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 • Primary hyperaldosteronism and 18-hydroxycortisol

Brief excursion 1

In – house DELFIA assays

Assay Principle

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

Antibody Biotinylation

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

In house “DELFIA” Salivary cortisol assay

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

Brief excursion 2 Cushing’s and salivary cortisols

Problems with Screening for Cushing’s Syndrome

• Urine collections unreliable • Some cases suppress with low-dose dexamethasone • Cyclical Cushing’s

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.

Cyclical Cushing’s

• Definition - 3 peaks and two troughs • Cycle lengths 12 hours to 85 days

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

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

Patient (EO)

19 consec. days

60 50 10 0 40 30 20 0900

clock time

2200

• Test Meal • No meal • 1ug CRH • 1ug Synacthen

75 year-old lady (3)

-

Plasma cortisol

Basal Peak 197 704 278 414 647

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 ?

GIP dependent Cushing’s

• GIP =

G

astric

I

nhibitory

P

eptide - now

G

lucose-dependent

I

nsulinotrphic

P

olypeptide • 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

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

Yaneva M

et al

JCEM

89

3345-3351 2004

Screening for Cushing’s syndrome

Sens/Spec (%) LDDST Combined evening saliva Raff

et al

1998 (N = 39) Castro

et al

1999 (N = 63) 92/95 93/93 (N = 33) Martinelli

et al

1999 100/95.2

(N = 11) Yaneva

et al

2004 100/96 91.4/94.4

100/95.2

100/93.3

100/100 -

Findling and Raff JCEM

91

(10) 3746-53 2006

Brief Excursion 3 Primary hyperaldosteronism / 18-hydroxycortisol

Primary Hyperaldosteronism(“PAL”)

• Adrenal adenoma ( Conn 1955 ) • Familial hyperaldosteronism Type II • As part of MEN1 • • Adrenocortical carcinoma ( v. rare) • Bilateral adrenal hyperplasia

G

lucocorticoid-

S

uppressible

H

yperaldosteronism (“GSH”) (Familial Hyperaldosteronism Type 1)

Primary Hyperaldosteronism • 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?

Prevalence of primary hyperaldosteronism Stowasser and Gordon (Trends in Endocrinol.Metab. 14 (7)310-317 2003) Patients diagnosed/year % hypokalaemic Conn’s adenoma Hyperplasia Pre- A/R ratios 1971-91 N=136 6.2

67% 78% 22% Post 1992-99 N=592 74.0

20% 31.6% 68%

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

Drug therapy and A/R ratios

b blockers ACE inhibitors AT receptor antag Ca Channel blockers Diuretics Spironolactone

A/R ratio

 (+62%)  (-30%)  (-43%  (-17% (  ) false +ve’s OK false ve’s BAH (Seifarth et al, Clin.Endocrinol. 57 457-465 2002 Mulatero et al, Hypertension 40 897-902 2002)

Confirmatory Tests

• Oral salt loading • Saline infusion • Fludrocortisone suppression test

Differentiating the subtypes

Questions: • Does the patient have GSH / FH-1 ?

• If GSH excluded, is autonomous aldo production unilateral or bilateral ?

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

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

The pathways of biosynthesis of aldosterone and cortisol in the adrenals Zona glomerulosa Zona fascicu lata 17

a

-H ydr oxylase (CYP17) 17,20-Lyase Cholesterol desmolase (CYP11A) 3

b

-Hydroxysteroid deh ydr ogenase 21-H yd roxylase (CYP21) ALDO-SYNTHASE 11

b

-Hydroxylase (CYP11B2) 18-H yd roxylase (CYP11B2) 18-O xidase (CYP11B2) CHOL ESTEROL PRE GNENOLONE PRO GEST ERONE 17-O H PREGNENOLO NE 17-O H PROGES TERONE DEOXYCORTICOS TERONE 11-DEO XYCORTISOL CORTICO STERONE 18-O H CORTICOSTE RO NE CORTISOL ALDOSTE RO NE DHEA ANDROST ENEDIO NE 11

b

-Hydroxylase (CYP11B1)

The pathways of biosynthesis of aldosterone and cortisol in the adrenals Adrenal Adenoma 17

a

-H ydr oxylase (CYP17) 17,20-Lyase Cholesterol desmolase (CYP11A) 3

b

-Hydroxysteroid deh ydr ogenase 21-H yd roxylase (CYP21) ALDO-SYNTHASE 11

b

-Hydroxylase (CYP11B2) 18-H yd roxylase (CYP11B2) 18-O xidase (CYP11B2) CHOL ESTEROL PRE GNENOLONE PRO GEST ERONE DEOXYCORTICOS TERONE 11-DEO XYCORTISOL CORTICO STERONE 18-O H CORTICOSTE RO NE 17-O H PREGNENOLO NE 17-O H PROGES TERONE CORTISOL 18-H YDROXYCO RT IS OL ALDOSTE RO NE 18-O XO CO RT IS OL DHEA ANDROST ENEDIO NE 11

b

-Hydroxylase (CYP11B1)

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

5’ 5’

Glucocorticoid Remediable Aldosteronism

5’ ALDO-S 1 1-OHa se ALDO-S une qual crossing ov er during meiosis ALDO-S Chi maeric G ene 1 1-OHa se 1 1-OHa se 3’ 3’ 3’

24 hr Urine 18-hydroxy cortisol nmol/day 5000 4000 3000 2000 1000 0

Southampton FIA

Conn’s adenoma BAH GSH EH

Edinburgh RIA

24 hr urine 18-hydroxy cortisol (nmol/day) 12000 10000 8000 6000 4000 2000 0 Conn’s adenoma BAH GSH EH