Biochemistry of Blood Lec. 3 PDF

South Valley University Faculty of Pharmacy Biochemistry Department Biochemistry of Blood Lec.3 Dr/ Shimaa Abd El-Nasser Lecturer of Biochemistry  Blood plasma proteins Proteins account for 6,5-8,5% out of the total 9-10% of dry blood p...

South Valley University Faculty of Pharmacy Biochemistry Department Biochemistry of Blood Lec.3 Dr/ Shimaa Abd El-Nasser Lecturer of Biochemistry  Blood plasma proteins Proteins account for 6,5-8,5% out of the total 9-10% of dry blood plasma residue. Total proteins – 65-85 g/L Albumins – 40-50 g/L Globulins – 20-40 g/L Fibrinogen – 1,5-3,5 g/L The most of blood serum proteins are synthesized in liver, but some of them are formed in other tissues. For example, γ-globulins are synthesized by lymphocytes; peptide hormones are mainly secreted by endocrine glands; peptide hormone erythropoietin is formed by kidney cells. Almost all the blood plasma proteins, with the exception of albumin, are glycoproteins. Functions of blood proteins: they take part in blood clotting; they provide viscous properties of blood; maintaining acid base balance; transport function; protective function; reserve of amino acids; regulation function; they maintain the oncotic pressure; maintaining a needed level of cations in blood.  Albumin Albumin level in blood plasma protein is 35-50 g/L. Albumins make up approximately 60% of the total plasma protein. Functions of albumins: 1) Albumins are responsible for 75 – 80% of oncotic pressure of human's plasma. The decreasing albumin concentration below 30 g/L leads to edema. 2) Transport function. It transports free fatty acids, calcium, certain steroids hormones, bilirubin, copper, different drugs etc.  Globulins Globulins: are divided into α1-globulins (3-6 g/L), α2-globulins (4-9 g/L), β-globulins (6-11 g/l) and γ-globulins (7-15 g/L). They perform transport and protective functions.  α-Globulins Haptoglobin (Hp) (is component of α2-globulin fraction). This glycoprotein binds extracorpuscular hemoglobin. The haptoglobin-hemoglobin complex can be absorbed by the macrophage system and cannot pass the glomerulus of the kidney. Thus Hp prevents a loss of free hemoglobin by kidney and provides the conservation and reutilization of iron.  α-Globulins Ceruloplasmin (α2–globulin): is a blue, copper-containing (0,32%) glycoprotein found in mammalian blood plasma. It contains about 3% of total amount of copper in organism and more 90% Cu of blood plasma. Cerruloplasmin exhibits a weakly pronounced catalytic activity in the oxidation of ascorbic acid, adrenaline, dihydrophenylalanine and a number of other compounds, and ferrooxidase activity (Fe2+→Fe3+). It is antioxidant. In Wilson's diseases the concentration of ceruloplasmin in the blood plasma is significantly lowered which a major diagnostic test for this pathology.  α-Globulins α1-Antitrypsin can inhibit trypsin and other proteolytic enzymes. The level of trypsin inhibitors is increased in inflammatory processes, in pregnancy and in a number of other states of organism. In inflammatory process the level of α1-antitrypsin increases in result of stimulation of its synthesis in hepatocytes. In acute pancreatitis the enchanced level of α1- antitrypsin arises from delivery of active pancreatic proteinases. Deficiency of α1-antitrypsin is associated with emphysema and one type of liver diseases (α1-antitrypsin deficiency liver disease).  α-Globulins α2- Macroglobulin is a large plasma glycoprotein (720 kDa). It is inhibitor of serine-, thiol-, carboxy- and metal proteinases. Therefore it is involved in regulation of blood clotting, immunologic processes, inflammatory processes. In addition, it binds many cytokines (eg. platelet-derived growth factor, transforming growth factor-β, etc.) and is involved in targeting them toward tissues.  β-Globulins β-Globulins: C-reactive protein is able to form a precipitate with the somatic C polysaccharide of pneumococcus C-reactive protein does not occur in the blood serum of healthy organisms. It is detected in many pathologic states attendant to inflammation and necrosis of the tissues. This protein is „acute phase” protein. Hemopexin binds free heme.  