measuring urinary free cortisol

Measuring Urinary Free Cortisol

Release of CortisolRelease of Cortisol

The hypothalamus

Pituitary Gland

Adrenal Cortex Cortisol

Increase Blood Glucose

Catabolism of proteins, fat.

Adrenocorticotropic Hormone (ACTH)

Suppress Immune

Response

Stress

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-

Corticotrophin Releasing Hormone (CRH)

Twenty-four-hour plasma cortisol profile

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)

Detection of Cushing’s Syndrome

Differential Diagnosis of Cushing’s Syndrome (cause: pituitary, adrenal, or other)

*High dose dexamethasone suppression test*ACTH levels

*CRH stimulation test*Imaging tests

Laboratory Tests

Presentation of excess cortisol*24-hour urine test

*Single low dose dexamethasone suppression test*Saliva or blood cortisol at 11 PM

. 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 testThe 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.

Presentation of excess cortisol

Differential Diagnosis of Cushing’s Syndrome (cause: pituitary, adrenal, or other)

. ACTH levels

. High dose dexamethasone suppression testStandard high dose DST: 2 mg 6-hourly for 8 doses. Plasma and/or urine cortisol are evaluated before, during and after dexamethasone administrationHigh dose glucocorticoids partially suppress ACTH secretion from most corticotroph adenomas (80-90%) whereas ectopic tumours are resistant to feedback inhibition.

. CRH Stimulation TestCRH (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.

hydroxycortisol

tetrahydrocortisol tetrahydrocortisone

Urinary metabolites of cortisol

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

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

Sample preparation

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

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.

Triple Quadrupole mass spectrometry

Electrospray ionization (ESI)

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

quadrupole

(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.

Cortisol 363.2/121.0

Standard cortisol + 11-deoxycortisol

11-deoxycortisol 347.3/97.1

C18, 98% methanol + 2% 5mM ammonium formate


Urine blank




Urine spiked with cortisol




Urine spiked with 11-deoxycortisol




Urine spiked with cortisol + 11-deoxycortisol




Urine blank




Urine blank, left over weekends in r.t.




Standard cortisol + 11-deoxycortisol




Urine spiked with 11-deoxycortisol




Urine spiked with cortisol




Urine spiked with cortisol+11-deoxycortisol



11-deoxycortisol is not a good internal standard in some cases

. Patients treated with MetyraponeMetyrapone 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?

measuring urinary free cortisol

Documents

blood cortisol

cortisol peaking

high levels of cortisol

hour urine cortisol

active free cortisol

urinary free cortisol

single cortisol result

overall cortisol production