Lab 4 Hormones v 2.0 Flashcards
What are hormones?
chemical substances produced by particular glands or groups of cells, secreted into the bloodstream, and that elicit specific responses fromdistant cells and organs.
Different types of molecules function as hormones; Derived from amio acids-examples Cholesterol derivatives-examples
Derived from amio acids(examples) -amino acid dervates: catecholamines, serotonin, thyroxine(T4) -Tripeptides: thyrotopin-releasinghormone(TRH) -Small peptides: vasopressin or antidiuretic hormone(AVP or ADH), somatostatin -Intermediate-size peptides; insuli, parathyroid hormone -Glycoproteins: gonadotropins, thyroid stimulating hormone(TSH), luteinizing hormone(LH) Cholesterol derivatives -cortisol, testosterone, estradiol, aldosterone
Some hormones function to transmit to the body important environmental cues, while others are more concerned with calibrating metabolic processes involved in maintaing ________. In either situation, it is critical that there is an effective translation of the magnitude of the ________ into the amount of hormone released. How this is achieved varies depending on the stimulus. The endocine systems which involve the hypothalamus, pituitary and downstream organs are usually termed what and are most usefully views as functional units for what purpose? What areas are controlled by hypothalamus?
-Homeostasis, stimulus -Termed axes, for purposes of clinical diagnosis and management -Both anterior and pituitary
Hormones
The hypothalmic hormone Thyrotropin-releasing hormone(TRH) stimulates secretion of which pituitary hormones?
TSH and PRL
Some hormones at on tissues whereas others, tropic hormones, act on other glands regulating secretion of their hormones. Diagram
The hypothalamic hormone corticotropin-releasing hormone(CRH) stimulates which pituitary hormone?
stimulates secretion of ACTH
Growth hormone- site of formation and action
Formation: anterior pituitary Action: liver, bone, other cells
The hypothalmic hormone gorwth hormone-releasing hormone(GHRH) stimulates which pituitary hormone?
Stimulates secretion of GH
The hypothalmic hormone gonadotropin-releasing hormone(GnRH) releases which pituitary hormone?
stimulates secretion of FSH nad LH
The hypothalamic hormone gonadotropin-inhibiting hormone or somatostatin(GHIH) inhibits release of which pituitary hormones?
inhibits release of GH and TSH
The hypothalmic hormone prolactine-inhibiting hormone(PIH), dopamine inhibits release of which pituitary hormone?
inhibits release of PRL
Thyroid stimulating hormoe(TSH) site of formation and action
Formation: Anterior pituitary Action: Thyroid
Thyroxine(T4) andTri-iodothyronine(T3) site of formation and action
Formation: Thyroid Action: Muscle, liver and other cells
Adrenocorticotropic hormone(ACTH) site of formation and action
Formation: Anterior pituitary Action: Adenal cortex
Cortisol, corticosterone and aldosterone site of formation and action
Formation: Adrenal cortex Action: Many tissues
Follicle-stimuating hormone(FSH) site of formation and action
Formation: Anterior pituitary Action: ovaries and testes
Luteinizing hormone(LH) site of formation and acttion
Fomation: Anerior pituitarry Action: ovaries/testes
Estradiol, progesterone, testosterone site of formation and action
Formation: ovaries/testes action; reproductive organs
Prolactin site of formation and action
Foration: anterior pituitary Action; mammary glands
Oxytocin site o formation and action
Formation: rouced by hypothalamus and released by posterior pituitary Action; smooth mysck and mammary glands
antidiuretic hormone(ADH) site of formation and action
formation; produced by hypotalamus and releaed by posterior pituitary Action: kidney, arterioles
What is a major feature of most endocrie systems? What does this mean? The effect?