β-Globulins Transferrin is glycoprotein with a molecular mass of approximately 80 kDa. Transferrin plays a central role in the body's metabolism of iron, because it transports iron (2 moles of Fe3+ per moll of transferrin) in the circulation to sites where iron is required, e.g. from the gut to the bone marrow and other organs.  γ-Globulins γ-Globulins: Cryoglobulin is absent in the blood serum of healthy individuals. It is found only in pathologic states. A specific feature of this protein is its solubility at a temperature 37°C and ability to form a precipitate or a gel in decreasing temperature to 4°C. It is detected in blood serum in myeloma, nephrosis, cirrhosis of the liver, rheumatism, lymphosarcoma, leucosis and other diseases.  γ-Globulins Interferons are specific proteins synthesized on the organism's cells invaded by virus. Interferon can inhibit viral multiplication in the cells however it has no effect on the viral particles that have been formed in the cell. Interferon is easy to leave the cell and to enter the blood stream in which it is carried over to tissues and organs. There are 3 types of interferons: IFN-α, IFN-β, and IFN-γ. IFN-α are mainly synthesized by leukocytes; IFN-β by fibroblasts; IFN-γ by T- and B- lymphocytes.  γ-Globulins Immunoglobulins (humoral antibodies) are synthesized mainly in plasmocytes, specialized cells of B-cell lineage that synthesize and secrete immunoglobulins into plasma. There are five immunoglobulin classes: IgG (γ-chains), IgA (α-chains), IgM (μ- chains), IgD (δ-chains), IgE (ε- chains).  Diagnosis by plasma protein In clinical practice, there have been reported states characterized by alteration in both the total content of blood plasma proteins and the percentage of individual protein fractions. Hypoproteinemia (a decrease in the total concentration of blood plasma proteins) is usually linked with the decreasing albumins. Hypoproteinemia occurs: 1) in nephrotic syndrome; 2) in liver disease (acute atrophy of the liver, toxic hepatitis, and other states); 3) in a drastically increased permeability of the capillary wall, 4) in protein deficiency (affected gastrointestinal tract, carcinoma, etc).  Diagnosis by plasma protein Paraproteinemia is the occurrence in the blood plasma of proteins, normally untypical to the healthy organism (for example, in myeloma). In the blood serum of patients with myeloma specific “myelomatous” proteins are detected. Hyperproteinemia is a pathologic condition manifested by an increased content of blood plasma proteins. Hyperproteinemia: 1) relative (it is caused by loss of liquid by organism (diarrhea in children; vomiting, due to an obstruction of the upper small intestine, or by extensive burns); 2) absolute (it is caused by an elevated level of γ-globulins).  Diagnosis by plasma protein Dysproteinemia is the changing ratio of individual protein fractions, while the total protein content in the blood serum is normal. - γ-Globulin fraction is increased in chronic inflammation, chronic polyartritis etc. - α2-Globulin fraction is increased in acute infections, acute rheumatism. Those proteins are called «acute phase» proteins, because they take part in development of inflammatory reaction of organism. Main inducer of the synthesis of the most acute phase proteins in hepatocytes is interleukin-1 liberated by mononuclear phagocytes. Haptoglobin, C-reactive protein, α1-antitrypsin, acid α1- glycoprotein, fibrinogen belong to proteins of acute phase.  Non-protein organic compounds of blood. Total nitrogen of the blood includes a protein nitrogen and nonprotein nitrogen (or residual nitrogen): N total = N prot. + N res. N res. = 14,3-25 mmol/L. Residual nitrogen of the blood includes: urea nitrogen (50%), amino acid nitrogen (25%), creatine nitrogen (5%), creatinine nitrogen, ammonia nitrogen, indican nitrogen, bilirubin nitrogen, uric acid nitrogen etc.  