg DST 1-mg DST
Impaired glucose tolerance, n (%) 31 (59.6)
Hypertension, n (%) 37 (71.2)
B 12
Dyslipidemia, n (%) 24 (46.2)
Fasting plasma glucose, mg/dL 105 (95, 136)
Glycoalbumin, % 20.9 (18.1, 26.8)
Plasma DEX level after 1-mg DST (ng/mL)

10
HbA1c, % 6.2 (5.7, 7.7)
AST, IU/L 21 (16, 25)
ALT, IU/L 18 (13, 27) 8
γ-GTP, IU/L 26 (17, 41)
Total cholesterol, mg/dL 183 (153, 205)
LDL-cholesterol, mg/dL 111 (87, 133) 6
HDL-cholesterol, mg/dL 39 (35, 47)
Triglycerides, mg/dL 104 (80, 145)
Serum Cr, mg/dL 0.77 (0.63, 0.93) 4
eGFR, ml/min/1.73m2 69.9 (61.9, 81.6)
Blood urea nitrogen, mg/dL 14 (11.3, 17.7)
Tumor size, mm 17.5 (12.8, 25.0) 2
0800 h ACTH, pg/mL 10.9 (5.3, 22.3)
2400 h ACTH, pg/mL 6.2 (2.7, 9.6)
0800 h Cortisol, μg/dL 9.2 (7.9, 11.9) 0
2400 h Cortisol, μg/dL 5.2 (3.8, 6.4) 0 2 4 6
Urinary free cortisol, μg/24 h 37.6 (26.2, 51.9) Plasma DEX level after 0.5-mg DST (ng/mL)
DHEA-S, μg/dl 59 (38.3, 113)
NF 30 (57.7%), Fig. 1 Correlation of individual plasma DEX levels after the
PA 14 (26.9%), 0.5-mg DST and 1-mg DST
Final diagnosis, n (%)
SCS 7 (13.5%), Fig. 1A shows box-and-whisker plots of plasma DEX
PA+SCS 1 (1.9%) levels after the 0.5-mg DST and 1-mg DST. Plasma
Data are expressed as the median and interquartile range (25 DEX values varied considerably. The lower (Q1) and
to 75%) for continuous variables or as percentages. γ-GTP, upper (Q3) quartile, representing observations outside
γ-glutamyltranspeptidase; HbA1c, glycated hemoglobin; AST, the 4 –96 percentile range. The red solid lines represent
aspartate aminotransferase; ALT, alanine aminotransferase; LDL, the median of plasma DEX values. Horizontal broken
low-density lipoprotein; HDL, high-density lipoprotein; Cr, lines (blue) represent the value of 2.2 ng/mL. However,
creatinine; eGFR, estimated glomerular filtration rate; ACTH, as shown in Fig. 1B, the individual plasma DEX levels
adrenocorticotropic hormone; DHEA-S, dehydroepiandrosterone were significantly correlated (ρ = 0.853, p < 0.001),
sulfate; NF, non-functioning adrenal tumor; PA, primary with a slope of 2.179 and intercept of 0.072. DEX,
aldosteronism; SCS, subclinical Cushing’s syndrome. dexamethasone; DST, dexamethasone suppression test.

Endocrine Journal Advance Publication

6 Sasaki et al.

5.1 ng/mL following the 0.5-mg DST and from 0.5 to and cortisol levels ≥3.0 μg/dL after DEX administra-
11.4 ng/mL following the 1-mg DST. In contrast, as tion at a dose of 0.5-mg and the same two subjects
shown in Fig. 1B, a highly significant statistical cor- (25.0%) showed DEX levels >2.2 ng/mL and corti-
relation was observed in plasma DEX levels between sol levels >1.8 μg/dL with the same DEX dose (Fig.
0.5-mg and 1-mg after loading. Spearman’s ρ and 2A). In contrast, four subjects (50.0%) had plasma
corresponding p value for these correlations were ρ DEX levels >2.2 ng/mL and cortisol levels ≥3.0 μg/
= 0.853 and p < 0.001, and slope and intercept were dL after a DEX dose of 1-mg, and the same four sub-
observed at 2.179 and −0.072, respectively. Thus, jects (50.0%) showed DEX levels >2.2 ng/mL and
these data clearly showed high inter-individual vari- cortisol levels >1.8 μg/dL (Fig. 2B). However, these
ability but low intra-individual variability of DEX lev- four SCS subjects showed positive findings in tests of
els after DST; furthermore, plasma levels after 1-mg HPA axis function other than the DST, suggesting that
loading in each patient were around two times higher low plasma DEX levels in the DST do not necessarily
than levels after 0.5-mg loading. exclude SCS (data not shown).

