Erythropoietin (EPO)

The role of red blood cells is to carry oxygen. Just like anything in the body, this is tightly regulated by a mechanism that monitors whether or not there is adequate oxygen getting to tissues and other cells. Hypoxia is detected by the peritubular fibroblasts of the kidneys which causes erythropoietin (EPO) to be released. The EPO gene has a hypoxia-sensing region in its 3’ regulatory component which causes hypoxia inducible factor-1 (HIF-1) transcription factor to be assembled and it interacts with the 3’ enhancer of the gene causing increased EPO mRNA and production of more EPO.  EPO is a true hormone, being produced in the kidneys, and acting upon another distant location being the bone marrow. When EPO binds to its ligand (receptor) on red blood cell progenitors it initiates a cascade which is mediated through the JAK2 signal transducers which ultimately effects the gene expression. EPO has three main physiological effects on the body; it allows early release of reticulocytes from the bone marrow, prevents apoptosis, and reduces the time needed for cells to mature in the bone marrow before release into the periphery. 

There are two mechanism for which EPO stimulates early release of red cell precurors into the bone marrow. It induces changes in the adventitial cell layer of the marrow sinuses that increases the width of the spaces that the red cells squeeze out of. It also down regulates red blood cell surface receptors for adhesive molecules that are located on the bone marrow stroma. As a result the red cells are able to pass through without the receptor so that they won’t bind to the stroma and delay release.

Apoptosis is programmed cell death. EPO inhibits apoptosis by removing the induction signal. Under normal physiology the bone marrow produces more CFU-Es than needed that are stored in the bone marrow which have a “head start” in the maturation process. About a 10 day head start in maturation. The CFU-Es (Colony-forming unit-erythroid) are red blood cell progenitor cells that develop from BFU-Es (Burst-forming unit-erythroid). Both BFU-E and CFU-E are red blood cell progenitor cells that develop into the pronormoblast, which is the first morphologically identifiable red blood cell precursor. If healthy, those cells live out there life span and undergo apoptosis. If there is a deficiency of red blood cell mass, those cells undergo maturation to be released, while simultaneously the apoptosis induction signal is inhibited. The normal death signal consists of a death receptor being FAS, on the membrane of the earliest red blood cell precursors (CFU-Es/BFU-Es), and FASL ligand on the maturing red blood cells precursors. When EPO levels are low, because there is adequate oxygen delivery the older FASL bearing cells cross-link with earlier FAS precursors which stimulates apoptosis. EPO is able to subdue apoptosis by stimulating the more mature precursors to be released from the marrow, especially in times of hypoxia. At which point there will no FASL bearing cells to cross-link the early FAS bearing precursors. Its a two fold effect, the more mature cells are released to help increase red cell mass in times of need, and the early precursor are allowed to mature and be released without undergoing apoptosis. When EPO binds to its ligand on the red blood cell activates the JAK2-STAT pathway, which ends in and up-regulation of transcription for BCL-2, which is an anti-apoptotic protein. This anti-apoptotic protein rests on the cell membrane and prevents the release of cytochrome c, which initiates apoptosis. 

JAKSTAT

EPO has an effect on the bone marrow transit time of a red blood cell precursor in two different ways; increased rate of cellular processes, and decreased cell cycle times. What this means is that EPO stimulates synthesis of red cell RNA, such as the production of hemoglobin. It also stimulates the production of egress-promoting surface molecules within the bone marrow which allow the red blood cells to flow through the marrow easier. EPO stimulates cells to enter cell cycle arrest earlier than normal, and as a result, spend less time maturing and are able to be released. These cells may appear larger in size and have a bluish tinge to their cytoplasm because of this.

Advertisements

Aspirin as a blood thinner?

