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%