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The Detoxification System Part I: The Human Liver by Mark J. Donohue The liver is one of the most biochemically complex organs in the body. It is the second largest organ (the largest being the skin) and the largest gland (synthesizes and secrets compounds) in the body. The liver serves as a gatekeeper between the intestines and the general circulation and performs an astonishingly large number of tasks that impact all body systems. Anatomy and Blood Flow of the Liver Averaging about the size of an American football in adults, the liver weighs about 3 lbs. (1.36 kg). It is reddish brown in color and is divided into four lobes of unequal size and shape. Medical terminology related to the liver often start in hepato- or hepatic- from the Greek word for liver (hepar). Blood is carried to the liver via two large vessels called the portal vein and the hepatic artery. The portal vein supplies approximately 75% of the livers blood supply and carries venous blood (returning to the heart). Three quarters of the portal vein’s blood supply originates from the small intestines and is rich with nutrients. The remaining one quarter originates from the spleen. The hepatic artery accounts for 25% of the blood flow to the liver and carries oxygen-rich arterial blood from the heart via the aorta (a major vessel of the heart). Both the portal vein and hepatic artery enter the liver dividing into smaller and smaller vessels - capillaries. These capillaries end in the thousands of lobules of the liver. Most livers have 50,000 to 100,000 lobules. Each lobule is composed of several different types of liver cells. Once the blood flow reaches the liver’s lobules it continues through the sinusoids of the liver lobules. Sinusoids are small blood vessels, similar to capillaries, and are highly permeable with fewer tight junctions and discontinuous endothelial cells. As blood passes through the sinusoids it comes into direct contact with the liver cell’s microvilli, which are packed with transport proteins. Here liver cells monitor and enzymatically act upon various substances within the blood. Afterwards, the blood empties into the central vein of each lobule. The central veins coalesce into the hepatic vein, which leaves the liver and empties into the inferior vena cava and returns to the heart. Image: Liver blood flow Normally, 10-15% of the total blood volume is in the liver, with roughly 60% of that in the sinusoids. When blood is lost, the liver dynamically adjusts its blood volume and can eject enough blood to compensate for a moderate amount of hemorrhage. Conversely, when vascular volume is acutely increased, as when fluids are rapidly infused, the hepatic blood volume expands, providing a buffer against acute increases in systemic blood volume. Image: Liver lobules Cells of the Liver There are four basic cell types that reside in the liver. These cells are the hepatocyte, the stellate cell, the liver endothelial cell and the Kupffer cell. These so-called resident cells control many of the key functions in the liver, with each specializing in a specific liver function. Hepatocytes – are the main cell type in the liver and make up 70 to 80% of the liver's volume. Hepatocytes are the work horses of the liver performing a majority of the livers daily tasks. Hepatocytes are involved in synthesizing amino acids, protein, cholesterol, bile salts, fibrinogen, phospholipids and glycoproteins. Other functions of the hepatocytes include the conversion and storage of carbohydrates and protein, the formation and secretion of bile and urea, and the detoxification of toxic substances. The hepatocyte is also the primary target of many liver diseases, including viral infections, excessive fat accumulation and drug induced liver damage. Injury to these cells initiates the cycle of inflammation that in turn perpetuates further liver damage. The liver is among the few internal human organs capable of natural regeneration of lost tissue. With as little as 25% remaining, a liver can regenerate into a whole liver again. This is predominantly due to hepatocytes acting as unipotential stem cells (ability to renew themselves). Hepatic Stellate Cells (HSC) or Ito Cells – reside in close proximity to the hepatocyte and make up 5 to 8% of the liver’s volume. Under normal conditions these cells just sit around in an inactive state, storing vitamin A (approximately 80% of the body’s supply) and a variety of other lipids. Under conditions of liver injury, hepatic stellate cells become activated and act as the liver’s reserve army. When activated, these cells promote ion movement, the production of antibodies, genesis of natural killer Tcells and the proliferation of chemical responses to stress. Researchers believe that hepatic stellate cells play a key role in releasing collagen scar tissue and encouraging liver scarring. Liver Endothelial Cells (LEC) – make up 3% of the liver’s volume and form the wall of the sinusoids, thereby separating hepatocytes from the blood flowing through the liver. Liver endothelial cells form a single layer with gaps between each cell known as