r.j. singh, ph.d. mayo clinic
TRANSCRIPT
Definition Diagnosis or Di`ag`nos´tics
• Identification of a disease, disorder, or syndrome through a method of consistent and accurate analysis.
11-Deoxycorticosterone Testosterone11-deoxycortisol
Aldosterone
CorticosteroneEstradiol
Cortisol
Cholesterol
Pregnenolone DHEA17- OH Pregnenolone
3βSDH- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
21 OH'ase- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - --
11 OH'ase- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - --
Biosynthesis of Steroids
18 OH'ase- - - - - - - - - - - - - - - - - - - - - -- - -
Progesterone 17-OH Progesterone Androstenedione17
OH
’ase
17,2
0 de
smol
ase
17b SDH
Aromatase
Estrone
Aromatase
17b SDH
Cortisone
Cushing’s SyndromeIntroduction
ACTH-Dependent Cushing’s Syndrome
ACTH-Independent Cushing’s Syndrome
Cushing’s DiseaseEctopic ACTH Syndrome
Adrenal adenomaAdrenal carcinoma
Pituitary
AdrenalGland
ACTH+
Cortisol-
Cortisol
Hypothalamus
CRH
http://www.pathology.vcu.edu/education/endocrine/endocrine/adrenal/micro/adrad1x.gif
1998
2006
Obesity Trends* Among U.S. AdultsBRFSS, 1990, 1998, 2006
(*BMI ≥30, or about 30 lbs. overweight for 5’4” person)
1990
No Data <10% 10%–14% 15%–19% 20%–24% 25%–29% ≥30%
21-Deoxycorticosterone Testosterone11-deoxycortisol
Aldosterone
CorticosteroneEstradiol
Cortisol
Cholesterol
Pregnenolone DHEA
17- OH Pregnenolone
3βSDH- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
21 OH'ase- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - --
11 OH'ase- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - --
Biosynthesis of Steroids
18 OH'ase- - - - - - - - - - - - - - - - - - - - - -- - -
Progesterone 17-OH Progesterone Androstenedione17
OH
’ase
17,2
0 de
smol
ase
17b SDH
Aromatase
Estrone
Aromatase
17b SDH
Cortisone
Lab Tests
• 24 Urine Free Cortisol• Plasma Cortisol• Plasma Free Cortisol• Midnight Plasma Cortisol• Midnight Salivary Cortisol
HO OH
OH
O
O
H
H
H
Cortisol
HO OH
OH
HO
O
H
H
H
5α-Tetrahydrocortisol
HO OH
OH
O
HO
H
H
H
20β-Dihydrocortisol
HO OH
OH
O
O
H
H
H
6β-Hydroxycortisol
HO OH
O
O
H
H
H
OH
OH
O
O
H
H
H
11-Deoxycortisol
O OH
OH
O
O
H
H
H
Cortisone
OH
H
O OH
OH
O
HO
H
H
H
20β-Dihydrocortisone
21-Deoxycortisol
O OH
OH
HO
O
H
H
H
5β-TetrahydrocortisoneH
O OH
OH
HO
O
H
H
H
5α-TetrahydrocortisoneH
HO OH
OH
HO
O
H
H
H
5β-TetrahydrocortisolH
HO OH
OH
HO
HO
H
H
H
20β-α-CortolH
HO OH
OH
HO
HO
H
H
H
α-CortolH
O OH
OH
HO
HO
H
H
H
20β-α-CortoloneH
O OH
OH
HO
HO
H
H
H
α-CortoloneH
•1935, 17-ketosteroids by Zimmermann
•1952, paper chromatography, and later column chromatography
•in mid-eighties, 50 steroid metabolites ('profile') in urine (GC-FID)
•radioimmuno-assays (RIA) were introduced for plasma steroids
•at low concentrations cross reactions and interferences recognized
•Nineties, isotope dilution/GC-MS produced highly accurate values for steroids and were considered as gold standard method
Clinical Steroidology-Coming of ageTeller W.M. Hormone Research Volume 50, Issue 1 1998
Immunoassays - central to current lab testing
• Immunoassays allow sensitive detection of a wide range of analytes
• Immunoassays can be very specific• Immunoassays come in a wide range of formats
and can be automated• Today’s reference ranges for a wide range of
analytes have been established with Immunoassays
• Immunoassay problems and limitations are well understood
Aldosterone ND* 11β-hydroxyprogesterone 0.50 Allotetrahydrocortisol 4.60 17α- hydroxyprogesterone 0.50 Androstenedione ND 17β-hydroxypregnanolone ND Corticosterone 2.80 11-keto-androsterone ND Cortisone 7.40 11-beta-etiocholanolone ND α-Cortol ND Prenanetriol ND α-cortolone ND Prenenolone ND β-Cortol ND Progesterone ND β-Cortolone ND Spironolactone ND Dehydrocorticosterone 0.50 Testosterone ND 11-deoxycorticosterone 0.40 Tetrahydrocortisol ND 11-deoxycortisol 7.30 Tetrahydrocortisone 0.54 21-deoxycortisol 4.50 Tetrahydro-11-
