CLIN. CHEM.
25/10, 1700-1703
(1979)
Serum Cortisoland 11-Deoxycortisolby LiquidChromatography:
ClinicalStudiesand Comparisonwith Radioimmunoassay
Ernesto Canalis,1’2 Antoinette M. Caldarella,’ and George E. Reardon”2
We describe a liquid-chromatographic
procedure for
techniques
separating and measuring cortisol and 11-deoxycortisol
in serum. We quantitated these steroids in patients who
7-9).
The
were undergoing various tests of pituitary and (or) adrenal
original
function and compared the results with those obtained by
two radioimmunoassays done in two different laboratories.
Results of 48 tests done in 37 functionally normal humans
are presented. Cortisol values for sera collected in the
morning as determined by liquid chromatography
were
tions,
suring
(mean ± SD) 134 ± 54 g/L. Serum cortisol concentrations increased from 136 ± 65 to 321 ± 80 j.g/L 60 mm
after injecting synthetic corticotropin and increased from
107 ± 46 to 242 ± 31 zg/L after insulin-induced hypoglycemia. Serum cortisol decreased from 142 ± 49 to 26
± 20 .zg/L after oral administration of metyrapone, while
11-deoxycortisol increased from <10 to 210 ± 53 tg/L.
Serum cortisol measured <10 g/L the morning after oral
ingestion of dexamethasone. Results of the dynamic tests
of adrenal function correlated well with previously reported
studies. However, the cortisol values obtained by our
technique were generally lower than those obtained by
radioimmunoassay, possibly owing to lack of specificity
of the latter methods used here for comparison. In contrast,
values
for
1 1-deoxycortisol
were
the same
by both
methods. The present studies confirm the usefulness of
liquid chromatography
for measuring
these two steroids
in serum during tests of pituitary and adrenal function.
Future refinements of the technique should continue to
increase its clinical applications.
#{149}
intermethod comparison
#{149}
and
il-deoxycortisol
steroids
and
measure
in a single
we can extract
them
serum
accurately
in serum.
(1).
there are other methods
for the clinical determinations
of
serum cortisol and 1 1-deoxycortisol
(2-6), liquid chromatography (LC) offers the advantages of greater specificity,
simplicity, and the capability to measure, simultaneously,
several steroids in a single assay. Of these advantages, the most
important
is the
‘Division
first,
especially
of Endocrinology,
because
Departments
common
114 Woodland
2
St., Hartford,
Division of Endocrinology,
laboratory
of Medicine,
and Laboratory Medicine, Saint Francis Hospital and
1700
CLINICAL
CHEMISTRY,
Pathology
Medical
Center,
CT 06105.
Department
of Medicine, University
of Connecticut
Health Center, Farmington,
CT 06032.
Received April 13, 1979; accepted June 11, 1979.
Vol. 25, No. 10, 1979
entirely
specific
studied and tests performed
small.
the clinical
cortisol
not
and
In the
present
use of LC more
11 -deoxycortisol
(4,
in our
investiga-
closely,
mea-
in functionally
normal human subjects undergoing tests of the pituitary
and
adrenal glands. We have examined the effects of corticotropin,
insulin,
and dexamethasone
administration
on serum
cortisol
concentrations
and the effects of metyrapone administration
on serum cortisol and 11 -deoxycortisol. In addition, we compared the measurements
obtained by LC to those obtained
by radioimmunoassay
(RIA).
Materials and Methods
Analytical
Methods
chromatography:
The method we used to measure
and li-deoxycortisol
in serum by LC has already been
described in detail (1). Blood was obtained by venipuncture
and the serum, separated by centrifugation
within 1 h after
the blood was drawn, was stored at -20 #{176}C
until assay. Serum
samples were extracted with methylene chloride, and the
extracts were washed, dried, and dissolved in methanol/water
(60/40 by vol) before analysis by LC.
Liquid
cortisol
The steroids used for standards
(from Sigma Chemical Co.,
St. Louis, MO 63178, and Steraloids,
Inc., Wilton, NH 03086)
(n
sample
and separate
are
Although
Recently, we developed a convenient and specific liquidchromatographic
procedure for separating and quantitating
With this method,
of patients
was relatively
we examined
serum
cortisol
these two
specificity of
.
cortisol
number
study
serum
were also dissolved in methanol/water
(60/40 by vol).
The chromatography
was done with a Model 204 liquid
chromatograph
purchased
from Waters Associates, Inc.,
Milford, MA 01757, equippe4 with a tBondapak
C,8 column.
