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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

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Investigating the relationship between serum 11-deoxycortisol levels and blood pressure

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  1. Investigating the relationship between serum 11-deoxycortisol levels and blood pressure Helen Turner MSc Project

  2. 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.

  3. 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

  4. 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

  5. Cholesterol Pregnenolone 17-OH Pregnenolone DHEA Progesterone 17-OH Progesterone Androstenedione Deoxycorticosterone 11-Deoxycortisol Corticosterone Cortisol 18-OH Corticosterone 21ß-Hydroxylase Aldosterone 11ß-Hydroxylase

  6. 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?

  7. 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

  8. 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

  9. 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

  10. 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

  11. Results: Females r=0.01, p=0.9348 r=-0.01, p=0.8837

  12. Results: Males r=0.01, p=0.8949 r=-0.01, p=0.2280

  13. 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

  14. Future Work • Measure cortisol • To compare 11deoxycortisol in the hypertensive patient to normal patients • Measure 11-deoxycortisol in a greater age range

  15. Acknowledgements • Pete Wood • Christine Glenn • MRC- Southampton • Karen Jameson • Martyn Egerton (Epsom and St Helier Hosptial)

  16. Cross Reactivity

  17. 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

  18. Hypertension 6th report of the Joint National Committee on the Prevention, Detection, Evaluation and treatment of Blood Pressure

  19. 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

  20. Lower Limit of Detection • 0.9nmol/L Recovery • Mean recovery 113%