Non-protein organic compounds of blood. Ammonia level (25-40 μmol/L) increases in liver diseases, inherited disturbances of ornithine cycle. Urea level (3,3-8,3 mmol/L) increases in chronic diseases of kidney, cancer of ureteral ducts, tuberculosis of kidney, some infectious diseases, sepsis and other. Its level decreases in liver diseases (hepatitis, cirrhoses), pregnancy, inherited disturbances of urea cycle. Creatinine (53-105 μmol/L) increases in retential azotemia, indicates the degree of chronic renal insufficiency. Uric acid level (149- 405 μmol/L) increases in gout.  Azotemia Azotemia is the increased level of residual nitrogen in blood. Productive azotemia is observed in an excessive delivery of nitrogenous products to the blood as result of accelerated degradation of tissues proteins in different states: inflammation, wounds, extensive burns, cachexia and other states. Retention azotemia is caused by incomplete urinary discharge of nitrogen containing products on their normal delivery to the blood stream.  Azotemia 1) Renal retention azotemia is caused by reduced excretory function of kidney (reduced renal clearance). Urea is mainly responsible for the increased residual nitrogen level in renal retention azotemia. Urea constitutes 90% of residual nitrogen of blood instead of 50% in normal conditions. 2) Extrarenal retention azotemia may arise from an acute circulatory insufficiency, low arterial pressure, or reduced renal blood flow. Also, the frequent cause of extrarenal retention azotemia is an obstruction to the urine outflow from the kidney.  Blood plasma enzymes Enzyme diagnostics is one of the branches of enzymology. It has two main directions: 1) use of enzymes as reagents for determination of normal and pathological components in serum, urine, gastric juice etc. 2) determination of enzyme activity in biological material with a diagnostic purpose.  Blood plasma enzymes Serum enzymes are divided into 3 groups: 1) Cellular enzymes enter the blood from different organs. Their activity in serum depends on enzyme content in organs, molecular weight, intracellular localization, rate of elimination. Cellular enzymes are divided into non-specific and organ specific. 2) Secretory enzymes are synthesized by cells, enter the bloodstream and fulfill their specific functions in the circulatory system. These are enzymes of coagulation system and fibrinolysis, choline esterase etc. 3) Excretory enzymes are synthesized by glands of GIT and enter the blood (amylase, lipase).  Blood plasma enzymes Aminotransferases (АLТ and АSТ). Aminotransferases catalyze the process of transamination, they are present in every organ and tissue. Isoenzymes of AST are localized both in cytoplasm and in mitochondrions. ALT predominates in cytoplasm. High concentration of AST is noted in heart and skeletal muscles, liver, kidneys, pancreas and erythrocytes. Damage of any of them leads to significant increase of AST in the blood serum. Highest concentration of ALT is noted in the liver cells. Skeletal muscles, kidneys and heart also contain ALT, but much less.  The most significant increase of AST is observed in myocardial damage. In myocardial infarction, AST activity in blood serum can increase 4-5 times. In acute myocardial infarction, 93-98% of patients have high AST activity; the latter has the same dynamic as Creatine Kinase MB (CK-MB). However, CK-MB increase is more significant. Increase in AST activity reveals hepatic pathology. The most significant increase is observed in acute viral and toxic hepatitis. From mild to moderate increase in AST occurs in liver cirrhosis (2-3 times), obstructive jaundice and liver metastasis. It can be also so in skeletal muscular pathology, for example progressive muscular dystrophy; in pancreatitis; intravascular haemolysis. Low AST activity usually reveals vitamin В6 deficiency, renal failure, pregnancy.  