Negative feedback. This meansthat a response induced by action of ahormone feeds back to inhibit the level of hormone production. The effect of this is to dampen down fluctuations in the process controlled by the hormone, thereby ehancing stability of that proccess ad hence homeostasis
If negative feedback is the only process at play then…. Many endocrine systems, especially ones controlled by hypothalamus, dislay…. and this is determined by….. In this situation, negative feedback serves to smooth out…… What is one potential mechanism of modulating the level of he output of the endocrine system
the output of the system remains constant display distinct rhythmicity determined by the net product of the neural inputs to the relevant secretory neurons In this situation negative feedback serves instead to smooth out the hormonal profile and to prevent short-term instability. Furthermore, modulation of the susceptibility of the controlling neuron to feedback inhibition represents one potential mechanism of modulating the level of the output of the endocrine sytem
CRH - coricotropin-releasing hormone diagram
- Hormones act by binding to specific receptors either on the _______or within the _______ cell. Classically, there is a very _____ degree of specificity in this binding and it is this ____________ interaction that triggers a wide range of biologic effects 2. Hormone receptors may be divided into different families according to their ________ and each family broadly shares _________ mechanisms 3. Many ______ hormones and peptides which cannot cross lipid bilayers act via ________ couples receptos while some larger peptides such as ______ and_________ act via tyrosine kinase receptors. Both of these recptors are inttrinsic ______________ and they rely on cascades of sequential phosphoryation events to alter ___________ and __________
- cell surface or target cell. high degree, this hormone-receptor interaction that triggers 2. according to their structure, signal transduction mechanisms 3. amine-like hormones, via G protein coupled receptors, insulin and insulin-like growth factor 1(IGF-1), intrinsic plasma membrane proteins, alter enzyme activity and gene expression
Describe steroid hormones
Steroid hormones, some hormones derived form fatty acids and tri-iodothyronine, which has physicochemical protpertes in common with steroids, enter cells before binding their receptors. THese so-called nuclear hormone receptors are effectively ligand-activated transcripion factors and provide a very direct link between the exposure of a ell to hormona ligand and alteration of patterns of gene expresseion
Levels of hormones are useful physiologic signals only if there is also an effective way of turning the _______off. Hormone inactivation usually occurs by further metabolism (eg,__________, __________ and _________), followed by excretion of the metabolites. Such degadation may occur in ______, in organs such as the _____ or in ________ tissues after receptor mediated internalization of the hormone. The rate of clearance of differernt hormones varies eenormously, from a few mintutes(______) through hours(______) to days(______)
signal off, (eg, proteolysis, hydroxylation and conjugation)
occur in plasma in organs such as liver or in target tissues
few minutes(insulin), through hours(steroids) to days(thyroxine)
Another feature of some endocrine systems particularly those involving steroid hormones, is local metabolism of the hormones or prohormones near their ________ receptor. This may involve conversion of an _______ circulating hormone to a more potent form (for example, testosterone to _________ by ___ reductase in androgen dependent hair follicles and prostrate gland) or creation of an acitve hormone from what is effectively a circulating _______(eg, thyroxine to tri-iodothyronine by _______, or synthesis of ________ from adrenal androgens in adipose tissue).
cognate receptor
active circulatng hormone
testesoterone to dihydrotestosterone
circulating prohormone
iodothyronine by de-iodinase
synthesis of estradiol
A similar mechanism is also employed to prevent ________ receptors in the kidney being exposed to high concentrations of cortisol, with ______ being inactivated by 11B-hydroxysteroid dehydrogenase type II which is expressed highly near the mineralocorticoid receptors. Many other examples of such intracellular interconversion of steroid hormones have also emerged and study of this phenomenon has sometimes been dubbed _______.
mineralcorticoid receptors
cortisol
intracrinology
Within ciruculation many small or hydrophobic hormones are transported bound to carrier proteins. Give examples.
What do transport proteins do and what does this mean?
For example, thyrxine and cortisol are transported on specific plasma-binding globulins such as thyroid-binding globulin(TBG) and cortisol-binding globulins(CBG).
The transport proteins extend the biologic half-life and increase the plasma concentration of the smaller hormones, which would otherwise be elminated rapidly in the liver or kidney. This also means that there may be large differences between the total plasma levels of hormone and the unbound or ‘free’hormone in solution which is geberally the biologically active form.
Clincal interpretation of plasma hormone levels is simplest if assays are developed to detect _____ hormone only, otherwise assumptions must be made about levels of binding proteins, which in turn may be significantly influenced by ______ and _______ milieu. It is not only _______ hormones that have plasma-binding proteins, however, both ____ hormone and_________have plasma-binding proteins which influence bioavailability and tissue activity of the hormones in a variety of different ways.
free hormone
nutritional and hormonal milieu
not only small lipophilic hormones
both hormone and insulin-like gorwth factor 1
What are 3 binding proteins and their % approximate bound and clinical note of Thyroxine?
