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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/