AND PATHOBIOCHEMISTRY OF BLOOD. RESPIRATORY FUNCTION OF ERYTHROCYTES. PATHOLOGICAL
FORMS OF HEMOGLOBIN.
Blood is a liquid tissue. Suspended in the watery plasma are seven types of cells and cell fragments.
• red blood cells (RBCs) or erythrocytes
• platelets or thrombocytes
• five kinds of white blood cells (WBCs) or leukocytes
Three kinds of granulocytes
Two kinds of leukocytes without granules in their cytoplasm
If one takes a sample of blood, treats it with an agent to prevent clotting, and spins it in a centrifuge,
• the red cells settle to the bottom
• the white cells settle on top of them forming the "buffy coat".
The fraction occupied by the red cells is called the hematocrit. Normally it is approximately 45%. Values much lower than this are a sign of anemia
Biological functions of the blood
The blood is the most specialized fluid tissue which circulates in vascular system and together with lymph and intercellular space compounds an internal environment of an organism.
The blood executes such functions:
1. Transport of gases – oxygen from lungs is carried to tissues and carbon dioxide from tissues to lungs.
2. Transport of nutrients to all cells of organism (glucose, amino acids, fatty acids, vitamins, ketone bodies, trace substances and others). Substances such as urea, uric acid, bilirubin and creatinine are taken away from the different organs for ultimate excretion.
3. Regulatory or hormonal function – hormones are secreted in to blood and they are transported by blood to their target cells.
4. Thermoregulation function - an exchange of heat between tissues and blood.
5. Osmotic function- sustains osmotic pressure in vessels.
6. Protective function- by the phagocytic action of leucocytes and by the actions of antibodies, the blood provides the most important defense mechanism.
7. Detoxification function - neutralization of toxic substances which is connected with their decomposition by the help of blood enzymes.
Blood performs two major functions:
All the various types of blood cells
· are produced in the bone marrow (some 1011 of them each day in an adult human!).
· arise from a single type of cell called a hematopoietic stem cell — an "adult" multipotent stem cell.
These stem cells
Which path is taken is regulated by
Two types of blood cells can be distinguished - white and red blood cells. White blood cells are called leucocytes. Their quantity in adult is 4-9 x 109/L.
Red blood cells are called erythrocytes. Their quantity in peripheral blood is 4,5-5 x 1012/L. Besides that, there are also thrombocytes or platelets in blood.
Leucocytes (white blood cells) protect an organism from microorganisms, viruses and foreign substances, that provides the immune status of an organism.
Leucocytes are divided into two groups: Granulocytes and agranulocytes. Granulocytes consist of neutrophils, eosinophils and basophils. Agranulocytes consist of monocytes and lymphocytes.
Neutrophils comprise of 60-70 % from all leucocytes. Their main function is to protect organisms from microorganisms and viruses. Neutrophils have segmented nucleus, endoplasmic reticulum (underdeveloped) which does not contain ribosomes, insufficient amount of mitochondria, well-developed Golgi apparatus and hundreds of different vesicles which contain peroxidases and hydrolases. Optimum condition for their activity is acidic pH. There are also small vesicles which contain alkaline phosphatases, lysozymes, lactopherins and proteins of cationic origin.
Glucose is the main source of energy for neutrophils. It is directly utilized or converted into glycogen. 90 % of energy is formed in glycolysis, a small amount of glucose is converted in pentosophosphate pathway. Activation of proteolysis during phagocytosis as well as reduction of phosphatidic acid and phosphoglycerols are also observed. The englobement is accompanied by intensifying of a glycolysis and pentosophosphate pathway. But especially intensity of absorption of oxygen for neutrophils - so-called flashout of respiration grows. Absorbed oxygen is spent for formation of its fissile forms that is carried out with participation enzymes:
1. NADP*Í -OXYDASE catalyzes formation of super oxide anion
2. An enzyme NADH- OXYDASE is responsible for formation of hydrogen peroxide
3. Ìyeloperoxydase catalyzes formation of hypochloric acid from chloride and hydrogen peroxide
Neutrophils are motile phagocyte cells that play a key role in acute inflammation. When bacteria enter tissues, a number of phenomena occur that are collectively known as acute inflammatory response. When neutrophils and other phagocyte cells engulf bacteria, they exhibit a rapid increase in oxygen consumption known as the respiratory burst. This phenomenon reflects the rapid utilization of oxygen (following a lag of 15-60 seconds) and production from it of large amounts of reactive derivates, such as O2-, H2O2, OH. and OCl- (hypochlorite ion). Some of these products are potent microbicidal agents. The electron transport chain system responsible for the respiratory burst contains several components, including a flavoprotein NADPH:O2-oxidoreductase (often called NADPH-oxidase) and a b-type cytochrome.
The most abundant of the WBCs. This photomicrograph shows a single neutrophil surrounded by red blood cells.
Neutrophils squeeze through the capillary walls and into infected tissue where they kill the invaders (e.g., bacteria) and then engulf the remnants by phagocytosis.
This is a never-ending task, even in healthy people: Our throat, nasal passages, and colon harbor vast numbers of bacteria. Most of these are commensals, and do us no harm. But that is because neutrophils keep them in check.
can reduce the numbers of neutrophils so that formerly harmless bacteria begin to proliferate. The resulting opportunistic infection can be life-threatening.
Some important enzymes and proteins of neutrophilis.
Myeloperoxidase (MPO). Catalyzed following reaction:
H2O2 + X-(halide) + H+® HOX + H2O (where X- = Cl-, Br-, I- or SCN-; HOX=hypochlorous acid)
HOCl, the active ingredient of household liquid bleach, is a powerful oxidant and is highly microbicidial. When applied to normal tissues, its potential for causing damage is diminished because it reacts with primary or secondary amines present in neutrophils and tissues to produce various nitrogen-chlorine (N-Cl) derivates; these chloramines are also oxidants, although less powerful than HOCl, and act as microbicidial agents (eg, in sterilizing wounds) without causing tissue damage. Responsible for the green color of pus.
2O2 + NADPH ® 2O2- + NADP + H+
Key component of the respiratory burst. Deficiency may be observed in chronic granulomatous disease.
Hydrolyzes link between N-acetylmuramic acid and N-acetyl-D-glucosamine found in certain bacterial cell walls. Abundant in macrophages.
Basic antibiotic peptides of 29-33 amino acids. Apparently kill bacteria by causing membrane damage.
Iron-binding protein. May inhibit growth of certain bacteria by binding iron and may be involved in regulation of proliferation of myeloid cells.
Neutrophils contain a number of proteinases (elastase, collagenase, gelatinase, cathepsin G, plasminogen activator) that can hydrolyze elastin, various types of collagens, and other proteins present in the extracellular matrix. Such enzymatic action, if allowed to proceed unopposed, can result in serious damage to tissues. Most of these proteinases are lysosomal enzymes and exist mainly as inactive precursors in normal neutrophils. Small amounts of these enzymes are released into normal tissues, with the amounts increasing markedly during inflammation. The activities of elastase and other proteinases are normally kept in check by a number of antiproteinases (a1-Antiproteinase, a2-Macroglobulin, Secretory leukoproteinase inhibitor, a1-Antichymotrypsin, Plasminogen activator inhibitor-1, Tissue inhibitor of metalloproteinase) present in plasma and the extracellular fluid.
Basophiles make up 1-5% of all blood leukocytes. They are actively formed in the bone marrow during allergy. Basophiles take part in the allergic reactions, in the blood coagulation and intravascular lipolysis. They have the protein synthesis mechanism, which works due to the biological oxidation energy . They synthesize the mediators of allergic reactions – histamine and serotonin, which during allergy cause local inflammation. Heparin, which is formed in the basophiles, prevents the blood coagulation and activates intravascular lipoprotein lipase, which splits triacylglycerin.
The number of basophils also increases during infection. Basophils leave the blood and accumulate at the site of infection or other inflammation. There they discharge the contents of their granules, releasing a variety of mediators such as:
which increase the blood flow to the area and in other ways add to the inflammatory process. The mediators released by basophils also play an important part in some allergic responses such as
They make up 3-6% of all leukocytes. Eosinophiles as well as neutrophiles defend the cells from microorganisms, they contain myeloperoxidase, lysosomal hydrolases. About the relations of eosinophiles with testifies the growth of their amount during the sensitization of organism, i.e. during bronchial asthma, helminthiasis. They are able to pile and splits histamine, “to dissolve” thrombus with the participation of plasminogen and bradykinin-kininase.
They are formed in the bone marrow. They make up 4-8% of all leukocytes. According to the function they are called macrophages. Tissue macrophages derive from blood monocytes. Depending on their position they are called: in the liver – reticuloendotheliocytes, in the lungs - alveolar macrophages, in the intermediate substance of connective tissue – histocytes etc. Monocytes are characterized by a wide set of lysosomal enzymes with the optimum activity in the acidic condition. The major functions of monocytes and macrophages are endocytosis and phagocytosis.
The amount – 20-25%, are formed in the lymphoid tissue or thymus, play important role in the formation of humoral and cellular immunity. Lymphocytes have powerful system of synthesis of antibody proteins, energy is majorily pertained due to glycolysis, rarely – by aerobic way.
There are several kinds of lymphocytes (although they all look alike under the microscope), each with different functions to perform . The most common types of lymphocytes are
Although bone marrow is the ultimate source of lymphocytes, the lymphocytes that will become T cells migrate from the bone marrow to the thymus where they mature. Both B cells and T cells also take up residence in lymph nodes, the spleen and other tissues where they
Monocytes leave the blood and become macrophages and dendritic cells.
This scanning electron micrograph (courtesy of Drs. Jan M. Orenstein and Emma Shelton) shows a single macrophage surrounded by several lymphocytes.
Macrophages are large, phagocytic cells that engulf
Thrombocytes (blood platelets)
Platelets are cell fragments produced from megakaryocytes.
Blood normally contains 150,000–350,000 per microliter (µl) or cubic millimeter (mm3). This number is normally maintained by a homeostatic (negative-feedback) mechanism .
The amount – less than 1%, they play the main role in the process of hemostasis. They are formed as a result of disintegration of megakaryocytes in the bone marrow. Their –life-time is 7-9 days. In spite of the fact that thrombocytes have no nucleus, they are able to perform practically all functions of the cell, besides DNA synthesis.
If this value should drop much below 50,000/µl, there is a danger of uncontrolled bleeding because of the essential role that platelets have in blood clotting.
When blood vessels are cut or damaged, the loss of blood from the system must be stopped before shock and possible death occur. This is accomplished by solidification of the blood, a process called coagulation or clotting.
A blood clot consists of
The most numerous type in the blood.
RBC precursors mature in the bone marrow closely attached to a macrophage.
Human blood contains 25 trillion of erythrocytes. Their main function – transportation of O2 and CO2 – they perform due to the fact that they contain 34% of hemoglobin, and per dry cells mass – 95%. The total amount of hemoglobin in the blood equals 130-160 g/l. In the process of erythropoesis the preceding cells decrease their size. Their nuclei at the end of the process are ruined and pushed out of the cells. 90% of glucose in the erythrocytes is decomposed in the process of glycolysis and 10% - by pentose-phosphate way. There are noted congenital defects of enzymes of these metabolic ways of erythrocytes. During this are usually observed hemolytic anemia and other structural and functional erythrocytes’ affections.
This scanning electron micrograph (courtesy of Dr. Marion J. Barnhart) shows the characteristic biconcave shape of red blood cells.
Thus RBCs are terminally differentiated; that is, they can never divide. They live about 120 days and then are ingested by phagocytic cells in the liver and spleen. Most of the iron in their hemoglobin is reclaimed for reuse. The remainder of the heme portion of the molecule is degraded into bile pigments and excreted by the liver. Some 3 million RBCs die and are scavenged by the liver each second.