Binding proteins: Thyroid-binding globulin(TBG) - 75%
Albumin- 10%-15%
Transthyretin- 10%-15%
Clinical note: clinically significant increase in early pregnancy
What is the binding protein and their % approximate bound and clinical note of testosterone?
Binding protein: sex hormone binding globulin(SHBG) - 60-70%
Clinical note: levels of SHBG are strongly hormonally regulated (eg, suppressed by insulin and increased by thyroid hormone)
What are 2 binding protein and their % approximate bound and clinical note of cortisol?
Binding protein: cortisol-binding globulins(CBG) - 75%, albumin 15-20%
Clinical note: variations in cortisol binding proteins ay be particularly isleading as it is total cortisol which is generally assayed. Thus women taking estrogens (increase CBG) may have high total but normal free cortisol, while in critical illness and malnutrition the converse is true
What is the binding proteins and their % approximate bound and clinical note of GH?
Binding protein of GH: Growth hormone binding protein(GHBP) - 50%
Clinical note: GHBP is a soluble fragment of the GH receptor. This is a common feature of many cytokine-like hormones
What is the binding proteins and their % approximate bound and clinical note of IGF-1?
Binding protein: insulin growth factor binding protein 3(IGFBP-3) -75%
Clinical note: There are 6 IGFBPs which modulate the paracrine activity of IGF-1 and may have signaling roles in their own right
Biochemical assesment of hormone action
The plasma concentrations of most hormones are exteremly ___. Therefore very sensitive and specific methods are required for measurement of hormone levels in the clinical laboratory. The classic procedure is ________(RIA).
What does RIA require?
How does RIA work?
RIA is used not only for hormones but also for many low abundance plasma proteins for ex:______ and ________
Low
radioimmunoassay (RIA)
It requires a specifc antibody to the hormone and a radiolabeled version of the hormone contaning tritium, radioactive iodine, or someother suitable isotope.
When the patient’s serum is assessed to a complex of the antbody with the radiolabeled hormone, the unlabeled hormone in the patient’s serum competes witht he radioactive hormone for binding to the antibody. After incubation for some minutes, the unbound hormone is seperated from the hormone-antibody complex. The higher the hormone concentration in the patient’s serum, the more radioactive hormone is displaced from the antibody.
For ex: C reactive protein and prostrate specific antigen
Biochemical assessment of hormone action
What are RIA procedures now being replaced by? Give examples
When an attached enzyme is used, the method is called ______
RIA procedures now being replaced by other immunoassay techniques that utilize fluorescence or chemiluminescence measurements rather than radioactivity.
For ex; an antibody to a hormone can be immobilized on a solid matrix or plate. The immobolized antibody binds the hormone in the patient’s sample. After washing, a second antibody, which binds to a second site on the hormone, is added to make an antibody “sandwich”. This second antibody has a fluorescent or chemiluminescent tag or an enzyme that is covalently attaches to it. The amount of the tag or enzyme is masure of the amount of hormone present.
When an attached enzyme is used, the method is reffered to as an enzyme-linked immunosorbent assay(ELISA). To detect the amount of enzyme present, a substrate is added that can be converted by the enzyme into a fluorescent or chemiluminescent product.
Biochemical assessment of hormone action
Measuremnt of ______ concentrations of hormones or their metabolites can be used in some diagnostic settings, including the diagnosis of ______and in pathologies such as secretor ______ tumors. As the concetration of hormone or metabolite in a single sample of urine depends both on the _______(mass) of this hormone or metabolite and on the______ of the sample of ____, one should perform __ hour urine collection and calculate an excretion of the hormone or the metabolite(mg/24h)
urinary concentrations
diagnosis of pregnancy
secretory adrenal tumors
amount(mass) of this hormone or metabolite and on the volume of the sample of urine
24 hour urine
Biochemical assessment of hormonal action
Laboratory testing of e______ systems aims first to determine whether the system is functioning_______, and second to localize the functional ______. Most commonly, this means that levels of the hormone that elicit the relevant physiologic response at target tissues are determined, usually by ______, together with measurement of one or more upstream ____ hormones, where they exist. The presence of _________regulation means that the system will attempt to correct perturbations in levels of the effector hormone with compensatory changes in levels of tropic hormones. Thus assessment of at least __ points in such an endocrine feedback loop is essential and permits focusing of later diagnostic imaging on the relevant gland.
endocrine systems
functioning abnormally, localize functional defect
immunoassay, tropic hormones
negative feedback regulation
2 points
Biochemical assessment of hormone action
Once the morst proper hormone to the clinical problem has been chosen, then appropriate method should be used for sampling. What should this take into account?
