This document discusses how certain diseases are characterized by the accumulation of toxic iron in specific cell compartments. It explores using the oral chelator deferiprone to redistribute accumulated iron from areas of excess to areas of deprivation for physiological reuse. Deferiprone is shown to shuttle iron between organelles within cells and donate iron to transferrin and other acceptors. This unique property could potentially be exploited clinically to treat diseases involving regional iron accumulation such as Friedreich ataxia and anemia of chronic disease.
Preserved blood cells undergo progressive functional and structural changes that reduce oxygen delivery to tissues
The release of extracellular vesicles and cell-free DNA during storage may cause a hypercoagulable state
STORAGE LESION : amalgamation of reversible and irreversible changes that begin after 2 to 3 weeks of storage, progress with duration of storage and reduce red-cell function and viability after transfusion
1) The document reviews calcium signaling in plant cell organelles delimited by a double membrane, such as mitochondria, chloroplasts, and nuclei.
2) It finds that these organelles are able to take up calcium from their environment and generate their own calcium signals. They contain calcium-dependent processes.
3) Specifically, chloroplasts and mitochondria have transport systems that allow calcium entry and transport within the organelle. The nucleus also shows calcium-dependent activities and responses to stimuli with calcium increases.
The document discusses the stages of hematopoiesis from early development in the yolk sac and liver to the later medullary phase where the bone marrow becomes the primary site. It also covers the role of the spleen, lymph nodes, thymus and other organs in blood cell production and maturation. The physiology of red blood cell production, destruction, and regulating factors like erythropoietin are examined in detail.
This document discusses tissue response to injury at the cellular level. It defines cell injury and outlines the cellular responses, which can include adaptations, reversible injury, or irreversible injury. The causes of cell injury are also categorized, including genetic, hypoxic/ischemic, physical, chemical, microbial, immunological, nutritional, and psychological factors. The key stages of reversible injury are decreased ATP generation, reduced pH, sodium pump damage, decreased protein synthesis, and functional/ultrastructural changes. Irreversible injury is marked by mitochondrial dysfunction and membrane damage, leading to cell death through autolysis, necrosis, apoptosis, or gangrene. Aging is also discussed in terms of genetic, environmental, and oxidative stress factors.
Red blood cells (RBCs) constitute 99% of blood cells and function to transport oxygen to tissues and carbon dioxide from tissues to the lungs. RBCs contain hemoglobin which gives blood its red color. RBCs are biconcave discs that are highly elastic and can change shape to pass through small blood vessels. RBCs have a lifespan of around 120 days before being broken down. The RBC membrane is selectively permeable and helps maintain the cell's shape through osmotic changes in the blood. RBCs transport oxygen by hemoglobin binding to oxygen to form oxyhemoglobin and transporting it to tissues where it is released.
Red blood cells, also known as erythrocytes, are biconcave discs that contain hemoglobin which transports oxygen and carbon dioxide throughout the body. Key functions of red blood cells include oxygen transport from the lungs to tissues and carbon dioxide transport from tissues back to the lungs. The document discusses normal red blood cell counts and dimensions in different age groups and gender. It also covers abnormal red blood cell shapes and conditions they are associated with, as well as factors that affect red blood cell sedimentation rate and hematocrit levels.
This document provides an overview of a lecture on blood and hematopoiesis. It discusses the composition of blood including the different cell types. It covers topics like hemopoiesis, erythropoiesis, classification of anemias, hemoglobin and related disorders, white blood cells, blood coagulation, and some common blood diseases. The key cell types discussed in more detail include erythrocytes, neutrophils, eosinophils, basophils, and their roles in the immune response. Common blood disorders like sickle cell anemia and thalassemia are also summarized.
This document summarizes key concepts about blood physiology. It describes the components of blood, including red blood cells and hemoglobin, which transports oxygen. It discusses blood groups and reasons for transfusion reactions. It also outlines the process of hemostasis that restricts blood loss from damaged vessels, and the consequences of thrombosis. Additionally, it covers immunity and defense against microbes, the roles of hematopoietic growth factors and cytokines, and the mechanisms of innate, acquired, humoral, and cellular immunity.
Preserved blood cells undergo progressive functional and structural changes that reduce oxygen delivery to tissues
The release of extracellular vesicles and cell-free DNA during storage may cause a hypercoagulable state
STORAGE LESION : amalgamation of reversible and irreversible changes that begin after 2 to 3 weeks of storage, progress with duration of storage and reduce red-cell function and viability after transfusion
1) The document reviews calcium signaling in plant cell organelles delimited by a double membrane, such as mitochondria, chloroplasts, and nuclei.
2) It finds that these organelles are able to take up calcium from their environment and generate their own calcium signals. They contain calcium-dependent processes.
3) Specifically, chloroplasts and mitochondria have transport systems that allow calcium entry and transport within the organelle. The nucleus also shows calcium-dependent activities and responses to stimuli with calcium increases.
The document discusses the stages of hematopoiesis from early development in the yolk sac and liver to the later medullary phase where the bone marrow becomes the primary site. It also covers the role of the spleen, lymph nodes, thymus and other organs in blood cell production and maturation. The physiology of red blood cell production, destruction, and regulating factors like erythropoietin are examined in detail.
This document discusses tissue response to injury at the cellular level. It defines cell injury and outlines the cellular responses, which can include adaptations, reversible injury, or irreversible injury. The causes of cell injury are also categorized, including genetic, hypoxic/ischemic, physical, chemical, microbial, immunological, nutritional, and psychological factors. The key stages of reversible injury are decreased ATP generation, reduced pH, sodium pump damage, decreased protein synthesis, and functional/ultrastructural changes. Irreversible injury is marked by mitochondrial dysfunction and membrane damage, leading to cell death through autolysis, necrosis, apoptosis, or gangrene. Aging is also discussed in terms of genetic, environmental, and oxidative stress factors.
Red blood cells (RBCs) constitute 99% of blood cells and function to transport oxygen to tissues and carbon dioxide from tissues to the lungs. RBCs contain hemoglobin which gives blood its red color. RBCs are biconcave discs that are highly elastic and can change shape to pass through small blood vessels. RBCs have a lifespan of around 120 days before being broken down. The RBC membrane is selectively permeable and helps maintain the cell's shape through osmotic changes in the blood. RBCs transport oxygen by hemoglobin binding to oxygen to form oxyhemoglobin and transporting it to tissues where it is released.
Red blood cells, also known as erythrocytes, are biconcave discs that contain hemoglobin which transports oxygen and carbon dioxide throughout the body. Key functions of red blood cells include oxygen transport from the lungs to tissues and carbon dioxide transport from tissues back to the lungs. The document discusses normal red blood cell counts and dimensions in different age groups and gender. It also covers abnormal red blood cell shapes and conditions they are associated with, as well as factors that affect red blood cell sedimentation rate and hematocrit levels.
This document provides an overview of a lecture on blood and hematopoiesis. It discusses the composition of blood including the different cell types. It covers topics like hemopoiesis, erythropoiesis, classification of anemias, hemoglobin and related disorders, white blood cells, blood coagulation, and some common blood diseases. The key cell types discussed in more detail include erythrocytes, neutrophils, eosinophils, basophils, and their roles in the immune response. Common blood disorders like sickle cell anemia and thalassemia are also summarized.
This document summarizes key concepts about blood physiology. It describes the components of blood, including red blood cells and hemoglobin, which transports oxygen. It discusses blood groups and reasons for transfusion reactions. It also outlines the process of hemostasis that restricts blood loss from damaged vessels, and the consequences of thrombosis. Additionally, it covers immunity and defense against microbes, the roles of hematopoietic growth factors and cytokines, and the mechanisms of innate, acquired, humoral, and cellular immunity.
1. The document discusses disorders of red blood cells including abnormalities in hemoglobin structure like hemoglobinopathies and thalassemia. It also discusses anemias which result from low red blood cell count or hemoglobin amount such as iron deficiency anemia, megaloblastic anemia, and aplastic anemia.
2. White blood cells are also examined, including their types (granulocytes like neutrophils, eosinophils and basophils and agranulocytes like lymphocytes and monocytes), functions in the immune system, and disorders like leukemia.
3. The roles of different white blood cells are outlined, such as neutrophils phagocytizing bacteria, basoph
Red blood cells, also known as erythrocytes, are produced through erythropoiesis in the red bone marrow. Their main function is to transport oxygen from the lungs to tissues via hemoglobin. Red blood cells are biconcave disks that are flexible enough to squeeze through small blood vessels. They have a lifespan of about 120 days before being broken down and recycled. Factors involved in red blood cell production include erythropoietin, iron, vitamins, and minerals. Abnormalities can result in anemias or polycythemia.
Red blood cells are produced exclusively in the bone marrow from early gestation until age 5. Between ages 5-20, long bones also produce red blood cells but production decreases after age 20, with the marrow of ribs, sternum, vertebrae and pelvis taking over production. Red blood cells are derived from pluripotential hematopoietic stem cells in the bone marrow that differentiate through committed stem cell stages into erythrocytes. Tissue oxygen levels are the main regulator of red blood cell production, stimulating erythropoietin hormone secretion from the kidneys which enhances red blood cell formation. Maturation of red blood cells requires vitamins B12 and folic acid.
1) The document discusses blood physiology, describing the components and functions of blood including plasma, red blood cells, white blood cells, and platelets.
2) Plasma contains water, proteins, blood sugar, lipids, inorganic salts, hormones, enzymes, and gases which help maintain homeostasis, transport nutrients, remove waste, and fight infections.
3) Red blood cells contain hemoglobin and transport oxygen and carbon dioxide throughout the body. White blood cells help fight infections and disease. Platelets help the blood clot and repair damaged tissues.
The document describes various pathological conditions involving different organ systems:
1. Cloudy swelling of the kidney is described as reversible hydropic degeneration of renal tubular cells caused by impaired ion transport. Microscopy shows swollen cells filled with granules and intact nuclei.
2. Acute hemorrhagic pancreatitis results from activation of pancreatic enzymes, leading to autodigestion, hemorrhage, and fat necrosis. Microscopy shows destruction of pancreatic tissue and fat.
3. Cerebral necrosis is discussed, with stages from pallor to softening and cyst formation. Microscopy shows necrosis, inflammation, and phagocytic cells called Gitter cells clearing debris.
This document discusses cell injury and adaptation. It covers various types of cell injury including ischemia/hypoxia, chemical injury, and free radical-induced injury. It describes mechanisms of injury like lipid peroxidation and ways cells can adapt through processes like hyperplasia, hypertrophy, atrophy, and metaplasia. Adaptations can be physiological or pathological in response to stresses and insults on cells. The document provides examples of cell injury and adaptation in different clinical scenarios.
RBCs are formed through a process called erythropoiesis where hematopoietic stem cells in the bone marrow differentiate into red blood cells over 4 days. The kidney regulates RBC production by releasing erythropoietin in response to low blood oxygen levels. As RBCs age over 120 days, they are destroyed by macrophages in the spleen and liver, recycling the iron while the heme portion is broken down into bilirubin and excreted in the feces and urine.