Red blood cells are responsible for the transport of oxygen and carbon dioxide.
In adult humans the hemoglobin (Hb) molecule
o two alpha (α) chains of 141 amino acids and
o two beta (β) chains of 146 amino acids
The reaction is reversible.
Carbon dioxide (CO2) combines with water forming carbonic acid, which dissociates into a hydrogen ion (H+) and a bicarbonate ions
Only about 5% of the CO2 generated in the tissues dissolves directly in the plasma. (A good thing, too: if all the CO2 we make were carried this way, the pH of the blood would drop from its normal 7.4 to an instantly-fatal 4.5!)
Figure 1 is a model of human deoxyhemoglobin. It was created in RasMol version 2.6 by Roger Sayle using the pdb coordinates from the pdb file 4hhb. The 3D coordinates were determed from x-ray crystallography by Fermi, G., Perutz, M. F., Shaanan, B., Fourme, R.: The crystal structure of human deoxyhaemoglobin at 1.74 A resolution. J Mol Biol 175 pp. 159 (1984)
is the protein that carries oxygen from the lungs to the tissues and carries
carbon dioxide from the tissues back to the lungs. In order to function most
efficiently, hemoglobin needs to bind to oxygen tightly in the oxygen-rich
atmosphere of the lungs and be able to release oxygen rapidly in the relatively
oxygen-poor environment of the tissues. It does this in a most elegant and
intricately coordinated way. The story of hemoglobin is the prototype example
of the relationship between structure and function of a protein molecule.
hemoglobin molecule consists of four polypeptide chains: two alpha chains, each
with 141 amino acids and two beta chains, each with 146 amino acids. The
protein portion of each of these chains is called "globin". The a and
b globin chains are very similar in structure. In this case, a and b refer to
the two types of globin. Students often confuse this with the concept of a
helix and b sheet secondary structures. But, in fact, both the a and b globin
chains contain primarily a helix secondary structure with no b sheets.
Figure 2 is a close up view of one of the heme groups of the human a chain from dexoyhemoglobin. In this view, the iron is coordinated by a histidine side chain from amino acid 87 (shown in green.)
Each a or b globin chain folds into 8 a helical segments (A-H) which, in turn, fold to form globular tertiary structures that look roughly like sub-microscopic kidney beans. The folded helices form a pocket that holds the working part of each chain, the heme.
A heme group is a flat ring molecule containing carbon, nitrogen and hydrogen atoms, with a single Fe2+ ion at the center. Without the iron, the ring is called a porphyrin. In a heme molecule, the iron is held within the flat plane by four nitrogen ligands from the porphyrin ring. The iron ion makes a fifth bond to a histidine side chain from one of the helices that form the heme pocket. This fifth coordination bond is to histidine 87 in the human a chain and histidine 92 in the human b chain. Both histidine residues are part of the F helix in each globin chain. t
The ability of hemoglobin to release oxygen, is affected by pH, CO2 and by the differences in the oxygen-rich environment of the lungs and the oxygen-poor environment of the tissues. The pH in the tissues is considerably lower (more acidic) than in the lungs. Protons are generated from the reaction between carbon dioxide and water to form bicarbonate:
This increased acidity serves a twofold purpose. First, protons lower the affinity of hemoglobin for oxygen, allowing easier release into the tissues. As all four oxygens are released, hemoglobin binds to two protons. This helps to maintain equilibrium towards the right side of the equation. This is known as the Bohr effect, and is vital in the removal of carbon dioxide as waste because CO2 is insoluble in the bloodstream. The bicarbonate ion is much more soluble, and can thereby be transported back to the lungs after being bound to hemoglobin. If hemoglobin couldn’t absorb the excess protons, the equilibrium would shift to the left, and carbon dioxide couldn’t be removed.
the lungs, this effect works in the reverse direction. In the presence of the
high oxygen concentration in the lungs, the proton affinity decreases. As
protons are shed, the reaction is driven to the left, and CO2 forms
as an insoluble gas to be expelled from the lungs. The proton poor hemoglobin
now has a greater affinity for oxygen, and the cycle continues.
Haemoglobin or hemoglobin (frequently abbreviated as Hb or Hgb) is the iron-containing oxygen-transport metalloprotein in the red blood cells of the blood in vertebrates and other animals; in mammals the protein makes up about 97% of the red cell’s dry content, and around 35% of the total content including water. Hemoglobin transports oxygen from the lungs or gills to the rest of the body, such as to the muscles, where it releases the oxygen load. Hemoglobin also has a variety of other gas-transport and effect-modulation duties, which vary from species to species, and which in invertebrates may be quite diverse.
The name hemoglobin is the concatenation of heme and globin, reflecting the fact that each subunit of hemoglobin is a globular protein with an embedded heme (or haem) group; each heme group contains an iron atom, and this is responsible for the binding of oxygen. The most common type of hemoglobin in mammals contains four such subunits, each with one heme group.
Mutations in the genes for the hemoglobin protein in humans result in a group of hereditary diseases termed the hemoglobinopathies, the most common members of which are sickle-cell disease and thalassemia. Historically in human medicine, the hemoglobinopathy of sickle-cell disease was the first disease to be understood in its mechanism of dysfunction, completely down to the molecular level. However, not all of such mutations produce disease states, and are formally recognized as hemoglobin variants (not diseases).
Hemoglobin (Hb) is synthesized in a complex series of steps. The heme portion is sythesized in both the the mitochondria and cytosol of the immature red blood cell, while the globin protein portions of the molecule are sythesized by ribosomes in the cytosol . Production of Hb continues in the cell throughout its early development from the proerythroblast to the reticulocyte in the bone marrow. At this point, the nucleus is lost in mammals, but not in birds and many other species. Even after the loss of the nucleus in mammals, however, residual ribosomal RNA allows further synthesis of Hb until the reticulocyte loses its RNA soon after entering the vasculature (this hemoglobin-synthetic RNA in fact gives the reticulocyte its reticulated appearance and name).
The empirical chemical formula of the most common human hemoglobin is C2952H4664N812O832S8Fe4, but as noted above, hemoglobins vary widely across species, and even (through common mutations) slightly among subgroups of humans.
In humans, the hemoglobin molecule is an assembly of four globular protein subunits. Each subunit is composed of a protein chain tightly associated with a non-protein heme group. Each protein chain arranges into a set of alpha-helix structural segments connected together in a globin fold arrangement, so called because this arrangement is the same folding motif used in other heme/globin proteins such as myoglobin. This folding pattern contains a pocket which strongly binds the heme group.
A heme group consists of an iron (Fe) atom held in a heterocyclic ring, known as a porphyrin. The iron atom, which is the site of oxygen binding, bonds with the four nitrogens in the center of the ring, which all lie in one plane. The iron is also bound strongly to the globular protein via the imidazole ring of a histidine residue below the porphyrin ring. A sixth position can reversibly bind oxygen, completing the octahedral group of six ligands. Oxygen binds in an "end-on bent" geometry where one oxygen atom binds Fe and the other protrudes at an angle. When oxygen is not bound, a very weakly bonded water molecule fills the site, forming a distorted octahedron.
The iron atom may either be in the Fe2+ or Fe3+ state, but ferrihemoglobin (methemoglobin) (Fe3+) cannot bind oxygen. In binding, oxygen temporarily oxidizes Fe to (Fe3+), so iron must exist in the +2 oxidation state in order to bind oxygen. The body reactivates hemoglobin found in the inactive (Fe3+) state by reducing the iron center.
In adult humans, the most common hemoglobin type is a tetramer (which contains 4 subunit proteins) called hemoglobin A, consisting of two α and two β subunits non-covalently bound, each made of 141 and 146 amino acid residues, respectively. This is denoted as α2β2. The subunits are structurally similar and about the same size. Each subunit has a molecular weight of about 17,000 daltons, for a total molecular weight of the tetramer of about 68,000 daltons. Hemoglobin A is the most intensively studied of the hemoglobin molecules.
Oxyhemoglobin is formed during respiration when oxygen binds to the heme component of the protein hemoglobin in red blood cells. This process occurs in the pulmonary capillaries adjacent to the alveoli of the lungs. The oxygen then travels through the blood stream to be dropped off at cells where it is utilized in aerobic glycolysis and in the production of ATP by the process of oxidative phosphorylation. It doesn't however help to counteract a decrease in blood pH. Ventilation, or breathing, may reverse this condition by removal of carbon dioxide, thus causing a shift up in pH.
Deoxyhemoglobin is the form of hemoglobin without the bound oxygen. The absorption spectra of oxyhemoglobin and deoxyhemoglobin differ. The oxyhemoglobine has significantly lower absorption of the 660 nm wavelength than deoxyhemoglobin, while at 940 nm its absorption is slightly higher. This difference is used for measurement of the amount of oxygen in patient's blood by an instrument called pulse oximeter.
Assigning oxygenated hemoglobin's oxidation state is difficult because oxyhemoglobin is diamagnetic (no net unpaired electrons), but the low-energy electron configurations in both oxygen and iron are paramagnetic. Triplet oxygen, the lowest energy oxygen species, has two unpaired electrons in antibonding π* molecular orbitals. Iron(II) tends to be in a high-spin configuration where unpaired electrons exist in eg antibonding orbitals. Iron(III) has an odd number of electrons and necessarily has unpaired electrons. All of these molecules are paramagnetic (have unpaired electrons), not diamagnetic, so an unintuitive distribution of electrons must exist to induce diamagnetism.
X-ray photoelectron spectroscopy suggests that iron has an oxidation state of approximately 3.2 and infrared stretching frequencies of the O-O bond suggests a bond length fitting with superoxide. The correct oxidation state of iron is thus the +3 state with oxygen in the -1 state. The diamagnetism in this configuration arises from the unpaired electron on superoxide aligning antiferromagnetically in the opposite direction from the unpaired electron on iron. The second choice being correct is not surprising because singlet oxygen and large separations of charge are both unfavorably high-energy states. Iron's shift to a higher oxidation state decreases the atom's size and allows it into the plane of the porphyrin ring, pulling on the coordinated histidine residue and initiating the allosteric changes seen in the globulins. The assignment of oxidation state, however, is only a formalism so all three models may contribute to some small degree.
Early postulates by bioinorganic chemists claimed that possibility (1) (above) was correct and that iron should exist in oxidation state II (indeed iron oxidation state III as methemoglobin, when not accompanied by superoxide .O2- to "hold" the oxidation electron, is incapable of binding O2). The iron chemistry in this model was elegant, but the presence of singlet oxygen was never explained. It was argued that the binding of an oxygen molecule placed high-spin iron(II) in an octahedral field of strong-field ligands; this change in field would increase the crystal field splitting energy, causing iron's electrons to pair into the diamagnetic low-spin configuration.
Binding and release of ligands induces a conformational (structural) change in hemoglobin. Here, the binding and release of oxygen illustrates the structural differences between oxy- and deoxyhemoglobin, respectively. Only one of the four heme groups is shown.
As discussed above, when oxygen binds to the iron center it causes contraction of the iron atom, and causes it to move back into the center of the porphyrin ring plane (see moving diagram). At the same time, the porphyrin ring plane itself is pushed away from the oxygen and toward the imidizole side chain of the histidine residue interacting at the other pole of the iron. The interaction here forces the ring plane sideways toward the outside of the tetramer, and also induces a strain on the protein helix containing the histidine, as it moves nearer the iron. This causes a tug on this peptide strand which tends to open up heme units in the remainder of the molecule, so that there is more room for oxygen to bind at their heme sites.
In the tetrameric form of normal adult hemoglobin, the binding of oxygen is thus a cooperative process. The binding affinity of hemoglobin for oxygen is increased by the oxygen saturation of the molecule, with the first oxygens bound influencing the shape of the binding sites for the next oxygens, in a way favorable for binding. This positive cooperative binding is achieved through steric conformational changes of the hemoglobin protein complex as discussed above, i.e. when one subunit protein in hemoglobin becomes oxygenated, this induces a conformational or structural change in the whole complex, causing the other subunits to gain an increased affinity for oxygen. As a consequence, the oxygen binding curve of hemoglobin is sigmoidal, or S-shaped, as opposed to the normal hyperbolic curve associated with noncooperative binding.