Most people who have had previous cardiac issues, those who have even had a minor heart attack or survived a major infarction have often been prescribed to take an aspirin daily. To tackle this issue, its important to understand what a heart attack or an infarction actually is. Usually blood travels to the lungs, it gets oxygenated, and then travels through the coronary arteries to oxygenate the heart muscle itself. People over time can develop plagues that thin the artery lumen, or opening, eventually to the point where only a small amount of oxygenated blood can actually pass through. As a result, the heart can’t keep itself oxygenated. Without oxygen, tissues become hypoxic and die. When they die they release toxic cytokines and chemicals that damage tissue further, which coincidently we can objectively measure to determine whether an individual has experienced a heart attack. Heart attacks can come from a deep vein thrombosis, or an emboli as well. In that scenario, the clot actually happens somewhere else in the body and a piece of it breaks off and circulates until it gets to the heart and blocks the blood flow in the heart, causing an infarct.

Aspirin works as a blood thinner. It impairs the bodies ability to form a clot. What is a clot formed out of? Platelets. So aspirin directly targets a precursor to thromboxane A2, which activates downstream signaling to aggregate platelets and form a clot in primary hemostasis.

Synthesis of TXA2

F1.large

The synthesis of thromboxane A2 is through the Arachidonic Acid, Cyclooxygenase (COX) pathway. Phospholipids are converted to Arachidonic Acid catalyzed by phospholipase C or phospholipase A2. Arachidonic acid can at that point go to two pathways; the Lipooxygenase pathway, or the Cyclooxygenase pathway. There are two Cyclooxygenase peroxidase; COX-1 and COX-2. COX-1 mediates the pathway through which thromboxane A2 is going to be synthesized, and COX-2 mediates another pathway that works to synthesize prostaglandins which directly counteract the function of thromboxane A2. Its the bodies way of keeping homeostasis. For every action, there has to be an equal reaction. In the next step in the pathway, Arachidonic Acid is converted to Prostaglandin H2 (PGH2) by PGH2 synthase and COX-1/COX-2 working synergistically. Prostaglandin H2 is then converted to thromboxane A2 (TXA2) by thromboxane synthase. TXA2 is a vasoconstrictor and potent hypertensive agent.

So, how does aspirin come into play at all? Good thing you asked. Aspirin as it turns out irreversibly binds to COX-1. This antagonist effect stops the pathway and does not allow for the synthesis of thromboxane A2. Without TXA2, there will be no platelet aggregation, and no clot. Without primary hemostasis being established, coagulation, or secondary hemostasis, can’t take over to stabilize the clot with fibrin.

 

Thyroid 101

The thyroid gland is one branch of the endocrine system that is located in the neck that has two lobes connected by an isthmus. The hypothalamus secretes Thyrotropin-releasing hormone (TRH) which stimulates the anterior pituitary to secrete Thyroid-stimulating hormone (TSH). TSH acts on the thyroid gland to secrete the hormones triiodothyronine (T3) and thyroxine (T4). Within the circulation T4 is converted to its active metabolite T3. There are two different types of thyroid cells. Follicular cells produce the thyroid hormones T3 and T4. The parafollicular cells produce calcitonin. Calcitonin causes calcium reabsorption and maintains calcium homeostasis.

Synthesis of Thyroid Hormones is from iodine and tyrosine. Triiodothyronine (T3) has three atoms of iodine per molecule and thyroxine (T4) has four atoms of iodine per molecule. The hormones are created from thyroglobulin which is a protein within the follicular space that is originally created within the rough endoplasmic reticulum (RER). Thyroglobulin contains 123 units of tyrosine with reacts with iodine within the follicular space. A sodium-iodide symporter pumps iodide actively into the cell where it enters the follicular lumen from the cytoplasm by the transporter pendrin. In the colloid, iodide is oxidized to iodine by the enzyme called thyroid peroxidase. Iodine is very reactive and iodinates the thyroglobulin at tyrosyl residues in its protein chain. This forms the precursors of the thyroid hormones monoiodotyrosine (MIT), and diiodotyrosine (DIT). The adjacent tyrosyl residues are then paired together and subsequently the entire complex re-enters the follicular cell by endocytosis. Proteolysis liberates triiodothyronine and thyroxine and they enter the blood stream. Of the hormones secreted from the gland, 80-90% is T4, and only about 10-20% is T3. The production of T3, and T4 is primarily regulated by thyroid-stimulating hormone (TSH) which is released by the anterior pituitary gland. The thyroid hormones provide negative feedback to the thyrotropes TSH and TRH; when the thyroid hormones are high, TSH production is suppressed, and when levels are low, TSH secretion is increased.

