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Ramblings in Endocrine Biochemistry

Ramblings in Endocrine Biochemistry. Pete Wood. To “ramble”. “to go as fancy leads” “to wander in mind or discourse” “to be desultory, incoherent or delerious” “an irregular excursion” (Chambers). Brief excursions:. Life after Iodine – 125 Cushing’s and salivary cortisol

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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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