The wavelength used was 254 nm, the sensitivity 0.005 absorbance unit (A) full scale, and the flow rate of the mobile
phase (methanol/water,
60/40 by vol) was 1 mL/min.
The analytical recovery of steroids added to serum had been
previously determined to be 95 to 105% for both cortisol and
11-deoxycortisol
(1). The coefficient of variation (CV) for
cortisol was 2.5% (n = 6) and for il-deoxycortisol
was <0.2%
AddItional Keyphrases: Pituitary/adrenal function assessment
#{149}
normal values
RIA
steroids
for
= 8).
Radioimmunoassay:
Serum cortisol and 11 -deoxycortisol
concentrations
were also determined
by RIA. Cortisol was
measured in the Radioisotope
Laboratory
of our hospital’
(RIA I) with a kit purchased from Beckman Instruments, Inc.,
Irvine, CA 92713, according to the manufacturer’s instructions
and at the Nichols Institute, San Pedro, CA 90731 (RIA II).
The percentage
recovery of added cortisol was 95 to 100% for
both RIA methods; the intra-assay CV was 5.4% for RIA I and
5.2 to 7.0% for RIA II; the interassay
CV was 7.8 to 15.2% for
RIA I and 6.3 to 10.9% for RIA II (Radioimmunoassay
Manual, Nichols Institute, 1977).
11 -Deoxycortisol was measured by Laboratory Procedures,
Inc., King of Prussia,
PA 19406.
Clinical Studies
Serum samples
tests
of pituitary
were obtained
and (or) adrenal
from patients
function
undergoing
at our hospital.’
All patients were evaluated by history, physical examination,
and routine biochemical and hematological
tests. Patients
undergoing
studies
of pituitary
function
were also assessed
(mean ± SD; n = 40). The values correlated well, but were
significantly (p <0.01) lower than those obtained by RIA (203
± 78, n = 40 for RIA I; 163 ± 61, n = 29 for RIA II) as performed in two different laboratories (Figure 1A).
Corticotropin
stimulation
test: Sixty minutes after the
administration
of synthetic corticotropin,
serum cortisol
by measurements
of somatotropin,
of thyroid and gonadal
reserve, and of serum prolactin. In addition, roentgenograms
concentrations
by LC increased
from a baseline value of 136
± 65 to 321 ± 80 tg/L. The delta value (difference
between
of the sella turcica,
pre- and post-injection)
was 185 ± 63 ftg/L, and there was no
difference between responses of patients in the morning and
afternoon. When the same serum samples were measured by
two RIA methods, the baseline cortisol values were higher
than those observed with LC. However, the serum cortisol
concentrations
measured by LC after the injection of corticotropin and the delta value were lower than those obtained
by one (RIA I) but not by the other laboratory (RIA II), indicating discrepancy in the values observed with RIA (Table
1A). The serum cortisol values obtained by LC after the injection of corticotropin correlated well with those by both RIA
methods (Figure 1B); the delta value correlated well with RIA
I (r = 0.93) but not with RIA II (r = 0.37).
Insulin tolerance
test: All the patients studied developed
significant
hypoglycemia
30 to 45 mm after the injection of
computerized
axial
tomography,
pneu-
moencephalography,
and visual field testing were done when
clinically indicated. Forty-two patients, 11 males and 31 females, were examined with various tests of pituitary and (or)
adrenal function; 37 of these had normal endocrine function,
five abnormal. Because the number of abnormal patients was
small, we present only the data on the subjects with normal
pituitary
and adrenal function.
Of these 37 functionally
nor-
mal patients, six had a pituitary tumor, five had galactorrhea-amenorrhea
syndrome without pituitary tumor, four had
empty-sella
syndrome,
and 22 had no demonstrable endocrine
disease. Four of these patients were later tested a second time,
increasing
the number of pituitary
and (or) ardrenal
evaluations to 41. A total of 48 dynamic tests of pituitary
and (or)
adrenal function was performed
during these 41 evaluations,
in addition
to 40 cortisol determinations
on specimens
col-
lected in the morning.
The following tests
performed:
of pituitary-adrenal
function
were
cortisol
in 12 patients by examining their cortisol responses to corticotropin. Serum was sampled, to measure cortisol, before and
60 mm after the intramuscular injection of 250 tg of synthetic
corticotropin
(Cosyntropin; Organon, Inc., West Orange, NJ
07052) according to established
protocols (10, 11). Six patients
were studied in the morning and six in the afternoon.
