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ENDOCRINOLOGICAL
ANALYSES
Kristina Hotakainen
Clinical Lecturer
Department of Clinical Chemistry
University of Helsinki
Endocrinology
Investigates the disorders of hormone secretion or
function
ENDOCRINE ORGANS
• Hypothalamus
• Pituitary gland
• Reproductive glands
– Testis
– Ovary
• Adrenal gland
• Thyroid gland
• Parathyroid glands
• Pancreas
• Gastrointestinal tract
• Kidney
• Bone
Hormones
AMINOACIDS
PEPTIDES
PROTEINS
LIPIDS
TRANSPORTATION OF
HORMONES IN BLOOD STREAM
Water-soluble hormones as free
molecules
Lipid-soluble thyroid hormones and steroid
hormones partially as free molecules and
partially as bound to carrier proteins
Albumin
Specific carrier proteins
Only the free fraction biologically active
PATHOPHYSIOLOGY OF
ENDOCRINE DISEASES
1. Disturbance in hormone production
The most common cause of endocrine diseases
– Reduced hormone production: lack of endocrine organ,
disturbance in hormone biosynthesis, secondary disruption of
endocrine tissue
– Excessive production: hyperplasia or tumor of the endocrine
organ, over stimulation of the endocrine organ (for example
autoimmune disease)
–
2. Altered hormone response
Abnormal receptor function
– Alteration in post-receptor signaling
– Rare causes of endocrine diseases
– Hormone resistance, constitutively active receptors
–
3. Tumors of endocrine glands
– hormone
overproduction
HORMONE MEASUREMENTS
• basal hormone levels
• evoked or suppressed hormone levels
• hormone-binding plasma proteins
Methods
• Immunochemical methods
• Gas and liquid chromatography
• Mass spectrometry
• (biological methods)
IMMUNOCHEMICAL METHODS
• antigen + antibody
• Various methods, labels, and detection methods
• Widely used in clinical chemistry analysis
• Most hormone tests are immunological
assays
– Laboratory testing
– Bed-side testing and self-testing
IMMUNOCHEMICAL METHODS
advantages:
• specific, sensitive, easily automated
disadvantages:
• cross-reactions, different variants of the hormone,
heterophilic antibodies, reference values methodspecific
CHROMATOGRAPHIC METHODS
• Physical separation
– Mobile phase gas: gas chromatography
– Mobile phase liquid: liquid chromatography
(HPLC, high performance liquid chromatography)
• bonded solid-phase column interacts with the analytes as they flow
past in a liquid solvent (mobile phase)
• The mobile and stationary phases are selected to optimize adherence
of the analytes to the stationary phase.
• adhered molecules can be eluted differentially from the solid phase
after washing to separate specific forms of the analyte from
interfering substances
• The efficiency of separation in a chromatography system is a function
of the flow rates of the different substances
CHROMATOGRAPHIC METHODS
Advantages:
• can be used to simultaneously measure multiple forms of
an analyte
• are not dependent on unique immunologic reagents
Disadvantages:
• complexity
• limited availability
MASS SPECTROMETRY
• fragmentation of target molecules
charged ions
• separation and measurement of the mass to charge ratio of the
components
• often combined with HPLC
• mass spectrum: a bar graph in which the heights of the bars
correspond to the relative abundance of a particular ion plotted as a
function of the mass-to-charge ratio
• structural information about the composition of individual solutes
• quantitation of the concentration of the eluting analytes
• possibility to quantitate multiple specific analytes in complex mixtures
• For example steroid hormone analyses
FACTORS INFLUENCING
HORMONE MEASUREMENTS
• age
• gender
• circadian rhythm
• menstrual cycle
• season
• sleep-wake rhythm
• pregnancy, lactation
• general diseases
• medication
• stress
• heterophilic antibodied
ENDOCRINE DISEASES DISCUSSED
• Diabetes mellitus
• Thyroid diseases
– Hyperthyroidism
– Hypothyroidism
• Hypocalcemia
• Adrenal diseases
Diabetes type I
LABORATORY DIAGNOSTICS OF
DIABETES MELLITUS
• diabetes mellitus type I and type II
• diagnosis is based solely on laboratory analysis, either
fasting blood glucose concentration or oral glucose
tolerance test (OGTT)
• point of care-testing + laboratory testing for follow up
• HbA1c
• C-peptide (proinsulin)
– estimation of insulin production in type II DM
HbA1c
(glycated hemoglobiini A1c or glycohemoglobin)
• ratio of glycated hemoglobin-A1c:n and total hemoglobin
