Biochemistry of Hormones 2025 PDF

Hormone definition ================== - A regulatory substance produced in an organism and transported in tissue fluids such as blood to stimulate specific cells or tissues into action. (Oxford dictionary) - A substance produced by **one tissue** and conveyed by the bloodstream to...

Hormone definition ================== - A regulatory substance produced in an organism and transported in tissue fluids such as blood to stimulate specific cells or tissues into action. (Oxford dictionary) - A substance produced by **one tissue** and conveyed by the bloodstream to another (cell or tissue) to affect physiological activity, such as growth or metabolism. Introduction to hormone biochemistry ==================================== What is the biochemical composition of the hormones? A. Carbs B. Lipids C. Proteins D. B and C E. None of the above Hormone biochemistry ==================== There are two main type of hormones based on their biochemistry composition - Peptides or proteins - hydrophilic molecules - relatively large molecular weight - mRNA-directed synthesis - stored in membrane-bound vesicles - can't diffuse freely across membranes - bind to receptors in cell membranes - Steroids or FA derivatives - hydrophobic molecules - relatively small molecular weight - synthesis by multi-step enzymemediated processes - not stored in signaling cells - cross membranes - bind to intracellular or membrane receptors Summary, peptide hormones ========================= How are peptide hormones produced? A. Synthesis from other protein B. Translation and transcription from a gene C. Synthesis from cholesterol D. Any of the above E. None of the above Summary, peptide hormones ========================= Is it possible to up or downregulate a peptide hormone production? A. No B. Yes How? Summary of Peptide hormones ===========================  Post transcriptional modifications ================================== - Why do you think that there is a post transcriptional modification process? - Could you give any example? POMC gene results in many hormones Ghrelin and Obestatin Ghrelin ======= - - CCK, one or many hormones? Steroids and fatty acids derivative =================================== - Synthesis by multi-step enzyme-mediated processes - Fatty acid (Arachidonic acid) → Prostaglandin → Thromboxane →Leukotrienes - Cholesterol → Progesterone → Estradiol → Testosterone Intro to steroids hormones ================================================ Can we up or downregulate a steroid hormone production? A. No B. Yes How? Fatty acids derivative synthesis ================================ - The first *enzymatic* step in steroid synthesis is the conversion of cholesterol into pregnenolone. - The enzyme that catalyzes this reaction is located in the inner mitochondrial membrane. - The rate-limiting step in this process is the transport of free cholesterol from the cytoplasm into mitochondria**.** This step is carried out by the Steroidogenic Acute Regulatory Protein (StAR) Cholesterol Metabolism for Steroid Production Steroids synthesis ================== **\"Old\" name** **Current name** -- ------------------ ------------------ P450~SCC~ CYP11A1 3 beta-HSD 3 beta-HSD P450~C17~ CYP17 P450~C21~ CYP21A2 P450~C11~ CYP11B1 P450~C11AS~ CYP11B2 P450~aro~ CYP19 Biochemistry of hormones ======================== Does more production/synthesis of a peptide hormone the same than more secretion? a. Yes b. No Do the changes in plasma concentration of a hormone represent changes in secretion? a. Yes b. No Do the changes in plasma concentration of a hormone represent more hormone activity? a. Yes b. No Measurements of hormones ======================== - Why do we have this section in the class? - Why we will have an open discussion about this? - How can we measure a hormone? - Are all the measurements the same? Hormone measurements ==================== 1. Biological methods 2. Immunological methods 3. Chemical methods Hormone measurements ==================== - Characteristics of the assays - Simple - Low cost - Relate to hormone activity or concentration - Calibrated to a known activity or concentration 1. Biological measurements ========================== Biological assays or bioassays: - Measures the activity of the hormone on a target organ or tissue - First develops - Injected plasma or extracts and see the biological response - Problem lack of sensitivity and specificity for low concentration hormones Now in vitro studies with cell assays Bioassay examples ================== 1. 2. 3. 4.    Immunoassay =========== - Endocrine assays are typically *immunological assays* that use [antisera] as reagents to detect the hormone. - Antisera: Antibody (Ig G specific for the hormone) - Antibodies can detect the low concentrations of hormones found in fluids. - Antisera must be both [sensitive] and [specific] - Do you think that is always like this? - Examples Immunoassay: [Competitive Binding Assay] - [Limited] (fixed) antibody is added to a tube containing a *labeled* (fixed) hormone and either sample [or] a standard - Labeled and un-labeled hormones compete for the *limited* antibody - Free hormone is removed from the tube - Antibody-bound *labeled* hormone is measured +-----------------------------------+-----------------------------------+ | -- ---------- -- | -- -- -- -- ---- | | \+ H\* + | Ab | | -- ---------- -- | -- -- -- -- ---- | +-----------------------------------+-----------------------------------+ Fixed amount of Ab Fixed amount of labeled hormone 1 2 --- Fixed amount of Ab Fixed amount of labeled hormone Variable amount of hormone 1 2 --- Competitive binding assay standard curve ======================================== - **Excess** of 1^st^ antibody is fixed to ta surface - Sample or standard are added and bond to the Ab - Labeled 2^nd^ Ab binds to capture hormone - After incubation excess of labeled Ab is removed - The signals are read from standards and samples are compared Std. curve is a direct relationship between hormone and amount of signal Validation of an assay ====================== - To Confirm that we are measuring the hormone that we claim that we measure we need to use a validated method. - There are 3 steps to validate a hormone assay 1. Parallelism. 2. Recovery 3. Cross-reactivity Validation of an assay ====================== Setting up an immunoassay ========================= Setting up an immunoassay ========================= Blank 0.1 ng/ml 1.0 ng/ml 10 ng/ml 100 ng/ml Positive control Parallel displacement ===================== - Serial dilutions should be parallel - if they are not, we need to evaluate how apart they are! Validation of an assay ====================== - On an unknown sample we add different known amounts of a standard. - We need to do them in at least 3 samples, example we add to the sample 50, 75 and 100 pM of the X hormone to a fix volume of plasma - The difference between the tubes should be 25 pM, independent of the actual reading (basal concentration + 50 or 75 or 100) Validation of an assay ====================== - Evaluate that the Ab for a particular hormone does not bind to a different hormone - Most of the time we do it with hormones with similar structure Common problems =============== - What happened if the plasma concentration of a hormone is less that the detectable for the assay? - If our model is swine, and there are no Ab against swine X hormone, what do we do? Immunoassay =========== - Antisera must be both [sensitive] and [specific] - Sometimes there is no specific antibody for the specie that we are working with. - CCK comertial kit recognize the first 8 AA (CCK-8), but there are other forms of circulating CCK that have different activity. - This is a big problem with immunoassays, they do not measure activity Chemical methods ================ - They are used mostly when there is no a bioassay or an immunoassay for a particular hormone. - They use unique aspect of the hormone structure - Size - Chemical composition - Liquid Chromatography - Gas Chromatography - Electrophoresis