Hemoglobin's oxygen-binding capacity is decreased in the presence of carbon monoxide because both gases compete for the same binding sites on hemoglobin, carbon monoxide binding preferentially in place of oxygen. Carbon dioxide occupies a different binding site on the hemoglobin. Through the enzyme carbonic anhydrase, carbon dioxide reacts with water to give carbonic acid, which decomposes into bicarbonate and protons:
Hence blood with high carbon dioxide levels is also lower in pH (more acidic). Hemoglobin can bind protons and carbon dioxide which causes a conformational change in the protein and facilitates the release of oxygen. Protons bind at various places along the protein, and carbon dioxide binds at the alpha-amino group forming carbamate. Conversely, when the carbon dioxide levels in the blood decrease (i.e., in the lung capillaries), carbon dioxide and protons are released from hemoglobin, increasing the oxygen affinity of the protein. This control of hemoglobin's affinity for oxygen by the binding and release of carbon dioxide and acid, is known as the Bohr effect.
The binding of oxygen is affected by molecules such as carbon monoxide (CO) (for example from tobacco smoking, cars and furnaces). CO competes with oxygen at the heme binding site. Hemoglobin binding affinity for CO is 200 times greater than its affinity for oxygen, meaning that small amounts of CO dramatically reduces hemoglobin's ability to transport oxygen. When hemoglobin combines with CO, it forms a very bright red compound called carboxyhemoglobin. When inspired air contains CO levels as low as 0.02%, headache and nausea occur; if the CO concentration is increased to 0.1%, unconsciousness will follow. In heavy smokers, up to 20% of the oxygen-active sites can be blocked by CO.
In similar fashion, hemoglobin also has competitive binding affinity for cyanide (CN-), sulfur monoxide (SO), nitrogen dioxide (NO2), and sulfide (S2-), including hydrogen sulfide (H2S). All of these bind to iron in heme without changing its oxidation state, but they nevertheless inhibit oxygen-binding, causing grave toxicity.
The iron atom in the heme group must be in the Fe2+ oxidation state to support oxygen and other gases' binding and transport. Oxidation to Fe3+ state converts hemoglobin into hemiglobin or methemoglobin (pronounced "MET-hemoglobin"), which cannot bind oxygen. Hemoglobin in normal red blood cells is protected by a reduction system to keep this from happening. Nitrogen dioxide and nitrous oxide are capable of converting a small fraction of hemoglobin to methemoglobin, however this is not usually of medical importance (nitrogen dioxide is poisonous by other mechanisms, and nitrous oxide is routinely used in surgical anesthesia in most people without undue methemoglobin buildup).
In people acclimated to high altitudes, the concentration of 2,3-bisphosphoglycerate (2,3-BPG) in the blood is increased, which allows these individuals to deliver a larger amount of oxygen to tissues under conditions of lower oxygen tension. This phenomenon, where molecule Y affects the binding of molecule X to a transport molecule Z, is called a heterotropic allosteric effect.
A variant hemoglobin, called fetal hemoglobin (HbF, α2γ2), is found in the developing fetus, and binds oxygen with greater affinity than adult hemoglobin. This means that the oxygen binding curve for fetal hemoglobin is left-shifted (i.e., a higher percentage of hemoglobin has oxygen bound to it at lower oxygen tension), in comparison to that of adult hemoglobin. As a result, fetal blood in the placenta is able to take oxygen from maternal blood.
Hemoglobin also carries nitric oxide in the globin part of the molecule. This improves oxygen delivery in the periphery and contributes to the control of respiration. NO binds reversibly to a specific cystein residue in globin; the binding depends on the state (R or T) of the hemoglobin. The resulting S-nitrosylated hemoglobin influences various NO-related activities such as the control of vascular resistance, blood pressure and respiration. NO is released not in the cytoplasm of erythrocytes but is transported by an anion exchanger called AE1 out of them.
When red cells reach the end of their life due to aging or defects, they are broken down, the hemoglobin molecule is broken up and the iron gets recycled. When the porphyrin ring is broken up, the fragments are normally secreted in the bile by the liver. This process also produces one molecule of carbon monoxide for every molecule of heme degraded ; this is one of the few natural sources of carbon monoxide production in the human body, and is responsible for the normal blood levels of carbon monoxide even in people breathing pure air. The other major final product of heme degradation is bilirubin. Increased levels of this chemical are detected in the blood if red cells are being destroyed more rapidly than usual. Improperly degraded hemoglobin protein or hemoglobin that has been released from the blood cells too rapidly can clog small blood vessels, especially the delicate blood filtering vessels of the kidneys, causing kidney damage
Decrease of hemoglobin, with or without an absolute decrease of red blood cells, leads to symptoms of anemia. Anemia has many different causes, although iron deficiency and its resultant iron deficiency anemia are the most common causes in the Western world. As absence of iron decreases heme synthesis, red blood cells in iron deficiency anemia are hypochromic (lacking the red hemoglobin pigment) and microcytic (smaller than normal). Other anemias are rarer. In hemolysis (accelerated breakdown of red blood cells), associated jaundice is caused by the hemoglobin metabolite bilirubin, and the circulating hemoglobin can cause renal failure.
Some mutations in the globin chain are associated with the hemoglobinopathies, such as sickle-cell disease and thalassemia. Other mutations, as discussed at the beginning of the article, are benign and are referred to merely as hemoglobin variants.
There is a group of genetic disorders, known as the porphyrias that are characterized by errors in metabolic pathways of heme synthesis. King George III of the United Kingdom was probably the most famous porphyria sufferer.
To a small extent, hemoglobin A slowly combines with glucose at a certain location in the molecule. The resulting molecule is often referred to as Hb A1c. As the concentration of glucose in the blood increases, the percentage of Hb A that turns into Hb A1c increases. In diabetics whose glucose usually runs high, the percent Hb A1c also runs high. Because of the slow rate of Hb A combination with glucose, the Hb A1c percentage is representative of glucose level in the blood averaged over a longer time (the half-life of red blood cells, which is typically 50-55 days).
Hemoglobin levels are amongst the most commonly performed blood tests, usually as part of a full blood count or complete blood count. Results are reported in g/L, g/dL or mol/L. For conversion, 1 g/dL is 0.621 mmol/L. If the total hemoglobin concentration in the blood falls below a set point, this is called anemia. Normal values for hemoglobin levels are:
Anemias are further subclassified by the size of the red blood cells, which are the cells which contain hemoglobin in vertebrates. They can be classified as microcytic (small sized red blood cells), normocytic (normal sized red blood cells), or macrocytic (large sized red blood cells). The hemaglobin is the typical test used for blood donation. A comparison with the hematocrit can be made by multiplying the hemaglobin by three. For example, if the hemaglobin is measured at 17, that compares with a hematocrit of .51.
Glucose levels in blood can vary widely each hour, so one or only a few samples from a patient analyzed for glucose may not be representative of glucose control in the long run. For this reason a blood sample may be analyzed for Hb A1c level, which is more representative of glucose control averaged over a longer time period (determined by the half-life of the individual's red blood cells, which is typically 50-55 days). People whose Hb A1c runs 6.0% or less show good longer-term glucose control. Hb A1c values which are more than 7.0% are elevated. This test is especially useful for diabetics.
Although bone marrow is the ultimate source of lymphocytes, the lymphocytes that will become T cells migrate from the bone marrow to the thymus where they mature. Both B cells and T cells also take up residence in lymph nodes, the spleen and other tissues where they
When blood vessels are cut or damaged, the loss of blood from the system must be stopped before shock and possible death occur. This is accomplished by solidification of the blood, a process called coagulation or clotting.
Albumins – multidispersed fraction of blood plasma which are characterized by the high electrophoretic mobility and mild dissolubility in water and saline solutions. Molecular weight of albumins is about 60000. Due to high hydrophilic properties albumins bind a significant amount of water, and the volume of their molecule under hydratation is doubled. Hydrative layer formed around the serum albumins provides to 70-80 % of oncotic pressure of blood plasma proteins, that can be applied in clinical practice at albumins transfusion to patients with tissue edemas. The decreasing of albumins concentration in blood plasma, for example under disturbance of their synthesis in hepatocytes at liver failure, can cause the water transition from a vessels into the tissues and development of oncotic edemas.
Albumins execute also important physiological function as transporters of a lot of metabolites and diverse low molecular weight structures. The molecules of albumins have several sites with centers of linkage for molecules of organic ligands, which are affixed by the electrostatic and hydrophobic bonds. Serum albumins can affix and convey fatty acids, cholesterol, cholic pigments (bilirubin and that similar), vitamins, hormones, some amino acids, toxins and medicines.
Globulins - heterogeneous fraction of blood proteins which execute transport (a1-globulins – transport of lipids, thyroxin, corticosteroid hormones; a2-globulins - transport of lipids, copper ions; b-globulins - transport of lipids, iron) and protective (participation of b-globulins in immune reactions as antitoxins; g-globulins as immunoglobulins) functions. They also support the blood oncotic pressure and acid-alkaline balance, provide amino acids for the organism requirements. The molecular weight of globulins is approximately 150000-300000.
The globulin level in blood plasma is 20-40 g/l. A ratio between concentrations of albumins and globulins (so called “protein coefficient”) in blood plasma is often determined in clinical practice. In healthy people this coefficient is 1,5-2,0.
Fibrinogen – important protein of blood plasma, precursor of fibrin, the structural element of blood clots. Fibrinogen participates in blood clotting and thus prevents the loss of blood from the vascular system of vertebrates. The approximate molecular weight of fibrinogen is 340000. It is the complex protein, it contains the carbohydrate as prosthetic group. The content of firinogen in blood is 3-4 g/l.
Immunoglobulins (Ig A, Ig G, Ig E, Ig M) - proteins of g-globulin fraction of blood plasma executing the functions of antibodies which are the main effectors of humoral immunity. They appear in the blood serum and certain cells of a vertebrate in response to the introduction of a protein or some other macromolecule foreign to that species.
Immunoglobulin molecules have bindind sites that are specific for and complementary to the structural features of the antigen that induced their formation. Antibodies are highly specific for the foreign proteins that evoke their formation.
C-reactive protein (g-fraction). This protein received the title owing to its capacity to react with C-polysaccharide of a pneumococcus forming precipitates. According to its chemical nature C-reactive protein is glycoprotein.
In blood plasma of healthy people the C-reactive protein is absent but it occurs at pathological states accompanied by an inflammation and necrosis of tissues. The availability of C-reactive protein is characteristic for the acute period of diseases – “protein of an acute phase”. The determination of C-reactive protein has diagnostic value in an acute phase of rheumatic disease, at a myocardial infarction, pneumococcal, streptococcal, staphylococcal infections.
Crioglobulin - the protein of the g-globulin fraction. Like to the C-reactive protein crioglobulin absent in blood plasma of the healthy people and occurs at leukoses, rheumatic disease, liver cirrhosis, nephroses. The characteristic physico-chemical feature of crioglobulin is its dissolubility at standard body temperature (37 oC) and capacity to form the sediment at cooling of a blood plasma up to 4 oC.
The biological role of a2-macroglobulin consists in regulation of the tissue proteolysis systems which are very important in such physiological and pathological processes as blood clotting, fibrinolysis, processes of immunodefence, functionality of a complement system, inflammation, regulation of vascular tone (kinine and renin-angiothensine system).
a1-antitrypsin (a1-globulin) – glycoprotein with a molecular weight 55 kDa. Its concentration in blood plasma is 2-3 ã/ë. The main biological property of this inhibitor is its capacity to form complexes with proteinases oppressing proteolitic activity of such enzymes as trypsin, chemotrypsin, plasmin, trombin. The content of a1-antitrypsin is markedly increased in inflammatory processes. The inhibitory activity of a1-antitrypsin is very important in pancreas necrosis and acute pancreatitis because in these conditions the proteinase level in blood and tissues is sharply increased. The congenital deficiency of a1-antitrypsin results in the lung emphysema.