This should take into account whether the hormone is very unstable(for ex: any small peptide hormones require blood samples to be taken inoto tubes on ice containing a protease inhibitos, and laso whether it is produced with a circadian or other rhythm. For some hormones, such as thryoxine which have very long half-life, timing of the smaple is not critical, whlle for tohers, such as cortisol, standardized timing of the sample is essential.
A further factor to take into account in hormone testing is whether or not secretion of the hormone is pulsatile. Nearly all of the hypothalmic and pituitary hormones show some degree of pulsatility in secretion. In most cases, this simply means that repeat testing in the case of mild abnormality is required while for some hormones, such as grwoth hormone, which has both a short half-life and very striking secretory spikes, single measurements are often meaningless
Biochemical assessment of hormone action
To avoid problems, endocrinologists commonly employ_______ testing to build up a profile of hormone levels at several points in the day. This can be an effective way of detecting subtle pertubations of ______ rhythm, and can also lessen the impact of ______ secretion of the hormone. A further approach is to use _______testing. In other words, hormone levels are measured not just in the resting state, but also after a relevant stimulus has been applied, in an attempt to assess the maximal capacity of the _______ gland or system being tested. Frequently the stimulus is high dose of tropic hormone (eg. ___, ___, ___ etc) however it may instead be a metabolic challenge(eg _______).
multiple testing
circadian rhythm
oscillatory secretion
provocative testing
endocrine gland
tropic hormone (eg. ACTH, TRH, GnRH etc)
metabolic challenge(eg with an oral glucose load or severe indulin-induced hypoglycemia to mimic stress)
Describe the stimulus, measurement and rational use of HP Adrenal provocative endocrine test
Stimulus: synthetic ACTH , Measuremnt: cortisol, Rational use: corstisol tests functional integrity of adrenal glands, which rely in turn on chronic tropic actionsof ACTH. This is an indirect test of pituitary/hypothalamus
Stimulus: insulin-induced hypoglycemis, Measurement: cortisol, Rational/use: severe hypoglycemia mimics physiologuc stress and robustyl tests the hypothalamus, unlike the Synacthen(synthetic hormone similar to ACTH) test
Describe the stimulus, measurement and rational use of HP Thyroid provocative endocrine test
Stimulus: TRH, Measurement: TSH, Rational/ use: the pattern of TSH release adter tTRH stimulation gives informtion which may be useful in diagnosing central hypothyroidism
Describe the stimulus, measurement and rational use of HP Growth provocative endocrine test
Stimulus: insulin induced hypoglycemia, measurement: GH, Rational/use: baseline pulsatility of GH is overcome by applying a strong stimulus to its release
Stimulus: oral glucose load, measuremnt: GH, Rationale/use: failure of suppresion of GH by glucose is used in the diagnosis of acromegaly
Solution for short-lived or unstable hormone
Take correct sample and store on ice: consider need for protease inhibitor such as aprotinin in tube
Solution for cyclical hormone levels
Sample only at defined time in cycle for which normal range has been established(eg 9 am sampling for cortisol, follicular phase sampling for LH/FSH/ estradiol)
Solution for highly pulsatile pattern of secretion (eg: GH)
Take average of multiple samples spaces out in time or use in provocative stimulus before measurement(eg hypoglycemia)
Solution for several possible points of dysfunction in system
Sample at 2 different points in the endocrine system enables anatomic localization of problem(ie tropic hormone plus effector hormone eg: TSH+T4, ACTH+cortisol)
Major types of endocrine pathology
Although each endocrine system may malfunction as a consequence of organ-specific damage or disease, for example relating to the anatomic site of the gland, there are also some general types of pathology to which endocrine cells are very susceptible. The frst of these is _______ destruction with detectable endocrine gland-specifc antibodies. This is a potential cause of loss of function of nearly all ______ glands and less commonly may also cause________. In fact, autoimmunity to the thyroid, adrenal and pancreatic islets accounts for more than __% of all organ-specific autoimmune disease. This appears in part due to genetic predisposition, accounted for by both ___ and ____ genes and also by poorly defined ________ factors
autoimmune destruction
endocrine glands
cause gland hyperfunction
50%
both MHC and non-MHC genes
poorly defined environmental factors
