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Ramblings in Endocrine Biochemistry

Join Pete Wood on a journey into endocrine biochemistry, covering topics like iodine deficiency, Cushing’s syndrome, and primary hyperaldosteronism. Explore in-house DELFIA assays and diagnostic salivary hormone testing.

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Ramblings in Endocrine Biochemistry

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  1. Ramblings in Endocrine Biochemistry Pete Wood

  2. To “ramble” • “to go as fancy leads” • “to wander in mind or discourse” • “to be desultory, incoherent or delerious” • “an irregular excursion” (Chambers)

  3. Brief excursions: • Life after Iodine – 125 • Cushing’s and salivary cortisol • Primary hyperaldosteronism and 18-hydroxycortisol

  4. Brief excursion 1 In – house DELFIA assays

  5. Assay Principle • Biotin-labelled detector antibody for immunometric assays • Biotin –labelled steroid for competitive immunoassays • Detect with Europium – labelled streptavidin

  6. Antibody Biotinylation • 1 mg Ab onto PD10 desalting column • Elute with bicarb. Buffer • Add biotinamidocaproyl-NHS ester • 4o C overnight • PD10 desalting column - elute with Tris-HCl • Add purified BSA stabiliser

  7. In-house “DELFIA” Salivary cortisol assay

  8. In-house DELFIA assays • Steroids Salivary cortisol *** Salivary cortisone Salivary 17 OHP Plasma 17 OHP Plasma and urine 18-hydroxycortisol*** • Peptides Intact insulin ( 2 assays) total proinsulin (for 32,33 split proinsulin) intact proinsulin

  9. Brief excursion 2 Cushing’s and salivary cortisols

  10. Problems with Screening for Cushing’s Syndrome • Urine collections unreliable • Some cases suppress with low-dose dexamethasone • Cyclical Cushing’s

  11. Cyclical Cushing’s • First described in 1971 • 1985 Atkinson and co-workers : • sequential urine samples collected in 9/14 successive patients with Cushing’s syndrome • 5/9 had evidence of cyclical Cushing’s • 2 patients showed considerable variation without a cyclical pattern being established.

  12. Cyclical Cushing’s • Definition - 3 peaks and two troughs • Cycle lengths 12 hours to 85 days

  13. 75 year-old lady • 12 kg weight loss and malaise • NIDDM diagnosed 18 months previously • Abdo CT - bilat. adrenal masses • Responded to empirical treatment with dexameth. • CT guided biopsy - adrenal hyperplasia • Centripetal weight distribution, prox. myopathy • No suppression of pl. cortisol to low and high dose dex ( 48 hrs each) • ACTH low but measurable (8-11 ng/L) • IPS/ CRH - 13/1 central/periph. gradient in ACTH • Transsphenoidal surgery - no adenoma identified

  14. 75 year-old lady (2) • High plasma cortisols persisted , but variable • 9 am and 10 pm salivary cortisols collected over a total of 19 days • 3 hour urines collected over 24 hours on three occasions

  15. 19 consec. days

  16. 75 year-old lady (3) Plasma cortisol Basal Peak • Test Meal 197 704 • No meal - 278 • 1ug CRH - 414 • 1ug Synacthen - 647

  17. 75 year-old lady (4) • Bilateral adrenalectomy • Histology - bilateral macronodular hyperplasia (”AIMAH”) • Why the false positive IPS/ CRH study ? • Why the initial features of hypoadrenalism ?

  18. GIP - dependent Cushing’s • GIP = Gastric Inhibitory Peptide - now Glucose-dependent Insulinotrphic Polypeptide • Expression of GIP receptors in the adrenal • To date, identified in 17 patients with AIMAH, and 7 patients with unilateral adenoma • not found in the adrenal cortex of normal subjects, fetuses, or in other forms of Cushing’s

  19. Criteria - routine diagnostic salivary hormone assays • Constant and predictable correlation must exist between salivary and serum hormone concentrations • Diagnostic value at least equal to serum hormone determinations • Single saliva samples as informative as single serum samples

  20. Yaneva M et al JCEM 89 3345-3351 2004

  21. Screening for Cushing’s syndrome Sens/Spec (%) eveningLDDSTCombined saliva Raff et al 1998 92/95 - - (N = 39) Castro et al 1999 93/93 91.4/94.4 100/93.3 (N = 33) Martinelli et al 1999 100/95.2 100/95.2 100/100 (N = 11) Yaneva et al 2004 100/96 - - (N = 63)

  22. Findling and Raff JCEM 91(10) 3746-53 2006

  23. Brief Excursion 3 Primary hyperaldosteronism / 18-hydroxycortisol

  24. Primary Hyperaldosteronism(“PAL”) • Adrenal adenoma ( Conn 1955 ) • Familial hyperaldosteronism Type II • As part of MEN1 • Adrenocortical carcinoma ( v. rare) • Bilateral adrenal hyperplasia • Glucocorticoid-Suppressible Hyperaldosteronism (“GSH”) (Familial Hyperaldosteronism Type 1)

  25. PrimaryHyperaldosteronism • Conventionally thought to be less than 1% of patients with hypertension. • Recently, prevalence of 10% or more has been reported in hypertensives • Not all patients are hypokalaemic • Are we missing cases?

  26. Prevalence of primary hyperaldosteronism Stowasser and Gordon (Trends in Endocrinol.Metab. 14 (7)310-317 2003) Pre- A/R ratios Post 1971-91 1992-99 N=136 N=592 Patients diagnosed/year 6.2 74.0 % hypokalaemic 67% 20% Conn’s adenoma 78% 31.6% Hyperplasia 22% 68%

  27. Screening Aldosterone/ renin ratio ( “ARR”) • Morning ambulant sample • Do while on drugs and assess result in light of known drug effects • Ratio > 25 ng/mU with aldo >150 suggests primary hyperaldosteronism

  28. Drug therapy and A/R ratios A/R ratio b-blockers (+62%) false +ve’s ACE inhibitors (-30%) AT receptor antag (-43% Ca Channel blockers (-17% Diuretics - OK Spironolactone () false -ve’s BAH (Seifarth et al, Clin.Endocrinol. 57 457-465 2002 Mulatero et al, Hypertension 40 897-902 2002)

  29. Confirmatory Tests • Oral salt loading • Saline infusion • Fludrocortisone suppression test

  30. Differentiating the subtypes Questions: • Does the patient have GSH / FH-1 ? • If GSH excluded, is autonomous aldo production unilateral or bilateral ?

  31. Unilateral vs bilateral • Adrenal CT unreliable - fails to detect >50% of adenomas • Posture response – limited value 50 % of adenomas may be posture-responsive • 18- hydroxycortisol may also be normal in some posture –responsive adenomas • Adrenal venous sampling best

  32. 18 - Hydroxy Cortisol ; A Hybrid Steroid  in Conn’s adenoma and GSH, normal in BAH

  33. Adenoma cell types • Aldo producing adenoma cell types can resemble ZG, ZF or ZR or hybrid ZF/ZG • One type of cell seems to predominate • If 80% non-ZF cells (ZG or hybrid), adenoma posture/ Angio II responsive • If 50-100 % ZF cells, then adenoma is posture/ Angio II unresponsive

  34. Southampton FIA 24 hr Urine 18-hydroxy cortisol nmol/day Conn’s adenoma BAH GSH EH

  35. Edinburgh RIA 24 hr urine 18-hydroxy cortisol (nmol/day) Conn’s adenoma BAH GSH EH

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