Measuring Urinary Free Cortisol
Release of Cortisol
Stress
-
The hypothalamus
-
Pituitary Gland
Adrenal Cortex
Increase
Blood Glucose
Suppress
Immune
Response
Corticotrophin Releasing
Hormone (CRH)
Adrenocorticotropic
Hormone (ACTH)
Cortisol
Catabolism of
proteins, fat.
Twenty-four-hour plasma cortisol profile
Detection of Cushing’s Syndrome
Cushing’s syndrome:
A group of signs and symptoms caused by abnormally high
levels of cortisol.
Causes of Cushing’s syndrome:
1. Ingestion of exogenous steroids
2. A pituitary tumor that produces ACTH (also called Cushing’s
disease)
3. Primary overproduction of cortisol by the adrenal gland due to
an adrenal tumor or to adrenal hyperplasia.
4. Tumor outside of the hypothalamic-pituitary axis that produces
ACTH (ectopic ACTH production)
Laboratory Tests
Presentation of excess cortisol
*24-hour urine test
*Single low dose dexamethasone suppression test
*Saliva or blood cortisol at 11 PM
Differential Diagnosis of Cushing’s Syndrome
(cause: pituitary, adrenal, or other)
*High dose dexamethasone suppression test
*ACTH levels
*CRH stimulation test
*Imaging tests
Presentation of excess cortisol
. 24-hour Urine Cortisol (or urine free cortisol)
sensitivity of 95-100%, specificity of 94-98% to detect cushing’s syndrome
Since cortisol levels vary greatly over the course of a day, a single cortisol result is of little value and other
ways of measuring overall cortisol production are used to evaluate overall cortisol production.
. Saliva or blood cortisol at 11 PM
. Single low dose dexamethasone suppression test
The overnight low dose test consists of dexamethasone (1mg) taken orally between 2300 and 2400
hours, and the measurement of fasting plasma cortisol between 0800 and 0900 hours the following
morning.
Patients with all types of Cushing’s syndrome will not show adequate suppression after a single low dose
of dexamethasone given at bedtime.
Differential Diagnosis of Cushing’s Syndrome
(cause: pituitary, adrenal, or other)
. ACTH levels
. High dose dexamethasone suppression test
Standard high dose DST: 2 mg 6-hourly for 8 doses. Plasma and/or urine cortisol are evaluated
before, during and after dexamethasone administration
High dose glucocorticoids partially suppress ACTH secretion from most corticotroph adenomas (8090%) whereas ectopic tumours are resistant to feedback inhibition.
. CRH Stimulation Test
CRH (corticotrophin releasing hormone) is injected and cortisol and ACTH are measured at baseline
(before CRH) and at 30 and 60 minutes. The normal response is a peak in ACTH levels at 30
minutes with cortisol peaking at 60 minutes. Most patients with Cushing’s syndrome caused by
adrenal tumors or ectopic ACTH secreting tumors do not respond to CRH.
. Imaging Tests
CT (computed tomography)
MRI (magnetic resonance imaging)
Ultrasound
Synthesis of cortisol
About 95% of cortisol is carried in the blood bound largely to
Corticosteroid Binding Globulin (CBG) or albumin. The
remainder occurs as physiologically active free cortisol.
Around 1 percent of plasma free cortisol is secreted unaltered in
the urine.
Urinary free cortisol levels reflect the biologically active
unbound hormone.
Urinary free cortisol normal values are 20 to 100ug per 24
hours.
Urinary metabolites of cortisol
hydroxycortisol
tetrahydrocortisol
tetrahydrocortisone
Routine methods for determination of urinary free cortisol
Immunoassy: . less expensive and easily available
. interfered with metabolites, precursor, other
steroids with similar structures and steroid drugs
. no internal standard to control extraction
process
HPLC:
. more specific, separate interfering substances
. internal standard to control extraction process
. extensive sample preparation
. long run time
Develop LC-MS/MS methods
Currently used in TML
Determination of urinary free cortisol by LC-MS/MS
Sample preparation
C18 SPE Column, 500mg
Wash with 3ml of methanol
3ml of H2O
Load samples
Wash with 3ml of acetone:H2O (1:4)
3ml of H2O
3ml of hexane
Elute with 3ml of diethyl ether
Dry and reconstitute in mobile phase
Journal of Chromatography, 426 (1988) 25-32.