This document discusses red blood cells (RBCs), including their definition, morphology, size, shape, counts, functions, nutritional requirements, hemoglobin structure and function, oxygen dissociation curve, production, metabolic pathways, and fate. RBCs, also known as erythrocytes, are biconcave discs that lack a nucleus and transport oxygen from the lungs to tissues via hemoglobin. Hemoglobin is an iron-containing protein made of globin and heme. RBC production occurs primarily in the bone marrow and is regulated by erythropoietin. RBCs undergo glycolysis and the pentose phosphate pathway for energy and biosynthesis.
This document discusses etiology and pathogenesis of cell injury. It defines cell injury as changes in a cell's internal and external environment due to various stresses from etiological agents. The cellular response depends on host factors like cell type and extent of injury. Injury can result in reversible or irreversible cell injury depending on factors like agent type/duration and cell adaptability. Common causes of cell injury include hypoxia, ischemia, toxins, microbes, nutrition imbalances, and aging. Ischemia and hypoxia are the most frequent causes of cell injury in humans. Reversible injury involves ATP depletion and membrane changes, while irreversible injury brings further damage including to mitochondria and nuclei, leading to cell death.
The document provides an overview of blood physiology, covering several key points in 3 or fewer sentences:
Blood serves the main functions of transport, homeostasis, and defense. It circulates constantly to carry out these roles. The document further discusses the composition of blood and the processes of hematopoiesis and hemostasis that generate and regulate blood cells.
Introduction
RBC
WBC
1. Granulocytes
Neutrophils
Eosinophil’s
Basophils
2. Agranulocytes
Lymphocytes
Monocyte
PLATELETS
Blood is a bright red, viscous, slightly alkaline fluid that accounts for approximately 7 % of total body weightThe average human has 5 litres of blood (Average Blood Volume is 4 to 6 liters).
It is a transporting fluid.
Red colour is due to the presence of oxyhaemoglobin.
Ph - 7.4 slightly alkaline.
Specific gravity - 1.060
Viscosity is 5 times greater then the water i.e thicker than water.
Blood is the only fluid tissue.
Blood is a complex connective tissue in which living cells, the formed elements, are suspended in fluid componenet called plasma.
The document discusses various types of cellular adaptation and injury:
1. Cells can adapt to increased demands through hypertrophy and hyperplasia, or reduced demand through atrophy.
2. Sublethal cellular damage includes swelling, fatty change, and is usually reversible. Prolonged stress leads to necrosis.
3. Necrosis is characterized by nuclear changes like pyknosis and karyorrhexis, loss of cell structure and integrity. Different patterns include coagulative, liquefactive and caseous necrosis.
Blood is connective tissue in fluid form that carries oxygen and nutrients throughout the body. It consists of plasma and formed elements including red blood cells, white blood cells, and platelets. Red blood cells contain hemoglobin and transport oxygen and carbon dioxide. White blood cells help fight infection and disease. The main types of white blood cells are neutrophils, lymphocytes, monocytes, eosinophils, and basophils. Erythropoiesis is the process where red blood cells are formed in the bone marrow. Hemoglobin in red blood cells binds to oxygen and carbon dioxide during respiration. Blood tests like complete blood count, ESR, and PCV are used to analyze blood components and detect diseases.
1) Hemoglobin research has been foundational to the development of molecular medicine over the last century. Studies of hemoglobin structure and function have contributed greatly to understanding human physiology and disease at the molecular level.
2) Hemoglobin transports oxygen through reversible binding to iron atoms in its heme groups. Recent research has also explored its interactions with other gases like nitric oxide (NO) that play important biological roles.
3) Continued study of hemoglobin seeks to further elucidate its molecular functions and implications for understanding related diseases, with the goal of developing new clinical applications and treatments. Research on hemoglobin has implications for molecular medicine broadly.
This document discusses ion channels, which are pore-forming membrane proteins that control the flow of ions across cell membranes. There are two main classes of ion channels: voltage-gated channels, whose gating is controlled by membrane polarization, and ligand-gated channels, whose gating is controlled by the binding of intracellular ligands. The document provides examples of different types of ion channels including potassium channels, calcium channels, chloride channels, GABA and glycine receptors. It also discusses some drugs that work by blocking specific ion channels and their potential applications.
This document discusses blood cells and immunity. It begins by describing the three main types of blood cells - red blood cells (RBCs), white blood cells (WBCs), and platelets. RBCs transport oxygen and carbon dioxide, WBCs fight infection and disease, and platelets help with blood clotting. The document then focuses on hematopoiesis, the formation of blood cells in the bone marrow, and the life cycles and functions of RBCs, WBCs like neutrophils and macrophages, and platelets. It also covers blood types, immunity, the inflammatory response, and various blood disorders like anemia.
This document outlines the various causes and mechanisms of cell injury. It discusses how oxygen deprivation, physical and chemical agents, infections, immune reactions, and nutritional imbalances can all lead to cell injury through mechanisms like ATP depletion, mitochondrial damage, calcium dysregulation, and oxidative stress. These mechanisms disrupt critical cellular processes and structures like membranes, ultimately leading cells to die through necrosis or apoptosis.
physiology of Blood and its current concepts in coagulationDr. Akash Ardeshana
This document provides information on blood, including its composition, functions, and properties. Some key points:
- Blood is composed of plasma and cells (red blood cells, white blood cells, platelets) and has a pH of 7.4.
- Plasma contains water (92-93%), proteins, gases, nutrients, hormones, and waste products.
- Red blood cells transport oxygen and carbon dioxide, help regulate pH, and are involved in blood typing. Hemoglobin is the iron-containing protein that gives blood its red color and allows for oxygen transport.
- White blood cells provide defense against infection and disease. There are various types with different functions.
- Blood properties like volume, pressure,
This document discusses white blood cells (WBCs), also known as leukocytes. It describes the 6 main types of WBCs, their concentrations in blood, how they are formed, and their functions. Neutrophils and macrophages defend against infections through processes like chemotaxis, phagocytosis, and releasing digestive enzymes. Eosinophils help fight parasitic infections and are involved in allergic reactions. Basophils also play a role in allergies. Lymphocytes are important for immunity. The document also discusses leukopenia, leukemia, and how these conditions affect the body.
A N A T O M Y & P H Y S I O L O G Y I I L A B Test 3 2 2michaeljordan
This document contains a detailed diagram and labeling of the major structures of the respiratory system, including the nose, pharynx, larynx, trachea, lungs, and related vasculature. Key structures that are labeled include the nasal cavities, epiglottis, vocal cords, trachea, primary and secondary bronchi, lungs, diaphragm, and pulmonary arteries and veins. Brief descriptions are also provided of the tissue types found in the trachea, bronchioles, and alveolar sacs.
Lcn webinar - from cliental to constituency with Natalie Finstadnisreenhaj
The document discusses a meeting between organizers from Tatua Kenya and the Leading Change Network to discuss addressing the dependency cycle through community organizing. The goals of the meeting were to learn about Tatua's work in ending dependency, identify actions to address dependency through organizing, and discuss collective responsibility. Participants discussed case studies and strategies for structuring work to empower communities to lead strategy creation. They also debated challenges of technical solutions versus adaptive change and how to build relationships on shared interests while redefining power dynamics. In conclusion, participants discussed how the issues affect their work and next steps to take collective responsibility in addressing dependency through organizing.
1. The document discusses disorders of red blood cells including abnormalities in hemoglobin structure like hemoglobinopathies and thalassemia. It also discusses anemias which result from low red blood cell count or hemoglobin amount such as iron deficiency anemia, megaloblastic anemia, and aplastic anemia.
2. White blood cells are also examined, including their types (granulocytes like neutrophils, eosinophils and basophils and agranulocytes like lymphocytes and monocytes), functions in the immune system, and disorders like leukemia.
3. The roles of different white blood cells are outlined, such as neutrophils phagocytizing bacteria, basoph
Red blood cells, also known as erythrocytes, are produced through erythropoiesis in the red bone marrow. Their main function is to transport oxygen from the lungs to tissues via hemoglobin. Red blood cells are biconcave disks that are flexible enough to squeeze through small blood vessels. They have a lifespan of about 120 days before being broken down and recycled. Factors involved in red blood cell production include erythropoietin, iron, vitamins, and minerals. Abnormalities can result in anemias or polycythemia.
Red blood cells are produced exclusively in the bone marrow from early gestation until age 5. Between ages 5-20, long bones also produce red blood cells but production decreases after age 20, with the marrow of ribs, sternum, vertebrae and pelvis taking over production. Red blood cells are derived from pluripotential hematopoietic stem cells in the bone marrow that differentiate through committed stem cell stages into erythrocytes. Tissue oxygen levels are the main regulator of red blood cell production, stimulating erythropoietin hormone secretion from the kidneys which enhances red blood cell formation. Maturation of red blood cells requires vitamins B12 and folic acid.
1) The document discusses blood physiology, describing the components and functions of blood including plasma, red blood cells, white blood cells, and platelets.
2) Plasma contains water, proteins, blood sugar, lipids, inorganic salts, hormones, enzymes, and gases which help maintain homeostasis, transport nutrients, remove waste, and fight infections.
3) Red blood cells contain hemoglobin and transport oxygen and carbon dioxide throughout the body. White blood cells help fight infections and disease. Platelets help the blood clot and repair damaged tissues.
The document describes various pathological conditions involving different organ systems:
1. Cloudy swelling of the kidney is described as reversible hydropic degeneration of renal tubular cells caused by impaired ion transport. Microscopy shows swollen cells filled with granules and intact nuclei.
2. Acute hemorrhagic pancreatitis results from activation of pancreatic enzymes, leading to autodigestion, hemorrhage, and fat necrosis. Microscopy shows destruction of pancreatic tissue and fat.
3. Cerebral necrosis is discussed, with stages from pallor to softening and cyst formation. Microscopy shows necrosis, inflammation, and phagocytic cells called Gitter cells clearing debris.
This document discusses cell injury and adaptation. It covers various types of cell injury including ischemia/hypoxia, chemical injury, and free radical-induced injury. It describes mechanisms of injury like lipid peroxidation and ways cells can adapt through processes like hyperplasia, hypertrophy, atrophy, and metaplasia. Adaptations can be physiological or pathological in response to stresses and insults on cells. The document provides examples of cell injury and adaptation in different clinical scenarios.
RBCs are formed through a process called erythropoiesis where hematopoietic stem cells in the bone marrow differentiate into red blood cells over 4 days. The kidney regulates RBC production by releasing erythropoietin in response to low blood oxygen levels. As RBCs age over 120 days, they are destroyed by macrophages in the spleen and liver, recycling the iron while the heme portion is broken down into bilirubin and excreted in the feces and urine.