Fibronectin – glycoprotein of blood plasma that is synthesized and secreted in intercellular space by different cells. Fibronectin present on a surface of cells, on the basal membranes, in connective tissue and in blood. Fibronectin has properties of a «sticking» protein and contacts with the carbohydrate groups of gangliosides on a surface of plasma membranes executing the integrative function in intercellular interplay. Fibronectin also plays important role in the formation of the pericellular matrix.
Haptoglobin - protein of a2-globulin fraction of blood plasma. Haptoglobin has capacity to bind a free haemoglobin forming a complex that refer to b-globulins electrophoretic fraction. Normal concentration in blood plasma - 0,10-0,35 g/l.
Haptoglobin-hemoglobin complexes are absorbed by the cells of reticulo-endothelial system, in particular in a liver, and oxidized to cholic pigments. Such haptoglobin function promotes the preservation of iron ions in an organism under conditions of a physiological and pathological erythrocytolysis.
Transferrin - glycoprotein belonging to the b-globulin fraction. It binds in a blood plasma iron ions (Fe3+). The protein has on the surface two centers of linkage of iron. Transferrin is a transport form of iron delivering its to places of accumulation and usage.
Ceruloplasmin - glycoprotein of the a2-globulin fraction. It can bind the copper ions in blood plasma. Up to 3 % of all copper contents in an organism and more than 90 % copper contents in plasma is included in ceruloplasmin. Ceruloplasmin has properties of ferroxidase oxidizing the iron ions. The decrease of ceruloplasmin in organism (Wilson disease) results in exit of copper ions from vessels and its accumulation in the connective tissue that shows by pathological changes in a liver, main brain, cornea.
Albumins, a1-globulins, fibrinogen are fully synthesized in hepatocytes. Immunoglobulins are produced by plasmocytes (immune cells). In liver cryoglobulins and some other g-globulins are produced too. a2-globulins and b-globulins are partly synthesized in liver and partly in reticuloendothelial cells.
Hypoproteinemia - decrease of the total contents of proteins in blood plasma. This state occurs in old people as well as in pathological states accompanying with the oppressing of protein synthesis (liver diseases) and activation of decomposition of tissue proteins (starvation, hard infectious diseases, state after hard trauma and operations, cancer). Hypoproteinemia (hypoalbuminemia) also occurs in kidney diseases, when the increased excretion of proteins via the urine takes place.
Absolute hyperproteinemia – accumulation of the proteins in blood. It occurs in infection and inflammatory diseases (hyperproduction of immunoglobulins), rheumatic diseases (hyperproduction of C-reactive protein), some malignant tumors (myeloma) and others.
Relative hyperproteinemia – the increase of the protein concentration but not the absolute amount of proteins. It occurs when organism loses water (diarrhea, vomiting, fever, intensive physical activity etc.).
Electrophoresis is the separation of proteins on the basis of their electric charge. It depends ultimately on their base-acid properties, which are largely determined by the number and types of ionizable R groups in their polipeptide chains. Since proteins differ in amino acid composition and sequence, each protein has distinctive acid-base properties. There are a number of different forms of electroforesis useful for analyzing and separating mixtures of proteins
If a precursor of an antibody-secreting cell becomes cancerous, it divides uncontrollably to generate a clone of plasma cells secreting a single kind of antibody molecule. The image (courtesy of Beckman Instruments, Inc.) shows — from left to right — the electrophoretic separation of:
Gamma globulins can be harvested from donated blood (usually pooled from several thousand donors) and injected into persons exposed to certain diseases such as chicken pox and hepatitis. Because such preparations of immune globulin contain antibodies against most common infectious diseases, the patient gains temporary protection against the disease.
• It is responsible
for the production of bile which is stored in the gallbladder and released when
required for the digestion of fats.
• The liver stores glucose in the form of glycogen which is converted back to glucose again when needed for energy.
• It also plays an important role in the metabolism of protein and fats. It stores the vitamins A, D, K, B12 and folate and synthesizes blood clotting factors.
• Another important role is as a detoxifier, breaking down or transforming substances like ammonia, metabolic waste, drugs, alcohol and chemicals, so that they can be excreted. These may also be referred to as "xenobiotic" chemicals. If we examine the liver under a microscope, we will see rows of liver cells separated by spaces which act like a filter or sieve, through which the blood stream flows. The liver filter is designed to remove toxic matter such as dead cells, microorganisms, chemicals, drugs and particulate debris from the blood stream. The liver filter is called the sinusoidal system, and contains specialized cells known as Kupffer cells which ingest and breakdown toxic matter.
From intestine glucose pass into the liver, where most part of it undergone the phosphorillation. Glucose-6-phosphate formed in result of this reaction, which catalyzed by two enzymes – hexokinase and glucokinase. When level of glucose in blood of v. porta and in the hepatocytes is normal activity of glucokinase is low. After eating activity of this enzyme increase and blood level of glucose decrease because glucose-6-phosphate cannot pass through membrane.
1. Synthesis of glicogen. Content in the liver – 70-100g. After eating amount of glicogen in the liver increase up to 150g. After 24 hours of starvation content of glicogen in the liver decreases to zero and glukoneogenesis started.
2. Glucose-6-phosphatase catalize dephosphorillation of glucose-6-phosphate and free glucose formed. This enzyme is present in the liver, kidney and small intestine. This process keep normal level of glucose in the blood.
3. Excess of glucose-6-phosphate, which not used for synthesis of glicogen and forming of free glucose, decomposites in glycolysis for pyruvate and for acetyl-CoA, which are used for fatty acids synthesis.
5. Part of glucose-6-phosphate oxidized in pentosophosphate cycle. This way of glucose decomposition supplyes reducted NADPH, which is necessary in fatty acid synthesis, cholesterin synthesis, and also pentosophosphates for nucleic acids. Near 1/3 of glucose in liver used for this pathway, another 2/3 – for glycolisis.
Hepatocytes content full set of gluconeogenesis necessary enzymes. So, in liver glucose can be formed from lactate, pyruvate, amino acids, glycerine. Gluconegenesis from lactate takes place during intensive muscular work. Lactate formed from glucose in muscles, transported to the liver, new glucose formed and transported to the muscles (Kori cycle).
1. Lipogenesis (synthesis of fatty acids and lipids). Substrate for this process – acetyl-CoA, formed from glucose and amino acids, which are not used for another purposes. This process is very active when the person eats a lot of carbohydrates. Liver more active than another tissues synthesizes saturated and monounsaturated fatty acids. Fatty acids then used for synthesis of lipids, phospholipids, cholesterol ethers. Glycerol-3-phosphate, which is necessary for lipids synthesis, formed in liver in result of two processes: from free glycerol under influence of glycerolkinase, or in reducing of dioxiacetone phosphate under influence of glycerolphosphate dehydrogenase. Active form of fatty acids interact with glycerol-3-phosphate and phosphatidic acid formed, which used for synthesis of triacylglycerines and glycerophospholipids.
2. Liver play a central role in synthesis of cholesterin, because near 80 % of its amount is synthesized there. Biosynthesis of cholesterin regulated by negative feedback. When the level of cholesterin in the meal increases, synthesis in liver decreases, and back to front. Besides synthesis regulated by insulin and glucagon. Cholesterin used in organism for building cell membranes, synthesis of steroid hormones and vitamin D. Excess of cholesterin leads out in the bile to the intestine. Another part of cholesterin used for bile acids synthesis. This process regulated by reabsorbed bile acids according to negative feedback principles.
3. Liver is a place of ketone bodies synthesis. These substances formed from fatty acids after their oxidation, and from liver transported to another tissues, first of all to the heart, muscles, kidneys and brain. These substances are main source of energy for many tissues of our organism excepting liver in normal conditions (heart) and during starvation (brain).
The high speed of protein synthesis and decomposition is typical for the liver. Hepatocytes catch different protein from blood (from hemolysated RBC, denaturated plasma proteins, protein and peptide hormones) and decomposite them to the free amino acids which used for new synthesis. When organism does not get necessary quantity of amino acids from food, liver synthesizes only high necessary proteins (enzymes, receptors).
Liver syntesizes 100 % of albumines, 90 % of α1-globulines, 75 % of α2-globulines, 50 % of β-globulines, blood clotting factors, fibrinogen, protein part of blood lipoproteins, such enzyme as cholinesterase. The speed of these processes is enough high, for example, liver synthesizes 12-16g of albumines per day.
After a life span of about 120 days the erythrocytes die. The dead erythrocytes are taken up by the phagocytes of the reticuloendothelial system of the body. About 7 gram of Hb is released daily from these phagocytosed erythrocytes. The Hb molecule is broken down into 3 parts:
2. Heme in the presence of the enzyme, heme oxygenase, loses one molecule of CO and one atom of iron in Fe3+ form producing biliverdin. In this reaction, the porphyrin ring is cleaved by oxidation of the alpha methenyl bridge between pyrrole rings. The enzyme needs NADPH+H+ and O2.
Biliverdin which is green in color is the first bile pigment to be produced; it is reduced to the yellow-colored bilirubin, the main bile pigment, by the enzyme biliverdin reductase requiring NADPH+H+.
Bilirubin is non-polar, lipid soluble but water insoluble. Bilirubin is a very toxic compound. For example, it is known to inhibit RNA and protein synthesis and carbohydrate metabolism in brain. Mitochondria appear to be especially sensitive to its effect. Bilirubin formed in reticuloendothelial cells then is associated with plasma protein albumin to protect cells from the toxic effects. As this bilirubin is in complex with plasma proteins, therefore it cannot pass into the glomerular filtrate in the kidney; thus it does not appear in urine, even when its level in the blood plasma is very high. However, being lipid soluble, it readily gets deposited in lipid-rich tissues specially the brain.
The detoxication of indirect bilirubin takes place in the membranes of endoplasmatic reticulum of hepatocytes. Here bilirubin interact with UDP-glucuronic acid and is converted to the water soluble form -bilirubin mono- and diglucoronids. Another name of bilirubin mono- and diglucoronids is conjugated bilirubin or direct bilirubin or bound bilirubin. This reaction is catalized by UDP-glucoroniltransferase.
Conjugated bilirubin is water soluble and is excreted by hepatocytes to the bile. Conjugated (bound) bilirubin undergoes degradation in the intestine through the action of intestinal microorganisms. Bilirubin is reduced and, mesobilirubin is formed. Then mesobilirubin is reduced again and mesobilinogen is formed. The reduction of mesobilinogen results in the formation of stercobilinogen (in a colon). Stercobilinogen is oxidized and the chief pigment (brown color) of feces stercobilin is formed. A part of mesobilinogen is reabsorbed by the mucous of intestine and via the vessels of vena porta system enter liver. In hepatocytes mesobilinogen is splitted to pyrol compounds which are excreted from the organism with bile. If the liver has undergone degeneration mesobilinogen enter the blood and is excreted by the kidneys. This mesobilinogen in urine is called urobilin, or true urobilin. Thus, true urobilin can be detected in urine only in liver parenchyma disease.
Another bile pigment that can be reabsorbed in intestine is stercobolinogen. Stercobolinogen is partially reabsorbed in the lower part of colon into the haemorroidal veins. From the blood stercobolinogen pass via the kidneys into the urine where it is oxidized to stercobilin. Another name of urine stercobilin is false urobilin.