deoxycortisol 0.20
20α-dihydrocortisol 0.50 20β-dihydrocortisol 0.70 20α-dihydrocortisone 0.40 20β-dihydrocortisone ND 11β-hydroxyandrosterone ND 6β-hydroxycortisol 2.40 11β-hydroxyetiocholanolone
ND
Synthetic Steroids Prednisolone (100 μg/dl) 27.00 6-methyl-prednisolone 20.90 Dexamethasone 0.20 Prednisone 6.60 Canrenone ND
Steroid interference in Immunoassay
http://www.mpbio.com/includes/technical/Corti-cote%20RIA%20-%2006B256440.PDF
Immunoassays for Cortisol
HPLC-UV
Normal Cortisol 5-55 µg/24 hrsCortisone 16-128 µg/24 hrs
Immunoassay
Cortisol 24-108 µg/24 hrs
Methods for Urine Cortisol Test
LC-MS
HPLC-UV
Normal Cortisol 5-55 µg/24 hrsCortisone 16-128 µg/24 hrs
Immunoassay
Cortisol 24-108 µg/24 hrs
Methods for Urine Cortisol Test
LC-MS
HPLC-UV
Normal Cortisol 5-55 µg/24 hrsCortisone 16-128 µg/24 hrs
Immunoassay
Cortisol 24-108 µg/24 hrs
Methods for Urine Cortisol Test
0.0 0.4 0.8 1.2 1.6 2.0
Cortisone
Cortisol
d4-Cortisol
Inte
nsity
cps
Cortisol and Cortisone BY LC MS/MS
Time minutes
y = 0.3555x + 3.9311R2 = 0.5441
01020
304050
0 20 40 60 80
Comparison of immunoassay vs. MS/MSC
ortis
ol µ
g/24
hr
by T
ande
m M
S
µg/24 hr Cortisol ACS 180
Sample Cortisol Concentration ug/24hr Steroid ConcentratID Immunoassay LC-UV LC-MS/MS Present ug/24hr
2 1 38.1 1.3 <1.0 Prednisone/Prednisolone 130
Adrenal Suppression with Synth. Steroids
Sample Cortisol Concentration ug/24hr Steroid ConcentratID Immunoassay LC-UV LC-MS/MS Present ug/24hr
2 1 38.1 1.3 <1.0 Prednisone/Prednisolone 130
11 2 1554 2237 10.1 Methylprednisolone 18875
SC6 3 1.8 <1.0 3.5 Dexamethasone 1404
SC7 4 2.8 <1.0 <1.0 Fludrocortisone 5
SC19 5 <6.0 <1.0 <1.0 Betamethasone 29
SC21 6 <7.5 <1.0 1.3 Dexamethasone 36
SC22 7 252 <1.0 <1.0 Prednisone/Prednisolone 776
Adrenal Suppression with Synth. Steroids
Iatrogenic Cushing’s SyndromeLiterature Reports
Kumar, S, Singh, RJ, Reed, AM, Lteif, AN. Pediatrics 113 (2004) 1820-1824
Iatrogenic Cushing’s SyndromeMechanism
Pituitary
AdrenalGland
ACTH+
SyntheticGlucocorticoid
Cortisol
Hypothalamus
CRH
How to confirm a diagnosis of iatrogenic Cushing’s Syndrome?
Measure level of synthetic steroids in plasma and/or urine
Cortisol
Synthetic SteroidsLaboratory Testing
HPLC Chromatogram
Taylor, RL, Grebe, SK, Singh, RJ. Clin. Chem. 50 (2004) 1-8
21-Deoxycorticosterone Testosterone11-deoxycortisol
Aldosterone
CorticosteroneEstradiol
Cortisol
Cholesterol
Pregnenolone DHEA
17- OH Pregnenolone
3βSDH- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
21 OH'ase- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - --
11 OH'ase- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - --
Biosynthesis of Steroids
18 OH'ase- - - - - - - - - - - - - - - - - - - - - -- - -
Progesterone 17-OH Progesterone Androstenedione17
OH
’ase
17,2
0 de
smol
ase
17b SDH
Aromatase
Estrone
Aromatase
17b SDH
Cortisone
History of Vit-D Testing in the Lab
• The HPLC assay in 1981– 10-50 samples/day
• Conversion to Diasorin RIA 3/20/2000– 50-150 samples/day
• Conversion to MS/MS 11/2/2004– 400 samples/day
• 3000 samples/day
25-OH Vit D3-d6internal
standard
25-OH Vitamin D3
25-OH Vitamin D2
Time (min)
Peak
Inte
nsity
LC-MS/MS VITAMIN D
21-Deoxycorticosterone Testosterone11-deoxycortisol
Aldosterone
CorticosteroneEstradiol
Cortisol
Cholesterol
Pregnenolone DHEA
17- OH Pregnenolone
3βSDH- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
21 OH'ase- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - --
11 OH'ase- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - --
Biosynthesis of Steroids
18 OH'ase- - - - - - - - - - - - - - - - - - - - - -- - -
Progesterone 17-OH Progesterone Androstenedione17
OH
’ase
17,2
0 de
smol
ase
17b SDH
Aromatase
Estrone
Aromatase
17b SDH
Cortisone