Insulin
tolerance
test: Serum cortisol was measured in 10
fasting patients before and 15,30,45,60, and 90 mm after the
intravenous
injection of insulin, 0.1 to 0.2 USP units/kg body
wt (12, 13). All patients developed evidence of hypoglycemia
(serum
glucose
decreased
by at least half from zero-time
val-
ues; absolute
concentrations
were lower than 0.5 g/L).
Met yrapone test: Samples to determine serum cortisol and
li-deoxycortisol
were obtained from 20 patients before and
after oral administration of standard doses of metyrapone (750
mg every 4 h, six doses),
of the hydroxylating
enzyme that converts il-deoxycortisol
to cortisol (5, 14).
Dexamethasone
suppression
test: To study whether the
a known
blocker
corticotropin-adrenal
axis could be suppressed, six patients
ingested 1 mg of dexamethasone
at 2300 h and, according to
established protocols,
blood for serum cortisol determinations
was obtained the following morning at 0800 h (15, 16).
Statistical
determined
by LC, increased
242 ± 31 tg/L
determinations:
Serum cortisol was
measured in 40 unselected serum samples obtained between
0800 and 1000 hours.
Corticotropin
stimulation
test: We tested adrenal reserve
Morning
insulin; the serum concentrations
of cortisol were highest
to 90 mm after the hormone
was given. Serum cortisol,
Methods
Data are expressed as mean ± standard
deviation
(SD) of
concentrations
of cortisol or li-deoxycortisol
in serum. The
significance of differences between LC and RIA was assessed
by paired t-test; correlation
between assays was tested by the
method of linear correlation.
Results
Confirming
our previous study, serum cortisol and 11deoxycortisol were separated, identified by their chromatographic retention times (8 and 12 mm, respectively),
and their
concentrations
determined by LC. The following are the results observed in functionally normal human subjects:
Morning
cortisol: Serum cortisol values obtained between
0800 h and 1000 h and measured by LC were 134 ± 54 g/L
after
from a baseline
hypoglycemia;
the delta
60
as
of 107 ± 46 to
value
was 135 ±
39 g/L. The values for serum cortisol obtained by LC, before
and after hypoglycemia,
correlated
well with both RIA
methods (Figure 1A and IB) but were lower than those obtained by RIA (Table 1B). The delta value by LC correlated
well and was not different from RIA II (r = 0.91), but was
lower than RIA I (r = 0.69) (Table 1B).
Metyrapone
apone, serum
test: After the oral administration
of metyrcortisol concentrations
as determined
by LC
decreased from 142 ± 49 to 26 ± 20zg/L, while li-deoxycortisol increased
from <10 zg/L to 210 ± 53 zg/L. Before me-
tyrapone administration,
serum cortisol by LC was 29 and 13%
lower than that obtained by RIA I and II, respectively,
whereas after metyrapone,
the values were 75 and 43% lower
and did not correlate well with RIA (Table 1C, Figure 1C). In
contrast to the results obtained for cortisol, LC determinations
of li-deoxycortisol
before and after metyrapone correlated
well and were not different from those obtained by RIA (Table
1C, Figure 2).
Dexamethasone
suppression
test: Serum cortisol measurements performed by LC the morning after dexamethasone
was administered
were <10 g/L; those performed by RIA
were <15 and <30 tg/L (RIA I and II, respectively).
Discussion
In the present
of cortisol
and
studies,
we describe
11 -deoxycortisol
the clinical
determinations
applications
by
LC and
present
data obtained
from a group of functionally
normal
patients
undergoing
tests of pituitary
and (or) adrenal function, comparing
LC with RIA. We measured
cortisol and
11 -deoxycortisol
in unselected morning serum samples and
in samples from patients undergoing tests of pituitary-adrenal
function such as stimulation with corticotropin,
the insulin
tolerance test, the metyrapone
test, and suppression with
dexamethasone.
After either the administration
of corticotropin
or insulin-induced
hypoglycemia,
cortisol
concentrations
in serum
were always increased by more than 80 zg/L. These results
resemble those previously reported by other investigators
using fluorometric
and competitive
binding assays (11-13, 17)
and confirm the clinical value of use of LC. After the administration of dexamethasone, serum cortisol as measured by LC
decreased to values <10 fzg/L, lower than those obtained by
CLINICALCHEMISTRY,Vol. 25, No. 10, 1979 1701
A
B
.
C
160
.
S
.
140
S
I
#{149}
K
S
I
#{149}
.
.
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#{149} K
20
S.
..
#{149} #{149}#{149}.K
#{149}
S
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S.
#{149}J
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K
S
I
#{149}#{149}.
K
S.
#{149}#{149}
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K
#{149} xnxx
a
a
.