As mentioned above, the conversion of bilirubin to mesobilirubin occurs under the influence of intestinal bacteria. These bacteria are killed or modified when broad-spectrum antibiotics are administered. The gut is sterile in the newborn babies. Under these circumstances, bilirubin is not-converted to urobilinogen, and the feces are colored yellow due to bilirubin. The feces may even become green because some bilirubin is reconverted to green-colored biliverdin by oxidation.
The total bilirubin content in the blood serum is 1,7-20,5 micromol/l, indirect (unconjugated) bilirubin content is 1,7-17,1 micromol/l and direct (conjugated) bilirubin content is 0,86-4,3 micromol/l.
Diazo reagent which is a mixture of sulfanilic acid, HCI and NaN02 is added to the serum. The conjugated bilirubin gives a reddish violet color with it and the maximum color intensity is obtained within 30 seconds; this is called direct test.
The unconjugated bilirubin does not give the direct test; however, it gives indirect test in which alcohol or caffeine is also added which sets free the bilirubin frum its complex with plasma proteins. Due to this difference in the type of diazo reaction given by these two forms of bilirubin, the term direct and indirect forms of bilirubin are also used to describe conjugated and unconjugated forms of bilirubin.
Jaundice or icterus is the orange-yellow discoloration of body tissues which is best seen in the skin and conjunctivae; it is caused by the presence of an excess of bilirubin in the blood plasma and tissue fluids. Depending upon the cause of an increased plasma bilirubin level, jaundice can be classified as
Pre-hepafic jaundice. This type of jaundice is due to a raised plasma level of unconjugated bilirubin. It is due to an excessive breakdown of red cells which leads to an increased production of uncongugated bilirubin; it is also called haemolytic jaundice. As the liver is not able to excrete into the bile all the bilirubin reaching it, the plasma bilirubin level rises and jaundice results. This type of jaundice was in the past called acholuric jaundice because the unconjugated bilirubin, being bound to plasma proteins, is not excreted in the urine despite its high level in the plasma; the urine is also without bile salts. Prehepatic jaundice is also seen in neonates (physiological jaundice) especially in the premature ones because the enzyme UDP-glucuronyl transferase is deficient. Moreover relatively more bilirubin is produced in-the neonates because of excessive breakdown of red blood cells.
Hepatic jaundice.This is typically seen in viral hepatitis. Several viruses are responsible for viral hepatitis and include hepatitis A, B, C and D viruses. The liver cells are damaged: inflammation produces obstruction of bile canaliculi due to swelling around them. This cholestasis causes the bile to regurgitate into the blood through bile canaliculi. The blood contains abnormally raised amount both of conjugated and unconjugated bilirubin and bile salts which are excreted in the urine.
Post hepatic jaundice. This results when there is extrahepatic cholestasis due to an obstruction in the biliary passages outside the liver. In this way, the bile cannot reach the small intestine and therefore the biliary passages outside as well as inside the liver are distended with bile. This leads to damage to the liver and bile regurgitates into the blood. The conjugated bilirubin and the bile salt levels of the blood are thus greatly raised and both of these are excreted in the urine. Liver function tests will vary according to the degree of obstruction, i.e complete or incomplete. If the obstruction is complete, the stools become pale or clay-colored and the urine does not have any stercobilin. The absorption of fat and fat soluble vitamins also suffers due to a lack of bile salts. Excess of bile salts in the plasma produces severe pruritus (itching).
A xenobiotics is a compound that is foreign to the body. The principal classes of xenobiotics of medical relevance are drugs, chemical cancerogens, and various compounds that have found their way into our environment by one route or another (insecticides, herbicides, pesticides, food additions, cosmetics, domestic chemical substances). Most of these compounds are subject to metabolism (chemical alteration) in the human body, with the liver being the main organ involved; occasionally a xenobiotics may be excreted unchanged.
Some internal substances also have toxic properties (for example, bilirubin, free ammonia, bioactive amines, products of amino acids decay in the intestine). Moreover, all hormones and mediatores must be inactivated.
Reactions of detoxification take place in the liver. Big molecules like bilirubin excreted with the bile to intestine and leaded out with feces. Small molecules go to the blood and excreted via kidney with urine.
In phase 1, the major reaction involved is hydroxylation, catalyzed by members of a class of enzymes referred to as monooxygenases or cytochrome P-450 species. These enzymes can also catalyze deamination, dehalogenation, desulfuration, epoxidation, peroxidation and reduction reaction. Hydrolysis reactions and non-P-450-catalyzed reactions also occur in phase 2.
In phase 2, the hydroxylated or other compounds produced in phase 1 are converted by specific enzymes to various polar metabolites by conjugation with glucuronic acid, sulfate, acetate, glutathione, or certain amino acids, or by methylation.
The overall purpose of metabolism of xenobiotics is to increase their water solubility (polarity) and thus facilitate their excretion from the body via kidney.Very hydrophobic xenobiotics would persist in adipose tissue almost indefinitely if they were not converted to more polar forms.
In certain cases, phase 1 metabolic reaction convert xenobiotics from inactive to biologically active compounds. In these instances, the original xenobiotics are referred to as prodrugs or procarcinogens. In other cases, additional phase 1 reactions convert the active compounds to less active or inactive forms prior to conjugation. In yet other cases, it is the conjugation reactions themselves that convert the active product of phase 1 to less active or inactive species, which are subsequently excreted in the urine or bile. In a very few cases, conjugation may actually increase the biologic activity of a xenobiotics.
RH above can represent a very widee variety of drugs, carcinogens, pollutants, and certain endogenous compounds, such as steroids and a number of other lipids. Cytochrome P-450 is considered the most versatile biocatalyst known. The importance of this enzyme is due to the fact that approximately 50 % of the drugs that patients ingest are metabolized by species of cytochrome P-450. The following are important points concerning cytochrome P-450 species:
2. They are present in highest amount in the membranes of the endoplasmic reticulum (ER) (microsomal fraction) of liver, where they can make up approximately 20 % of the total protein. Thay are also in other tissues. In the adrenal, they are found in mitochondria as well as in the ER; the various hydroxylases present in that organ play an important role in cholesterol and steroid biosynthesis.
3. There are at least 6 closely related species of cytochrome P-450 present in liver ER, each with wide and somewhat overlapping substrate specificities, that act on a wide variety of drugs, carcinogens, and other xenobiotics in addition to endogenous compounds such as certain steroids.
6. Most species of cytochrome P-450 are inducible. For instance, the administration of phenobarbital or of many other drugs causes a hypertrophy of the smooth ER and a 3- to 4-fold increase in the amount of cytochrome P-450 within 4-5 days. Induction of this enzyme has important clinical implications, since it is a biochemical mechanism of drug interaction.
7. One species of cytochrome P-450 has its characteristic absorption peak not at 450 nm but at 448 nm. It is often called cytochrome-448.This species appears to be relatively specific for the metabolism of polycyclic aromatic hydrocarbons (PAHs) and related molecules; for this reason it is called aromatic hydrocarbon hydroxylase (AHH). This enzyme is important in the metabolism of PAHs and in carcinogenesis produced by this agents.
8. Recent findings have shown that individual species of cytochrome P-450 frequently exist in polymorphic forms, some of which exhibit low catalytic activity. These observation are one important explanation for the variations in drug responses noted among many patients.
In phase 1 reactions, xenobiotics are generally converted to more polar, hydroxylated derivates. In phase 2 reactions, these derivates are conjugated with molecules such as glucuronic acid, sulfate, or glutatione. This renders them even more water-soluble, and they are eventually excreted in the urine or bile.
1. Glucuronidation. UDP-glucuronic acid is the glucuronyl donor, and a variety of glucuronyl transferases, present in both the ER and cytosol, are the catalysts. Molecules such as bilirubin, thyroxin, 2-acetylaminofluorene (a carcinogen), aniline, benzoic acid, meprobromate (a tranquilizer), phenol, crezol, indol and skatol, and many steroids are excreted as glucuronides. The glucuronide may be attached to oxygen, nitrogen, or sulfur groups of substrates. Glucuronidation is probably the most frequent conjugation reaction.
Glucuronidation, the combining of glucuronic acid with toxins, requires the enzyme UDP-glucuronyl transferase (UDPGT). Many of the commonly prescribed drugs are detoxified through this pathway. It also helps to detoxify aspirin, menthol, vanillin (synthetic vanilla), food additives such as benzoates, and some hormones. Glucuronidation appears to work well, except for those with Gilbert's syndrome--a relatively common syndrome characterized by a chronically elevated serum bilirubin level (1.2-3.0 mg/dl). Previously considered rare, this disorder is now known to affect as much as 5% of the general population. The condition is usually without serious symptoms, although some patients do complain about loss of appetite, malaise, and fatigue (typical symptoms of impaired liver function). The main way this condition is recognized is by a slight yellowish tinge to the skin and white of the eye due to inadequate metabolism of bilirubin, a breakdown product of hemoglobin. The activity of UDPGT is increased by foods rich in the monoterpene limonene (citris peel, dill weed oil, and caraway oil). Methionine, administered as SAM, has been shown to be quite beneficial in treating Gilbert's syndrome.
2. Sulfation. Some alcohols, arylamines, and phenols are sulfated. The sulfate donor in these and other biologic sulfation reactions is adenosine 3´-phosphate-5´-phosphosulfate (PAPS); this compound is called active sulfate.
Sulfation is the conjugation of toxins with sulfur-containing compounds. The sulfation system is important for detoxifying several drugs, food additives, and, especially, toxins from intestinal bacteria and the environment. In addition to environmental toxins, sulfation is also used to detoxify some normal body chemicals and is the main pathway for the elimination of steroid and thyroid hormones. Since sulfation is also the primary route for the elimination of neurotransmitters, dysfunction in this system may contribute to the development of some nervous system disorders.
Many factors influence the activity of sulfate conjugation. For example, a diet low in methionine and cysteine has been shown to reduce sulfation. Sulfation is also reduced by excessive levels of molybdenum or vitamin B6 (over about 100 mg/day). In some cases, sulfation can be increased by supplemental sulfate, extra amounts of sulfur-containing foods in the diet, and the amino acids taurine and glutathione.
Sulfoxidation is the process by which the sulfur-containing molecules in drugs and foods are metabolized. It is also the process by which the body eliminates the sulfite food additives used to preserve many foods and drugs. Various sulfites are widely used in potato salad (as a preservative), salad bars (to keep the vegetables looking fresh), dried fruits (sulfites keep dried apricots orange), and some drugs. Normally, the enzyme sulfite oxidase metabolizes sulfites to safer sulfates, which are then excreted in the urine. Those with a poorly functioning sulfoxidation system, however, have an increased ratio of sulfite to sulfate in their urine. The strong odor in the urine after eating asparagus is an interesting phenomenon because, while it is unheard of in China, 100% of the French have been estimated to experience such an odor (about 50% of adults in the U.S. notice this effect). This example is an excellent example of genetic variability in liver detoxification function. Those with a poorly functioning sulfoxidation detoxification pathway are more sensitive to sulfur-containing drugs and foods containing sulfur or sulfite additives. This is especially important for asthmatics, which can react to these additives with life-threatening attacks. Molybdenum helps asthmatics with an elevated ratio of sulfites to sulfates in their urine because sulfite oxidase is dependent upon this trace mineral.
3. Conjugation with Glutathione. Glutathione (γ-glutamylcysteinylglycine) is a tripeptide consisting of glutamic acid, cysteine, and glycine. Glutathione is commonly abbreviated to GSH; the SH indicates the sulfhydryl group of its cysteine and is the business part of the molecule. A number of potentially toxic electrophilic xenobiotics (such as certain carcinogens) are conjugated to the nucleophilic GSH. The enzymes catalyzing these reactions are called glutathione S-transferases and are present in high amounts in liver cytosol and in lower amounts in other tissues. glutathione conjugates are subjected to further metabolism before excretion. The glutamyl and glycinyl groups belonging to glutathione are removed by specific enzymes, and an acetyl group (donated by acetyl-CoA) is added to the amino group of the remaining cystenyl moiety. The resulting compound is a mercapturic acid, a conjugate of L-acetylcysteine, which is then excreted in the urine.