Increased ALT activity is most frequently revealed in acute liver and biliary ducts diseases. ALT activity rises significantly in the early stages of acute viral hepatitis: in 50% of patients ALT increases 5 days before jaundice and hepatomegaly appear, in 90% of patients – 2 days before these symptoms. AST/ALT ratio is called de Ritis ratio. Its normal value 1-1,3. It decreases in liver diseases and increases in heart diseases. In toxic (alcoholic) liver damage AST activity rises predominantly, where de Ritis ratio exceeds 2. In viral hepatitis de Ritis ratio decreases. This ratio increases in obstructive jaundice, cholecystitis, liver cirrhosis, while ALT and AST activity increase slightly.  Respiratory function of erythrocytes. Erythrocytes constitute about 44% of the total blood volume (4,5-5x1012/L). The life of erythrocytes is 120 days. New synthesized erythrocytes contain ribosomes and elements of endoplasmic reticulum. Mature erythrocytes don't contain ribosomes, mitochondria, lysosomes, Golgi apparatus. Synthesis of erythrocytes is regulated by erythropoietin. Erythropoietin is synthesized in kidney. It is liberated to blood in hypoxia and is transported to bone marrow.  Erythrocytes metabolism 1) Main source of energy is glucose. Glucose enters red blood cells by facilitated diffusion, a process mediated by the glucose transporter (GLUT1), also known as glucose permease. 2) Source of ATP is anaerobic glycolysis. 3) The formation of 2,3-bisphosphoglycerate from 1,3-bisphosphoglycerate by Rappoport-Leubering shunt is very important for regulation of affinity of Hb to O2.  Rapoport-Lubering cycle Rapoport-Lubering cycle is a side-pathway (a shunt) from glycolysis that aims at dissipating or waste the excess ATP since RBCs produces ATP through glycolysis which far exceeds its needs. The cells lack many metabolic reactions that require energy. This cycle is a shunt from glycolysis that is formed of two steps: 1) 1,3-diphosphoglycerate is mutated into 2,3-diphosphoglycerate (2,3-DPG), by diphosphoglycerate mutase enzyme. 2) By 2,3-diphosphoglycerate phosphatase, the phosphate group at C2 of 2,3-DPG is hydrolyzed producing 3-phosphoglycerate. This reaction is also activated by phosphoglycerate mutase. 3-phosphoglycerate rejoins the ordinary pathway of glycolysis. Biochemical significance of 2,3-Diphosphoglycerate (2,3-DPG): A. 2,3-DPG acts as a cofactor in the conversion of 3-phosphoglycerate to 2- phosphoglycerate by mutase. B. It decreases the affinity of hemoglobin (Hb) to oxygen helping oxygen dissociation and unloads oxygen in tissue capillaries for oxygenation. Therefore, the levels of 2,3-DPG increases markedly in peripheral hypoxic tissues and hypoxic conditions (high altitudes, hypoxic hypoxia, stagnant hypoxia and anemic hypoxia). - Red blood cells have a high concentration of 2,3-DPG (4 - 5 mM, equimolar to Hb) in contrast to traces amount in most other cells. During storage of blood in blood banks, 2,3-DPG concentration decreases gradually to reach traces at ten days. So, Hb of this blood has a high affinity to O2 and is not suitable for blood transfusion to hypoxic patients suffering from, e.g., respiratory disorders and severely ill patients. - RBCs membrane is impermeable to 2,3-DPG. So, instead of adding 2,3-DPG, inosine is added which penetrates RBCs easily and its ribose is changed very slowly into 2,3-DPG by HMP shunt and glycolysis. - Fetal hemoglobin binds 2,3-DPG less strongly than adult hemoglobin and therefore has a higher O2 affinity to extract O2 from mother's blood.  Erythrocytes metabolism 5) 5-10% of glucose are metabolized by means of pentose phosphate pathway. NADPH is necessary for reduction of glutathione. Deficiency of glucose-6-phosphate dehydrogenase is the cause of drug-induced hemolytic anemia (Favism). 6) Glutathione may be synthesized in erythrocytes. It is necessary for elimination of peroxides. 7) Autooxidation of Hb results in formation of metHb (1,7% under normal conditions). This is accompanied by formation of O2 (superoxide radical). NADH dependent methemoglobin reductase converts metHb to Hb.  Erythrocytes metabolism 8) The synthesis of glycogen, fatty acids, proteins, nucleic acids does not occur in erythrocytes. Some lipids, for example cholesterol, may be exchanged with corresponding lipids of plasma. 9) Erythrocytes have some enzymes of nucleotide metabolism.