fenestra. These gaps allow for an efficient flow of essential materials to pass from the blood to hepatocytes and play an important role in hepatic microcirculation. In addition, these endothelial cells are rich in lysosomal enzymes which are used to digest worn out organelles, food particles and engulf viruses and bacteria. Kupffer cells – make up 2% of the liver’s volume and are located within the sinusoidal lining of the liver. Kupffer cells are liver specific macrophages - a type of white blood cell (immune system) - which are phagocytic. Meaning kupffer cells primary function is to engulf and digest and therefore, represent the main cellular system for removal of particulate materials and microbes from the circulation. Their location just downstream from the portal vein allows Kupffer cells to efficiently scavenge bacteria, aging red blood cells, unnecessary proteins and foreign microbes that get into portal venous blood through breaks in the intestinal lining. In a way, the kupffer cells are like bodyguards and assassins for the hepatocytes, protecting them from invaders and cell refuse. However, if Kupffer cells become overburdened the filtration system will slow down. This allows a lot of bowel microorganisms, antigens, and foreign proteins along with other waste products to enter the circulatory system as they pass from the liver. Therefore, these cells are a significant factor in the host resistance to primary and secondary infections. Kupffer cells facilitate and amplify the response by secreting cytokines (signaling molecules) that recruit and expand the population of other pro-inflammatory cells in the liver. The Biliary System The biliary system is a series of channels and ducts within the liver that conveys bile from the liver into the duodenum (first section of the small intestine). Bile is a complex yellowish, blue/green fluid secreted by hepatocytes. Bile consists of water, electrolytes and a battery of organic molecules including bile acids, cholesterol, phospholipids and bilirubin. Hepatocytes secrete bile into the canaliculi - the dilated intercellular space between adjacent hepatocytes (see lobule image above). In the canalicul, bile secretions flow parallel to the sinusoids, but in the opposite direction that blood flows. At the ends of the canaliculi, bile flows into bile ducts, which are true ducts lined with epithelial cells. From there bile can either drain directly into the duodenum via the common bile duct or be temporarily stored in the gallbladder via the cystic duct. The gallbladder is a pear shaped organ that stores bile (or “gall”) between meals until needed for digestion. Typically, bile is concentrated five-fold in the gallbladder by absorption of water and small electrolytes - virtually all of the organic molecules are retained. During meals, especially those containing fat, the gallbladder pumps bile into the intestine. Once in the small intestine bile’s main purpose is to emulsify dietary fats (increase surface area to help enzyme action). Image: The Biliary System Bile also prevents intestinal contents from decaying, enhances transit time of the stool, and is the main elimination route for cholesterol as well as for toxins that have been broken down or detoxified by the liver. Liver Functions The liver serves more than 500 vital functions in the human body. These functions can be organized and grouped in a variety of different ways. Though there is some cross over, I’ve taken the liberty of organizing the functions of the liver into six areas: 1. 2. 3. 4. 5. 6. Digestion Metabolism Synthesis Storage Immunological Biotransformation Digestive Function of the Liver As described above one of the main functions of the liver is to assist in the digestion and absorption of fats via the biliary system. This process begins when chyme (partially digested mass of food) from an ingested meal enters the small intestine. Once in the small intestine acid (from stomach HCL) and partially digested fats stimulate secretion of cholecystokinin and secretin. These hormones have important effects on both bile and pancreatic secretions. The most potent stimulus for release of cholecystokinin is the presence of fat in the duodenum. Once released, it stimulates contractions of the gallbladder and common bile duct, resulting in delivery of bile into the small intestine. Secretin is secreted in response to acid in the duodenum. It simulates biliary duct cells to secrete bicarbonate and water, which expands the volume of bile and increases its flow out into the intestine. The secretion of bile into the small intestines functions as a detergent on particles of dietary fat which causes fat globules to break down or be emulsified into minute, microscopic droplets. Emulsification is not digestion per se, but is of importance because it greatly increases the surface area of fat, making it available for digestion by lipase, a pancreatic enzyme used to digest fats. Bile acids are also critical for transport and absorption of the fat-soluble vitamins (A, D, E & K). Bile is continuously secreted by the liver and stored in the gallbladder until a meal. Even though large amounts of