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40
‘C
20
20
Lc
40
60
80
100
Fig.1.Correlation
between cortisol
as measured (J.Lg/L)
by LC and two RIA procedures
A, Cortisol values obtained between 0800 hand 1000 h:RIA l(#{149})y
= 1.2x + 4, r = 0.84:RIA II (x)y
B, Pooledcortisol
valuesobtained after the Injection ofcorticotropin and after hypoglycemia:RIA I()
0.81
C. Cortisol values obtained after the oral administration of metyrapone: AlA I(#{149})
y O.5x + 8.9,r
lx + 2.7, r
=
y
=
=
0.91
3.2x + 1.3,r
=
0.7x+ 10.6,r
0.94;RIA 11(x)
y
=
Table 1. Cortisol and li-Deoxycortisol
Concentrations in Serum as Determined by Liquid
Chromatography (LC) and Radloimmunoassay
(RIA) before and after the Indicated Treatment
After
Before
A. Corticotropin administrationb
Cortisol,
tg/L
LC
136±65
321±80
442±107c
RIA I
215 ± 98C
RIA II
164 ± 83#{176}
306±68
B. Insulin-induced hypoglycemia d
107±46
242±31
RIAI
159±76c
333±37#{176}
RIAII
138±43#{176}
278±35C
C. Metyraponeadministration0
Cortisol, J2g/L
142 ± 49
LC
26 ± 20
RIA I
200 ± 63#{176} 102 ± 29#{176}
RIA II
164 ± 50#{176}
46 ± 18#{176}
studies
#{149}
‘Delta’ is the difference
between values before and after the Indicated
terminations
in normal humans, before and 1 h after the Injection of synthetic
corticotropin
(250 zg).
C Significantly
different from LC, p < 0.05.
5Values are means ± SD for 10 (LC and AlA I) and seven (RIA II) serum determinations
in normal humans, before and after InsulIn-induced
hypoglycemia.
Values are means ± SD for19-20(IC, AlA I and AlA) and Ii (RIA II) serum
determinations
obtained before and after the administration of metyrapone (750
mg every 4 h. six doses).
CLINICAL CHEMISTRY,
Vol. 25, No. 10, 1979
in our laboratory
on the use of serum
11-
when
crossreacting
steroids,
such as 11-
300
135 ± 39
174 ± 59#{176}
140 ± 48
treatment.
b Values are means ± SD for 12 (LC and RIA I) and nine (AlA II) serum de-
1702
performed
by RIA, especially
210 ± 53
198 ± 45
<10
<10
0.64
deoxycortisol measurements by LC obtained before and after
Delta8
metyrapone. In accordance with our previous results and those
obtained by other investigators,
il-deoxycortisol
always increased to values exceeding 100 g/L in individuals with
185±63
normal pituitary reserve (1, 5).
In a previous communication
(1), we suggested
that cortisol
227±89#{176}
measurements
obtained
by
LC
were
lower
than
those
obtained
142±44
11-Deoxycortisol, /Lg/L
LC
RIA
=
RIA and by other methods previously reported (15, 16). This
discrepancy could be due to greater specificity of LC than
other assays. The present investigations also extend previous
Cortisol, tg/L
LC
0.35;AlA 11(x)
y = 0.7x+ 3.2,r
280
e
260
‘
240
S
220
I
S
.
.
200
0
.
.
180
0
I.,
S
160
e
0
O
.
140
120
100
0
“
100
120
140
ii-
ISO
180
DEOXYCORTISOL.
200
220
IC
240
260
280
300
“9/I
Fig. 2. Correlation between 1 1-deoxycortisol as measured
by LC and RIA
Valuesare forsamples obtainedafteroraladministration
ofmetyrapone (y
0.6x + 6.3,r 0.76)
=
deoxycortisol, were present in appreciable amounts in serum.
Our current investigation
indicates that morning values for
serum cortisol obtained by LC correlate well with those by
RIA. However, the degree of correlation observed in the dynamic tests varied. Furthermore,
most of the values obtained
by LC were significantly lower than those obtained by RIA.
The difference between LC and RIA was marked in samples
obtained after the administration
of metyrapone, which, by
blocking the li-hydroxylating
enzyme, causes an increase in
11 -deoxycortisol and other crossreacting cortisol precursors.