Plasma dexamethasone and cortisol levels after the Plasma dexamethasone and ACTH levels after the
0.5-mg or 1-mg DST 0.5-mg or 1-mg DST
Fig. 2 displays serum cortisol and plasma DEX lev- Detectable ACTH levels after the 0.5-mg and 1-mg
els after DST. The number of subjects with DEX lev- DST were present in 30 (57.7%) and 19 (36.5%) sub-
els ≤2.2 ng/mL after the 0.5-mg and 1-mg DST was jects, respectively. Twenty-eight (93.3%) of the subjects
45 (86.5%) and 17 (32.7%), respectively (p < 0.001). with detectable ACTH levels after the 0.5-mg DST did
Among eight subjects with a diagnosis of SCS, two not exceed a DEX level of 2.2 ng/mL; in contrast, only
subjects (25.0%) had plasma DEX levels >2.2 ng/mL four (21.1%) of the subjects did so after the 1-mg DST.

A 20 B 20
Serum cortisol level after 0.5-mg DST (μg/dL)

Serum cortisol level after 1-mg DST (μg/dL)

15 15

10 10

5 5

0 0
0 3 6 9 12 0 3 6 9 12
Plasma DEX level after 0.5-mg DST (ng/mL) Plasma DEX level after 1-mg DST (ng/mL)
Fig. 2 Correlation between individual DEX and cortisol levels after DST
Both panels show the individual distribution of DEX and cortisol levels after the 0.5-mg and 1-mg DST. Among eight subjects
with a diagnosis of subclinical Cushing’s syndrome (SCS), two subjects (25.0%) had plasma DEX levels >2.2 ng/mL and
cortisol levels ≥3.0 μg/dL after DEX administration at a dose of 0.5 mg and the same two subjects (25.0%) showed DEX levels
>2.2 ng/mL and cortisol levels >1.8 μg/dL with the same DEX dose (Fig. 2A). In contrast, four subjects (50.0%) had plasma
DEX levels >2.2 ng/mL and cortisol levels ≥3.0 μg/dL after a DEX dose of 1 mg, and the same four subjects (50.0%) showed
DEX levels >2.2 ng/mL and cortisol levels >1.8 μg/dL (Fig. 2B). Vertical broken lines (black) represent the value of 2.2 ng/
mL, upper horizontal broken lines (red) indicate the cut-off value of 3 μg/dL, lower horizontal broken lines (blue) indicate the
cut-off value of 1.8 μg/dL, and red closed circles represent patients diagnosed as having SCS. DEX, dexamethasone; DST,
dexamethasone suppression test.

Endocrine Journal Advance Publication

 1-mg DST is superior to 0.5-mg DST 7

Assessment of the relationship between plasma dexa- Discussion

methasone levels and clinical parameters
We next performed univariate and multivariate
analyses to identify clinical parameters related to
plasma DEX levels after the 0.5-mg or 1-mg DST
(Table 2). In the Spearman’s rank correlation analy-
sis, there was a weak correlation between DEX lev-
els after the 0.5-mg DST and body mass index (BMI)
(ρ = 0.273, p = 0.049). There were also weak cor-
relations between DEX levels after the 1-mg DST
and body weight (ρ = 0.285, p = 0.041), BMI (ρ =
0.328, p = 0.018), γ-GTP (ρ = 0.339, p = 0.014), and
glycated hemoglobin (ρ = 0.290, p = 0.037). The
plasma DEX levels in the subject taking CYP3A4
inhibitors tended to be slightly higher, but not to
a statistically significant level (data not shown).
Furthermore, multiple linear analysis revealed that
there were no independent variables for 0.5-mg DST,
while γ-GTP was selected as a significant indepen-
dent variable associated with the plasma DEX lev-
els after the 1-mg DST. However, the association
between γ-GTP and DEX levels was lost after exclud-
ing subjects with γ-GTP levels above the sex-matched
reference range.
There were two main features of the present study.
First, we reported the development of an accurate
and precise LC-MS/MS assay for measuring plasma
DEX levels. Second, the 1-mg DST was superior to
0.5-mg loading for the diagnosis of adrenal SCS based
on the plasma DEX levels measured using the LC-MS/
MS assay.
As has been indicated, because inadequate plasma
DEX levels may cause false-positive results on the
DST, quantitative determination of synthetic gluco-
corticoid is indispensable verification for comparing
the accuracy between DST procedures. LC-MS/MS
offers superior analytical specificity compared with
conventional immunoassays. Moreover, an antibody
to DEX is not currently available in Japan. Therefore,
we developed a LC-MS/MS method to quantify DEX.
DEX levels displayed acceptable standard devi-
ations (2.4–5.5%) and a low LLQ (0.74 ng/mL).
Results of the additional recovery test and the repro-
ducibility test were also satisfactory, showing that
the assay system we developed is reliable enough to
undergo further investigation.

