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Blood Biochemistry Composition of Blood Blood is the body’s only fluid tissue It is composed of liquid plasma and formed elements Formed elements include: Erythrocytes, or red blood cells (RBCs) Leukocytes, or white blood cells (WBCs) Platelets – the percentage of RBCs out of the total blood volume Hematocrit Components of Whole Blood Plasma (55% of whole blood) 1 Withdraw blood and place in tube 2 Centrifuge Buffy coat: leukocyctes and platelets (<1% of whole blood) Erythrocytes (45% of whole blood) Formed elements Plasma The blood fraction obtained after removal of the cellular components About 77%-81% in the total blood values Hydrometer is 1.050-1.060, pH is 7.35-7.45, osmotic pressure is 770kPa (37°C) in the normal human relevant to coagulation factors, immunoglobulins and complements Serum The blood fraction after separation of the protein fibrinogen from plasma Generally obtained by allowing the blood to clot In this process, fibrinogen is converted to an insoluble protein, fibrin, which is easily removed Serum does contain some degradation products of clotting factors Plasma composition Clotting factors Liquid: water protein plasma Nonprotein nitrogen (NPN) Serum solids Low-molecular-weight organic substances such as Serum solids glucose, lipids, vitamins, hormones and so on Na+, K+, Ca2+, Mg2+ electrolytes Gases:O2, CO2 CI-, HCO32-, HPO42- Non-Protein Notrogen (NPN) Non-protein nitrogenous compounds urea, uric acid, creatinine, creatine, nucleotides, amino acids, bilirubin, polypeptides, glutathione and many others The Concentration of NPN 14.28~24.99 mmol/L, 50% of NPN is blood urea nitrogen (BUN) Source of NPN derived from the metabolism of nucleic acid and proteins Excretion of NPN transported to the kidneys fro excretion from the urin Significance act as an index of renal function Male versus female Hematocrit (% volume that is red cells) 40-50% in males 35-45% in females Function of Blood Blood as a transport system transport nutrients and oxygen to the cells and carries away cellular waster products Blood as a regulative system maintaining normal acid-base balance in the body; Regulating the water balance and body temperature Blood as a defense system white blood cells and the circulating antibodies Coagulation and fibrinolysis Section 1 Plasma Proteins Plasma Proteins More than 200 Most abundant Albumin - 4-5 g/100 mL g-glubulins - ~1 g/100 mL fibrinogen - 0.2-0.4g/100 mL Original classification by zone electrophoresis at pH 8.6 Separation by pI with several molecular weight species within each group Zone Electrophoresis of Plasma Proteins + globulins g pI 6.0 b a1 a2 5.6 5.1 albumin 4.7 Protein Separation Size Exclusion Chromatography (SEC) Porous matrix (sephadex) Affinity chromatography molecule attached to a column that specifically binds the protein of interest Coenzyme / enzyme Antigen / Antibody SDS-PAGE (polyacrylamide gel electrophoresis) Separates by size Proteins are complexed with SDS to give the same charge density Two Dimensional Electrophoresis Decreasing Mr Decreasing pI Characteristics of Plasma Proteins Most plasma proteins are synthesized in the liver, however, certain proteins are synthesized in other sides Generally synthesized on membrane –bound polyribosomes With the exception of albumin, almost all plasma proteins are glycoproteins Many plasma proteins exhibit polymorphism Each plasma protein has a characteristic half-life in the circulation The levels of certain proteins in plasma increase during acute inflammatory states or secondary to certain types of tissue damage Functions of Plasma Proteins (1) Functional enzymes of the plasma Have catalysis in the plasma, such as thrombin, lipoprotein lipase, LCAT etc (2) non-functional enzymes of the plasma Maintenance of: Colloid osmotic pressure (COP) (p) pH electrolyte balance COP relates to blood volume DP = p Protein sol’n Water Transport of ions, fatty acids, steroids, hormones etc. Albumin (fatty acids), ceruloplasmin (Cu2+), transferrin (Fe), lipoproteins (LDL, HDL) Nutritional source of amino acids for tissues Hemostasis (coagulation proteins) Prevention of thrombosis (anticoagulant proteins) Defense against infection (antibodies, complement proteins) Albumin MW 66 000 Single chain, 580 amino acids, sequence is known Dimensions - Heart shaped molecule 50% a helix [He and Carter, Nature, 358 209 (1992)] Modeled as: 80 Å 30 Å Synthesis Mainly liver cells then exported Assembly time on ribosome ~ 1-2 min t0.5 in circulation - 19 days 14 g lost per day 0.4 mg synthesized per hour per g of liver Need liver of approximately 1.5 kg in weight to maintain Functions Maintaining colloid osmotic pressure of blood (80% due to albumin) Colloid osmotic pressure is generated by plasma proteins The most abundant of the plasma proteins The lowest molecular weight of the major protein molecules in the plasma High negative charge Regulates water distribution Transportation Albumin can act as a carrier molecule for bilirubin, fatty acids, trace elements and many drugs Section 3 Metabolism