After secretion, there is a very small percentage of the thyroid hormones that travel freely in the blood and that are metabolically active. Most are bound to thyroxine-binding globulin (TBG), transthyretin, and albumin. They act upon their respective tissues by crossing the cell membrane and binding to intracellular nuclear thyroid hormone receptors, which bind with hormone response elements and transcription factors to modulate DNA transcription. This modulation is what drives protein synthesis within the target tissue to actively project its physiological function within on the tissue and body.

Calcitonin is secreted by the parafollicular cells which helps maintain calcium homeostasis. Calcitonin is produced in response to high blood calcium. This causes inhibition of the release of calcium from the bone by decreasing the activity of osteoclasts. Osteoclasts are cells which break down bone. Bone is constantly reabsorbed by osteoclasts and created by osteoblasts, so calcitonin is effectively stimulates movement of calcium into bone. The effects of calcitonin are opposite of those of the parathyroid hormone (PTH) produced by the parathyroid gland.

Function

The primary function of the thyroid gland is the production of the thyroid hormones that have downstream metabolic, cardiovascular, and developmental effects. The basal metabolic rate is increased which effects all tissues. Gut adsorption and motility is increased. The generation, uptake by cells and breakdown of glucose is increased. The thyroid hormones also increase the breakdown of fats which increases free fatty acids, but contrary to believe, thyroid hormones decrease cholesterol levels.

There is an increase in cardiac output, as well as rate of breathing, intake and consumption of oxygen and increase in oxidative respiration within the mitochondria. These factors combine increase vascular pressure and elevate the bodies temperature.

The thyroid hormones are important for normal development. The cells of the developing brain are a major target for the thyroid hormones. They play a crucial role in brain maturation during fetal development. The thyroid hormones also play a role in maintaining normal sexual function, circadian sleep rhythm, and thought patterns.

The overarching effect is an augmented flight-or-fight response. It increases the release of the catecholamines which drives sympathetic innervation.

Clinical Significance

Hyperthyroidism is an excessive production of the thyroid hormones, most commonly a result of Graves Disease. Graves disease, also known as toxic diffuse goiter is an autoimmune disease that results in an enlarged thyroid. The exact cause is unknown, however it appears to be a combination of both genetic and environmental factors. An antibody, called the thyroid-stimulating immunoglobulin (TSI) which mimics the effect of TSH. This causes an increased T3 and T4, and an increased radio iodine uptake. Laboratory tests will show a decreased level of TSH, because there is negative feedback on the pituitary, but because of the antibody, production of the thyroid hormones continuous. One of the classic findings of Graves disease is exophthalmos, which is bulging of the eyes. This is often accompanied by irritability, muscle weakness, sleeping problems, increased heart rate and blood pressure and unintentional weight loss. Patients may complain of being “hot” all the time, illustrating a poor tolerance of heat.

Hypothyroidism is an underactive thyroid gland which results in decreased secretion of thyroid hormones. One of the most common causes is an autoimmune disorder called Hashimotos thyroiditis or chronic lymphocytic thyroiditis. The disease is characterized by gradual destruction of the thyroid gland. There are various antibodies that have been identified targeting against thyroid peroxidase, thyroglobulin, and TSH receptors. There is activation of cytotoxic T-cells in response to a cell mediated immune response affected by helper T-cells that drives thymocyte destruction. Cytokine release recruits macrophages within the gland to further drive destruction. Early on in the disease there may be no clinical evidence or symptoms of Hashimotos, but as the disease progresses, so does the clinical presentation. The most common symptoms are fatigue, weight gain, feeling cold, joint and muscle pain, depression, and bradycardia. This disease is about seven times more common in women than in men. Diagnosis is from TSH and T4 levels, imaging, along with other clinical symptoms. The thyroid gland may become firm, large and lobulated. There is lymphocytic infiltration and fibrosis that is seen.

