General principles of endocrine functions Integration systems of the organism • Integration and coordination = maintaing the integrity and activity of the organism on alf levels in the relation to the changing external and internal environments • Hormonal system • Nervous system Immune system ENVIRONMENTAL STIMULI ■ sensory nerves Hippocampus ^ 1 ^ Amygdala Hypothalamus Anterior pituitary I gland (3) Regulation of brain and behavior - stress - arousal - learning and memory GLUCOCORTICOIDS (1) PHYSIOLOGICAL RESPONSES Brain: neurochemical changes Skeletal muscle: decreases protein synthesis decreases glucose uptake Adipose tissue: increases lipid mobilization decreases glucose uptake Liver: increases gluconeogenesis Immune system: immunosuppression No system works independently = functional integration Physical; emotional; chemical; etc. Hypothalamus CRH neurons Pituitary gland Hormones • Neurohormones Neurotransmitters • Paracrine (autocrine) effectors Adrenal gland Medulla • • • • Epinephrine and O Norepinephrine • • • Cytokines such as IL-1 Viruses; /q, bacteria; W/ tumors How do cells communicate? • Intracrine • Autocrine • Paracrine • Neurocrine • Endocrine • Neuroendocrine source environment target cell 1. INTRACRINE ((^^) IJ 2. AUTOCRINE 3. PARACRINE TARGET CELLS ^ 0 SIGNALING CELL f 1 4. NEUROCRINE ) T // \V *^®) /® a) NEURONS 5. ENDOCRINE ^^^^^^^^^^^^SSEL ENDOCRINE CELLS **— M ^^^^^^ TARGET CELLS 6. NEUROENDOCRINE J\. NEUROSECRETORY ~"S «» V NEURON ^„ ,„-»„,-, ^y^*^^^^^^^^^ BLOOD VESSEL ^^^^^^^^ ANTERIOR PITUITARY CELLS source synthesis/secretion no influence on specificity of effect - synthesis/secretion - main determinant of target eel (determined by localization) environment blood - universal environment - dilution and interactions r matrix/interstitial fluid diffusion binding proteins proteases components of extracellular matrix - receptor = specificity - cell response - number of receptors - signaling pathways - other ligands - metabolisation of ligand/receptor specificity and sensitivity diffusion barrier determinants of gradient inhibition signaling pathways effect of other ligands binding proteins Interstitial fluid A A BMP4 A A A^ ^ proteases, chordin, noggin Interstitial fluid/IVF > 1-^ differentiation bone formation antimicrobial peptides l,25-[OH]2D3 autocrine paracrine endocrine macrophage l,25-[OH]2D3 l,25-[OH]2D3 kidney - proximal tubule Hormones • Starling 1905 - secretin • Glandotropic hormones • Aglandotropic hormones • Target cells Limited time of effect Hypothalamus Releasing hormones: GHRH, CRH, TRH, GnRH Inhibitory hormones: somatostatin, dopamine, vasopressin oxytocin Thyroid gland T3, T4, & calcitonin Adrenal glands Cortisol Aldosterone Adrenal androgens Epinephrine Norepinephrine Ovaries Estrogens Progesterone ř Pituitary gland Growth hormone, Prolactin. ACTH, MSH. TSH. FSH, & LH Parathyroid glands Parathyroid hormone Pancreas Insulin Glucagon Somatostatin Testes Testosterone Source: Molina PE: Endocrine Physiology, 4th Edition: www.accessmediclne.com Copyright c The McGraw-Hill Companies, Inc. All rights reserved. Chemical nature of hormones DERIVED FROM AMINOACIDS -Adrenaline -Noradrenaline -Dopamine -Melatonine -T3/T4 T o STEROID -Cortisol -Aldosterone -Testosterone -Progesterone -Estradiol -Calcitriol PEPTIDES AND PROTEINS -Hypothalamic hormones -Adenohypophyseal hormones -Insulin, glucagon, somatostatin -Gastrin, cholecystokinin, secretin -Natriuretic peptides -Erythropoietin, thrombopoietin -PTH, PHrP -etc HO > O H2N O NH N- NH 0= P. -NH HN—' \ V- NH i O V) / HN^/ 0^-NH H JiN.