LC MS/MS System
Applied Biosystems/MDS
Sciex API 3000™
LC/MS/MS System
Triple quadrupole mass
spectrometer
Triple Quadrupole mass spectrometry
Series of three quadrupoles can be used, this is known as Triple Quadrupole mass spectrometry. The
first and third quadrupoles are mass filters, and the middle one is a collision cell. This allows the study
of fragments, useful in structural studies.
For example, the first quadrupole may be set to "filter" for a drug ion of a known mass, which is
fragmented in the second quadrupole. The third quadrupole can then be set to scan the entire m/z
range, giving information on the sizes of the fragments made. Thus, the structure of the original ion
can be deduced.
Electrospray ionization (ESI)
quadrupole
The quadrupole consists of four parallel metal rods. Each opposing rod pair is connected together
electrically and a radio frequency voltage is applied between one pair of rods, and the other. A
direct current voltage is then superimposed on the R.F. voltage. Ions travel down the quadrupole
in between the rods. Only ions of a certain m/z will reach the detector for a given ratio of
voltages: other ions have unstable oscillations and will collide with the rods. This allows
selection of a particular ion, or scanning by varying the voltages
(1) Product ion scan. In this case, the
precursor ion is focussed in Q1 and
transferred into Q2 - the collision cell where it interacts with a collision gas and
fragments. The fragments are then measured
by scanning Q3. This results in the typical
MS/MS spectrum and is the method most
commonly employed with ESI ionisation
and/or LC-MS.
(2) Precursor ion scan. In this case Q3 is held
to measure the occurrence of a particular
fragment ion and Q1 is scanned. This results
is a spectrum of precursor ions that result in
that particular product ion - this is especially
useful when used with EI or CI ionisation
and/or GC-MS.
(3) Neutral loss scan. In this case Q1 is
scanned as in (2) but this time Q3 is also
scanned to produce a spectrum of precursor
ions that undergo a particular neutral loss.
Again this mode is especially useful for EI
and CI ionisation.
Standard cortisol + 11-deoxycortisol
C18, 98% methanol + 2% 5mM ammonium formate
Cortisol 363.2/121.0
11-deoxycortisol 347.3/97.1
Urine blank
C18, 98% methanol + 2% 5mM ammonium formate
Cortisol 363.2/121.0
11-deoxycortisol 347.3/97.1
Urine spiked with cortisol
C18, 98% methanol + 2% 5mM ammonium formate
Cortisol 363.2/121.0
11-deoxycortisol 347.3/97.1
Urine spiked with 11-deoxycortisol
C18, 98% methanol + 2% 5mM ammonium formate
Cortisol 363.2/121.0
11-deoxycortisol 347.3/97.1
Urine spiked with cortisol + 11-deoxycortisol
C18, 98% methanol + 2% 5mM ammonium formate
Cortisol 363.2/121.0
11-deoxycortisol 347.3/97.1
Urine blank
C18, 98% methanol + 2% 5mM ammonium formate
Cortisol 363.2/121.0
11-deoxycortisol 347.3/97.1
Urine blank
C8, 98% methanol + 2% 5mM ammonium formate
Cortisol 363.2/121.0
11-deoxycortisol 347.3/97.1
Urine blank, left over weekends in r.t.
C8, 98% methanol + 2% 5mM ammonium formate
Cortisol 363.2/121.0
11-deoxycortisol 347.3/97.1
Standard cortisol + 11-deoxycortisol
C8, 98% methanol + 2% 5mM ammonium formate
Cortisol 363.2/121.0
11-deoxycortisol 347.3/97.1
Urine spiked with 11-deoxycortisol
C8, 98% methanol + 2% 5mM ammonium formate
Cortisol 363.2/121.0
11-deoxycortisol 347.3/97.1
Urine spiked with cortisol
C8, 98% methanol + 2% 5mM ammonium formate
Cortisol 363.2/121.0
11-deoxycortisol 347.3/97.1
Urine spiked with cortisol+11-deoxycortisol
C8, 98% methanol + 2% 5mM ammonium formate
Cortisol 363.2/121.0
11-deoxycortisol 347.3/97.1
11-deoxycortisol is not a good internal standard in
some cases
. Patients treated with Metyrapone
Metyrapone is a potent inhibitor of the conversion of 11-deoxycortisol
(11-DOC) to cortisol and is used in the treatment of Cushing's
syndrome.
. Patients with high ratio of 11-deoxycortisol to cortisol (adrenocortical
carcinoma).
Work to be done:
D2-cortisol as internal standard?
Calibration curve?
Sensitivity?
Precision?
Interference?
Comparison with immunoassay?