of the Blood Cells Cellular Elements of Blood Red cells 40 - 50% of blood volume 5 x 106 cells /mL Composed of a membrane surrounding a solution of hemoglobin non-nucleated, no intracellular organelles no proliferation cell membrane in excess so that deformation does not rupture Shape Biconcave disc 8 mm in diameter, 2.7 mm thick, volume ~ 90 mm3, area ~ 160 mm2 Scanning Electron Micrograph of Red Blood Cells Why this shape? Area to volume ratio is high Facilitates diffusion of O2 and CO2 minimal distance of contents from surface Originates in bone marrow (hematopoiesis) Molecular explanation based on the properties of the proteins in the cell membrane is found in Elgsaeter et al. Science, 234, 1217 (1986) Production of Erythrocytes – blood cell formation Hematopoiesis occurs in the red bone marrow of the: Hematopoiesis Axial skeleton and girdles Epiphyses of the humerus and femur Hemocytoblasts elements give rise to all formed Production of Erythrocytes: Erythropoiesis A hemocytoblast is transformed into a committed cell called the proerythroblast Proerythroblasts develop into early erythroblasts The developmental pathway consists of three phases Phase 1 – ribosome synthesis in early erythroblasts Phase 2 – hemoglobin accumulation in late erythroblasts and normoblasts Phase 3 – ejection of the nucleus from normoblasts and formation of reticulocytes Reticulocytes then become mature erythrocytes Production of Erythrocytes: Erythropoiesis Figure 17.5 The major function of the red cells Delivering oxygen to the tissues, helping in the disposal of carbon dioxide and protons formed by tissue metabolism Normal red cell breakdown haemoglobin haem iron transferrin globin protoporphyrin CO Expired air Amino acids Bilirubin (free) Liver conjugation erythroblast Bilirubin glucuronides Urobilin(ogen) Urine Stercobilin(ogen) faeces Hemoglobin synthesis Heme synthesis starts with the condensation of glycine and succinyl coenzyme A under the action of a rate limiting enzyme δ-aminolevulinic acid (ALA) synthase. δ -ALA will be formed. Pyridoxal phosphate (vit. B6) is a coenzyme for this reaction. COOH COOH H2C CH 2 C¡«SCoA CH 2NH 2 HSCoA + CO2 H2C + CH 2 COOH ALA synthase ( Pyridoxal phosphate ) C O This step takes place in the mitochondria O CH 2NH 2 A series of biochemical reactions will follow. Two molecules of δ-ALA condense to form a pyrrole called porphobilinogen (PBG) COOH O OH CH 2 HO CH 2 O O C H C H H N H ALA dehydratase 2H2O H2N This step occurs in the cytoplasm N H Four PBG condense to form a tetrapyrrole uroporphyrinogen III. UPG III is then converted to coproporphyrinogen. Deaminase Four PBG Linear tetrapyrrole UPG III isomeiase UPG III decarboxylase coproporphyrinogen Ⅲ uroporphyrinogen III This step occurs in the cytoplasm Haemoglobin synthesis CPG then changes to protoporphyrin which ultimately combines with iron in the ferrous state (Fe2+) to form haem. Iron is brought to the developing red cells by a carrier protein ( transferrin) which attaches to special binding sites on the surface of these cells. Transferrin releases iron and returns back to circulation. Haemoglobin synthesis Each molecule of haem combines with a globin chain. A tetramer of four globin chains each with its own haem group in a pocket is formed to make up a haemoglobin molecule. Haemoglobin structure Haem consists of a protoporphyrin ring with an iron atom at its centre. The protoporphyrin ring consists of four pyrrole groups which are united by methane bridges (=C-). The hydrogen atoms in the pyrrole groups are replaced by four methylene (CH3-), two vinyl (-C=CH2) and two propionic acid (-CH2-CH2COOH) groups. Metabolic Characteristics of Mature Erythrocytes Can not carry out synthesis of nucleic acid and proteins Can not obtain energy by oxidative phosphorylation of the mitochondria ATP is synthesized from glycolysis and is important in process that help the red blood cell maintain its biconcave shape and also in the regulation of the transport of ions and of water in and out of the cell The principal modes of glucose metabolism are anaerobic glycolysis and the pentosephosphate pathway Glycolysis Obtain energy by glycolysis of glucose Utilize 2ATP moleculars, produces 4ATP moleculars with a net gain of 2ATP - The function of ATP To maintain the correct ion balance, brought about by the pumping out of sodium in exchange for potassium To maintain the correct conformation of the cell To protect against the formation of methaemoglobin To synthesize NAD+ and glutathione The pathway of 2,3-bisphosphoglycerate (2,3-BPG) Formation of 2,3-BPG Glucose 1, 3-BPG Diphosphoglyceromutase Phosphoglycerate kinase 2, 3-BPG Glycerate 3-phosphate Diphosphoglycerate phosphatase Lactate The role of 2,3-BPG Plays an important role in the binding of oxygen to hemoglobin in erythrocytes Combine with hemoglobin, causing a decrease affinity of hemoglobin for oxygen pO2 2,3-DPG (lungs) Oxyhemoglobin Hemoglobin pO2 2,3-DPG (tissues) (HbO2) (Hb) The role of the pentose phosphate pathway Produce the NADH which is essential for the regeneration of reduced glutathione