This document discusses red blood cells (RBCs), including their definition, morphology, size, shape, counts, functions, nutritional requirements, hemoglobin structure and function, oxygen dissociation curve, production, metabolic pathways, and fate. RBCs, also known as erythrocytes, are biconcave discs that lack a nucleus and transport oxygen from the lungs to tissues via hemoglobin. Hemoglobin is an iron-containing protein made of globin and heme. RBC production occurs primarily in the bone marrow and is regulated by erythropoietin. RBCs undergo glycolysis and the pentose phosphate pathway for energy and biosynthesis.
This document discusses etiology and pathogenesis of cell injury. It defines cell injury as changes in a cell's internal and external environment due to various stresses from etiological agents. The cellular response depends on host factors like cell type and extent of injury. Injury can result in reversible or irreversible cell injury depending on factors like agent type/duration and cell adaptability. Common causes of cell injury include hypoxia, ischemia, toxins, microbes, nutrition imbalances, and aging. Ischemia and hypoxia are the most frequent causes of cell injury in humans. Reversible injury involves ATP depletion and membrane changes, while irreversible injury brings further damage including to mitochondria and nuclei, leading to cell death.
The document provides an overview of blood physiology, covering several key points in 3 or fewer sentences:
Blood serves the main functions of transport, homeostasis, and defense. It circulates constantly to carry out these roles. The document further discusses the composition of blood and the processes of hematopoiesis and hemostasis that generate and regulate blood cells.
Introduction
RBC
WBC
1. Granulocytes
Neutrophils
Eosinophil’s
Basophils
2. Agranulocytes
Lymphocytes
Monocyte
PLATELETS
Blood is a bright red, viscous, slightly alkaline fluid that accounts for approximately 7 % of total body weightThe average human has 5 litres of blood (Average Blood Volume is 4 to 6 liters).
It is a transporting fluid.
Red colour is due to the presence of oxyhaemoglobin.
Ph - 7.4 slightly alkaline.
Specific gravity - 1.060
Viscosity is 5 times greater then the water i.e thicker than water.
Blood is the only fluid tissue.
Blood is a complex connective tissue in which living cells, the formed elements, are suspended in fluid componenet called plasma.
The document discusses various types of cellular adaptation and injury:
1. Cells can adapt to increased demands through hypertrophy and hyperplasia, or reduced demand through atrophy.
2. Sublethal cellular damage includes swelling, fatty change, and is usually reversible. Prolonged stress leads to necrosis.
3. Necrosis is characterized by nuclear changes like pyknosis and karyorrhexis, loss of cell structure and integrity. Different patterns include coagulative, liquefactive and caseous necrosis.
Blood is connective tissue in fluid form that carries oxygen and nutrients throughout the body. It consists of plasma and formed elements including red blood cells, white blood cells, and platelets. Red blood cells contain hemoglobin and transport oxygen and carbon dioxide. White blood cells help fight infection and disease. The main types of white blood cells are neutrophils, lymphocytes, monocytes, eosinophils, and basophils. Erythropoiesis is the process where red blood cells are formed in the bone marrow. Hemoglobin in red blood cells binds to oxygen and carbon dioxide during respiration. Blood tests like complete blood count, ESR, and PCV are used to analyze blood components and detect diseases.
1) Hemoglobin research has been foundational to the development of molecular medicine over the last century. Studies of hemoglobin structure and function have contributed greatly to understanding human physiology and disease at the molecular level.
2) Hemoglobin transports oxygen through reversible binding to iron atoms in its heme groups. Recent research has also explored its interactions with other gases like nitric oxide (NO) that play important biological roles.
3) Continued study of hemoglobin seeks to further elucidate its molecular functions and implications for understanding related diseases, with the goal of developing new clinical applications and treatments. Research on hemoglobin has implications for molecular medicine broadly.
This document discusses ion channels, which are pore-forming membrane proteins that control the flow of ions across cell membranes. There are two main classes of ion channels: voltage-gated channels, whose gating is controlled by membrane polarization, and ligand-gated channels, whose gating is controlled by the binding of intracellular ligands. The document provides examples of different types of ion channels including potassium channels, calcium channels, chloride channels, GABA and glycine receptors. It also discusses some drugs that work by blocking specific ion channels and their potential applications.
This document discusses blood cells and immunity. It begins by describing the three main types of blood cells - red blood cells (RBCs), white blood cells (WBCs), and platelets. RBCs transport oxygen and carbon dioxide, WBCs fight infection and disease, and platelets help with blood clotting. The document then focuses on hematopoiesis, the formation of blood cells in the bone marrow, and the life cycles and functions of RBCs, WBCs like neutrophils and macrophages, and platelets. It also covers blood types, immunity, the inflammatory response, and various blood disorders like anemia.
This document outlines the various causes and mechanisms of cell injury. It discusses how oxygen deprivation, physical and chemical agents, infections, immune reactions, and nutritional imbalances can all lead to cell injury through mechanisms like ATP depletion, mitochondrial damage, calcium dysregulation, and oxidative stress. These mechanisms disrupt critical cellular processes and structures like membranes, ultimately leading cells to die through necrosis or apoptosis.
physiology of Blood and its current concepts in coagulationDr. Akash Ardeshana
This document provides information on blood, including its composition, functions, and properties. Some key points:
- Blood is composed of plasma and cells (red blood cells, white blood cells, platelets) and has a pH of 7.4.
- Plasma contains water (92-93%), proteins, gases, nutrients, hormones, and waste products.
- Red blood cells transport oxygen and carbon dioxide, help regulate pH, and are involved in blood typing. Hemoglobin is the iron-containing protein that gives blood its red color and allows for oxygen transport.
- White blood cells provide defense against infection and disease. There are various types with different functions.
- Blood properties like volume, pressure,
This document discusses white blood cells (WBCs), also known as leukocytes. It describes the 6 main types of WBCs, their concentrations in blood, how they are formed, and their functions. Neutrophils and macrophages defend against infections through processes like chemotaxis, phagocytosis, and releasing digestive enzymes. Eosinophils help fight parasitic infections and are involved in allergic reactions. Basophils also play a role in allergies. Lymphocytes are important for immunity. The document also discusses leukopenia, leukemia, and how these conditions affect the body.
A N A T O M Y & P H Y S I O L O G Y I I L A B Test 3 2 2michaeljordan
This document contains a detailed diagram and labeling of the major structures of the respiratory system, including the nose, pharynx, larynx, trachea, lungs, and related vasculature. Key structures that are labeled include the nasal cavities, epiglottis, vocal cords, trachea, primary and secondary bronchi, lungs, diaphragm, and pulmonary arteries and veins. Brief descriptions are also provided of the tissue types found in the trachea, bronchioles, and alveolar sacs.
Lcn webinar - from cliental to constituency with Natalie Finstadnisreenhaj
The document discusses a meeting between organizers from Tatua Kenya and the Leading Change Network to discuss addressing the dependency cycle through community organizing. The goals of the meeting were to learn about Tatua's work in ending dependency, identify actions to address dependency through organizing, and discuss collective responsibility. Participants discussed case studies and strategies for structuring work to empower communities to lead strategy creation. They also debated challenges of technical solutions versus adaptive change and how to build relationships on shared interests while redefining power dynamics. In conclusion, participants discussed how the issues affect their work and next steps to take collective responsibility in addressing dependency through organizing.
Este documento describe el desarrollo del ensayo inmunoenzimático (E.L.I.S.A.), desde su creación en 1960 hasta su uso actual. El E.L.I.S.A. permite detectar y cuantificar antígenos, anticuerpos u otras sustancias biológicas mediante la unión específica antígeno-anticuerpo y el uso de un marcador enzimático. El E.L.I.S.A. se ha aplicado ampliamente en medicina, industria alimentaria y estudios ambientales debido a
El documento presenta el menú semanal de comidas y meriendas de martes a domingo, incluyendo desayuno, media mañana, comida, merienda y cena. Cada día también incluye los puntos extras de frutas y verduras, calcio, grasas vegetales y bebidas. El sábado solo presenta el desayuno. Al final se incluye un enlace a la página web donde se encuentra el menú completo.
The document discusses the career and interests of an individual who spent 7 years at Microsoft and is passionate about digital health, technology innovation, new business models, and start-ups. It also explores trends in areas like wearable devices, mobile health sensors, digital games for medical adherence and training, and communities for patients and health professionals to connect, share, and learn.
Culture shock is a natural part of adapting to a new culture. While it can cause some initial discomfort, gradually becoming involved with local activities and people helps the differences and similarities become clearer. Living abroad necessarily exposes one to new lifestyles and cultures, which can be difficult to adjust to but also leads to a new appreciation for cultural differences. With an open mind and positive attitude, one can overcome culture shock through this process of cultural adaptation.
El documento habla sobre la enfermedad coronaria, incluyendo sus factores de riesgo, síndromes coronarios agudos, biomarcadores, tratamientos médicos, percutáneos y quirúrgicos. Define la angina estable e inestable, así como el infarto agudo de miocardio con y sin elevación del segmento ST. Resalta que la enfermedad coronaria es una de las principales causas de muerte y que su tratamiento ha mejorado gracias a avances tecnológicos.
Anemia is a common condition of cancer patients. This is because cancers cause inflammation that decrease red blood cell production. In addition, many chemotherapies are myelosuppressive, meaning they slow down the production of new blood cells by the bone marrow.
Anemia is a common condition of cancer patients. This is because cancers cause inflammation that decrease red blood cell production. In addition, many chemotherapies are myelosuppressive, meaning they slow down the production of new blood cells by the bone marrow.
This document describes several methods used to assess iron status in the body. It discusses how iron is incorporated into hemoglobin and transported by transferrin. Key methods mentioned include measuring hemoglobin concentration, red blood cell indices, serum iron, total iron binding capacity, transferrin saturation, serum transferrin receptor, red cell zinc proporphyrin, red cell ferritin, hypochromic red cells, serum ferritin, and tissue biopsy iron staining of bone marrow. Each method is outlined with normal reference ranges, diagnostic uses, and potential confounding factors.
The cell membrane is semi-permeable, allowing some substances to pass through while blocking others. It consists of a phospholipid bilayer with hydrophobic tails facing inward and hydrophilic heads facing outward. Proteins embedded in the membrane control what enters and exits the cell. The fluid bilayer structure allows for lateral movement of components but flipping from one side to the other requires significant energy due to hydrophobic and hydrophilic interactions. Lipid rafts are domains within the membrane involved in specific functions. Changing temperature can increase fluidity but the bilayer structure is maintained and regions cannot flip inside out.
Copper induces cell death by targeting lipoylated TCA cycle proteins. The document summarizes a study that found copper ionophores induce regulated cell death dependent on intracellular copper accumulation. Genome-wide CRISPR screens identified that killing by copper ionophores was rescued by knockout of genes including FDX1 and those encoding components of the lipoic acid pathway. The study found copper directly binds and induces oligomerization of the lipoylated TCA cycle protein DLAT, resulting in a toxic gain of function. This identifies a novel mechanism where copper-induced cell death is mediated by targeting protein lipoylation in the TCA cycle.