There was disparity
between results by the two RIA methods
used, the difference between LC and RIA being more pronounced with RIA I than with RIA II, which is more specific
(1). In fact, the results obtained with RIA II were generally
closer to those obtained with LC than with RIA I, suggesting
that the specificity of RIA varies among different assay systems. The higher cortisol values obtained with RIA than with
LC are probably referable to the cross reactivity of anti-cortisol antibodies with other steroids present in serum, such as
progesterone,
17-hydroxyprogesterone,
cortisone, 11 -deoxycorticosterone,
and 11-deoxycortisol
(7). Because the cross
reactivity of the antibodies differs among assays, the specificity will also vary. In the present case, the package insert
furnished with the kit used in the assay designated RIA I lists
cross reactivity
with several
of the above-mentioned
steroids,
as was confirmed in the laboratory (Dr. Judith Hopkins, unpublished observations),
while that of RIA II appears to be
small. From our data, we conclude that LC is more specific
than either of the two RIA procedures. Although our results
cannot be extended to cortisol RIA in general without further
study
of each system,
they suggest
that LC is a more accurate
method than RIA in various clinical situations. RIA has one
advantage
over LC: the capability to test many samples
quickly. However, new developments
in LC equipmentspecifically,
automatic
sample injectors-are
rapidly increasing the clinical capability of the assay. In addition,
methods designed to measure multiple steroids in a single
serum extract, which currently are being developed (Reardon,
Caldarella, and Canalis, unpublished observations), will also
increase the clinical utility of LC. The greatest value of LC is
to provide physicians with an accurate assay for various steroids, and the newer developments
here alluded to will make
it a useful tool for many clinical laboratories.
In contrast to the results obtained with cortisol and confirming our previous observations,
concentrations
of 11deoxycortisol in serum as measured by LC and RIA were not
different, probably owing to the high specificity of both the
LC and RIA methods for 11-deoxycortisol
(1, 6).
In conclusion, our studies confirm that LC is a useful
method for measuring cortisol and li-deoxycortisol
in patients
who are undergoing various tests of pituitary and (or) adrenal
function. As new refinements of the LC assay become available, more steroids could be measured simultaneously
in a
single serum sample, increasing the capability,
and the clinical applications of the method.
We thank members of The Nichols Institute,
the efficiency,
San Pedro, CA, for
performing cortisol assays at a reduced cost, Mrs. Judith Thibeault
for expert technical assistance, and Mrs. Gail Kissell for valuable
secretarial help.
Supported in part by a grant from the Women’s Auxiliary of Saint
Francis Hospital
and Medical Center.
References
1. Reardon, G. E., Caldarella, A. M., and Canalis, E., Determination
of serum cortisol and 11-deoxycortisol
by liquid chromatography.
Clin. Chem. 25, 122 (1979).
2. Steenburg,
R. W., and Thomasson, B. H., Fluorometric determination of corticoids
in human blood: Comparison of the results with
the Silber-Porter Method. J. Clin. Endocrinol. Metab. 24, 875
(1964).
3. Murphy, B. E. P., Some studies of the protein-binding
of steroids
and their applications to the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein-binding
J. Clin. Endocrinol. Metab. 27,973 (1967).
B., and Kalty, R., Radioimmunological determination of plasma cortisol. Experientia 28, 1104 (1972).
5. Spark, R. F., Simplified assessment of pituitary-adrenal reserve.
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4. Vecsei,
P., Penke,
Measurement of serum il-deoxycortisol and cortisol after metyraAnn. Intern. Med. 75,717 (1971).
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pone.
1644 (1975).
7. Antoniades,
versity Press,
H. N., Hormones in Human
Cambridge,
MA, 1976, p 727.
Blood. Harvard Uni-
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R. E., and DeLuna,
R. F., Assessment
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(1968).
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R., Interference of 21-deoxycortisol
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insufficiency.
Clin. Endocrinol. Metab. 1, 549 (1972).
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control subjects. J. Clin. Invest. 45, 429 (1966).
13. Jacobs, H. A., and Nabarro, J. D., Tests of hypothalamic-pituitary-adrenal
function in man. Q. J. Med. 38,475 (1969).
14. Liddle, G. W., Estep, E. L., Kendall, J. W., et al., Clinical application of a new test of pituitary reserve. J. Clin. Endocrinol. Metab.
19, 875 (1959).
15. Nugent, C. A., Nichols, T., and Tyler, F. H., Diagnosis of Cushing’s
syndrome. Single dose dexamethasone test. Arch. Intern. Med. 116,
172 (1965).
16. Pavlatos, F. C., Smilo, R. P., and Forsham, P. H., A rapid
screening test for Cushing’s syndrome. J. Am. Med. Assoc. 193, 720
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CLINICAL CHEMISTRY,
Vol. 25, No. 10, 1979
1703