^-^ 0 N-^0 SNH ' ~"C o-vNH2 -NH \ N J.. . u O^NH 0 = HN Chemical nature of hormones Hormone -characteristics Peptides -proteins Catecholamines Steroid hormones Thyroid hormones Ph-CH properties hydrophilic hydrophilic lipophilic lipophilic synthesis proteosynthesis Tyr modification CH precursors Tyr modifications storage secretory granules secretory granules not present colloid secretion controlled exocytosis controlled exocytosis diffusion diffusion transport free free/weakly bound bound bound elimination half-life short (4-40-170min) very short (2-3 min) moderate (up to 180 min) long (20 hours - 7 days) receptors membrane membrane cytosol nuclear effect short-term very short-term long-term long-term cell response quick very quick slow slow CHEMICAL STRUCTURE OF HORMONES DETERMINES THEIR BIOSYNTHESIS, STORAGE, RELEASE, TRANSPORTATION, ELIMINATION HALF-LIFE, WAY OF ELIMINATION AND THE MECHANISM OF EFFECT ON TARGET CELLS Hormones aldosterone Pleiotropic effects Multiplicity • Permissive effect Arterioles - a2 receptors Complement factors (adipsin) Blood pressure factors (angiotensinogen] Endocrine organs specialised cells - specialised organs („endocrine") „secretory" cells - organs with endocrine function cells without specialised secretory function cells converting hormone precursors Cytoki (IL-6, Tf adipose tissue Adipokines Clinical aspects • Production of hormones by tumors - PARANEOPLASTIC SYNDROMES Lung tumors Liver and kidney tumors GIT tumors - ADH (hyponatremia) - erythropoietin - ACTH (Cushing syndrome) - ACTH (Cushing syndrome) (polycythemia) - PTHrP (hypercalcaemia) Secretion of hormones and its regulation • Neuronal control • hypothalamus • sympathetic/parasympathetic nervous system • Hormonal control • Regulation od secretion by ions or substrates (Glu, AA) INSULIN Hormone secretion is controlled by feedback system hypotalamus ^^^^^^^^ i© adenohypofýza 1 (i) troPni h°rmon hormon cílové žlázy I biologický účinek biologická odpověď er endokrinní buňka I hormon Feedback negative X positive simple X complex Taken from Kittnar et al. Lékařská fyziologie. 1st edition. Grada 2011. kalcemie Positive feedback - why? Estrogen ■ from ^^ovaries Oxytocin from fetus and pituitary Induces oxytocin receptors on uterus Stimulates uterus to contract Stimulates placenta to make Prostaglandins Stimulate more contractions of uterus ■ ■ i ■ Pinilla, L, Aguilar, E., Dieguez, C, Millar, R. P., Tena-Sempere, M., 2012. KISSPEPTINS AND REPRODUCTION: PHYSIOLOGICAL ROLES AND REGULATORY MECHANISMS. Physiological Reviews. 92,1235-1316. FIGURE 1. Neurobiology of the hypothalamio-pituitBry-gonadal [HPG] axis, Schematic presentation of the major elements of the neuroendocrine axis controlling reproduction: trie HPG axis. Hypothalamic GnRH neurons, which receive trans-synaptic and glial inputs, release GnRH to the hypophysial portal blood system, In turn, GnRH dictates the pulsatile secretion of gonadotropins, LH and FSH, that stimulate the maturation and regulate the function of the gonads; note that in the scheme, both the ovary and testis are presented. These major hormonal elements are connected via feed-forward and feedback regulatory loops. The function of the HPG axis is under the regulation of several peripheral signals that include gonadal steroids, responsible for feedback control: testicular