• several assays - variable results - international
standardization
• represents blood glucose balance of preceeding 2-8 week
• follow-up of DM
• can be used in diagnostics (>48 mmol/mol or 6,5%)
• immunological assays, chromatographic assays
Thyroid disorders
THYROID GLAND
• Hormones:
– T4 (tetrajodithyronine = thyroxine)
– T3 (trijodithyronine)
• Transportation:
– thyroid-binding globuline (TBG)
– transthyretine (prealbumin)
– albumin
– as free hormone: 0.04% T4 and 0.4% T3
• Biological effects:
– anabolic in many tissues
– crucial for normal development and growth
– regulation of carbohydrate, lipid and protein metabolism
LABORATORY DIAGNOSTICS
OF THYROID DISEASE
1. Basic tests
– TSH and T4-V
2. Additional tests
– T3-V in hyperthyroidism with normal T4-v (T3tyreotoxicosis)
– TRH test
3. Thyroid antibodies (autoimmune etiology)
Calcium metabolism
Liver
PARATHYROID HORMONE (PTH)
• peptide hormone secreted by parathyroid glands
• regulates calcium and phosphate metabolism,
activation of vitamin D, bone metabolism (and
hematopoiesis)
• Overproduction causes hypercalcemia
• Underproduction causes hypocalcemia
• measurement of intact PTH in plasma by
immunological methods
HYPERCALCEMIA
PTH dependent hypercalcemia
• Primary hyperparathyreosis (adenoma, hyperplasia,
carsinoma)
– Plasma/serum PTH level invreased
PTH independent hypercalcemia
• PTH level low or unmeasurable
– bone metastases
• prostate cancer, breast cancer
– Paraneoplastic syndrome
• PTH-related peptide
– vitamin D intoxication, immobilization, thyreotoxicosis,
sarcoidosis
VITAMIN D
• from diet or synthesized on skin (UV light)
• D2 and D3
• activation in two steps:
1. 25-hydroxylation in liver
25(OH)D3
2. 1-hydroxylation in kidney
and 1,25(OH)D3
25(OH)D2 and
1,25(OH)D2
VITAMIN D
Serum vitamin D concentration [25(OH)D] is measured when vitamin D
deficiency or over-dose is suspected or when examining disorders of
calcium metabolism
• 25(OH)D correlates with symptoms and findings of vitamin D deficiency as
well as with gained and stored vitamin D
• 25(OH)D concentration:
– recommendations vary between 40-80 nmol/l
– severe deficiency 20 nmol/l
– toxicity 400 nmol/l
• seasonal variation according to sun light exposure
• recommendations for vitamin D supplementation updated in Finland: children
in 2011 and over 60-year-old in 2010
Adrenal gland
ADRENAL GLANDS
1. Cortex
• aldosterone
• glucocorticoids, androgens
2. Medulla
• catecholamines
GLUCOCORTICOIDS
• cortisol
• biological effects:
– regulation of carbohydrate, lipid and protein
metabolism
– regulation of immune system
– neuro-psychiatric effects
– regulation of calcium metabolism and bone
CORTISOL
• majority (90%) bound to plasma proteins
– cortisol-binding globulin (CBG, transcortin) 75%
– Albumin 15%
– 10% as free hormone: biologically active
• Circadian rhythm
– morning 150-650 nmol/l
– at mid-night below 100 nmol/l
• stress hormone
MINERALOCORTICOIDS
• aldosterone
• renin-angiotensin-system and serum potassium
(and ACTH) regulate production
• enhances sodium up-take in kidney
• enhances excretion of K+ and H+
• in blood stream 40% of aldosterone as free
hormone, 40% bound to albumin and 20%
bound to transcortin
DISORDERS OF ADRENAL CORTEX
1. overproduction
– hypercortisolism (Cushing)
• ACTH-dependent
• ACTH independent
– hyperaldosteronism
2. underproduction
– primary (Addison’s disease)
– secondary (injury, metastases etc)
CORTISOL: ASSAYS
1. serum cortisol
– total cortisol
– immunological methods
2. dexamethasone test
– synthetic glucocorticoid dexamethasone administrated at 11
p.m.
serum cortisol measured at 8 a.m.
3. urinary (dU-) cortisol
– reflects mean serum free cortisol
– less affected by stress than serum cortisol
– diagnostics of hypercortisolism
– HLPC (or mass spectrometry) best methods
4. salivary cortisol (reflects serum free cortisol)
ALDOSTERONE: ASSAYS
• total serum aldosterone measured by
mass spectrometry or immunological methods
• renin measured by immunological or
enzymological methods
– renin concentration or renin activity
Take home message
Several physiological factors influence hormone
concentrations, f.ex. time of day, month or year, stress,
age, pregnancy...
Hormone secretion is regulated tightly and often by the
hormone itself
Endocrinological disorders are caused by
over/underproduction of the hormone and/or altered
response to the hormone
LABORATORY GUIDE
http://huslab.fi/ohjekirja/