Ramblings in Endocrine Biochemistry

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Ramblings in Endocrine Biochemistry

• Pete Wood

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To ramble

• to go as fancy leads
• to wander in mind or discourse
• to be desultory, incoherent or delerious
• an irregular excursion
• (Chambers)

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Brief excursions

• Life after Iodine 125
• Cushings and salivary cortisol
• Primary hyperaldosteronism
• and 18-hydroxycortisol

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Brief excursion 1

• In house DELFIA assays

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Assay Principle

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

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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

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In-house DELFIA Salivary cortisol assay
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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

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Brief excursion 2

• Cushings and salivary cortisols

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Problems with Screening for Cushings Syndrome

• Urine collections unreliable
• Some cases suppress with low-dose dexamethasone
• Cyclical Cushings

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Cyclical Cushings

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

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Cyclical Cushings

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

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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

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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

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19 consec. days
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75 year-old lady (3)

• Plasma cortisol
• Basal Peak
• Test Meal 197 704
• No meal - 278
• 1ug CRH - 414
• 1ug Synacthen - 647

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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 ?

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GIP - dependent Cushings

• 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 Cushings

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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

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Yaneva M et al JCEM 89 3345-3351 2004
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Screening for Cushings syndrome

• Sens/Spec ()
• evening LDDST Combined
• 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)

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Findling and Raff JCEM 91(10) 3746-53 2006
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Brief Excursion 3

• Primary hyperaldosteronism
• / 18-hydroxycortisol

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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)

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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?

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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
• N136 N592
• Patients diagnosed/year 6.2 74.0
• hypokalaemic 67 20
• Conns adenoma 78 31.6
• Hyperplasia 22 68

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Screening

• Aldosterone/ renin ratio ( ARR)
• Morning ambulant sample
• Do while on drugs and assess result in light of
  known drug effects
• Ratio gt 25 ng/mU with aldo gt150 suggests primary
  hyperaldosteronism

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Drug therapy and A/R ratios

• A/R ratio
• b-blockers ?(62) false ves
• ACE inhibitors ?(-30)
• AT receptor antag ?(-43
• Ca Channel blockers ?(-17
• Diuretics - OK
• Spironolactone (?) false -ves BAH
• (Seifarth et al, Clin.Endocrinol. 57 457-465 2002
• Mulatero et al, Hypertension 40 897-902 2002)

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Confirmatory Tests

• Oral salt loading
• Saline infusion
• Fludrocortisone suppression test

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Differentiating the subtypes

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

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Unilateral vs bilateral

• Adrenal CT unreliable
• - fails to detect gt50 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

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18 - Hydroxy Cortisol A Hybrid Steroid
? in Conns adenoma and GSH, normal in BAH
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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

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Southampton FIA
24 hr Urine 18-hydroxy cortisol nmol/day
Conns adenoma
BAH
GSH
EH
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Edinburgh RIA
24 hr urine 18-hydroxy cortisol (nmol/day)
Conns adenoma
BAH
GSH
EH