This document discusses hepcidin, a peptide hormone that plays a central role in regulating iron metabolism. It is predominantly synthesized by hepatocytes and works by binding to ferroportin, a cellular iron exporter, causing its internalization and degradation. This decreases iron absorption from the diet and iron release from cells. The document reviews hepcidin's structure, kinetics, function, and regulation. It also discusses how hepcidin levels are altered in various iron disorders and inflammatory states. Reliable assays have been developed to measure hepcidin levels, showing promise for diagnosing iron disorders, though more research is needed before clinical use.
This document discusses anemia of chronic disease (ACD), beginning with two case scenarios of patients presenting with anemia. It then provides an overview of anemia classification and the pathophysiology of ACD. ACD is the most common type of anemia in hospitalized patients. It results from immune activation and chronic inflammation, which stimulate hepcidin production. Hepcidin restricts iron absorption and macrophage iron release, leading to iron retention and impairment of erythropoiesis. Cytokines also directly impair erythropoiesis and erythropoietin production. Together, these factors contribute to the reduced red blood cell survival characteristic of ACD.
This document discusses red blood cells and erythropoiesis. It provides information on:
- The primary function of red blood cells is to carry oxygen from the lungs to tissues and carbon dioxide from tissues to the lungs.
- Erythropoiesis is the process where stem cells in the bone marrow mature into red blood cells. It is regulated by the hormone erythropoietin which stimulates red blood cell production.
- Important micronutrients required for erythropoiesis include iron, vitamin B12, and folate. Deficiencies in these "haematinics" can lead to megaloblastic or iron deficiency anemia.
The document summarizes cell injury, adaptation, and death. It discusses various types of cell death including necrosis, apoptosis, necrapoptosis, and anoikis. Causes of cell injury include internal stresses and external factors. Cells can respond to injury through recovery, adaptation mechanisms like atrophy, hypertrophy, hyperplasia, metaplasia, and storage, or death. Adaptation responses aim to restore homeostasis. The stages of cell injury and mechanisms of apoptosis and necrosis are described in detail.
Iron is required by every cell and can interconvert between ferrous and ferric forms, making it useful for oxygen-binding molecules and enzymes. Free iron is highly toxic, but proteins bind iron to reduce this effect. The main roles of iron are to carry oxygen as part of hemoglobin and provide oxygen delivery to tissues. Iron circulates in the blood bound to transferrin and is stored in cells bound to ferritin. Eighty percent of iron passing through the plasma transferrin pool is recycled from broken-down red blood cells, and storage iron is derived from phagocytosis of senescent erythrocytes by macrophages in the liver, bone marrow, and spleen.
Iron chelators in treatment of iron overload syndromesDR RML DELHI
This document reviews different iron chelators used to treat iron overload syndromes. It discusses the main iron chelators - deferoxamine, deferiprone, and deferasirox. Each chelator has advantages and disadvantages in terms of target diseases, levels of iron deposition, and patient symptoms, making the best choice complex. Proper evaluation of iron overload is important for monitoring chelation therapy effectiveness through measures like serum ferritin, liver biopsy, MRI. Chelation aims to prevent excess iron accumulation and related organ dysfunction through safely removing iron from the body.
The document provides information on Wilson's disease, including:
1) Wilson's disease is an inherited disorder of copper metabolism caused by mutations in the ATP7B gene. This leads to copper accumulation in the liver, brain, kidney, and cornea.
2) Clinical presentations can include hepatic (e.g. cirrhosis), neurological (e.g. movement disorders), psychiatric, and ocular features like Kayser-Fleischer rings.
3) Diagnosis involves low serum ceruloplasmin, elevated urinary copper, genetic testing, and liver biopsy. Treatment involves chelation therapy and sometimes liver transplantation for severe cases.
This review article discusses ion channels, which are proteins that generate and regulate electrical signals in cells. Defects in ion channel proteins can cause diseases like cystic fibrosis, long QT syndrome, and various inherited muscle disorders. The article focuses on the physiology of ion channels and how recent advances in molecular cloning have identified the genes encoding several ion channels. It also discusses two prominent diseases caused by defective ion channels - cystic fibrosis and long QT syndrome - as well as two specific ion channels, ENaC and CLCN5, whose identification raises prospects for new treatments.
Modulation of MMP and ADAM gene expression in human chondrocytes by IL-1 and OSMpjtkoshy
The document examines the effects of interleukin-1 (IL-1) and oncostatin M (OSM) on the expression of matrix metalloproteinase (MMP), ADAM, and ADAM-TS genes in human chondrocytes. The study finds that IL-1 and OSM synergistically induce expression of the collagen-degrading enzymes MMP-1, MMP-8, MMP-13, and MMP-14 as well as the aggrecan-degrading enzyme ADAM-TS4. In particular, MMP-1, MMP-3, and MMP-13 expression is induced early, while MMP-8 expression occurs later. IL-1 and OSM also synergistically induce MMP
This document discusses iron refractory iron deficiency anemia (IRIDA). It defines key terms like anemia and iron deficiency. It describes normal iron metabolism and the role of transport proteins like DMT1 and ferroportin in intestinal iron absorption. Hepcidin regulates iron transport by binding to ferroportin and inducing its degradation. Mutations in ferroportin can cause iron overload by preventing its binding to hepcidin. The document also discusses rare conditions like atransferrinemia caused by near absence of plasma transferrin.
This document discusses iron overload and its management. It begins by describing normal iron physiology and causes of excess iron accumulation, including hereditary hemochromatosis and multiple blood transfusions. Signs of iron overload include organ damage to the liver, heart, endocrine glands, and joints. Diagnosis involves blood tests of ferritin and transferrin saturation as well as MRI to measure iron levels. Treatment involves iron chelation therapy using drugs like deferoxamine, deferiprone, and deferasirox to remove excess iron from the body.
1) Red blood cells undergo numerous biochemical changes during storage known as the "storage lesion" that may impair their function. These include loss of nitric oxide, potassium leakage into plasma, decreased 2,3-DPG and pH.
2) Retrospective studies show patients receiving older stored blood have worse outcomes like increased mortality, morbidity and organ dysfunction.
3) A case study describes a patient who developed hyperkalemia and cardiac arrest after receiving very old stored blood, highlighting the risks of potassium leakage from aged red blood cells.
1) The study examined the role of Rho kinase in T cell activation and immune responses.
2) Inhibition of Rho kinase attenuated T cell proliferation, cytokine gene expression, actin polymerization, and aggregation of T cell receptors.
3) Treatment with a Rho kinase inhibitor prolonged survival of allogeneic heart transplants in mice and diminished cytokine mRNA expression in the transplants.
4) Rho kinase promotes structural rearrangements in T cells that are critical for T cell signaling and activation during cellular immune responses.
The skin is the largest organ and its health plays a vital role among the other sense organs. The skin concerns like acne breakout, psoriasis, or anything similar along the lines, finding a qualified and experienced dermatologist becomes paramount.
Computer in pharmaceutical research and development-Mpharm(Pharmaceutics)MuskanShingari
Statistics- Statistics is the science of collecting, organizing, presenting, analyzing and interpreting numerical data to assist in making more effective decisions.
A statistics is a measure which is used to estimate the population parameter
Parameters-It is used to describe the properties of an entire population.
Examples-Measures of central tendency Dispersion, Variance, Standard Deviation (SD), Absolute Error, Mean Absolute Error (MAE), Eigen Value
Spontaneous Bacterial Peritonitis - Pathogenesis , Clinical Features & Manage...Jim Jacob Roy
In this presentation , SBP ( spontaneous bacterial peritonitis ) , which is a common complication in patients with cirrhosis and ascites is described in detail.
The reference for this presentation is Sleisenger and Fordtran's Gastrointestinal and Liver Disease Textbook ( 11th edition ).
Travel vaccination in Manchester offers comprehensive immunization services for individuals planning international trips. Expert healthcare providers administer vaccines tailored to your destination, ensuring you stay protected against various diseases. Conveniently located clinics and flexible appointment options make it easy to get the necessary shots before your journey. Stay healthy and travel with confidence by getting vaccinated in Manchester. Visit us: www.nxhealthcare.co.uk
Giloy in Ayurveda - Classical Categorization and SynonymsPlanet Ayurveda
Giloy, also known as Guduchi or Amrita in classical Ayurvedic texts, is a revered herb renowned for its myriad health benefits. It is categorized as a Rasayana, meaning it has rejuvenating properties that enhance vitality and longevity. Giloy is celebrated for its ability to boost the immune system, detoxify the body, and promote overall wellness. Its anti-inflammatory, antipyretic, and antioxidant properties make it a staple in managing conditions like fever, diabetes, and stress. The versatility and efficacy of Giloy in supporting health naturally highlight its importance in Ayurveda. At Planet Ayurveda, we provide a comprehensive range of health services and 100% herbal supplements that harness the power of natural ingredients like Giloy. Our products are globally available and affordable, ensuring that everyone can benefit from the ancient wisdom of Ayurveda. If you or your loved ones are dealing with health issues, contact Planet Ayurveda at 01725214040 to book an online video consultation with our professional doctors. Let us help you achieve optimal health and wellness naturally.
STUDIES IN SUPPORT OF SPECIAL POPULATIONS: GERIATRICS E7shruti jagirdar
Unit 4: MRA 103T Regulatory affairs
This guideline is directed principally toward new Molecular Entities that are
likely to have significant use in the elderly, either because the disease intended
to be treated is characteristically a disease of aging ( e.g., Alzheimer's disease) or
because the population to be treated is known to include substantial numbers of
geriatric patients (e.g., hypertension).
Are you looking for a long-lasting solution to your missing tooth?
Dental implants are the most common type of method for replacing the missing tooth. Unlike dentures or bridges, implants are surgically placed in the jawbone. In layman’s terms, a dental implant is similar to the natural root of the tooth. It offers a stable foundation for the artificial tooth giving it the look, feel, and function similar to the natural tooth.
The biomechanics of running involves the study of the mechanical principles underlying running movements. It includes the analysis of the running gait cycle, which consists of the stance phase (foot contact to push-off) and the swing phase (foot lift-off to next contact). Key aspects include kinematics (joint angles and movements, stride length and frequency) and kinetics (forces involved in running, including ground reaction and muscle forces). Understanding these factors helps in improving running performance, optimizing technique, and preventing injuries.
Breast cancer: Post menopausal endocrine therapyDr. Sumit KUMAR
Breast cancer in postmenopausal women with hormone receptor-positive (HR+) status is a common and complex condition that necessitates a multifaceted approach to management. HR+ breast cancer means that the cancer cells grow in response to hormones such as estrogen and progesterone. This subtype is prevalent among postmenopausal women and typically exhibits a more indolent course compared to other forms of breast cancer, which allows for a variety of treatment options.
Diagnosis and Staging
The diagnosis of HR+ breast cancer begins with clinical evaluation, imaging, and biopsy. Imaging modalities such as mammography, ultrasound, and MRI help in assessing the extent of the disease. Histopathological examination and immunohistochemical staining of the biopsy sample confirm the diagnosis and hormone receptor status by identifying the presence of estrogen receptors (ER) and progesterone receptors (PR) on the tumor cells.