Glutathione is also an important antioxidant. This combination of detoxification and free radical protection, results in glutathione being one of the most important anticarcinogens and antioxidants in our cells, which means that a deficiency is cause of serious liver dysfunction and damage. Exposure to high levels of toxins depletes glutathione faster than it can be produced or absorbed from the diet. This results in increased susceptibility to toxin-induced diseases, such as cancer, especially if phase I detoxification system is highly active. Disease states due to glutathione deficiency are not uncommon.
A deficiency can be induced either by diseases that increase the need for glutathione, deficiencies of the nutrients needed for synthesis, or diseases that inhibit its formation. Smoking increases the rate of utilization of glutathione, both in the detoxification of nicotine and in the neutralization of free radicals produced by the toxins in the smoke. Glutathione is available through two routes: diet and synthesis. Dietary glutathione (found in fresh fruits and vegetables, cooked fish, and meat) is absorbed well by the intestines and does not appear to be affected by the digestive processes. Dietary glutathione in foods appears to be efficiently absorbed into the blood. However, the same may not be true for glutathione supplements.
In healthy individuals, a daily dosage of 500 mg of vitamin C may be sufficient to elevate and maintain good tissue glutathione levels. In one double-blind study, the average red blood cell glutathione concentration rose nearly 50% with 500 mg/day of vitamin C. Increasing the dosage to 2,000 mg only raised red blood cell (RBC) glutathione levels by another 5%. Vitamin C raises glutathione by increasing its rate of synthesis. In addition, to vitamin C, other compounds which can help increase glutathione synthesis include N-acetylcysteine (NAC), glycine, and methionine. In an effort to increase antioxidant status in individuals with impaired glutathione synthesis, a variety of antioxidants have been used. Of these agents, only microhydrin, vitamin C and NAC have been able to offer some possible benefit.
Over the past 5-10 years, the use of NAC and glutathione products as antioxidants has become increasingly popular among nutritionally oriented physicians and the public. While supplementing the diet with high doses of NAC may be beneficial in cases of extreme oxidative stress (e.g. AIDS, cancer patients going through chemotherapy, or drug overdose), it may be an unwise practice in healthy individuals.
4. Acetylation. These reactions is represented by X + Acetyl-CoA → Acetyl-X + CoA, where X represents a xenobiotic. These reactions are catalyzed by acetyltransferases present in the cytosol of various tissues, particularly liver. The different aromatic amines, aromatic amino acids, such drug as isoniazid, used in the treatment of tuberculosis, and sulfanylamides are subjects to acetylation. Polymorphic types of acetyltransferases exist, resulting in individuals who are classified as slow or fast acetylators, and influence the rate of clearance of drugs such as isoniazid from blood. Slow acetylators are more subject to certain toxic effects of isoniazid because the drug persists longer in these individuals.
Conjugation of toxins with acetyl-CoA is the primary method by which the body eliminates sulfa drugs. This system appears to be especially sensitive to genetic variation, with those having a poor acetylation system being far more susceptible to sulfa drugs and other antibiotics. While not much is known about how to directly improve the activity of this system, it is known that acetylation is dependent on thiamine, pantothenic acid, and vitamin C.
5. Methylation. A few xenobiotics (amines, phenol, tio-substances, inorganic compounds of sulphur, selen, mercury, arsenic) are subject to methylation by methyltransferases, employing S-adenosylmethionine as methyl donor. Also catecholamines and nicotinic acid amid (active form of vitamin PP) are inactivated due to methylation.
Methylation involves conjugating methyl groups to toxins. Most of the methyl groups used for detoxification come from S-adenosylmethionine (SAM). SAM is synthesized from the amino acid methionine, a process which requires the nutrients choline, vitamin B12, and folic acid. SAM is able to inactivate estrogens (through methylation), supporting the use of methionine in conditions of estrogen excess, such as PMS. Its effects in preventing estrogen-induced cholestasis (stagnation of bile in the gall bladder) have been demonstrated in pregnant women and those on oral contraceptives. In addition to its role in promoting estrogen excretion, methionine has been shown to increase the membrane fluidity that is typically decreased by estrogens, thereby restoring several factors that promote bile flow. Methionine also promotes the flow of lipids to and from the liver in humans. Methionine is a major source of numerous sulfur-containing compounds, including the amino acids cysteine and taurine.
Antioxidant vitamins like vitamin C, beta-carotene, and vitamin E are obviously quite important in protecting the liver from damage as well as helping in the detoxification mechanisms, but even simple nutrients like B-vitamins, calcium, and trace minerals are critical in the elimination of heavy metals and other toxic compounds from the body. The lipotropic agents, choline, betaine, methionine, vitamin B6, folic acid, and vitamin B12, are useful as they promote the flow of fat and bile to and from the liver. Lipotropic formulas have been used for a wide variety of conditions by nutrition-oriented physicians including a number of liver disorders such as hepatitis, cirrhosis, and chemical-induced liver disease. Lipotropic formulas appear to increase the levels of SAM and glutathione. Methionine, choline, and betaine have been shown to increase the levels of SAM.
There is a long list of plants which exert beneficial effects on liver function. However, the most impressive research has been done on silymarin, the flavonoids extracted from silybum marianum (milk thistle). These compounds exert a substantial effect on protecting the liver from damage as well as enhancing detoxification processes. Silymarin prevents damage to the liver through several mechanisms: by acting as an antioxidant, by increasing the synthesis of glutathione and by increasing the rate of liver tissue regeneration. Silymarin is many times more potent in antioxidant activity than vitamin E and vitamin C. The protective effect of silymarin against liver damage has been demonstrated in numerous experimental studies. Silymarin has been shown to protect the liver from the damage produced by such liver-toxic chemicals as carbon tetrachloride, amanita toxin, galactosamine, and praseodymium nitrate.
One of the key mechanisms by which silymarin enhances detoxification is by preventing the depletion of glutathione. Silymarin not only prevents the depletion of glutathione induced by alcohol and other toxic chemicals, but has been shown to increase the level of glutathione of the liver by up to 35%, even in normals. Inhuman studies, silymarin has been shown to have positive effects in treating liver diseases of various kinds, including cirrhosis, chronic hepatitis, fatty infiltration of the liver, and inflammation of the bile duct. The standard dosage for silymarin is 70-210 mg three times/day.
Several amino acids (glyucine, taurine, glutamine, arginine, and ornithine) are used to combine with and neutralize toxins. Of these, glycine is the most commonly utilized in phase II amino acid detoxification. Patients suffering from hepatitis, alcoholic liver disorders, carcinomas, chronic arthritis, hypothyroidism, toxemia of pregnancy, and excessive chemical exposure are commonly found to have a poorly functioning amino acid conjugation system. For example, using the benzoate clearance test (a measure of the rate at which the body detoxifies benzoate by conjugating it with glycine to form hippuric acid, which is excreted by the kidneys), the rate of clearance in those with liver disease is 50% of that in healthy adults.
Even in apparently normal adults, a wide variation exists in the activity of the glycine conjugation pathway. This is due no only to genetic variation, but also to the availability of glycine in the liver. Glycine, and the other amino acids used for conjugation, become deficient on a low-protein diet and when chronic exposure to toxins results in depletion.
Adding certain supplements to your diet can stimulate detoxification. Fiber, vitamin C and other antioxidants, chlorophyll, and glutathione (as the amino acid L-cysteine) will all help. Herbs such as garlic, red clover, echinacea, or cayenne may also induce some detoxification. Saunas, sweats, and niacin therapy have been used to cleanse the body.
Simply increasing liquids and decreasing fats will shift the balance strongly toward improved elimination and less toxin buildup. Changes might include increased consumption of filtered water, herb teas, fruits, and vegetables, as well as reducing fats, especially fried food, meat and milk products. In general, moving from an acid-generating diet to a more alkaline one will aid the process of detoxification. Acid-forming foods, such as meats, milk products, breads and baked goods, and especially the refined sugar and carbohydrate products, will increase body acidity and lead to more mucus production and congestion, whereas the more alkaline vegetarian foods enhance cleansing and clarity in the body.
A deeper level of detoxification diet is made up exclusively of fresh fruits and vegetables, either raw and cooked, and whole grains, both cooked and sprouted. This diet keeps fiber and water intake high and helps colon detoxification. Most people can handle this well and make the shift from their regular diet with a few days transition. Some people do well on a brown rice fast (a more macrobiotic plan), usually for a week or two, eating three to four bowls of rice daily along with liquids such as teas."
Different methods are used for evaluating of liver’s functions. Base for some of them is role of liver in proetin metabolism (e.g. thymol’s test), for another – role of liver in detoxification (indican’s test) or in excretion (checking of bilirubin level in blood). In all cases physician must make a conclusion about disorder of liver’s functions after complex investigation, because, as mentioned above, all metabolic ways are present in liver.
The liver filter can remove a wide range of microorganisms such as bacteria, fungi, viruses and parasites from the blood stream, which is highly desirable, as we certainly do not want these dangerous things building up in the blood stream and invading the deeper parts of the body. Infections with parasites often come from the contaminated water supplies found in large cities, and indeed other dangerous organisms may find their way into your gut and blood stream from these sources. This can cause chronic infections and poor health, so it is important to protect your liver from overload with these microorganisms. The safest thing to do is boil your water for at least 5 minutes, or drink only bottled water that has been filtered and sterilized. High loads of unhealthy microorganisms can also come from eating foods that are prepared in conditions of poor hygiene by persons who are carrying bacteria, viruses or parasites on their skin. Foods, especially meats that are not fresh or are preserved, also contain a higher bacterial load, which will overwork the liver filter if they are eaten regularly.
Recently, it has become very fashionable for people to detoxify their bodies by various means, such as fasting or cleansing the bowels with fiber mixtures. Fasting can by its extreme nature, only be a temporary method of cleansing the body of waste products, and for many people causes an excessively rapid release of toxins which can cause unpleasant, acute symptoms. The liver filter, like any filter, needs to be cleansed regularly, and it is much easier and safer to do it everyday. This is easily and pleasantly achieved by adopting a daily eating pattern that maintains the liver filter in a healthy clean state. By following the methods and guidelines on this site, you will be able to keep the liver filter healthy and clean. Although it is important to keep the intestines moving regularly and to sweep their walls with high fiber and living foods, it is important to remember that the bowels are really a channel of elimination and not a cleansing organ per se. In other words the bowels cannot cleanse, filter or remove toxic wastes from the blood stream.
The liver is the most important organ in detoxification, as it is the body's premier cleansing organ. All the blood in the body passes through the liver, which removes toxins, impurities, and debris from the bloodstream.
The liver stores fat-soluble substances; these can include chemicals, which can be stored in the liver for years. Using enzymes, the liver transforms these chemicals into water-soluble substances that can be excreted though the kidneys or the gastrointestinal tract.
Hormones are metabolized by the liver. Estrogen produced by the body and from hormone replacement therapies is broken down. If estrogen is not adequately processed, excess estrogen can result in endometriosis; high blood pressure; PMS; and breast, uterine, and vaginal cancer.
The liver also manufactures bile to digest fats; chemically changes many foods into vitamins and enzymes; converts carbohydrates and proteins into glucose for brain fuel and glycogen for muscular energy; and stores nutrients to be secreted as needed by the body to build and maintain cells.
If the liver cannot perform these jobs well, you may exhibit a number of symptoms. These include gas; constipation; a feeling of fullness; loss of appetite; nausea after fatty meals; an oily taste in the mouth; revulsion to fatty foods; frequent headaches not related to stress; weak ligaments, tendons, and muscles; skin problems; and emotional excesses.