bile acids are secreted into the intestine every day, only a small quantity is lost from the body. This is because approximately 95% of the bile acids delivered to the duodenum are absorbed back into blood within the ileum (final section of the S.I.). Venous blood from the ileum goes straight into the portal vein, and hence through the sinusoids of the liver. Hepatocytes extract bile acids very efficiently from sinusoidal blood, and little escapes the healthy liver into systemic circulation. Bile acids are then transported across the hepatocytes to be re-secreted into canaliculi. The net effect of this enterohepatic recirculation is that each bile salt molecule is reused about 20 times, often two or three times during a single digestive phase. Metabolic Function of the Liver There is no organ that is more important to healthy metabolism than the liver - in many ways, it is as central to metabolism as the heart is to the circulation of blood. Because of the crucial importance of healthy metabolism to overall health, diseases of the liver can be devastating, leading to fatigue, malaise, and even to death. There are three types of fuel consumed by the human cell - carbohydrate, fat, and protein - and living cells derive large amounts of energy from the "burning" (oxidation) of these fuels. Carbohydrate Metabolism: It is critical for humans to maintain concentrations of glucose in blood within a narrow, normal range. Maintenance of normal blood glucose levels over both short (hours) and long (days to weeks) periods of time is one particularly important function of the liver. Hepatocytes house many different metabolic pathways and employ dozens of enzymes that are alternatively turned on or off depending on whether blood levels of glucose are rising or falling out of the normal range. This function is vital because the primary source of energy for the human cell, especially nerve cells, is glucose. Glucose Glucose itself cannot be stored therefore excess glucose entering the blood after a meal is rapidly taken up (via the insulin independent transporter - GLUT 2) by hepatocytes and stored as the polymer (large molecule) glycogen. This process of converting glucose to glycogen is called glycogenesis. Later, between meals, when blood concentrations of glucose begin to decline, the liver activates other pathways which breakdown and convert the polymer of glycogen back to glucose. The process of converting glycogen to glucose is called glycogenolysis. The human body can store only about 4-500 grams or about 10% of the livers weight in glycogen. This can be converted to glucose in less than one day of normal activity, which can easily become exhausted during a fast or intense physical activity. When this occurs hepatocytes activate additional groups of enzymes that begin synthesizing glucose out of fatty acids and glycerol. When these molecules become depleted amino acids are used last. The ability of the liver to synthesize this new glucose from either fatty acids or amino acids is called gluconeogenesis. Fat Metabolism: Because glucose is very hydrophilic (water loving), this results in co-storage of large amounts of water. This form of energy storage takes up too much weight and volume to be efficient. Therefore surplus glucose is metabolized into fatty acids, via a process called lipogenesis, which are subsequently combined with glycerol to form triglycerides (fat). Fatty acids from lipogenesis or triglycerides directly from the diet are important metabolic fuel sources - particularly for muscle tissue. Under normal dietary circumstances fats are used by all the body’s tissues – except the brain (CNS) – for fuel. Fats that are not immediately oxidized and used for energy production are stored in adipose tissue (body fat). Triglyceride The liver is extremely active and is the body’s primary site for oxidizing fats (either from diet or adipose tissue), to produce energy. The liver breaks down fats for energy through a series of steps called beta-oxidation which takes place in the mitochondria of hepatocytes. Protein Metabolism: The liver is the chief regulator of protein synthesis and metabolism. The other biological fuels discussed (carbohydrates & fats) contain only the elements carbon, hydrogen and oxygen. Proteins contain nitrogen as well and sometimes sulfur. Protein is obtained through the diet or the human body has a metabolic pool of amino acids derived from plasma proteins (proteins in blood). The first step in protein metabolism is the break-down of large complex molecules, via digestion of protein foods or the catabolism of plasma protein, into simpler molecules - amino acids. The second step in protein metabolism is the removal of the amino group (-NH2) from the molecule, referred to as deamination. After removal of the amino group, the non-nitrogenous part of the molecule, referred to as the “carbon skeleton” is converted to either acetyl CoA or pyruvate. Carbon skeletons that are converted to Acetyl CoA are committed to the citric acid / kreb cycle, which functions solely to produce ATP – cellular energy. The carbon skeletons that are converted to pyruvate may be used for