Staging involves determining the size of the tumor (T), the involvement of regional lymph nodes (N), and the presence of distant metastasis (M). The American Joint Committee on Cancer (AJCC) staging system is commonly used. Accurate staging is critical as it guides treatment decisions.
Treatment Options
Endocrine Therapy
Endocrine therapy is the cornerstone of treatment for HR+ breast cancer in postmenopausal women. The primary goal is to reduce the levels of estrogen or block its effects on cancer cells. Commonly used agents include:
Selective Estrogen Receptor Modulators (SERMs): Tamoxifen is a SERM that binds to estrogen receptors, blocking estrogen from stimulating breast cancer cells. It is effective but may have side effects such as increased risk of endometrial cancer and thromboembolic events.
Aromatase Inhibitors (AIs): These drugs, including anastrozole, letrozole, and exemestane, lower estrogen levels by inhibiting the aromatase enzyme, which converts androgens to estrogen in peripheral tissues. AIs are generally preferred in postmenopausal women due to their efficacy and safety profile compared to tamoxifen.
Selective Estrogen Receptor Downregulators (SERDs): Fulvestrant is a SERD that degrades estrogen receptors and is used in cases where resistance to other endocrine therapies develops.
Combination Therapies
Combining endocrine therapy with other treatments enhances efficacy. Examples include:
Endocrine Therapy with CDK4/6 Inhibitors: Palbociclib, ribociclib, and abemaciclib are CDK4/6 inhibitors that, when combined with endocrine therapy, significantly improve progression-free survival in advanced HR+ breast cancer.
Endocrine Therapy with mTOR Inhibitors: Everolimus, an mTOR inhibitor, can be added to endocrine therapy for patients who have developed resistance to aromatase inhibitors.
Chemotherapy
Chemotherapy is generally reserved for patients with high-risk features, such as large tumor size, high-grade histology, or extensive lymph node involvement. Regimens often include anthracyclines and taxanes.
Full Handwritten notes of RA by Ayush Kumar M pharm - Al ameen college of pha...
Blood 2008-sohn-1690-9
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2008 111: 1690-1699
Prepublished online November 1, 2007;
doi:10.1182/blood-2007-07-102335
Redistribution of accumulated cell iron: a modality of chelation with
therapeutic implications
Yang-Sung Sohn, William Breuer, Arnold Munnich and Z. Ioav Cabantchik
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BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3 REDISTRIBUTION OF IRON AS THERAPEUTIC STRATEGY 1691
Table 1. Iron binding parameters of agents used in this study tion onto 96-well plates, or onto microscopic slides attached to perforated
Iron ligands (abbreviations) pFe(III)* tissue culture 3-cm plates.
Probe loading into cells. For cytosolic loading of CALG, cells in
Calcein green (CALG) 20.3†
DMEM plus 10mM Na-HEPES (DMEM-HEPES) at 37°C were exposed
Deferrioxamine (DFO) 26.6
for 10 minutes to 0.25 M CALG-AM, washed and incubated in
Deferiprone (DFP) 19.3
DMEM-HEPES containing 0.5 mM probenecid (to minimize probe leak-
Diethylene-triamine-pentaacetic acid (DTPA) 28.2
age). For CALG-Fe(III) (1:1) complexes and for sulforhodamine loading
Nitrilotriacetic acid (NTA) 18.1
into endosomes, cells were exposed to 50 M to 100 M complex in
Transferrin (Tf) 22.3
DMEM-HEPES for 30 minutes at 37°C and washed extensively with the
Rhodamine B- (1, 10-phenanthrolin-5-yl) 7.7 (11.5 for Fe(II))‡
same probe-free medium and subsequently with HEPES-buffered saline
aminocarbonyl benzyl ester (RPA)
(HBS; 20 mM HEPES, 135 mM NaCl; pH 7.4).20,21 RPA was loaded into
*pFe(III) values are log M , where M is the concentration of the free metal ion mitochondria as described in earlier studies.21-23
when ligand 10 M and Fe(III) 1 M at pH 7.4. Data from Harris.17 Histone-CALG. For loading into the nucleus, CALG was coupled to
†Data from Thomas et al.18 core histones from calf thymus (H) 7.2 mg/mL, in 50 mM Na-MES,
‡Values shown are for 1,10-phenanthroline.
100 mM NaCl, pH 5.5, containing 1.25 mM CALG:Cobalt (Co protects
CALG metal-binding groups). Coupling was initiated by adding 12 mM
EDC, followed by incubation for 1 hour at room temperature and overnight
found to reduce iron accumulation in the dentate nuclei of at 5°C. After exhaustive dialysis, 11 mM DTPA was added (to remove
Friedreich ataxia patients,13 indicating its ability to cross the Co(II)) and histone-CALG (H-CALG) was further dialyzed against HBS.
blood-brain barrier in humans, which is consistent with previous CALG coupling to H was estimated at 1.1:1 by fluorescence ( excitation
findings in animals.14-16 480 nm, emission 520 nm). H-CALG-Fe was prepared by titrating CALG
In the present study we assess DFP’s capacity to act as an with FAS. Loading 10 M H-CALG or H-CALG-Fe into cells was done in
iron-relocating agent at the cellular level. To trace labile iron DMEM-HEPES containing 100 M chloroquine (for improved FMS
mobilized by chelators, we used model cardiac and macrophage targeting to cell nuclei),24 followed by HBS washes.
cells in culture and fluorescent metal sensors (FMS) targeted to Fluorescein-DFO-histone. The probe was prepared by coupling of
cellular and extracellular compartments. We show that DFP can N-(fluorescein-5-thiocarbamoyl)-desferrioxamine (Evrogen, Moscow, Rus-
sia) to H using fluorescein-DFO (FlDFO)–Fe (1:1) complex generated by
relocate iron accumulated in cell compartments within and across
adding 1.1 mM FeSO4 to 1 mM FlDFO in HBS. To 2.64 mL of FlDFO-Fe
the cell, and we demonstrate that iron withdrawn from sites of cell
complex was added 8.5 mg H, followed by 0.18 mL of 1 M MES (Na form),
accumulation can be safely transferred to extracellular transferrin pH 6.5, and 15 mg of EDC. After overnight agitation at 5°C, the mixture
for physiologic reuse. was dialyzed (3.5 kDa cut-off tubing) against 10 mM acetic acid;
1 mM EDTA; 150 mM NaCl, pH 4.5 (to remove bound iron); and finally
HBS. The fluorescein-DFO-histone (H-FlDFO) ratio was 1:1, based on
stoichiometric iron-quenching of H-FlDFO fluorescence ( excitation
Methods 494 nm, emission 520nm). H-FlDFO, like H-CALG, was loaded into cells
in DMEM-HEPES.
Materials
Rhodamine-labeled apotransferrin. Human apotransferrin (4 mg/mL
Calcein green (CALG) 3,3 -bis[N,N-bis(Carboxymethyl) aminomethyl]fluo- in 25 mM Na2CO3, 75 mM NaHCO3; pH 9.8), was incubated at 5°C
rescein and its acetomethoxy (AM) precursors CALG-AM, lissamine overnight with 1 mM lissamine rhodamine sulfonyl chloride and the labeled
rhodamine sulfonyl chloride, and sulforhodamine were from Molecular protein isolated by gel filtration on Sepharose G25 (Sigma-Aldrich)
Probes (Eugene, OR). Human serum holotransferrins and apotransferrins preequilibrated with 150 mM NaCl, 20 mM MES; pH 5.3.
(aTf) were from Kamada (Rehovot, Israel). Diethylene-triamine- Transfer of iron from DFP-Fe to apotransferrin. DFP-Fe complexes
pentaacetic acid (DTPA), nitrilotriacetate (NTA), EDC (1-ethyl-3-(3- were generated by incubating Fe-NTA (50:150 M) with either 150 M or
dimethylaminopropyl) carbodiimide), ferric ammonium citrate (FAC), 250 M DFP in HBS for 20 minutes, and completion of complex formation
ferrous ammonium sulfate (FAS), succinyl acetone, HEPES (N-2- was assessed by absorption at 455 nm. The complexes were mixed with
hydroxyethylpiperazine-N -2-ethanesulfonic acid), 4-morpholine ethanesul- 50 M aTf and 25 mM NaHCO3 and incubated for 1.5 hours in a 5% CO2
fonic acid (MES), core histone mixture from calf thymus (H), N,N - incubator. Because of the large spectral overlap of DFP-Fe and transferrin-
hexamethylene-bis-acetamide, and octyl-glucoside were from Sigma- Fe, we added DTPA (10 mM) to selectively scavenge iron from DFP-Fe.
Aldrich (St Louis, MO). Rhodamine isothiocyanate was from Fluka (Buchs, Transferrin that was free of low-molecular components was obtained by
Switzerland). DFP (1,2-dimethyl-3-hydroxypyridin-4-one) was from Apo- either filtering 0.5 mL through 30-kDa cut-off spin filters (Pall, East Hills,
Pharma (Toronto, ON). DFO was from Novartis (Basel, Switzerland). The NY) via centrifugation (2900g for 20 minutes), or by use of a 5-mL
red fluorescent mitochondrial metal–sensor rhodamine B-[(1, 10-phenanth- dry-spin–gel filtration-centrifugation at 1100g over precentrifuged Seph-
rolin-5-yl aminocarbonyl] benzyl ester (RPA), was a kind gift from U. adex G-50 medium columns (Sigma-Aldrich). The complex-free transferrin
Rauen and R. Sustmann, University of Duisburg-Essen, Essen, Germany. was diluted in HBS, pH 7.4. Tf-Fe was analyzed by absorbance at 465 nm,
A summary of the probes used and their properties is given in Table 1. and aTf by tryptophan fluorescence (280 nm excitation, 306 nm emission;
Felix spectrofluorimeter station version 2.5; Photon Technology Interna-
tional, Lawrenceville, NJ).