Table 2 Results of univariate (upper) and multiple linear (lower) regression analysis of factors affecting plasma
DEX levels after the 0.5-mg or 1-mg DST
0.5-mg DST 1-mg DST
ρ p value ρ p value
Age, years 0.105 0.458 0.133 0.347
Body weight, kg 0.207 0.141 0.285 0.041
Body mass index, kg/m2 0.273 0.049 0.328 0.018
Height, cm -0.002 0.988 0.122 0.389
eGFR, mL/min/1.73m2 -0.011 0.939 -0.141 0.320
Serum Cr, mg/dL 0.063 0.655 0.251 0.073
Blood urea nitrogen, mg/dL 0.092 0.516 0.153 0.279
AST, IU/L 0.136 0.337 0.111 0.432
ALT, IU/L 0.163 0.249 0.255 0.068
γ-GTP, IU/L 0.223 0.111 0.339 0.014
HbA1c, % 0.146 0.301 0.290 0.037
0.5-mg DST 1-mg DST
B β p value B β p value
Body mass index, kg/m2 0.013 0.011 0.946 0.030 0.090 0.830
γ-GTP, IU/L 0.006 0.187 0.271 0.027 0.330 0.049
HbA1c, % -0.013 -0.056 0.704 -0.032 -0.048 0.734
Upon multiple linear regression analysis, γ-GTP was a significant parameter for affecting plasma DEX levels after
1-mg loading. Because body mass index (BMI) shows an interaction with body weight and had a smaller p value
on univariate analysis, BMI was used as an explanatory variable. Values of p <0.05 were considered statistically
significant. B, partial regression coefficient; β, standardized partial regression coefficient; DEX, dexamethasone;
DST, dexamethasone suppression test; AST, aspartate aminotransferase; ALT, alanine aminotransferase; γ-GTP,
γ-glutamyltranspeptidase; Cr, creatinine; eGFR, estimated glomerular filtration rate.