energy production via the citric acid / kreb cycle or they may be used to synthesize glucose (gluconeogenesis). The amino group (-NH2) is the nitrogen-containing part of the amino acid. Once removed it will either be converted to ammonia - elevated levels are toxic especially for CNS - and therefore is further converted by liver enzymes to urea for easy excretion. Or the amino group will be used to synthesize and make a new amino acid. The further metabolism of the amino group to ammonia or another amino acid is called transamination. Synthesizing Function of the Liver The liver’s hepatocytes are responsible for the synthesizing of numerous compounds such as: Plasma Proteins: The human body contains approximately 5 liters (5.3 quarts) of blood. Blood is a mixture of two components: 1) cells – red blood cells, white blood cells and platelets, 2) plasma – which makes up 55% or 2.75 to 3 liters of the blood volume and is the medium in which blood cells travel. Plasma is a clear yellowish fluid made up of 90% water which is important for the hydration of body tissue. The remaining 10% of plasma consist of hormones, mineral ions, glucose, clotting factors, waste products, immunoglobulins and proteins. All plasma proteins, with the exception of gamma globulins (antibodies), are synthesized in the liver’s hepatocytes. Some of the more common plasma proteins and their functions are: Albumin – accounts for 60% of total plasma protein and is essential for maintaining osmotic pressure needed for proper distribution of body fluids to all body tissues. It also acts as a plasma carrier or transporter of various substances (hormones, lipids, vitamins, minerals, etc.). Globulins – make up 35% of total plasma protein. There are four types of globulins alpha1, alpha 2 and beta which and are also involved in the transporting of various substances. The fourth type - gamma are involved in immune processes, but are not synthesized by the liver rather by lymphocytes (plasma cells). Fibrinogen or Factor I – makes up 4% of total plasma protein and is a sticky, fibrous coagulant essential in the formation of blood clots. This is done by thrombin (another plasma protein) reacting with fibrinogen to create fibrin a stringy substance which creates a clot. Hormones: A hormone is a chemical compound that is produced and secreted in one part of the body that effect cells in other parts of the body. Essentially, hormones are chemical messengers that transport a signal from one cell to another via the blood stream. The liver synthesizes and secretes several important hormones. Angiotensinogen – technically a pro-hormone (precursor to a hormone). Angiotensinogen plays an important role in the rennin-angiotensin system whose primary function is to regulate blood pressure and water balance. Estrogen – though the hormone estrogen is found at significantly higher levels in females (being produced primarily in the ovaries), estrogen is also found in males. This is because estrogen is also synthesized, in smaller amounts, in the liver and adrenal gland of both men and women. In both sexes estrogen is responsible for regulating certain functions in the reproductive system (e.g. menstrual cycle, sperm maturation). Hepcidin – is the master regulator of iron homeostasis in the body. Hepcidin does this by preventing the absorption of iron from the digestive tract and by also inhibiting the release of stored iron from macrophages and hepatocytes. This function not only helps to maintain homeostatic levels of iron in the body fluids but also serves as a defense against pathogenic bacteria. Many pathogens require substantial amounts of iron for their virulence and the release of hepcidin in response to infection starves them of this needed iron. Tri-iodothyronine (T3) – is a metabolically active thyroid hormone that is produced from thyroxine (T4) - a prohormone produced by the thyroid gland. The conversion of T4 to T3 takes place in the thyroid, brain, liver and various other body tissues. Thyroid hormones act as the body’s thermostat, regulating the rate at which virtually all biochemical reactions occur in the body - metabolism. Thrombopoietin (TPO) – a hormone produced primarily in the liver, whose function is to regulate platelet production which is essential for blood clotting. Insulin-like growth factor 1 (IGF-1) - is a hormone synthesized in the liver which is similar in molecular structure to insulin. Growth hormone released from the pituitary gland binds to receptors on the surface of hepatocytes which stimulates the synthesis and release of IGF-1. IGF-1 then stimulates systemic body growth. The levels of IGF-1 in the blood are highest during the years of puberty which is a time of rapid growth. Nutrients: In the liver some nutrients are directly synthesized while others are converted into their more biologically active forms. B Vitamins – a compound is called a vitamin when it cannot be synthesized in sufficient quantities by the body and must be obtained from the diet. Therefore, the liver does not directly synthesize B-vitamins. Rather the liver converts some B vitamins from the diet, which are in a form that cannot