Methods
Iron-loading of cells was done by overnight incubation of cells with
Complexes of iron with NTA (Fe-NTA) were generated by mixing FAS and 100 M FAC in culture conditions followed by washing with HBS
NTA (1:3 molar ratio) in water and allowing iron to oxidize in ambient containing 100 M DFO, and with HBS alone. To achieve selective
conditions. The DFP-Fe(III) complexes were generated by mixing Fe-NTA accumulation of iron in mitochondria, cells were treated with 1 mM
with DFP in water; formation of fully substituted DFP-Fe complexes was succinyl acetone for 3 hours at 37°C.
confirmed by measuring absorbance at 455 nm.19 Cell hemoglobin synthesis. MEL cells cultured for 2 days in media
Cell-culture lines. Rat H9C2 cardiomyocytes, J774 mouse macro- supplemented with 5mM hexamethylene-bis-acetamide were washed and
phages, and erythroleukemia (MEL) were grown in 5% CO2 Dulbecco- lysed with octyl-glucoside (1.5%). After 2 minutes centrifugation at
modified Eagle medium (DMEM) supplemented with 10% fetal calf serum, 12 000g, 100 L of lysate supernatants was transferred to a 96-well
4.5 g/L D-glucose, glutamine, and antibiotics (Biological Industries, microplate for estimating hemoglobin (by measuring absorbance at
Kibbutz Bet Haemek, Israel). Cells were plated 1 day before experimenta- 410 nm). Alternatively, lysates were mixed with 100 L freshly prepared
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1692 SOHN et al BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3
Figure 1. Transfer of iron from extracellular DFP-iron
complexes to mitochondria. H9C2 cells loaded with the
mitochondrial iron-sensor RPA were incubated with or
without DFP-Fe (15:5 M) and epifluorescence micro-
scopic images were recorded every 5 minutes under
settings for rhodamine. Representative fields of initial cell
fluorescence at time 0 (A,C) and after incubation for 1 hour
in the presence (B) or absence (D) of 5 M DFP-Fe
complex. Magnification was 600; oil objective was a
(Plan Apo) 60 /1.40 NA. (E) Mean fluorescence values in
relative units (r.u.) plus or minus SD of 5 cells per field
calculated for each time-point image and normalized to the
initial fluorescence, representing cells incubated without
(None) and with 5 M Fe-complex (DFP-Fe or NTA-Fe);
the lines denoted as DFP-Fe and NTA-Fe were cor-
rected for spontaneous decay given by the control (None).
Arrow indicates time the Fe complex was added. (F) Effect
of various iron chelates on the fluorescence (f normalized
to initial value, f0, in r.u.) of 0.5 M RPA in solution (HBS
buffer). The iron chelates all contained 5 M Fe complexed
to NTA (1:3 ratio) in the absence (f) and presence ( ) of
100 M ascorbate (Fe:NTA, Fe:NTA ASC) or to DFP,
with 100 M ascorbate, at DFP:Fe ratios of 3:1 (F), 5:1 (E),
and 7:1 (‚). The values of fluorescence intensity are
means of triplicate samples run in parallel, plus or
minus SEM.
tetramethylbenzidine (0.5 mg/mL in 10% acetic acid) and finally 8 L of endogenous labile iron and/or to probe photobleaching. On the
30% H2O2. Absorption (604 nm) was read after 90 seconds. other hand, addition of preformed DFP-Fe(III) complexes (DFP-
Data acquisition and analysis. Epifluorescence imaging (Nikon TE Fe, ratio 3:1) evoked a time-dependent (t ⁄ 15 minutes) quenching
12
2000 microscope; Melville, NY; and Orca-Era CCD camera; Hamamatsu,
of mitochondrial RPA fluorescence (Figure 1E). Consistent with
Bridgewater, NJ) coupled to a Volocity 4 system (Improvision, Coventry,
United Kingdom) was used for image data acquisition and analysis.20 For RPA’s selectivity for Fe(II), no quenching of RPA by Fe-NTA
high-throughput fluorescence monitoring we used fluorescence plate read- occurred in solution unless ascorbate was added. Transfer of iron
ing (Tecan-Safire; Neotec, Mannedorf, Austria) essentially as described.20
¨ from DFP-Fe complexes to RPA in the presence of ascorbate was
detected over a wide range of DFP to Fe ratios (1:1 to 7:1).
Transfer of iron from extracellular DFP-Fe complexes to
Results nuclei. With the view of targeting a fluorescent iron sensor into
the cell nucleus, we constructed a conjugate of H with the
To follow DFP-mediated translocation of iron from the medium to high-affinity iron chelator and iron sensor H-FlDFO.11 To minimize
cells, within cells, and from cells to the medium, we used uptake via the endocytic pathway, incubation was carried out at
fluorescent acceptors and donors of iron that undergo quenching or room temperature and in the presence of chloroquine, as described
dequenching upon metal binding or removal. An additional feature in “Histone-CALG.” H-FlDFO within nuclei was rapidly
of these probes was their localization in specific cell compartments (t ⁄
12 5 minutes) quenched by DFP-Fe (3:1) added to the extracel-
or in the extracellular medium. lular medium (Figure 2E), consistent with the high cell permeabil-
ity of DFP-Fe complexes shown in Figure 1. In solution, H-FlDFO
Transfer of iron from extracellular DFP-iron complexes to
is efficiently and rapidly (t ⁄12 1 minute) quenched by DFP-Fe
mitochondria
complexes even at a 9:1 DFP:Fe ratio (Figure 2F), as expected from
Ingress of ionic iron from the extracellular medium to the DFP’s capacity to shuttle iron to DFO both in vivo and in vitro.11
mitochondria of H9C2 cardiomyocytes was followed with the DFP-mediated redistribution of iron between cellular compart-
high-affinity Fe(II)–acceptor probe, RPA22,23 (Figure 1). The hydro- ments: from nuclei to mitochondria. DFP’s facilitation of iron
phobic cationic rhodamine B is responsible for RPA’s potentiomet- movement between discrete cell compartments was followed in
ric partitioning in the mitochondria, and phenanthroline is respon- H9C2 cells sequentially labeled in cell nuclei with the iron-donor
sible for detection of Fe(II).22 Exposure of cells to Fe(III)-NTA probe H-CALG-Fe(III) and in mitochondria with the Fe(II)-
evoked no significant changes in RPA fluorescence relative to the acceptor probe RPA. H-CALG-Fe penetrates the plasma membrane
spontaneous decrease (t ⁄ 60 minutes), probably due to binding of
12 and accumulates initially in the cytosol and subsequently in the
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BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3 REDISTRIBUTION OF IRON AS THERAPEUTIC STRATEGY 1693
Figure 2. Transfer of iron from extracellular DFP-iron complexes to nuclei.
H9C2 cells loaded with the nuclear iron sensor H-FlDFO were incubated with or Figure 3. DFP-mediated relocation of iron between cellular compartments:
without 5 M DFP-Fe complex (DFP:Fe ratio 3:1) and epifluorescent microscopic from nuclei to mitochondria. H9C2 cells were loaded with the nuclear iron sensor
images were recorded every 5 minutes under settings for fluorescein. Representative H-CALG that had been precomplexed to iron (H-CALG-Fe), followed by loading with
fields of initial cell fluorescence at time 0 (A,C) and after incubation for 1 hour in the the mitochondrial iron sensor RPA. The double-labeled cells were then exposed to
absence (B) or presence (D) of 5 M DFP-Fe complex. (E) Mean fluorescence values 50 M DFP and epifluorescence images were recorded every 5 minutes. Representa-
in r.u. plus or minus SD of 5 cells per field, calculated for each time-point image and tive fields of cell fluorescence observed under settings for fluorescein (A,B) and
normalized to the initial fluorescence (f/f0), representing cells incubated without rhodamine (C,D). Images are shown at time 0 (A,C) and after incubation for 1 hour in
(None) and with 5 M DFP-Fe complex (DFP-Fe). Arrow indicates time the Fe the presence of 50 M DFP (B,D). (E) Mean fluorescence values plus or minus SD in
complex was added. (F) Effect of DFP-Fe chelates (DFP:Fe at ratios of 1:1, 3:1, 5:1, r.u. of 5 cells per field, calculated for each time-point image and normalized to the
9:1) on the fluorescence (normalized to initial value, f0, in r.u.) of 0.5 M H-FlDFO in initial fluorescence (f/f0), representing cells incubated with 50 M DFP (circles) and
solution (HBS buffer). The values of fluorescence intensity are means of triplicate with no addition (squares). Fluorescence of H-CALG is indicated by filled symbols
samples run in parallel, plus or minus SEM. (F and f), and fluorescence of RPA is indicated by open symbols (E and ). The
scheme illustrates entry of DFP into the cytosol (C), nuclei (N) containing H-CALG-Fe
(iron donor) and mitochondria (M) containing RPA (iron acceptor), and transfer of iron
from nuclei to mitochondria. E refers to endosomes.
nucleus, particularly in nucleolus-like sites.21 H-CALG-Fe was
used subsaturated with iron so that its accumulation in the nuclei
could be followed via changes in the residual fluorescence of the
CALG-Fe into endosome/pinosomes and its accessibility to DFP, as
unquenched fraction of the probe (revealed by chelation; Figure
evidenced by dequenching, in a previous study.20 Moreover, CALG-Fe
3A). Loading of RPA into mitochondria was apparent from the
coendocytosed with the classical fluid phase markers sulforhodamine
characteristic perinuclear staining pattern (Figure 3C), from colo-
and rhodaminated dextran, both of which showed less than 65% cell
calization with other potentiometric probes and sensitivity to
colocalization as obtained with the Volocity program (not shown).
protonophores.22,23 Addition of 50 M DFP produced an increase in
the fluorescence of H-CALG-Fe in the nuclei and a concomitant H-FlDFO was detectable in the nuclear area, while CALG-Fe was
decrease in the fluorescence of RPA in the mitochondria, both of concentrated in punctuate, perinuclear spots, attributable to endosome-
which occurred within 5 to 8 minutes and were complete within trapped CALG-Fe (Figure 4). However, after incubation with 50 M
20 minutes (Figure 3E). The changes are attributable to dequench- DFP, the fluorescence in the endosomes increased significantly, while
ing of H-CALG-Fe due to removal of iron by DFP and to that in the nuclei decreased relative to the respective untreated controls.
quenching of RPA due to transfer of iron from DFP-Fe. Only minor The kinetics of the changes in the 2 compartments followed similar
changes in fluorescence were observed in untreated control cells. It patterns, although in opposite directions. To ascertain whether the iron
is likely that nuclear H-CALG-Fe was not the only source of iron acquired by DFP was exclusively intracellular, the cells were washed
delivered to RPA by DFP, because the addition of DFP to with 0.1 mM DFO to remove all extracellular iron before the addition of
RPA-loaded cells also led to quenching of mitochondrial probe DFP. Furthermore, as the addition of DFP to cells loaded with H-FlDFO
(data not shown), although to a much lesser extent than in the alone failed to produce a decrease in its fluorescence (data not shown),
presence of H-CALG-Fe. the quenching of H-FlDFO in cells coloaded with CALG-Fe is most
DFP-mediated relocation of iron between cellular compart- likely due to DFP-mediated mobilization of iron from endosomes.
ments: from endosomes to nuclei. H9C2 cells were labeled However, the possibility that in the course of the experiment some
sequentially in the nuclei with the iron-acceptor probe H-FlDFO and in iron might have spontaneously leaked from endosomes to cytosol
the endosomes with the iron-donor probe CALG-Fe (Figure 4). We and rendered it transferable to mitochondria by added DFP cannot
showed the bulk-phase pinocytic uptake of the iron-quenched probe be ignored.
6. From bloodjournal.hematologylibrary.org by guest on September 28, 2011. For personal use only.
1694 SOHN et al BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3
Figure 4. DFP-mediated relocation of iron between
cellular compartments: from endosomes to nuclei.