These are not all the causes of hyper/hypothyroidism, but these are the most common, and in most cases, the most severe.

 

 

 

Pheochromocytoma Workup

A pheochromocytoma is a catecholamine-secreting tumor that arises from the chromatin cells of the adrenal medulla. Extraadrenal pheochromocytoma arise from the sympathetic ganglia and are referred to as catecholamine-secreting paragangliomas. These neoplasms are very rare, occurring in less than 0.2% of the patients. Most catecholamine-secreting tumors are sporadic and occur in the fourth to fifth decade of life, effecting both men and women equally. However, about 40% of the patients that present with a pheochromocytoma there is a familial inheritance. These familial tumors arise earlier in life and typically associated with several familial disorders. Such disorders are Von Hippel-Lindau (VHL) syndrome, multiple endocrine neoplasia type 2 (MEN2), and neurofibromatosis (NF1).

Clinical Presentation

Symptoms and signs only occur in about 50% of patients and paroxysmal in nature. There is a classic triad of symptoms that is observed which consists of episodic headache, sweating, and tachycardia with either paroxysmal hypertension or primary hypertension. Paroxysmal hypertension is the most common sign of pheochromocytoma. The headache associated with pheochromocytoma can vary from mild to severe and occurs in 90% of patients. Generalized sweating occurs accompanied by forceful palpitations, tremors, dyspnea, fatigue, and often anxiety and panic attack-type symptoms. There is increased secretion of catecholamines; epinephrine, norepinephrine, and dopamine which cause abnormalities in carbohydrate metabolism which leads to insulin resistance, and an impaired fasting glucose which mimics type 2 diabetes mellitus. In rare cases there is episodic hypotension and rapid cyclic fluctuations of hypertension and hypotension.

Initial Evaluation

The diagnosis of pheochromocytoma is made upon biochemical testing for catecholamine hypersecretion, followed by imaging studies to identify an adrenal tumor. There are many indications for testing that a physician may take into consideration before subjecting patients to many tests and appointments. Some of the indications for testing include the classic triad of symptoms (headache, sweating, and tachycardia), hyperadrenergic spells (palpitations, diaphoresis, tremor, pallor), onset of paroxysmal hypertension or primary hypertension at an early age, or any familial syndromes or history of pheochromocytoma.

Biochemical Testing

The range of biochemical testing that is performed is based upon the index of suspicion that the patient in fact has a pheochromocytoma. Low index of suspicion includes a 24-hour urinary fractionated catecholamines and metanephrines. If there is a high index of suspicion, it is recommended to use a plasma fractionated metanephrines. These tests are performed by high-performance liquid chromatography (HPLC) with tandem mass spectroscopy or electrochemical detection. The more recent techniques have overcome the traditional problems that are associated with drug interference and contrast agents.

Catecholamines

Catecholamines are an organic compound that are derived from the amino acid tyrosine. Tyrosine can either be derived from diet or synthesized from phenylalanine. Epinephrine, norepinephrine, and dopamine are the primary catecholamines that are secreted from the adrenal medulla during the sympathetic flight-or-fight response.

Norepinephrine is a neuromodulator and a hormone that circulates in the blood. Its primary function is to mobilize the brain and the body for action as part of the peripheral sympathetic system. In the flight-or-fight response norepinephrine causes arousal and alertness. It enhances the formation and retrieval of memory, and focuses attention. As a hormone it increases cardiac output by increasing the heart rate and blood pressure. It triggers glycolysis and increases vascular blood flow to the skeletal muscles.

Dopamine functions as a neurotransmitter that is released by neurons to send signals to other functioning nerve synapses. Dopamine plays a critical role in reward-motivated behavior. The anticipation of most types of rewards increase the levels of dopamine in the brain. Many addictive drugs mimic this pathway while simultaneously blocking the reuptake of it. Dopamine is also functional in the motor control pathway as a neuromodulator which controls the release of many other hormones. In circulation outside of the brain, dopamine functions as a chemical messenger. It inhibits norepinephrine release and acts as a vasodilator. It increases renal excretion of sodium. It acts to reduce insulin secretion and gastrointestinal motility.

