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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

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investigating the relationship between serum 11 deoxycortisol levels and blood pressure

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
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 serum
  • 11-deoxycortisol and blood pressure in an
  • adult population.
hypertension
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
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
slide5

Cholesterol

Pregnenolone

17-OH Pregnenolone

DHEA

Progesterone

17-OH Progesterone

Androstenedione

Deoxycorticosterone

11-Deoxycortisol

Corticosterone

Cortisol

18-OH Corticosterone

21ß-Hydroxylase

Aldosterone

11ß-Hydroxylase

11 deoxycortisol and 11 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
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

Plate Wall

Coating Antibody

Patient Sample

Highly fluorescent Eu-(2-NTA)3(TOPO)2-3

Enhancement Solution

Eu3+ labelled Avidin

Anti 11-deoxycortisol

Biotinylated 11-deoxycortiol

DELFIA Set up
validation
Validation
  • Recovery 113%
  • Cross reactivity 11% 17OHP

<0.1 other steroids

  • Lower limit of Detection 0.9nmol/L
  • Precision within assay 16%

between assay 20%

  • Comparison to RIA r=0.72 p=<0.0001
materials
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
Results: Females

r=0.01, p=0.9348

r=-0.01, p=0.8837

results males
Results: Males

r=0.01, p=0.8949

r=-0.01, p=0.2280

conclusions
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
Future Work
  • Measure cortisol
  • To compare 11deoxycortisol in the hypertensive patient to normal patients
  • Measure 11-deoxycortisol in a greater age range
acknowledgements
Acknowledgements
  • Pete Wood
  • Christine Glenn
  • MRC- Southampton
  • Karen Jameson
  • Martyn Egerton (Epsom and St Helier Hosptial)
precision

Low QC

Low QC

Medium QC

Medium QC

High QC

High QC

Mean (nmol/L)

Mean (nmol/L)

4.83

2.4

32.85

12.9

56.44

56.5

SD (nmol/L)

SD (nmol/L)

1.36

0.5

4.70

2.1

9.31

7.5

%CV

%CV

31.0

20.4

14.31

16.3

16.50

13.2

Precision

Intra Assay

Variation

Inter Assay

Variation

hypertension1
Hypertension

6th report of the Joint National Committee on the Prevention,

Detection, Evaluation and treatment of Blood Pressure

steroids hypertension 2
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
slide21
Lower Limit of Detection
  • 0.9nmol/L

Recovery

  • Mean recovery 113%