H9C2 cells were loaded with the nuclear iron-sensor
H-FlDFO and subsequently exposed to preformed
complexes of CALG and iron (CALG-Fe, 1:1 ratio) that
were taken up by adsorptive pinocytosis into endo-
somes. The cells containing both labels were then
exposed to 50 M DFP and epifluorescent images
were recorded every 5 minutes under fluorescein set-
tings. Representative fields of cell fluorescence are
shown at time 0 (A) and after incubation for 20 minutes
(B) in the presence of 50 M DFP. (C) The nuclei
(N) and endosomes (E) are indicated by arrows.
(D) Mean fluorescence values in r.u. within selected
subcellular areas corresponding to endosomes
(Di) and nuclei (Dii), using 5 cells per field, calculated
for each time-point image and normalized to the initial
fluorescence (f/f0).
Transfer of iron from DFP-Fe complexes to transferrin. We The rationale was that intracellular iron mobilized to the extracellu-
followed iron complexation by transferrin using DFP-Fe com- lar medium by DFP would be transferred to the fluorescent Tf,
plexes (3:1 and 5:1, 50 M in Fe) and NTA-Fe complexes (3:1, which, upon binding iron, would then bind to cell-surface trans-
50 M in Fe) as iron sources and apotransferrin (50 M) as ferrin, and after receptor-mediated endocytosis, would become
acceptor. To remove from the reaction mixture any traces of iron concentrated in endosomes. To increase the level of mitochondrial
associated with low molecular weight complexes or free ligands, labile iron, the cells were pretreated with the heme-synthesis
we added DTPA after 1 hour of reaction and followed it by size inhibitor succinylacetone, which causes selective accumulation of
filtration. Filtration and differential chelation allowed the assess- unused, chelatable iron in the mitochondria.22 After 1 hour of
ment of DFP-Fe transfer of iron to aTf both by light absorption of incubation with DFP, the level of cell-bound fluorescent aTf rose
Tf-Fe complexes at 465 nm (Figure 5) and by tryptophan fluores- by 2.3-fold over controls without DFP (Figure 6A,B). The binding
cence of aTf that is quenched after specific binding of iron25 was specific and iron dependent, as it was fully blocked by excess
(Figure 5). While the 2 analytical methods monitored binding of holotransferrin (Figure 6C,D) and enhanced by addition of saturat-
iron to transferrin, the optical changes are mirror images of each ing concentrations of Fe-NTA (Figure 6E,F), both in the presence
other. Transfer of iron from DFP-Fe to transferrin ensued at both and in the absence of DFP. In control cells not treated with
3:1 and 5:1 stoichiometries of DFP:Fe and was of a stable nature: it succinylacetone, DFP failed to enhance fluorescent-Tf binding
persisted after addition of chelators that differentially dissociate (data not shown), indicating that under normal conditions where
DFP-Fe complexes (but not Fe-transferrin). On the other hand, iron incorporation into heme is not blocked, the concentration of
DFP alone and NTA alone evoked only minor changes that were DFP-mobilized iron is insufficient to be detectable in this system.
eliminated by addition of DTPA before the reaction with aTf, DFP-mediated iron delivery for hemoglobin synthesis. The
indicating that the changes were associated with traces of iron potential of DFP-Fe complexes as donors of iron for physiologic
present in the medium. Essentially similar results were obtained by use was assessed using a model system of cellular iron incorpora-
assessing the transfer of iron to apotransferrin using fluorescein- tion, synthesis of hemoglobin (Hb) by differentiating preerythroid
labeled aTf (not shown). However, with lissamine rhodamine- cells. The well-documented induction of Hb synthesis in MEL cells
labeled aTf (R-aTf), the acquisition of iron evoked insignificant by hexamethylene bisacetamide (HMBA) is highly dependent on
changes in fluorescence. the supply of iron.26 It is repressed in serum-free medium lacking
transferrin, but it is significantly enhanced by the addition of
DFP-mediated mobilization of iron from H9C2 cells to
Fe-NTA irrespective of the presence of Tf (Figure 7). DFP-Fe (3:1)
extracellular transferrin: receptor-mediated uptake of
supported Hb synthesis to a degree similar to that seen with
holotransferrin as an index of iron shuttling
Fe-NTA, both in the absence and presence of added Tf. Fully
Mobilization of cellular iron by DFP and its transfer to extracellular saturated holotransferrin (15 M) was equally effective as Fe-NTA
transferrin are critical features of DFP’s potential function as an and DFP-Fe (data not shown), indicating that various forms of iron
iron shuttle. This capability was assessed using H9C2 cells can equally support Hb synthesis in this experimental system. On
incubated with lissamine R-aTf in the presence of DFP (Figure 6). the other hand, DFP without added iron depressed Hb synthesis
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BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3 REDISTRIBUTION OF IRON AS THERAPEUTIC STRATEGY 1695
Figure 5. Transfer of iron from DFP-Fe complexes
to transferrin. DFP-Fe complexes (1:3 and 1:5 ratios;
50 M Fe(III)) were mixed with apotransferrin aTf
(50 M) and incubated for 1.5 hours in a humidified 5%
CO2 incubator at 37°C. At the conclusion of the reac-
tion, 10 mM DTPA was added and the low–molecular
weight material was removed by filtration as described
in “Histone-CALG.” The tryptophan (trp) fluorescence
at 280 nm excitation/306 nm emission (top graph) was
obtained from the emission spectra (inset). The high–
molecular weight fractions were diluted in HBS, pH 7.4,
and the absorbance was read at 465nm (bottom graph).
Data are given as means plus or minus SD of
3 independent experiments. The labels below the bars
indicate the complexes tested and their concentrations.
The values above the bars represent the percentage
change in absorbance or fluorescence.
below control levels, indicating that the chelator can have an (data not shown), an effect similar to DFO-induced iron depriva-
iron-withdrawing effect under conditions of limited iron supply. tion in other hematopoietic cells.27 To demonstrate that transfer of
In the serum-free medium used in these experiments, DFP iron from DFP-Fe to aTf gave rise to functionally active holotrans-
( 100 M) without added iron, as well as DFO (100 M), ferrin, DFP-Fe was allowed to interact with aTf for 1 hour, and then
prevented differentiation and ultimately caused massive cell death the mixture was exhaustively dialyzed (cut-off 12 kDa) against
Figure 6. DFP-mediated mobilization of iron from
H9C2 cells to extracellular apotransferrin: receptor-
mediated uptake of holotransferrin as an index of iron
transfer. H9C2 cells were pretreated with succinylac-
etone as described in “Methods” to increase mitochon-
drial labile iron levels. They were then incubated at
37°C in DMEM-HEPES medium containing 20 M
lissamine R-aTf with various additions, and epifluores-
cence microscopy images were obtained under rhodamine
settings after 60 minutes of incubation. Representative
images of cells with no addition (A), 30 M DFP (B),
100 M unlabeled holotransferrin (C), 100 M unlabeled
holotransferrin with 30 M DFP (D), 20 M Fe-NTA, 1:3
ratio (E), and 20 M Fe-NTA with 30 M DFP (F). (G) The
illustration represents entry of DFP into cells, mobilization
of iron from the cytosol (C), nuclei (N) and mitochondria
(M), followed by exit of DFP-Fe complexes from the cells
(step 1). This is followed by transfer of iron from DFP-Fe to
R*-aTf to form R*-Tf-Fe (step 2), which then binds to
transferrin receptors on the cell surface (step 3), concen-
trates in the endosomes, and is detected by fluorescence
microscopy as punctuate fluorescence typical of mi-
crovesicles. (H) Mean cell-associated fluorescence values
in r.u. of 5 cells per field ( SD, from 3 separate experi-
ments), calculated from snapshots such as shown in
panels A through F, obtained after 1 hour of incubation.
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1696 SOHN et al BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3
Figure 7. Stimulation of Hb synthesis in murine erythroleukemia cells. (A) DFP-mediated iron delivery to murine erythroleukemia (MEL) cells synthesizing hemoglobin is
schematically depicted. (B) MEL cells suspended in serum-free DMEM containing 1.25 mg/mL bovine serum albumin and 5 mM HMBA were supplemented with various iron
complexes (step 1 in panel A) and cultured for 48 hours. Hemoglobin synthesis (step 2 in panel A) was assessed in terms of hemoglobin content in cell lysates as described in
“Cell hemoglobin synthesis” and is expressed relative to the hemoglobin content in control cells with no additions (set as 100%). The additions included 10 M Fe complexed to
30 M NTA without (Fe-NTA) and with 15 M human apotransferrin (Fe:NTA aTf); 10 M Fe complexed to 30 M DFP without (DFP-Fe) and with 15 M human
apotransferrin (DFP-Fe aTf); 30 M DFP alone (DFP); 15 M human apotransferrin alone (aTf). Generation of holotransferrin from DFP and apotransferrin (DFP-Fe aTf
Dial.): 10 M Fe:30 M DFP complex was preincubated with 15 M human for 1 hour and dialyzed. Results shown are averages of 3 separate experiments plus or minus SD;
the average Abs604 of control cells was 0.39. (C) DFP-mediated mobilization of iron from J774 (Fe donor; step 1a in panel A) cells to the extracellular medium (step 1b in panel
A), followed by entry of DFP-Fe complexes into MEL (Fe acceptor) cells (step 1c in panel A) and intracellular donation of iron for hemoglobin (Hb) synthesis (step 2 in panel A).
MEL cells were cultured for 48 hours in DMEM containing 1.25 mg/mL bovine serum albumin and 5 mM HMBA without (Con) or with 10 M Fe-NTA (NTA-Fe), or with various
supernatants of J774 cell lysates that were supplemented with 5 mM HMBA, and lysates were assayed for hemoglobin content. To obtain J774 mouse macrophage
supernatants, J774 cells were cultured overnight without (untreated J774) or with 100 M FAC (Fe-loaded J774), washed with 100 M DFO to remove all extracellular iron, and
incubated for 2 hours at 37°C in serum-free DMEM containing 1.25 mg/mL bovine serum albumin, without ( DFP) or with 30 M DFP ( DFP). The cell supernatants were
collected and centrifuged to remove detached cells, and HMBA (5 mM) was added to MEL cells. Shown are values of hemoglobin (Hb; from a representative experiment)
obtained in MEL cells exposed for 48 hours to the various conditions.
buffer containing 80 mg/L aTf to remove DFP-Fe without gaining support for the proposed use of DFP as an iron shuttle even without
contaminant iron from the solution. When added to differentiating the mediation of transferrin, such as might occur in the central
MEL cells, this preparation, generated from DFP-Fe and aTf, fully nervous system.
supported Hb synthesis (Figure 7B).