from oxidized glutathione NADP+ 2GSH Glutathione reductase NADP++H+ Pentose phosphate pathway GSSG The role of glutathione are as follows The role in the destruction of hydrogen peroxide (H2O2) in erythrocytes NADP+ 2GSH Glutathione reductase NADP++H+ H2O2 Glutathione peroxidase GSSG 2H2O Reduction of methemoglobin Methemoglobin does not combine with molecular oxygen and does not have the function of transporting oxygen Normally, methemoglobin is reduced to the ferrous state by the NADH-dependent methrmoglobin reductase Methrmoglobin reductase MHb (Fe3+) ½ O2 Hb (Fe2+) NADH+H+ NAD H2O Genetic abnormality-deficiency of glucose-6phosphate dehydrogenase glucose-6-phosphate dehydrogenase is the first enzyme of the pentose phosphate pathway A deficiency of this enzyme will lead to failure of restoring GSSG to GSH in the erythrocytes, a step essential for the removel of H2O2 Cell damage is likely to result from oxidation of the membranes by the H2O2, leading to hemolytic anemia White Blood Cells (Leukocytes) Total count - approximately 7000/mL Various types Neutrophils 62% Eosinophils 2.3% granulocytes Basophils 0.4% Monocytes 5.3% Lymphocytes 30% Plasma cells (mainly in the lymph) Monocytes in tissue become macrophages Function Defense against foreign invaders bacteria viruses foreign materials (including biomaterials) Phagocytosis Neutrophils, macrophages Move to foreign particle by chemtaxis Chemicals induce migration Toxins, products of inflamed tissues, complement reaction products, blot clotting products Response is extremely rapid (approx 1 h) Lymphocytes B cells - responsible for humoral immunity T cells - responsible for cell mediated immunity B cells responsible for production of antibodies Receptor matches antigen Cells multiply Antibodies Abs are just immunoglobulins discussed earlier T cells Cytotoxic T cells (Killer T cells) Bind to cytotoxic cells (eg infected by virus) Swell Release toxins into cytoplasm Helper T cells Most numerous Activate B cells, killer T cells Stimulate activity by secretion of IL2 Stimulate macrophages Suppressor T cells Regulate activities of other cell types Erythropoietin Mechanism Start Normal blood oxygen levels Increases O2-carrying ability of blood Stimulus: Hypoxia due to decreased RBC count, decreased availability of O2 to blood, or increased tissue demands for O2 Reduces O2 levels in blood Enhanced erythropoiesis increases RBC count Erythropoietin stimulates red bone marrow Kidney (and liver to a smaller extent) releases erythropoietin Figure 17.6 Haemoglobin catabolism *normal red cell destruction* Red cell destruction usually occurs after a mean life span of 120 days. The cells are removed extravascularly by macrophages of the reticuloendothelial system (RES), specially in the bone marrow but also in the liver and spleen. Red cell metabolism gradually deteriorates as enzymes are degraded and not replaced, until the cells become non viable, but the exact reason why the red cells die is obscure. Haemoglobin catabolism *normal red cell destruction* The breakdown of red cells liberates 1- iron for recirculation via plasma transferrin to marrow erythroblasts 2- protoporphyrin which is broken down to bilirubin. 3- globins which are converted to amino acids. Normal red cell destruction - The bilirubin circulates to the liver where it is conjugated to glucuronides which are excreted into the gut via bile and converted to stercobilinogen and stercobilin(excreted in faeces). - Stercobilinogen and stercobilin are partly reabsorbed and excreted in urine as urobilinogen and urobilin. Normal red cell destruction A small fraction of protoporphyrin is converted to carbon monoxide (CO) and excreted via the lungs. Globin chains are broken down to amino acids which are reutilized for general protein synthesis in the body. Normal red cell breakdown haemoglobin haem iron transferrin globin protoporphyrin CO Expired air Amino acids Bilirubin (free) Liver conjugation erythroblast Bilirubin glucuronides Urobilin(ogen) Urine Stercobilin(ogen) faeces Haemoglobin abnormalities There are mainly two types of abnormalities, these are : Quantitative abnormalities: where there is reduction in the production of certain types of globins e.g. a thalassaemia b thalassaemia Qualitative abnormalities: where there is production of abnormal haemoglobin e.g. sickle cell anaemia. Composition and Function of Blood Blood composition - 5-6 L in an adult - 70 mL/kg of body weight - Suspension of cells in a carrier fluid (plasma > Cells - 45% by volume (cellular fraction) > Plasma - 55% by volume (non-cellular fraction) Cells Red cells (erythrocytes) 5x106/mL White cells (leukocytes) 7x103/mL Platelets (thrombocytes) 3x105/mL Oxygen Binding of Hb Blood must carry 600 L of O2 from lungs to tissues each day Very little carried in plasma since O2 only sparingly soluble Nearly all bound and transported by Hb of RBC Possible for Hb to carry four O2 molecules, one on each a chain, one on each b chain O2 depleted Hb solution placed in contact with O2(g) Equilibrium reaction Fraction (s) of Hb converted to oxyhemoglobin