Epinephrine, also known as adrenaline or adrenalin functions as a neurotransmitter, hormone, and as a medication. It plays an important role in increasing cardiac output, pupil dilation, and increasing insulin release to stimulate glycolysis. It acts on the alpha and beta receptors to ultimate activate the flight-or-fight response. Epinephrine is also used medically to treat a number of conditions including anaphylaxis, cardiac arrest, and bleeding.

Glycolysis

Glycolysis is the first phase of a series of reactions for the catabolism of carbohydrates. Catabolism is the breakdown of larger molecules into its respective smaller constituents. Glycolysis is the first part of cellular respiration that generates pyruvate to be used in either anaerobic respiration in the absence of oxygen or in the TCA cycle in aerobic respiration which yields useable energy for cells. This will be a general outline of the steps in glycolysis.

The whole process can be broken down into an energy investment phase where ATP is being used and an energy payoff phase where ATP is being generated. Fructose-1,6-biphosphate is where the energy investment phase ends. That is where the last ATP has to be used for energy to drive glycolysis.

A simple equation can be remembered as a summary of glycolysis.

Glucose + 2 ADP + 2 phosphate ions + 2 NAD+ —-> 2 Pyruvate + 2 ATP + 2 NADH + 2 H20 + 2 H+.

main-qimg-44f67c7dc25d5d4813b834c12922170d

In the first step of glycolysis an addition of a high energy phosphate from ATP yields glucose-6-phosphate and ADP. This step is initialized by the enzyme hexokinase. G6P is more reactive than glucose.

In the next step, glucose-6-phosphate is converted to its isomer, fructose-6-phosphate by phosphoglucose isomerase.

In the third step of glycolysis, fructose-6-phosphate is converted to fructose-1,6-biphosphate by phosphofructokinase (PFK) and the addition of ATP. This is the committed step, meaning that fructose-1,6-biphosphate MUST be converted to pyruvate. This is also the end of the energy investment phase of glycolysis.

Fructose-1,6-biphosphate is converted to glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate catalyzed by aldolase. Glyceraldehyde-3 phosphate maintains a reversible reaction with dihydroxyacetone phosphate through triode phosphate isomerase. The resulting reaction generates two molecules of glyceraldehyde-3-phosphate. A key point going forward is that two molecules of each substrate are produced.

Each G3P molecule gains an inorganic phosphate and with the addition of NAD+ to form the energized carrier molecules NADH. The resulting reaction catalyzed by glyeraldehyde-3-phosphate dehydrogenase generates two molecules of 1,3-bisphophoglycerate which yield two high energy phosphates.

Through the addition of two low energy ADP molecules and the enzyme phosphoglycerate kinase, the two molecules of 1,3-bisphophoglycerate are converted to 3-phosphoglycerate and yields two molecules of ATP. This reaction is called the break even reaction because at this point the energy input is equal to the energy output. Two molecules of ATP were expended and at this step there was a generation of two ATP molecules.

In the next step the two molecules of 3-phosphoglyercate are converted to 2-phosphoglycerate through the enzymatic properties of phosphoglycerate mutase.

The molecules of 2-phosphoglycerate are converted to phosphoenolpyruvate catalyzed by enolase. This step yields H20 molecules.

In the final step of glycolysis, the molecules of phosphoenolpyruvate are converted to pyruvate catalyzed by pyruvate kinase. ATP is generated from the addition of ADP and the two high energy phosphates from the molecules of phosphoenolpyruvate.

Upon the completion of glycolysis, the pyruvate molecules can be oxidized to carbon dioxide in cellular respiration to generate 28 molecules of ATP.

The NADH that is produced is turned back into NAD+ to drive further glycolysis. There are two ways to accomplish this. In the presence of oxygen NADH passes it electrons into the electron transport chain, which regenerates NAD+ for use in glycolysis. In the absence of oxygen, cells regenerate NAD+ by undergoing fermentation.