Briefly put, living. What you eat, where you live, and what you do all can affect the liver's performance. If you consume a lot of processed foods, the additives can eventually affect the liver. If you live in an area that is highly polluted, exposure to chemicals in the air and water affects the liver. All of this can hurt the liver's performance.
An impaired liver does not process food or detoxify substances as rapidly or as completely as a healthy liver. If the liver is not producing enough bile, it cannot adequately digest fats. If the liver is detoxifying more slowly than it should, it can result in more toxic substances circulating in the body.
If toxins continue to accumulate, the liver may not be able to work fast enough to clean the blood. It is like being on a treadmill that is going a little too fast: try as you might, you cannot go forward, but instead are swept back into greater toxicity. Instead of being converted into something useful or being eliminated, toxins remain unchanged. They are eventually stored in fatty body tissue and in the cells of the brain and central nervous system. The stored toxins may be slowly released to recirculate in the blood, contributing to many chronic illnesses.
A toxin is basically any substance that creates irritating and/or harmful effects in the body; stressing and undermining one's biochemical health and organ function. Toxins can come from by products of normal cell metabolism or from the outside environment e.g. pollution, drugs, pesticides, dyes, chemicals, microbes, heavy metals, tobacco smoke and so on.
Toxicity occurs when we take in more then we can utilize and eliminate. Toxic chemicals can be a real problem, since after years of exposure to these substances the body’s ability to eliminate them can slow down. They can get recirculated into the bloodstream or stored in the liver, body fat or other parts of the body. These types of buildups and problems throughout the body can contribute to the development of serious illnesses. Many chemicals are so widespread that we are unaware of them. But they have worked their way into our bodies faster than they can be eliminated, and are causing allergies and addictions in record numbers. The body's built in detoxification apparatus include the respiratory, gastrointestinal, urinary, skin and lymphatic systems.
Symptoms of Toxicity
Cancer and cardiovascular disease are two of the main toxicity-related diseases. Arthritis, allergies, obesity, and many skin problems are others. In addition, a wide range of symptoms, such as headaches, fatigue, pains, coughs, gastrointestinal problems and problems from immune weakness can all be related to toxicity.
Common indications of toxicity include frequent, unexplained headaches, back or joint pain, tight or stiff neck, arthritis, chronic respiratory or sinus problems, asthma, abnormal body odor, bad breath, coated tongue, food allergies, poor digestion, chronic constipation with intestinal bloating or gas, brittle nails and hair, psoriasis, adult acne, unexplained weight gain over 10 pounds, unusually poor memory, chronic insomnia, anxiety, depression, irritability, chronic fatigue, and environmental sensitivities, especially to odors.
Detoxification is the process of clearing toxins from the body or neutralizing them. Energy balancing and detoxification herbal baths prompt the body to eliminate toxins from specific areas of the body. As these toxins are released from the areas where they have been stored, they move into the blood, lymph and other body fluids out of the body through the urinary, gastrointestinal, lymphatic and respiratory systems and the skin. The period of detoxification can be a few days, a few weeks or a months depending on the extent, location and type of the toxins in the body. As a person is detoxifying they may experience uncomfortable symptoms including depression, mood changes, nausea, diarrhea , foggy head, fatigue, lack of energy, bad breathe, foul urine odour, foul perspiration odour, body odour, sores, rashes, acne, cold or flu like symptoms, headaches or any other symptom. This period where symptoms may seem to worsen is sometimes called a healing crisis, but is actually just the body's reacting to the presence of the toxins in the bloodstream and the movement of the toxins out of the body. .
"The liver is one of the most important organs in the body when it comes to detoxifying or getting rid of foreign substances or toxins. The liver plays a key role in most metabolic processes, especially detoxification. The liver neutralizes a wide range of toxic chemicals, both those produced internally and those coming from the environment. The normal metabolic processes produce a wide range of chemicals and hormones for which the liver has evolved efficient neutralizing mechanisms. However, the level and type of internally produced toxins increases greatly when metabolic processes go awry, typically as a result of nutritional deficiencies. These non-end-product metabolites have become a significant problem in this age of conventionally grown foods and poor diets.
Many of the toxic chemicals the liver must detoxify come from the environment: the content of the bowels and the food, water, and air. The polycyclic hydrocarbons (DDT, dioxin, 2,4,5-T, 2,3-D, PCB, and PCP), which are components of various herbicides and pesticides, are an example of chemicals that are now found in virtually all fat tissues measured. Even those eating unprocessed organic foods need an effective detoxification system because all foods contain naturally occurring toxic constituents.
The liver plays several roles in detoxification: it filters the blood to remove large toxins, synthesizes and secretes bile full of cholesterol and other fat-soluble toxins, and enzymatically disassembles unwanted chemicals. This enzymatic process usually occurs in two steps referred to as phase I and phase II. Phase I either directly neutralizes a toxin, or modifies the toxic chemical to form activated intermediates which are then neutralized by one of more of the several phase II enzyme systems.
Proper functioning of the liver's detoxification systems is especially important for the prevention of cancer. Up to 90% of all cancers are thought to be due to the effects of environmental carcinogens, such as those in cigarette smoke, food, water, and air, combined with deficiencies of the nutrients the body needs for proper functioning of the detoxification and immune systems. The level of exposure to environmental carcinogens varies widely, as does the efficiency of the detoxification enzymes, particularly phase II. High levels of exposure to carcinogens coupled with slow detoxification enzymes significantly increases susceptibility to cancer.
One of the liver's primary functions is filtering the blood. Almost 2 quarts of blood pass through the liver every minute for detoxification. Filtration of toxins is absolutely critical as the blood from the intestines contains high levels of bacteria, bacterial endotoxins, antigen-antibody complexes, and various other toxic substances. When working properly, the liver clears 99% of the bacteria and other toxins during the first pass. However, when the liver is damaged, such as in alcoholics, the passage of toxins increases by over a factor of 10.
The liver's second detoxification process involves the synthesis and secretion of bile. Each day the liver manufactures approximately 1 quart of bile, which serves as a carrier in which many toxic substances are dumped into the intestines. In the intestines, the bile and its toxic load are absorbed by fiber and excreted. However, a diet low in fiber results in inadequate binding and reabsorption of the toxins. This problem is magnified when bacteria in the intestine modify these toxins to more damaging forms.
When the excretion of bile is inhibited (i.e. cholestasis), toxins stay in the liver longer. Cholestasis has several causes, including obstruction of the bile ducts and impairment of bile flow within the liver. The most common cause of obstruction of the bile ducts is the presence of gallstones. Currently, it is conservatively estimated that 20 million people in the U.S. have gallstones. Nearly 20% of the female and 8% of the male population over the age of 40 are found to have gallstones on biopsy and approximately 500,000 gall bladders are removed because of stones each year in the U.S. The prevalence of gallstones in this country has been linked to the high-fat, low-fiber diet consumed by the majority of Americans.
Impairment of bile flow within the liver can be caused by a variety of agents and conditions. These conditions are often associated with alterations of liver function in laboratory tests (serum bilirubin, alkaline phosphatase, SGOT, LDH, GGTP, etc.) signifying cellular damage. However, relying on these tests alone to evaluate liver function is not adequate, since, in the initial or subclinical stages of many problems with liver function, laboratory values remain normal. Among the symptoms people with enzymatic damage complain of are:
Perhaps the most common cause of cholestasis and impaired liver function is alcohol ingestion. In some especially sensitive individuals, as little as 1 ounce of alcohol can produce damage to the liver, which results in fat being deposited within the liver. All active alcoholics demonstrate fatty infiltration of the liver. Methionine, taken as SAM, has been shown to be quite beneficial in treating two common causes of stagnation of bile in the liver--estrogen excess (due to either oral contraceptive use or pregnancy) and Gilbert's syndrome.
Oranges and tangerines (as well as the seeds of caraway and dill) contain limonene, a phytochemical that has been found to prevent and even treat cancer in animal models. Limonene's protective effects are probably due to the fact that it is a strong inducer of both phase I and phase II detoxification enzymes that neutralize carcinogens.
Curcumin, the compound that gives turmeric its yellow color, is interesting because it inhibits phase I while stimulating phase II. This effect can be very useful in preventing certain types of cancer. Curcumin has been found to inhibit carcinogens, such as benzopyrene (found in charcoal-broiled meat), from inducing cancer in several animal models. It appears that the curcumin exerts its anti-carcinogenic activity by lowering the activation of carcinogens while increasing the detoxification of those that are activated. Curcumin has also been shown to directly inhibit the growth of cancer cells.
As most of the cancer-inducing chemicals in cigarette smoke are only carcinogenic during the period between activation by phase I and final detoxification by phase II, curcumin in the turmeric can help prevent the cancer-causing effects of tobacco. Those exposed to smoke, aromatic hydrocarbons, and other environmental carcinogens will probably benefit from the frequent use of curry or turmeric.
The activity of phase I detoxification enzymes decreases in old age. Aging also decreases blood flow through the liver, further aggravating the problem. Lack of the physical activity necessary for good circulation, combined with the poor nutrition commonly seen in the elderly, add up to a significant impairment of detoxification capacity, which is typically found in aging individuals. This helps to explain why toxic reactions to drugs are seen so commonly in the elderly.
Liver failure, or hepatic failure, is severe deterioration of liver function resulting from extensive damage of liver cells. The syndrome respresents a severe clinical condition and is associated with high mortality; therefore, a great challenge to intensive care management.
Kidney – the couple organ, which is responsible for excriting of final products of metabolism and for homeostasis. They regulate water and mineral metabolism, acid-base balance, excriting of nitrogenous slags, osmotic pressure. Also they regulate arterial pressure and erhythropoesis.
Urine – fluid with different organic and inorganic compouds, which must be excreted (excess of water, final products of nitrogen metabolism, xenobiotics, products of protein’s decay, hormones, vitamins and their derivates). Most of them present in urine in a bigger amount than in blood plasma. So, urine formation – is not passive process (filtration and diffusion only).
Glomerulal filtration. Water and low weight molecules go to the urine with help of following powers: blood hydrostatic pressure in glomerulas (near 70 mm Hg), oncotic pressure of blood plasma proteins (near 30 mm Hg) and hydrostatic pressure of plasma ultrafiltrate in glomerulal capsule (near 20 mm Hg). In normal conditions, as You see, effective filtration pressure is about 20 mm Hg.
Primary urine formed in result of filtration (about 200 L per day). Between all blood plasma substances only proteins don’t present in a primary urine. Most of these substances are undergone to the following reabsorbtion. Only urea, uric acid, creatinin, and other final products of different metabolic pathways aren’t undergone to the reabsorbtion.
Drugs which stimulate blood circulation in kidney (theophyllin), also stimulate filtration. Inflammatory processes of renal tissue (nephritis) reduce filtration, and azotaemia occurred (accumulation of urea, uric acid, creatinin, and other metabolic final products).
Reabsorbtion. Lenght of renal tubules is about 100 km. So, all important for our organism are reabsorbed during passing these tubules. Epitelium of renal tubules reabsorb per day 179 L of water, 1 kg of NaCl, 500 g of NaHCO3, 250 g of glucose, 100 g of free amino acids.
Na+ reabsorbed by active transport to the epitelium cell, then – into the extracellular matrix. Cl- and HCO3- following Na+ according to the electroneutrality principle, water – according to the osmotic gradient. From extracellular matrix substances go to the blood vessels. Mg2+ and Ca2+ are reabsorbed with help of special transport ATPases. Glucose and amino acids use the energy of Na+ gradient and special carriers. Proteins are reabsorbed by endocytosis.