Finally, we assessed DFP’s ability to shuttle iron from cell to
cell (Figure 7C). In vivo, direct intercellular transfer of iron via Discussion
DFP is not likely, due to the presence of more than 50 M due to
the presence of more than 25 M transferrin in plasma.28 However, The recognition that intracellular iron accumulation is one of the
intercellular transfer of iron via DFP could occur in the brain where underlying causes of some clinical syndromes has led to the
the iron-binding capacity of transferrin in cerebrospinal fluid (CSF) suggestion that it might be amenable to treatment with iron
is estimated to be in the submicromolar range.29,30 In the experi- chelators.3,33,34 These syndromes include Parkinson disease, neuro-
ment illustrated in Figure 7A, J774 mouse macrophages were degeneration with NBIA, and Friedreich ataxia, where iron depos-
chosen as the iron-donating cells because of their high capacity for its in specific areas of the brain have been identified by histologic or
iron accumulation.31 DFP at a concentration of 30 M, achieved in imaging techniques34 and are thought to be a central factor in the
vivo with moderate oral doses,1,32 mobilized sufficient iron from development of the diseases.35 Similarly, in anemia of chronic
iron-loaded J774 cells, but not from untreated ones, to significantly disease, iron is retained within erythrocyte-phagocytosing macro-
enhance Hb synthesis in MEL cells (Figure 7C). Spontaneous phages, presumably to prevent access by invading pathogens to
efflux of iron from iron-loaded J774 cells took place, as evidenced iron from the circulation.7-9,31 While regional iron mobilization
by the increased MEL cell Hb levels even in the absence of might be essential for stabilizing or reversing the toxic effects of
DFP (although increased, these levels were significantly lower in labile iron, in some pathologies it might be equally important to
the absence of DFP than in its presence). This result provides render the mobilized iron available for metabolic reuse. This is
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BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3 REDISTRIBUTION OF IRON AS THERAPEUTIC STRATEGY 1697
especially important in those conditions in which regional iron hemoglobin synthesis when presented together with DFP or after
accumulation is accompanied by regional or systemic deprivation. DFP’s removal (Figure 7).
All the chelators in clinical use have been designed for massive The direct bioavailability of DFP-bound iron was investigated
removal and excretion of iron from organs of iron-overloaded using hemoglobin synthesis by cells in culture as a model. DFP-Fe
patients.2,10,33 Here we assessed the potential application of an iron supported hemoglobin synthesis even in the absence of transferrin
chelator in clinical use (DFP) as a shuttling agent capable of (Figure 7), consistent with a similar activity of 1:1 complexes of
redistributing iron within and among cells, while satisfying certain iron with salicylaldehyde isonicotinoyl hydrazone.43 More impor-
basic requirements. tant, DFP enhanced the transfer of iron from iron-loaded macro-
Permeability across cell membranes in the free and iron- phages to preerythroid cells for hemoglobin synthesis, without the
bound form. The high membrane permeability of the iron-free intermediary presence of transferrin (Figure 7). Such direct dona-
form of DFP is well documented, as shown by its capacity to tion to intracellular iron-requiring or iron-metabolizing enzymes
rapidly access and deplete intracellular labile iron pools.36,37 On the may not be a necessary feature if iron is efficiently transferred from
other hand, the ability of DFP-Fe complexes to traverse intracellu- the shuttle to circulating transferrin. However, it may be desirable
lar and plasma membranes of cells has not been studied in or even essential in the brain, where the iron-binding capacity of
detail.20,21,36,37 One indication is the capacity of orally administered transferrin in the CSF is negligible and has been estimated in the
DFP to increase serum transferrin saturation12 and labile iron11 submicromolar range.29,30
within an hour after intake in thalassemia patients, which is Low toxic potential of iron-shuttling agent complexes.
attributable to the rapid exit of DFP-Fe complexes from cells into A major concern in the use of chelators is the formation of iron
the circulation. At the intracellular level, it is shown in this work complexes that undergo redox cycling and thereby catalyze ROS
that DFP can facilitate the removal of labile iron from nuclei generation. This tendency is present in chelators with relatively low
(Figure 3), from endosomes (Figure 4), and from mitochondria redox potentials, such as those based on amine- and carboxyl-
(Figure 6), and can transfer iron to acceptors in the mitochondria liganding groups but not with the bidentate 3:1 complexing
(Figure 3), nuclei (Figure 4), and extracellular medium (Figure 6). hydroxypyridone DFP.20,21,44,45 However, speciation studies10 and
The transfer might involve dissociation of the DFP-Fe complexes recent examination of ROS production42 indicated that complete
abolition of labile iron by DFP is attained only at DFP:Fe(III) ratios
and/or formation of ternary complexes with other ligands.
close to 5:1. Whether DFP levels reached therapeutically are
Ability to compete effectively for intracellular labile iron with
consistently high enough to avoid formation of substoichiometric
other intermediate affinity species. Labile iron that is likely to be
redox-active complexes is difficult to predict. This is particularly
accessible to DFP is assumed to be bound to several possible
important in susceptible, DNA-containing organelles such as
ligands (eg, citrate, ATP) and to be in dynamic equilibrium between
nuclei and mitochondria. The same applies to intracellular signal-
Fe(II) and Fe(III) as dictated by the redox status of the intracellular
ing pathways activated by oxidative stress that could be generated
environment.38,39 DFP competes effectively for labile cell iron both
by DFP-mediated redistribution of cell iron.
in vitro and in vivo,32 and in individuals with normal iron balance it
Permeability across the blood-brain barrier (BBB) for thera-
raises serum iron levels, indicating mobilization of tissue iron.12
peutic applications to neurodegenerative diseases. The capacity
This was also observed with isolated cells (Figures 3,4), where
of an iron-shuttling agent to redistribute iron in the central nervous
DFP readily chelated iron bound to the EDTA analog CALG in
system (CNS) may be an essential requirement for iron redistribu-
endosomes and nuclei and was indicated in other systems.40 An
tion in Parkinson disease, Friedreich ataxia, or NBIA. A high rate of
undesirable property of a chelating molecule would be interference BBB penetration is expected for free DFP, based on its low
with normal cellular iron metabolism by overchelation. In this molecular weight (below 300) and lipophilicity. This was con-
respect, DFP ought to be used at restricted doses, as at high levels it firmed by direct measurement of DFP accumulation in the perfused
appears to inhibit iron-requiring tyrosine and tryptophan rat brain.15,16 In rats given DFP doses several times higher than
hydroxylases.10,14 analogous doses given to thalassemia patients, brain tyrosine and
Ability to donate iron to physiologic acceptors. The rationale tryptophan hydroxylase activities were significantly inhibited, as
behind DFP-mediated redistribution of iron leans on DFP’s ability measured by the accumulation of 3,4-dihydroxyphenylalanine
to transfer the metal to extracellular transferrin, thereby minimizing (DOPA) and 5-hydroxytryptophan, respectively, presumably via
undesired iron loss by excretion via the urinary or biliary pathways. coordination to iron bound by these enzymes.14 Yet, despite
We surmised that under physiologic conditions iron transfer to aTf DFP’s brain accessibility, major effects on CNS function have not
is likely to occur (1) spontaneously, as the pFe value of Fe(III) for been reported.32,44
transferrin is 3 orders of magnitude higher than for DFP41 (Table 1), Whether DFP can alter the iron balance in the brain by shuttling
and (2) directly as Fe(III), because the thermodynamically favored iron across the BBB remains an open question. The absence of iron
DFP-Fe complexes are (DFP)2-Fe(III) and (DFP)3-Fe(III).10,25,42 In loading of the CNS in iron-overload conditions indicates that
vitro studies based on an indirect analytical method11 corroborated neither transferrin-bound nor non–transferrin-bound iron is able to
earlier observations that a single oral dose of 3 g DFP raised within freely cross the BBB.35 Because DFP treatment has not been
6 hours the transferrin saturation (determined by urea-gel electro- reported to be associated with increased iron loading of the CNS in
phoresis) of a healthy individual from a normal level of 20% to thalassemia patients, it is conceivable that DFP-Fe complexes do
80%.12 In this work we provided direct demonstration of iron not readily permeate the BBB. The permeabilities of free and
transfer from DFP-Fe to aTf at physiologic concentrations (Figure iron-bound DFP may differ due to differences in their molecular
5). Using spectrophotometric and fluorimetric methods, we showed weights (473 for 3DFP:1Fe compared with 139 for DFP). In a
that efficient transfer occurs at various DFP:Fe ratios and within recent study, 6 months of treatment with DFP was shown to result
1 to 2 hours in culture conditions. The resulting holotransferrin in a decrease in the iron-associated magnetic resonance imaging
formed from DFP-Fe was biologically active, as it was recognized (MRI) transverse relaxation rate (R2*) signals in dentate nuclei of
by the transferrin receptor (Figure 6) and provided iron to cells for Friedreich ataxia patients.13 Whether this decrease was caused by
10. From bloodjournal.hematologylibrary.org by guest on September 28, 2011. For personal use only.
1698 SOHN et al BLOOD, 1 FEBRUARY 2008 VOLUME 111, NUMBER 3
egress of DFP-Fe complexes from the brain or by redistribution of iron loss.48,49 Optimization of such strategies with novel chelators
iron from an area of accumulation to areas of lower concentration for treating the various conditions of regional iron accumulation
within the brain is unclear. demand further laboratory and clinical work.
We show that redistribution of iron in multiple directions and to
multiple recipients is experimentally feasible, particularly from
areas of accumulation to areas of iron need, via donation to Acknowledgments
transferrin or by direct metal donation. A modality of iron chelation
based on iron redistribution has several therapeutic implications. In This work was supported by the Association Française contre les
the CNS, it could potentially be used to relocate local iron deposits Myopathies, the Israel Science Foundation (ISF), the EEC Frame-
work 6 (LSHM-CT-2006-037296 Euroiron1) and French-Israeli
that may be an important factor in the etiology of several
Organization for Research in Neuroscience (AFIRN). Z.I.C. is the
neurodegenerative diseases. In ACD, it could be used to release
Sergio and Adelina Della Pergolla Professor of Life Sciences.
iron trapped within macrophages. Current experimental approaches
to alleviation of ACD tend to be directed at depressing the levels of
hepcidin, the iron-regulator protein responsible for macrophage
Authorship
iron retention. However, recent evidence for hepcidin-independent
down-regulation of the iron exporter ferroportin by the inflamma- Contribution: Y.-S.S. and W.B. performed experimentation and
tory mediators46 could obviate such an approach. As shown in this analysis; A.M. and Z.I.C. designed and supervised the research. All
work, an alternate or complementary approach could be based on authors contributed to the writing of the paper.
iron-shuttling agents that relocate misdistributed iron and safely Conflict-of-interest disclosure: The authors declare no compet-
bypass impaired internal routes of iron trafficking. Early pilot ing financial interests.
studies with iron chelators in rheumatoid arthritis patients with Correspondence: Z. I. Cabantchik, Alexander Silberman Insti-
ACD47 provided clinical evidence that agents like DFP might be tute of Life Sciences, Hebrew University of Jerusalem, Safra
adopted as part of a short-term strategy of regional metal detoxifi- Campus at Givat Ram, Jerusalem, Israel 91904; e-mail:
cation and systemic relocation that generates no significant urinary ioav@cc.huji.ac.il.
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