be used by the body, into their biologically active co-enzyme forms. For example the liver converts: Dietary B Vitamin Thiamin B1 Riboflavin B2 Pyridoxal B6 Folate B9 Coenzyme Form Thiamin pyrophosphate (TPP) Flavine mononucleotide (FMN) Flavine adenine dinucleotide (FAD) Pyridoxal phosphate (PLP) Tetrahydrofolate (THFA) Iron and Copper – in order for iron and copper to be bio-available they must be bound to their appropriate bloodstream transport or storage proteins. The synthesis of these proteins, such as transferring, ferritin and ceruloplasmin and the binding of the minerals takes place in the liver. Carnitine – the liver synthesizes carnitine from the amino acid lysine and methionine. Carnitine escorts fats into the mitochondria, where the fats may be burned to generate ATP – bio-energy. NOTE – virtually every nutrient, whether it is a vitamin, mineral or amino acid, must be converted by the liver into its proper biochemical form in which the nutrient may be stored, transported or used in cellular metabolism. If the liver fails to do this, then even the most well absorbed, high potency, broad spectrum supplements will be useless at best and possibly even mildly toxic. The forms in which nutrients are found in supplements and foods are NOT the final, active biochemical forms used by the cells. And even if you do get the active coenzyme form of a nutrient from a food or supplement, it will usually be broken down during digestion. So there's no getting around the critical role of the liver in bio-activating the nutrients we get from foods or supplements. Cholesterol & Lipoproteins: Cholesterol is an essential structural component of cell membranes, where it is required to establish proper membrane permeability and fluidity. Within the cell membrane cholesterol also functions in intracellular transport, cell signaling and nerve conduction. In addition, cholesterol is an important component for the manufacture of bile acids, steroid hormones (estrogen, progesterone, testosterone, cortisol, aldosterone), and several fat-soluble vitamins (vit.D). Cholesterol is either obtained from the diet - cheese, egg yolk, beef, pork, poultry and shrimp - or synthesized in a variety of tissues – adrenal cortex, skin, intestine, testes and liver. About 10 - 20% of total daily cholesterol production occurs in the liver. The liver is the main organ responsible for the regulation of cholesterol levels circulating throughout the bloodstream. The liver does this by: Image: Cell membrane Synthesizing cholesterol Varying the amount of cholesterol it synthesizes based on the amount of cholesterol intake from dietary sources (negative feedback loop). Synthesizes the various lipoproteins involved in transporting cholesterol and lipids throughout the body. Lipoproteins are a biochemical assembly that contains both lipids (fat) and protein, for example – high density lipoprotein (HDL) and low density lipoprotein (LDL). Removes cholesterol from the body by converting it to bile salts and excreting it in the bile. Storage Function of the Liver The liver acts as a storage facility for certain vitamins, minerals and sugars such as: Glycogen Lipids Cholesterol Vitamin Vitamin Vitamin Vitamin Folate A D K B12 Iron Copper Zinc Immunological Function of the Liver The liver is an immunologically distinct organ, though as such the liver receives little attention, despite the fact that it fulfills a number of roles in the defense of the body. Case in point; the reticuloendothelial system, which most people have never heard of, is an integral part of the body’s defenses – the immune system. The reticuloendothelial system consists of a special class of cells widely distributed in the body. The function of these cells is to engulf, digest and destroy bacteria, viruses, foreign substances and worn out or abnormal cells. This special class of cells are called macrophages (immune cells). Eighty percent of the body’s resident macrophages are found in the liver and once inside the liver are referred to as Kupffer cells. Picture: Macrophage engulfing foreign particle Described earlier, Kupffer cells are essential to the liver’s primary function, which is cleansing the blood of foreign materials and toxic substances. When no foreign materials are present, Kupffer cells are in a resting state. Kupffer cells can be activated by numerous molecules, for example: bacterial endotoxins. An endotoxin is a toxin that forms part of the cell wall of certain bacteria and is only released upon destruction of the bacterial cell. As endotoxins cross the intestinal barrier and enters the bloodstream, it interacts with the Kupffer cells in the liver, thereby activating them. When activated, Kupffer cells secrete a variety of cytokines, including a molecule called tumor necrosis factor alpha (TNF–α) and several types of interleukins. All of these molecules can act as inflammatory cytokines—that is, they induce an inflammatory response necessary to remove the offending toxic or foreign molecules and initiate the healing process. In addition to producing cytokines, activated Kupffer cells are the major source of reactive oxygen species (ROS) in