testosterone fT] conducts inhibitory actions on GnRH/gonadotropin secretion [negative feedback), whereas ovarian steroids, mainly estradiol [E2] and progesterone [P], can carry out both negative-and positive-feedback actions depending on the stage of the ovarian cycle. Other peripheral regulators of the HPG axis are metabolic hormones; among those, the prominent stimulatory/permissive roles of leptin, produced by the white adipose tissue [WAT], are depicted. Some of the central transmitters involved in the control of the HPG axis are also shown: predominant inhibitory transmitters are depicted in red, whereas excitatory factors are labeled in blue. Among the excitatory signals to GnRH neurons, Kissl neurons are highlighted. Please note that to concise presentation, discrimination between direct and indirect afferents to GnRH neurons is not made in the figure. Likewise, for sake of simplicity, some of the stimulatory and inhibitory signals to GnRH neurons are depicted inthesame neurons; exceptforthe Kissl/NKB/Dyn neurons, this does not denote necessarily coexpression of these molecules in the same cells. Glu, glutamate; GABA, ■y-aminobu-tyric acid; EOP, endogenous opioid peptides; ME, norepinephrine; NKB, neurokinin-B; Dyn, dynorphin; RFRP, RF-related peptides. [Adapted from Roa and Tena-Sempere [377).] SCN: - Afferent - retina - Efferent - hypothalimic nucleus Melatonin ADH ACTH - Cortisol Insulin Ghrelin Adiponectin Leptin Cyclic changes in hormone secretion External 24 h light-dark cycle Endogenous circadian rhythm SCN (Master clock) Photic Zeitgeber Synchronization I Nonphotic Zeitgeber ■ Sleep-wake cycle Peripheral oscillators ■ Physical activity ■ Social time ■ Meals Cellular oscillators i ^> Neuronal/hormonal = SNC-dependent (o Q Satiety/fasting Body temperature environment Hypothalamus Adenohypophysis Adipose tissue Hormone transport Chemical properties of hormone Transport protein(s) bond and its significance • Albumin • Globulins • Specific proteins - TBG, SHBG, CBG Bond strength ^Alternative" binding - TBG versus transthyretin •Protection • Reservoir •Ubiquitous distribution •Transport across plasmatic membrane (SHBG-megalin) DYNAMIC BALANCE BETWEEN HORMONE AND TRANSPORT PROTEIN Hormone elimination Different length of time in circulation Metabolisation by • Target cells • Enzymatic systems in blood • Organs - mainly liver Elimination • Liver • Kidneys PHASE I Hydroxylation, decarboxylation Oxidation, reduction PHASE II Glucuronidation Sulphatation Methylation Conjugation with glutathione \7 Vascular system bile urine Hormones and cell response Target cells Specificity High affinity Selectivity hormone O QO #°o0o°o' MECHANISMS Conformation changes Phosphorylation/dephosphorylation + protein recruitment GTP binding (G proteins) cAMP binding (efector proteins) Precursor molecule generation in PM Non-covalent Ca2+ bond SIGNALING PATHWAYS I CELL RESPONSE Receptor binding Signal amplification and transduction efector molecules % of occupied receptors conformation change synergy antagonism possible loss of sensitivity feedback-loop regulation CELL RESPONSE IS MEDIATED BY RELEVANT RECEPTORS Receptor level of cell response regulation Active receptor Desensitized receptor Receptor protein Downregulation Upregulation Homologous desensitization Heterologous desensitization Phosphorylation (specific kinases) Dephosphorylation (specific phosphatases) Modification by proteins of inhibited signaling pathway Agonist (a) Receptor inactivation (b) Receptor internalization Lysosome (c) Receptor down-regulation Figure 13.10. Major mechanisms fur the termination of receptor-dependent signal transduction. Textbook of Biochemistry With Clinical Correlations. Sixth Edition. Edited by Thomas M. Devlin. Copyright © 2006 John Wiley & Sons. Inc. Sensitisation and desensitisation of G protein-coupled protei a subunit with GTPase activity resensitisation desensitisation . GTPase ^and receptor resensitization Synthesis and targeting of components Receptor Effector GDP Arrestin Receptor kinase Receptor +■ CP Receptor Arrestin Receptor kinase G protein activation Receptor activation by agonist GTP GDP Arrestin Receptor kinase Hormones - proteins and peptids „classic" hormones Hormones produced by non-specialised cells (e.g. adipokines) Paracrine/autocrine peptides teceptors associated with plasmatic membrane Nucleus Cisternae Transition elements Capillary lumen Secretory vesicles ° o O 0 C -K) Immature secretory granules Mature secretory granules 0, Plasma membrane Lysosome preprohormone - prohormone - hormone (+ fragments) hormones as a part of preprohormones počet aminokyselin v prekurzoru 166 prekurzorový peptid prepropresofyzin počet aminokyselin v hormonu AVP 242 prepro TRH TRH TRH TRH TRH TRH TRH 267 265 preproenkefalin A 5,7,8 | JMet-enk | Leu-enk preproopiomelanokortin *Met-enk oktapeptid *Met-enk heptapeptid MSH ACTH MSH konec 39 ACTH 31fi-endorfin 236 preprodynorfin (preproenkefalin B) 17 dynorfin Leu-enk N-konec Dyn Obr. 1-22. Příklady velkých prekurzoru (preprohormonů) malých peptidových hormonů. Viz také obr, 14-12. TRH - hormon uvolňující thyrotropin; AVP - argininvazopresin, Met-enk - met-enkefalin, Leu-enk - leu-enkefalin, MSH - hormon stimulující melanocyty, ACTH - adrenokortikotropní hormon, konec - p-endorfin, Dyn - dynorfin, N-konec - neoendorfin Taken from Ganong, W. Přehled lékařské fyziologi 20th edition. Galén 2005. Ligand-gated ion channels SECRETION OF HYPOTHALAMIC HORMONES AFTER BINDING OF CORRESPONDING TYPE OF LIGAND (NEUROTRANSMITTER) G protein-coupled receptors (GPCR) GPCR (inactive) GDP- " %ygtp~® O Hormone ▼ GPCRHormone complex O (active) Adenylyl cyclase GDP GTP amplification ■>■ GTP-© - Effector proteins \ Phospholipase I [Others Increased level of 2nd messengers •GIRKs •P/Q and N type voltage-dependent Ca2+ channels •Some AC isoforms •Some PLC isoforms cAMP (Fig 1-13) DAG (Fig 1-15) IP3 —> Ca2+ (Fig 1-15) 4 Specific cellular response to specific hormone-GPCR signal G protein-coupled receptors (GPCR) Gs - Gs, G0|f - activation of AC inhibition of AC •G0 (2, brain) »Gt (2, photorec. - cAMP-PDE) »GZ (inhibition of K+ channels) Gq/11 - activation of G12/13~ inhibition and activation of RhoGEF Peptide and protein: Glucagon, Angiotensin, GnRH, SS, GHRH, FSH, LH, TSH, ACTH Amino acid derived: Epinephrine, norepinephrine Ion channels, PI3Ky, PLC-ß, adenylate cyclases Biological responses 1 ' (2) CS) GTP GTP GTP GTP Adenylate PLC-p Adenylate RhoGEFs cyclase DAG cyclase Rho 1 CAMP Ca++ PKC 1 cAMP Gene expression regulation / Transcription factors End of activation and limitation of cell response GPCR-► Hormone • GPCR Hormone • GPCR • P Receptor tyrosinkinases EGF receptor PDGF receptor Insulin receptor Cys-rich domain lg-like domain I Tyrosine kinase domain • 58 RTKs/20 subfamilies • Usually dimerisation after ligand binding • ATP as a source of P for phosphorylation of intracellular domains/associated