Investigating the relationship between
serum 11-deoxycortisol levels and blood pressure
Helen Turner MSc Project
Do levels of 11-deoxycortisol affect blood pressure in a normal
population?
Objectives:•To optimise and validate a novel method for the measurement of serum 11-deoxycortisol.
•To establish the relationship between serum11-deoxycortisol and blood pressure in an adult population.
Hypertension• Without treatment
• increased risk of cardiovascular and renal complications
• Can be split into essential or secondary hypertension
• 95% of patients have essential hypertension• which can be attributed to both environmental and
genetic factors
• 5% have secondary hypertension • Where the cause is known, and if treated will alleviate
the symptoms
Steroids & Hypertension• Supportive evidence
– Abnormal steroid production can lead to hypertension-e.g in PA, CAH, Cushings & GSH
– Male presenting with hypertension, may have elevated glucocorticoid metabolites in their urine
– Alterations in the 11-deoxycortisol: cortisol ratio has been seen in hypertensive patients
Cholesterol
17-OH Pregnenolone DHEA
Aldosterone
18-OH Corticosterone
Corticosterone
Deoxycorticosterone
Progesterone
Pregnenolone
Androstenedione
Cortisol
17-OH Progesterone
11-Deoxycortisol
11ß-Hydroxylase
21ß-Hydroxylase
11-deoxycortisol and 11ß hydroxylase
• Mutations in CYB11B1 alter 11ß hydroxylase activity
• 11ß hydroxylase deficient patients have increased levels of 11-deoxycortisol
• Present with hypertension
Therefore…could slight changes in 11ß hydroxylase activity, cause slight variations in blood pressure?
Method• Extraction
– 400ul sample + 100ul assay buffer, add to 2.5mls CCL4
– Discard aqueous layer
• Drying down– 1ml extract dried under low pressure N2
@37ªC
• Reconstitution– With 200ul assay buffer, @RT for 30mins
• DELFIA– Dissociation enhanced lanthanide
fluoroimmunoassay
DELFIA Set up
Patient Sample
Plate Wall
Coating Antibody
Highly fluorescent Eu-(2-NTA)3(TOPO)2-3
Enhancement Solution
Anti 11-deoxycortisol Biotinylated 11-
deoxycortiol
Eu3+ labelled Avidin
Validation• Recovery 113%
• Cross reactivity 11% 17OHP
<0.1 other steroids
• Lower limit of Detection 0.9nmol/L
• Precision within assay 16%
between assay20%
• Comparison to RIA r=0.72 p=<0.0001
Materials• Patient samples
• From MRC Hertfordshire study• 271 patients: 140males and 131 females• Born between 1931 and 1939 in
Hertfordshire • No major medical conditions• Clinics between June 2003 and June 2004
• Blood Pressure• Recorded at clinics• Lying and seated positions
Results: Females
0
5
10
15
20
25
30
90 110 130 150 170 190
Sitting systolic BP (mm/Hg)
11-d
eoxy
cort
iso
l nm
ol/L
0
5
10
15
20
25
30
45 55 65 75 85
Sitting diastolic BP (mmHg)
11-d
eoxy
cort
iso
l nm
ol/L
r=-0.01, p=0.8837
r=0.01, p=0.9348
Results: Males
0
5
10
15
20
25
90 110 130 150 170
Sitting systolic BP (mmHg)
11-d
eoxy
cort
iso
l nm
ol/L
0
5
10
15
20
25
45 55 65 75 85 95
Sitting diastolic BP (mmHg)
11-d
eoxy
cort
iso
l nm
ol/L
r=-0.01, p=0.2280
r=0.01, p=0.8949
Conclusions• We produced a simple method for
measuring 11-deoxycortisol in serum
• There is no relationship between 11-deoxycortisol and blood pressure in a normal adult population
Future Work
• Measure cortisol• To compare 11deoxycortisol in the
hypertensive patient to normal patients
• Measure 11-deoxycortisol in a greater age range
Acknowledgements• Pete Wood• Christine Glenn• MRC- Southampton• Karen Jameson• Martyn Egerton (Epsom and St
Helier Hosptial)
Cross ReactivitySteroid Cross reactivity
(%)
Cortisone 0.37
Progesterone 0.6
Prednisone 0.39
21-deoxycortisol <0.1
Aldosterone <0.1
Adione <0.1
Cortisol 0.6
17 OHP 14%
Precision Low QC Medium
QCHigh QC
Mean (nmol/L)
2.4 12.9 56.5
SD (nmol/L)
0.5 2.1 7.5
%CV 20.4 16.3 13.2
Low QC Medium QC
High QC
Mean (nmol/L)
4.83 32.85 56.44
SD (nmol/L)
1.36 4.70 9.31
%CV 31.0 14.31 16.50
Intra Assay Variation
Inter Assay Variation
HypertensionStage Systolic BP
(mmHg)Diastolic BP (mmHg)
Normal <120 <80
Pre-hypertensive
120-139 80-89
Stage 1 140-159 90-99
Stage 2 160> 100>
6th report of the Joint National Committee on the Prevention, Detection, Evaluation and treatment of Blood Pressure
Steroids & Hypertension 2• Non- supportive evidence:
– No significant correlation between cortisol levels in plasma and the degree of hypertension
– Deoxycorticosterone levels shows no correlation with blood pressure
– The metabolites of deoxycorticosterone, are potent mineralocorticoids but require 11-hydroxylase for their production
Lower Limit of Detection• 0.9nmol/L
Recovery• Mean recovery 113%