The second major group of pathologies that present as endocrine disease is ______ which may be either benign or malignant. Neoplastic cells arise as _______ clones within endocrine glands due to somatic mutations in growth factor _________ or commonly in G-protein coupled signaling pathways. They may produce disease due to either excessive and dysregulated production of biologically ______ hormones or damage to neighbouring, normal _______ cells with attendant loss of ________. Very small and benign _______ which would remain undetected in nonendocrine tissues may produce ____ disease by virtue of hormone hypersecretion coupled to the potency of hormones in eliciting biologic response
neoplasia
autonomous clones
growth factor signaling pathways or commonly in G-protein coupled signaling pathways
active hormones or damage to neighbouring, normal endocrine cells with attendant loss of hormone secretion
Very small and benign adenomas
florid disease
Diagnostic applications of immunglobulins
Describe enzyme-linked immunosorbent assay(ELISA)
ELISA method allows to analyze the presence of antigen(or sometimes antibodies) in different solutions, including serum, plasma, cell culture supernates, cell lysates, salivea, tissue lysates and urine. Smaple is placed on a plastic dish, Some plastics will irreversibly bind many types of macromolecules. Subsequently, the plate is washed and labeled(radioactive or enzyme tag) reactant is added, After washing again, the amount of bound labeled antibody is measured. Binding of the complex(antigen-antibody) may be detected with labeled anti-human globulin from an animal
Diagnostic applications of immunglobulins
Describe Direct ELISA
In a direct ELISA an antigen is immobilized directly on the plate and a conjugated detection antibody binds to the target protein. Substrate is then addded, producing a signal that is proportional to the amount of analyte in the sample
Diagnostic applications of immunoglobulins
Indirect ELISA
similar to a direct ELISA in taht an antigen is immobilized on a plate, but it includes an additional amplicfication detection step. First, an unconjugated primary detection antibody is added and binds to the spefic antigen. A conjugated secondary antibody directed afainst the host species of the primary antibody is then added, Substrate then produces a signal proportional to the amount of antigen bound in the well
Diagnostic applications of immunoglobulins
Sandwich ELISA
Sandwich ELISAs are the most common type of ELISA. 2 specific antibodies are used to sandwich the antigen commonly reffered to as amtched antibody pairs. Capture antibodyis coated on a microplate, sample is added, and the protein of interest binds and is immobilized on the plate. A conjugated-detection antibody is then added and binds to an additional epitope on the target protein, Substrate is added and produces a signal that is proportional to the amount of analyte present in the sample. Snadwich ELISAs are highly specific, since 2 antibodies are required to bind to the protein of interest
ELISA has found wide and ever increasing apllications in immunodiagnosius in clinical labs. ELISA tests are used as in in vitro diagnositcs for instance in:
detection of mycobacterium antibodies in tuberculosis
detection of rotavirus in feces
detection of hepatitis B markers in serum
detection of enterotoxin of E coli in feces
detection of HIV antibodies in blood samples
Describe the western blot
- technique the identified specific antibodies proteins that have been sperated from one another according ot their size bt gel elecreophoresis
- the blot is amembrane, composed of nitrcellulise or PVDF(polyvinylidene fluoride)
- gel is placed next membrane and application of an electric current induces the proteins in gel to move to the membrae where they adhere
- membrane becomes a replica of gel’s protein pattern and is detected by antibody which is linked to a reporter enzyme
- antibody is usulally linked to biotin or to a reporter enzyme such as alkaline phosphatase or horseradish peroxidase
Describe the western blot
When specific enzyme is exposed to an appropriate substrate dirves a colorimetric reaction and produces color in proportion to the amount of protein. A senstive sheet of photographic film is placed against the membrane and exposure to the light fromthe reaction creates an image os the antibodes bound to the blot. The diagnostic applications of Western blot are: confirmatory HIV test, confirmatory test for Hep Binfection, test for bovine pongiform encephalopathy (BSE, commonly refered to as mad cow disease).
Western blot image
The western blot procedure includes the following steps: gel elctrophoresis, transfer from gel to membrane and immunostaning of the blot