proteins • Insulin • IGF-1/2 Insuline receptor - genomic effects Insulin • IRS = insulin receptor substrate • Grb = adaptor protein (growth factor receptor-bound protein) • SoS = Son of sevenless homologue • Ras = small GTPase-like proteins (ability to bind GTP) • Raf = serin/threonin- Mek -> MekP MAPK-► MAPKP Transfer to nucleus I Phosphorylation of transcription factors I Change in gene expression I Cellular response (Primarily mitogenic actions of insulin) nsulin receptor - metabolic effects P13K = phosphatidylinositol-3-kinase Akt = proteinkinase B í>ÍBé^ Insulin P1P2-P1P3 V IRS J Also recruitment of activation of PKC isoforms Activation of small G protein TCIO GLUT 4 (in vesicle) Active Akt/PKB \ > Protein phosphorylation \ Cellular response (primarily metabolic actions of insulin) Insertion of GLUT4 into cell membrane Increased uptake of glucose Glucose Receptors associated with cytosolic TK GH, prolactin, leptin, erythropoietin Dimeric receptor without TK activity Association with JAK kinase After ligand binding -dimerisation, transphosphorylation, activation Hormone/cytokine signal transducers and activators of transcription SOCS proteiny Nucleus^sX STAT regulation of \A gene expression Receptor serin/threonin kinases Anti-Müllerian hormone, inhibitin Form of dissociated heterodimer SMAD = „latent transcription factors" TGF-ß-related hormones RII/RI dimer Nucleus Regulation of specific gene expression gamma-activated sequence-like elements (GLEs, promotor region of some genes) Receptor guanylate cyklases Signal transduction - system of second messengers HORMONE = FIRST MESSENGER INTRACELLULAR SIGNALING MOLECULE GENERATED AFTER HORMONE-RECEPTOR BONDING = SECOND MESSENGER • CAMP • TSH, glucagon, ACTH, hypothalamic hormones, ADH etc. • Proteinkinase A • Modulation of signaling pathways by compartmentalization (A-kinase anchoring proteins (AKAPs)) cGMP • ANP, BNP, CNP NO as a signaling molecule • Proteinkinase G DAG and IP • PIP2 - phospholipase C system Ca2+ ^^^^^H • Ca2+/Ca2+- calmodulin EXTRACELLULAR SIGNAL MUST BE CONVERTED TO INTRACELLULAR RESPONSE AC-cAMP system • PKA • CREB (cAMP-responsive element-binding protein) • Epac (E) as an another effector molecule (exchange protein activated by cAMP) • cyclic nucleotide gated (CNG) channels • hyperpolarization-activated cyclic nucleotide modulated (HCN) channels • phosphodiesterases Ionic cui (e.g., H GTP Protein phosphorylation (membrane, cytosolic, & nuclear proteins) Activation of effector proteins Cellular response PLC - DAG and IP3 system Ca2+ - calmodulin system O Calmodulin binds Ca2+ four calcium ions Calmodulin Calcium- calmodulin complex Q Calmodulin change conformation, resulting in an active complex Target protein Q The two globular hands" of the complex wrap around a binding site on a target protein O 3 o a. c_ ■ Q. n> -a n> 3 Q. n> 3 Calmodulin-binding site (a) Structure of Ca2~-calmodulin complex (b) Function of Ca2~-calmodulin complex Copyright © 2009 Pearson Education. Inc. ,3 in Extracellular fluid Channel closed Change in electrical properties of cell Calcium enters cell through open channel As second messenger Muscle Secretion Calmodulin contraction / / "I \ Ca ER O i line Ca-calmodulin Activates enzymes Protein- P Cytosol I—I I— Response in cell (muscle contraction, altered metabolism, altered transport) Extracellular signals (hormones, neurotransmitters) NO as a signalling molecule - cGMP NOS Cellular response (I smooth