the liver. Reactive oxygen species, also known as free radicals, are oxygen containing molecules that are highly reactive with other complex molecules in the cell (e.g., proteins, fat molecules and DNA). Reactive oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radicals have been implicated in the development of liver damage. Excessive levels of reactive oxygen species within a cell and/or the lack of molecules that can eliminate them (i.e., antioxidants) leads to a state called oxidative stress that is detrimental to the cell. The liver is also a major contributor to the formation of lymph. Lymph is a clear, watery fluid that resembles the plasma of blood. Lymph flows through an elaborate network of channels and ducts called lymph vessels. Lymph, lymph vessels and lymph organs such as - lymph nodes, thymus, spleen and bone marrow- are collectively referred to as the lymphatic system. The lymphatic system has several functions, but one of the primary functions is to fight infections and defend the body against diseases. It does this by producing special white blood cells called lymphocytes that produce antibodies. Lymph is the medium in which lymphocytes, along with macrophages, travel through the lymphatic system where they come in contact with foreign particles and germs - hopefully destroying them. The function of the lymphatic system is considered by many to really be part of the immune system. It is estimated that 25 to 50% of lymph flowing through the major lymphatic vessel – the thoracic duct - is produced by the liver. This occurs due to the large pores or fenestrations in the liver’s sinusoidal endothelial cells. These openings allow fluid and proteins in the blood to flow freely into the space between the endothelium and hepatocytes – “the space of Disse" - forming lymph. Lymph flows through the space of Disse to collect in small lymphatic capillaries associated with portal triads. Another important immunological function of the liver is the production and secretion of a specific immunoglobulin. Immunoglobulins, also known as antibodies, are gamma globulin proteins that are found in blood and other bodily fluids and are used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. Immunoglobulins come in different varieties known as isotypes of which there are five known types in the human body – IgA, IgD, IgE, IgG and IgM. “Ig” stands for immunoglobulin and each one differs in their biological properties, functional locations and ability to deal with different antigens (foreign particle). The immunoglobulin we are concerned with is IgA, which is found in mucosal areas – intestine, respiratory tract, urogenital tract, prostate, saliva, tears, and breast milk - and prevents colonization by pathogens (germs). A selective lack of immunoglobulin A, which constitutes the most common type of immunoglobulin deficiency, appears in about 1 in 400 individuals. Common symptoms are recurring infections of mucosal surfaces – nose, throat, lungs, intestines as well as diseases of the mucosal surfaces - respiratory allergies, gastrointestinal diseases, autoimmune diseases, and malignant tumors. The liver plays a major role in secretion and / or clearance of circulating IgA into the upper gastrointestinal tract. IgA in the sinusoidal blood combines with secretory components (SC) on the surface of hepatocytes before being transported into the bile. It has also been found that IgA containing plasma cells are numerous in human biliary mucosa where they release IgA into the bile ducts. Together the proportion of IgA in human bile that is derived from local synthesis within hepato-biliary tissues is high – about 50%. Biotransformation Function of the Liver The main function of the small intestine is to absorb and it is not picky about what it absorbs. Consequently, it absorbs many substances that are potentially noxious (harmful) or at least of questionable value. Therefore, it is up to the liver to get rid of them. This is not an easy task, especially for lipophilic molecules (affinity for lipids /fats), as everything that leaves the body (via body fluids and waste) is more or less hydrophilic (watery). The liver’s solution is a process called biotransformation, which is covered in more detail in part II of this report. References Human Physiology, by Wikibooks contributors, 2007 Nagura H. Phillip D.S., Nakane P.K., Brown W.R., IgA in Human Bile and Liver, The Journal of Immunology, 126(2): 587-594 (1981) Nagura H. Tsutsumi Y. Hasegawa K. Watanabe P.K., IgA plasma cells in biliary mucosa: a likely source of locally synthesized IgA in human hepatic bile, Clinical Experimental Immunology, 54: 671-680 (1983) Ohtani O. Ohtani Y., Lymph Circulation in the Liver, The Anatomical Record, 291: 643-652 (2008) Wheeler M.D., Endotoxin and Kupffer Cell Activation in Alcoholic Liver Disease, National Institute of Health, (2004) World Wide Web: Brown University Colorado State University General Biology by Jim Kimball PhD, 2009 Lecture – Arno Helmberg, Innsbruck Medical University, Austria National Anemia Action Council On-Line Biology Book by Michael Farabee 2006 University of South Australia Wikipedia