Endocrine Journal Advance Publication

8 Sasaki et al.
No study has directly compared plasma DEX lev-
els measured by using LC-MS/MS and a conventional
method. Nonetheless, plasma DEX levels determined
using LC-MS/MS at 0800 h following 1-mg DST were
similar to those determined using an RIA method, with
plasma levels in most subjects within the range of 1.0
ng/mL to 10 ng/mL [8, 21-23].
The present study shows the superiority of the
1-mg DST to 0.5-mg DST; this conclusion is sup-
ported by the following reasons: 1) plasma DEX lev-
els after the 1-mg DST in each patient were consis-
tently higher than those after 0.5-mg loading, 2) use
of 1-mg DEX can more frequently achieve the rec-
ommended value by Endocrine Society guidelines,
and 3) ACTH secretion was more strongly suppressed
by 1-mg DEX than by 0.5-mg DEX. Nevertheless, a
lower loading dose and simplification of DST is ben-
eficial and desirable from a safety standpoint, and the
use of recent less-stringent criteria for the use of cor-
tisol in testing is expected to prevent underestimation
of subtle hypercortisolism, including SCS. However,
these alterations may increase the risk for false-pos-
itive DST results; additionally, using a recent proce-
dure, the diagnostic specificity in some studies of CS
or pseudo-CS was lower than that previously reported
[24]. Accordingly, it is important to develop proce-
dures for simultaneous determination of post-test
DEX and cortisol levels. On the 1-mg DST, the fre-
quency of false-positive results remained approxi-
mately 10%, but reached 20–40% on the 0.5-mg DST.
Thus, there is no rational basis for changing the dose
of DEX from 1 mg to 0.5 mg.
DEX levels after a low-dose DST in the current
study did not attain a predictably effective value (2.2
ng/mL) in a higher proportion of patients than previ-
ously reported [21]. One possible explanation for the
unexpected results is that the Endocrine Society rec-
ommendations regarding DEX levels may be inad-
equate. The recommendation was based on results
indicating that when the plasma DEX levels follow-
ing overnight low dose DST were <2.2 ng/mL, there
was an overlap of the range of cortisol levels between
patients with documented CS and those with suspected
CS but who exhibited no progression of Cushingoid
features on follow-up [8]. Furthermore, the report
used the classic threshold value of 5 µg/dL for inad-
equate cortisol suppression on DST; hence, it is uncer-
tain whether the results can also apply to the diagno-
sis of SCS. Accordingly, further research is needed to
Endocrine Journal Advance Publication
clarify the optimal cut-off plasma DEX value for eval-
uation of autonomous, albeit subtle, cortisol secretion
from adrenal adenoma.
There is considerable inter-individual variation in
plasma DEX levels after DST [8]. This may be due to
several factors that may affect oral DST; such factors
include variable intestinal absorption and decreased or
increased DEX metabolism due to other compounds.
Moreover, age, sex, and adiposity might influence the
outcome of DST, but results vary among studies [10, 25,
26]. From physical differences between Japanese and
Western people, Oki et al. [7] suggested that in Japanese
patients, 1-mg DST might suppress plasma cortisol lev-
els too strongly. Using a receiver operator characteris-
tic analysis, Oki et al. [7] concluded that 0.5-mg DST
is a more sensitive and specific screening tool for diag-
nosis of ACTH-dependent CS compared to 1-mg DST.
However, this prior study did not mention DEX levels.
In the present study, body weight, body surface area,
and BMI were not associated with plasma DEX levels
on multiple regression analysis, indicating that physical
differences do not provide a rationale for adjustment of
DEX dose in patients with suspected adrenal SCS. In
agreement with our results, most studies showing differ-
ences in body weight fail to account for the marked vari-
ability in post-oral administration DEX levels [23, 25].
O’Sullivan et al. indicated no single pharmacoki-
netic factor was responsible for the lower DEX lev-
els in depressed patients without adequate cortisol
suppression by DST [26]. Moreover, Weiner et al.
reported that healthy subjects with DEX ≤ 2.2 ng/mL
were rare (5%), but all were >50 years old, indicating
that DST may be invalid more often in older individ-
uals [27]. Although the cause of differences among
studies is not clear, these findings indicate that there
may be an increased need for simultaneous measure-
ment of cortisol and DEX in older individuals.
Additionally, degradation of DEX by hepatic
CYP3A4 also may be responsible for the variation in
DEX levels; several drugs markedly accelerate glu-
cocorticoid clearance, resulting in much lower levels
than expected [5]. We tried to exclude patients tak-
ing CYP3A4 inhibitors as much as possible, but six
patients wished to continue taking them. However, in
this study plasma DEX levels in these subjects were
not significantly higher than that in other subjects, sug-
gesting that the agents may not have affected the pres-
ent findings. This further suggests that medication use
should be considered when administering DST.
 1-mg DST is superior to 0.5-mg DST
Furthermore, Huizenga et al. [10] reported a posi-
tive correlation between AST, ALT, and γ-GTP levels
with DEX levels after 1-mg DEX administration in
men from a population-based cohort. Nonetheless, the
relationship was no longer significant when exclud-
ing subjects with supra-normal levels of at least one
of the three enzymes. Although we excluded patients
with positive hepatitis B surface antigen and hepatitis
C virus antibody, chronic hepatitis, liver cirrhosis, or
alcohol abuse, we likewise found a significant asso-
ciation between γ-GTP and plasma DEX levels after
the 1-mg DST. Interestingly, the association was also
lost after excluding subjects with γ-GTP above the
sex-matched reference range. These findings suggest
that subjects without chronic alcoholic or viral liver
disorder but with mild liver dysfunction may demon-
strate false-positive results on a DST.
In a previous study, nonalcoholic fatty liver disease
(NAFLD) occurred in 20% of patients with CS, and
glucocorticoid excess appeared to increase hepatic de
novo lipogenesis [28]. Recently, Woolsey et al. [29]
showed that CYP3A4 activity was significantly reduced
in human NAFLD as well as in mouse and cell cul-
ture models of NAFLD. Thus, mild liver dysfunction
resulting from NAFLD may be present in some SCS
patients and may delay DEX metabolism via reduced
CYP3A4 function [30]. Further investigation to deter-
mine the importance of this issue will be required.
This study has several limitations. First, we did
not determine whether the validity of the conven-
tional cut-off value for plasma DEX levels (2.2 ng/
mL), as previously set forth by the RIA method [8],
also applies to results obtained using the LC-MS/MS
method. However, the purpose of this study was to
compare the precision of the 1-mg and 0.5-mg DST
based on plasma DEX levels or magnitude of ACTH
suppression. Notwithstanding, even if the cut-off
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Acknowledgments
This study was supported by a Health and Labor
Science Research Grant, Research on Intractable
Diseases, Research Committee on Disorder of Adrenal
Hormones from the MHLW, Japan. The authors wish to
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None of the authors have any potential conflicts of
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