muscle tone) Donald JA, Forgan LG, Cameron MS: The evolution of nitric oxide signalling in vertebrate blood vessels. J Comp Physiol B-Biochem Syst Environ Physiol 2015, 185(2):153-171. Summary - membrane receptors and associated systems 4 DAG Ca2+ cGMP 4 4 JJ PKC kalmodulin PKG proteiny 4 proteinkinázy proteinkinázy proteinové substráty cílové kinázy multifunkční kinázy proteinové substráty y'' \ *♦* \ proteinové substráty další fosfolipázy Clinical aspects Syndromes of resistance to hormones (i.e. IR, IGF-1, TR(3) Syndromes caused by CPCRs and G proteins mutations ADH - nephrogenic diabetes insipidus ACTH - familiar ACTH resistance GnRH - hypogonadotrophic hypogonadism FSH - hypergonadotrophic ovarial dysgenesis LH - male pseudohermaphroditism Melanocortin 4 - obesity PTH/PTHrP - Blomstrand lethal chondrodysplasia Hormones acting through nuclear receptors HORMONES -Thyroid hormones - TRa/ß <^ i heterodimers -Estrogens - ERa/ß -Testosterone - AR -Progesterone - PR homodimers -Aldosterone - MR -Cortisol - GR VITAMINS -1,25-[0H]2D3 - VDR -All-trans-retinoic acid - RA receptors a, ß, y -9-c/s-retinoic acid - retinoid X receptor RXR a, ß, y PRODUCTS OF METABOLISM AND XENOBIOTICS -Fatty acids- PPAR a, P, y -Oxysterols - liver X receptor LXR a, P -Bile acids - BAR -Hem - RevErb a, P -Phospholipids - homologue of liver receptor LRH-1, SF-1 -Xenobiotics - pregnane X receptor PXR - constitutive androstane receptor CAR -Orphan receptors -Variable receptors Explanation of some effects and pathologies General mechanism of effect of hormones acting through nuclear receptors Prohormone (ligand precursor) Hormone (ligand) -High affinity of ligand bond = due to R structure -Recognition of specific promotor region -Dimerisation of receptors (homodimers, heterodimers) -Remodelation of chromatin for gene expression (HDAC) -Gene expression at the end decreased or increased WHY ONLY NUCLEAR RECEPTORS? -Synthesis in cytoplasm -Stay until ligand binding or until transport to nucleus -Regulation mechanism - modification, count of receptors -Important parameter - selectivity of target cells -Tissue-specific factors, coactivators and corepressors Nuclear receptors ATD DBD LBD (amino terminus domain) (DNA binding domain) (ligand binding domain) -Coregulatory proteins binding (independent on ligand) - Phosphorylation sites -DNA binding (zinc fingers) -Dimerisation -ERE, PRE, GRE, MRE, ARE -Ligand binding (agonist, antagonist) -Coregulatory proteins binding (dependent on ligand) -Dimerisation -Nuclear translocation -Chaperone association (HSP) Example - steroid hormones GTFs = general transcription factors (remodulators of chromatin) HAT = histon acetyltransferase Pathway 1 (Steroid hormones) (-) Hormone (+) Hormone Gene T Basal transcription Recruitment of co-activators Recruitment and activation of general transcription factor Chromatin structure Gene T Stimulated transcription Pathway 2 (Thyroid hormones, vitamin D. PPARs) (-) Hormone Example - thyroid hormones THRs, VDR, PPARs, RXRs THR = heterodimer hormone | hormone + RA Blocking general transcription factor -j HRE Chromatin structure Gene Repressed transcription (+) Hormone Dissociation of co-repressors basic transcription (+) Hormone Basal transcription Recruitment of co-activators Recruitment of activation of ^ general transcription factors Gene Chromatin structure stimulated transcription i Stimulated transcription Termination of hormone action Receptor-mediated endocytosis