Henle’s loop play important role in the reabsobtion process. Its descendent and ascendent parts create anti-stream system, which has big capacity for urine concentration and dilution. Fluid which passes from proximal part of renal tubule to the descendent part of Henle’s loop, where concentration of osmotic active substances higher than in kidney cortex. This concentration is due to activity of thick ascendent part of Henle’s loop, which is non-penetrated for water and which cells transport Na+ and Cl- into the interstitium. Wall of descendent part is penetrated for water and here water pass into the interstitium by osmotic gradient but osmotic active substances stay in the tubule. Ascendent part continue to reabsorb salt hypertonically, even in the absence of aldosteron, so that fluid entering the distal tubule still has a much lower osmolality than does interstitial fluid.
Kidney have a very high level of metabolic processes. They use about 10 % of all O2, which used in organism. During 24 hours through kidney pass 700-900 L of blood. The main fuel for kidney are carbohydrates. Glycolysis, ketolysis, aerobic oxidation and phophorillation are very intensive in kidney. A lot of ATP formed in result.
Metabolism of proteins also present in kidney in high level. Especially, glutamine deaminase is very active and a lot of free ammonia formed. In kidney take place the first reaction of creatin synthesis.
Utilization of glucose in cortex and medulla is differ one from another. Dominative type of glycolysis in cortex is aerobic way and CO2 formed in result. In medulla dominative type is anaerobic and glucose converted to lactate.
Two sources contribute to the renal ammonia: blood ammonia (is about one-third of excreted ammonia), and ammonia formed in the kidney. The predominant source for ammonia production within the kidney is glutamine, the most abundant amino acid in plasma, but a small amount may originate from the metabolism of other amino acids such as asparagine, alanine, and histidine. Ammonia is secreted into the tubular lumen throughout the entire length of the nephron. Secretion occurs both during normal acid-base balance and in chronic acidosis.Metabolic acidosis is accompanied by an adaptive increase in renal ammonia production with a corresponding increase in urinary ammonium excretion.
Kidney cortex like liver appear to be unique in that it possess the enzymatic potential for both glucose synthesis from noncarbohydrate precursors (gluconeogenesis) and glucose degradation via the glycolytic pathway. Gluconeogenesis is important when the dietary supply of glucose does not satisfy the metabolic demands. Under these conditions, glucose is required by the central nervous system, the red blood cells, and possibly other tissues which cannot obtain all their energy requirements from fatty acids or ketone body oxidation. Also, gluconeogenesis may be important in the removal of excessive quantities of glucose precursors from the blood (lactate acid after severe exercise for example). Although the biosynthetic pathways are similar, there are several important differences in the factors which regulate gluconeogenesis in the two organs. 1) The liver utilizes predominately pyruvate, lactate and alanine. The kidney cortex utilizes pyruvate, lactate, citrate, α-ketoglutarate, glycine and glutamine. 2) Hydrogen ion activity has little effect upon hepatic gluconeogenesis, but it has marked effects upon renal gluconeogenesis. Thus, when intracellular fluid pH is reduced (metabolic acidosis, respiratory acidosis or potassium depletion), the rates of gluconeogenesis in slices of renal cortex are markedly increased. The ability of the kidney to convert certain organic acids to glucose, a neutral substance, is an example of a nonexcretory mechanism in the kidney for pH regulation.
2. Formation of hydrocarbonates. Inside renal cells carboanhydrase forms from CO2 and H2O H2CO3, which dissociated to H+ and HCO3-. H+ excreted from cell into urine (antiport with Na+) and leaded with urine. Na+ connect with HCO3-, NaHCO3 formed and go to the blood, thereupon acidity decreased.
Excessive entrance of water leads to dilution of extracellular fluid. Decreasing of osmolality inhibits secretion of antidiuretic hormone. Walls of collective tubules stay non-penetrated to water and dilutive urine formed.
If volume of blood circulation increases, circulation in kidney increases also and hyperosmotic medium of kidney medulla removed. Some substances in these conditions return into blood. So, excess of water carried with urine and a lot of soluble substances are reabsorbed into blood. After water loading stopped, hyperosmolality in kidney medulla returns for previous stage during some days.
Normal colour of urine is yellow (like hay or amber), what is due to presence of urochrom (derivate of urobilin or urobilinogen). Some another colour substances are uroerythrin (derivate of melanine), uroporphyrines, rybophlavine and other. Colour depends from urine concentration.
Creatinin and creatin. Near 1-2 g of creatinin is excreted per day, what depended from weight of muscles. This is the constant for each person. Men excrete 18-32 mg of creatinin per 1 kg of body weight per day, women – 10-25 mg. Creatinin is non-reabsorbable substance, so this test used for evaluating of renal filtration.
Couple substances. Hypuric acid (benzoyl glycine) is excreted in amount 0,6-1,5 g per day. This index increases after eating a lot of berries and fruits, and in case of protein’s decay in the intestines.
Indican (potassium salt of indoxylsulfuric acid). Per day excrition of indican is about 10-25 g. Increasing of indican’s level in urine is due to inrtensification of decay proteins in the intestines and chronic diseases, which are accompanied by intensive decopmosition of proteins (tuberculosis, for example).
Vitamines. Almost all vitamines can be excreted via kidney, especially, water-soluble. Approximately 20-30 mg of vit C, 0.1-0.3 mg of vit B1, 0.5-0.8 mg of vit B2 and some products of vitamine’s metabolism. These data can be used for evaluating of supplying our organism by vitamines.
Hormones. Hormones and their derivates are always present in urine. Their amount depends from functional state of endocrinal glands and liver. There is a very wide used test – determination of 17-ketosteroids in urine. For healthy man this index is 15-25 g per day.
Phosphorus. In urine are present one-substituted phosphates of potassium and sodium. Their amount depends from blood pH. In case of acidosis two-substituted phosphates (Na2HPO4) react with H+ and one-substituted phosphates (NaH2PO4) formed. In case of alkalosis one-substituted phosphates react with bases and two-substituted phosphates formed. So, in both cases amount of phosphates in urine increases.
ABSTRACT: The clearance concept is central to three critical areas of nephrology; it was a key feature to early conceptual analysis of the nature of urine formation, it was utilized as a measure of kidney function in advancing renal diseases, and it was a pivotal concept to elucidate the physiology of the kidneys. This paper describes the clearance concept as currently understood and then it examines how clearance was utilized to understand these various aspects of kidney function.
The concept of clearance is central to three major areas of nephrology. First, the nature of urine for mation was explored to a great extent using clearance techniques. Second, the early search for measures of kidney function with advancing disease resorted to clearance procedures, particularly involving urea and creatinine. Third, the physiology of the kidney was examined and developed with great power and sophistication by the deepening theoretical understanding of the concept of clearance accompanied by ingenious analytical techniques and procedures. There is an additional domain which hovered over the studies of urine formation. This had to do with the per vasive resort to vitalism as an explanation of physiologic regulation. My task was to examine the birth and evolution of the clearance concept. For purposes of exposition, it may be helpful to describe first the clearance concept as it is currently understood and then recount how the concept emerged and developed during the 19th and early 20th centuries, as it was repeatedly invoked to analyze the process of urine formation, the failure of renal function, and the nature of physiologic regulation.
Figure 1 gives the definition of clearance in currently conventional units of time and concentration. It is evident from the formula that the numerator is a rate of excretion (mg/mm); the denominator is a plasma concentration (mg/mL). Therefore, the clearance of any substance is expressed as mL/min (mg/min x mL/mg=mL/min). Clearance, therefore, has the dimensions of a volume per unit time. This simultaneous measurement of the excretion rate of a solute and a flow rate of fluid from which the solute is derived has resulted in some confusion. Fig 2, modified from Cassin and Vogh, emphasizes that the kidney removes (clears) a small fraction of a substance from each mL of total flow. The clearance, therefore, of any substance is the virtual volume of plasma flow required to supply the amount of the substance excreted in any one minute.
The historical evolution of the clearance concept is intimately connected with studies examining the nature of urine formation. Particularly noteworthy reviews have been published by Smith (2), Bradley (3), Thurau, Davis and Haberle (4), Gottschalk (5), Schuster and Seldin (14). In the early 19th centur y, Johannes Muller (18011858) advanced a theor y of urine formation that rested on two prevalent concepts current at the time: 1) fluid movement was a secretory process mediated by glands; 2) the activity of the secretor y system required vitalistic forces that could not be reduced to physical processes. Despite enormous contributions to microscopic anatomy, he denied that the glomerulus was directly connected with the renal tubules, and ascribed urine formation to the secretory activity of the tubules, regarding the kidney as a gland. Notwithstanding the powerful currents of vitalism at the time, Carl Ludwig (1816-1895) came to the study of kidney function with an uncompromising physicochemical orientation. He appreciated the role of the afferent and efferent arteriole in elevating the hydrostatic pressure in the intervening glomerulus, thereby facilitating the movement of a protein-free ultrafiltrate, containing all the elements to be found in the urine, and restraining the passage of protein and formed elements. To account for the different composition of blood and urine, Ludwig proposed that some unspecified chemical force promoted active sodium chloride reabsorption while some property of the tubular wall restrained urea back-diffusion. No vital force was postulated, although the nature of the “chemical force” promoting reabsorption was unspecified. Simultaneously and independently, William Bowman (1816-1892) also postulated that water was separated from blood at the glomerulus, but he assumed that solutes remained in the blood and were subsequently secreted into the urine by the tubules. This was an expression of the prevalent view of glandular secretion mediating solute movement. The central feature of Ludwig’s theory that urine formation was critically linked with glomerular pressure, was challenged by Rudolph Heidenhain (1834-1897). On the basis of calculations of a clearance type, he concluded that to attribute urea excretion to filtration alone would require 70 liters of filtrate and 68 liters of water reabsorption per day. Heidenhain concluded that such circumstances were inconceivable, that urine formation was linked with renal blood flow, not filtration pressure, and that urine was derived from secretion by both glomerulus and tubules. When Cushny (1866-1926) introduced his “modern view” in his monograph of 1917, he accepted Ludwig’s thesis that the ultimate source of urine was filtration at the glomerulus of non-colloid constituents by a purely physical process. He rejected Heidenhain’s calculation as involving far too low an estimate of renal blood flow; in addition, when the immense number of renal tubules are taken into account, the requirement for water reabsorption could be reasonably accounted for. Cushny rejected entirely the notion of tubular secretion. To account for reabsorption, Cushny postu1ated some “vital activity” on the part of the epilthelium – a postulate that Ludwig had steadfastly rejected. Thurau and his associates (4) have pointed out that these various theories of urine formation were unifed in a perceptive analysis by Metzner (1858-1935) published in 1906, some ten years before Cushny’s “modern theory”.
His conception of a trifold process of ultrafiltration at the glomerulus by physical forces, reabsorption of most of the filtrate in part by active tubular processes, and active secretion of certain solutes by the tubular epithelia is remarkably close to modern views. His summary is worth quoting (Fig. 3). It should be emphasized that the use of clearance calculations by Heidenhain and their reinterpretation by Cushny served to make creditable the conceptual model of the comparatively modest magnitude of urine flow in a setting of huge volumes of glomerular filtration.
Although Cushny may have over-emphasized the commitment of Heidenhain and others to vitalism, there is no question that the concept of vital activity, not reducible to physical forces, was a powerful conceptual factor that infected the theories of renal function. For most of the 19th centur y, a basic problem in biology was conceived to be the distinction between living and non-living matter. A mechanistic explanation assumed that organic and non-organic matter were not irreducibly different. A vitalistic explanation assumed that a reduction of living to non-living phenomena is in principle impossible. Embryology was a dominating biologic discipline. To provide a flavor of the intellectual climate surrounding the study of renal function, it may be helpful to review briefly the prevailing embryologic studies. Landmark studies exemplified by the work of Hans Driesch are summarized in a comprehensive publication in 1914 (7). In a series of studies on embr yonic sea-urchins, he demonstrated that rearrangement of cells at the blastomere stage had no effect on normal development. Moreover, a single blastomere, isolated from the rest at the two - or fourcell stage, can develop into a normal sea-urchin embr yo.