and subsequent lysosome degradation Phosphorylation/ dephosphorylation of receptor or proteins of signaling pathway Ubiquitination and proteosomal degradation Binding of regulatory factor on corresponding protein (enzyme) Inner enzymatic activity and its regulation J i Clinical aspects Hormone overproduction Hormone underproduction Changes in sensitivity of target tissues and/or change in cell response Higher rate of inactivation or degradation of hormones Insufficient production or higher degradation of transport proteins Changes of transport hormones production during physiological conditions (pregnancy) Clinical aspects A. Decreased hormone responsiveness u 100 qj 3= 0) c o X SO 0 Maximal response-^ ff -> B. Decreased hormone sensitivity Í 100 o o 50 0 / 4 Hormone / * concentration /■ required to elicit half- /Y maximal response. * s • Vjl-> Log [hormone] Log [hormone] Source: Molina PE: Endocrine Physiology, 4th Edition: www.ac.cessmediciiie.com Source: Molina pe: Endocrine Physiology, 4lh Edition: vjww_atcessniediciiie.com Copyright & Tlie Mcüraw-Hill Companies, Inc. All rights reserved. Copyright & Tlie McGraw-Hill Companies, Inc. All rights reserved. Decreased number of receptors Decreased concentration of hormone-activating enzyme(s) Increased concentration of non-competitive inhibitor Decreased number of target cells Decreased affinity of hormone to receptor Decreased number of receptors Increased rate of hormone degradation Increased concentration of antagonists/competitive inhibitors Determination of hormone levels in blood -HIGH SENSITIVITY DEMANDS -WIDE CONCENTRATION RANGE Antigen-antibody interaction-based methods -Anibody requirements (poly- X monoclonal) -Monoclonal antibodies = specific epitopes -Radioactive labeled antibodies -Necessity of quantification! -RIA, ELISA Methods based on HPLC-MS Nucleic acid-based methods -hybridization techniques -restriction fragmentation, electrophoresis, sequencing Separation techniques - free X bound hormones - dialysis Cortisol Vitamin D-25-OH -Progesterone -DHE A -T3 - Testosterone—male -Norepinephrine -FSH -Prolactin Testosterone—female -Vitamin D 1,25-OH Estradiol—female Estradiol—mate . H Aldosterone TSH - FT* - Insulin - Epinephrine Growth hormone 100 10.000 Concentration in picomoles/L 1,000,000 EXTREMELY LOW LEVELS OF HORMONES IN BLOOD Competitive binding RIA = radioimmunoassay Antibody affinity = K,/K , = [AbAg]/[Ab][Ag] 125 l-Analyte 6 Ab • Ag* 10 Ab* 8 Ab + 16 Ag* + 4 Agc 7* 2Ab«Ag° 2Ag° -< -< *★*★ j * * o i **** + 00 — ;-<*•«*; * * * I -< "< ★ ★** 0 0 + s * * * -< -< * * * * :-<0 -<*i I* 0 *i Bound Free Calibration of standards Ab ♦ Ag* + Ag° Ab» Ag*+Ab»Ag° + Ag* + Ag° 8 16 0 ! • • « 8 0 j j 8 0 8 16 4 • • 1 I 6 2 | j 10 2 8 16 12 ■ I 1 4 4 | 12 8 8 16 36 1 : • 2 6 | ..............J j 14 30 Constant Vanable Bound Free B i-r 8 12 16 20 24 28 32 36 Concentration }NSB HPLC-MS 100 l 0*-100 -I 0"-r 100 1 0* 1 100-1 o*- °1 100 100 mo 1 1 o*L t-r 0^—I-1-r 100 "I I r t-r t-1-r -1-1-r 100 i 1 r 7.46 1 DHE AS -1— 1 I--1-r-1--1-1-1-1-1-1-1 t-1-r 9.56 Cortisol T-1-1-1-1-1-1-1-1 11.08 t—i—i—i—r JL" Deoxycortisol 11.07 r—I-1-1-1-1-r 11.38 ^ ^A^A^ And roste nedione 1 I T l r I l-T-1 I f I I 7.48 12,67 A T-1 I-1-1- —I-1—™T 13.22 Testosterone Estradiol A t—I—r-1— I f I I I ■ 17-Hydroxyproge ste rone i—I—r ■7A6 II DI ITA i—I—ii h " r " I I X 13.99 1-1-1-1 l-1-r 16:65 Progestt t-r-r l-1—1-rr 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Time (min)