Plasma proteins.pdf

Plasma Proteins, Immunoglobulins, Acute Phase Reactants Prof. Dr. Ufuk Çakatay Department of Medical Biochemistry Cerrahpaşa Faculty of Medicine Serum versus plasma:Difference between serum and plasma • Serum samples are most commonly used by clinical laboratory. • Plasma is more easily seperated...

Plasma Proteins, Immunoglobulins, Acute Phase Reactants Prof. Dr. Ufuk Çakatay Department of Medical Biochemistry Cerrahpaşa Faculty of Medicine Serum versus plasma:Difference between serum and plasma • Serum samples are most commonly used by clinical laboratory. • Plasma is more easily seperated from the blood cells with the anticoagulant (e.g heparin, citrate, EDTA) containing tubes. • Serum has the similar composition as plasma except for the absence of fibrinogen. • Clothing factors that are consumed during clothing. Why have the blood collection tubes got different colored tops? Different colored tops are used to take blood samples. The tubes will be colored according to be substances that they contain. Each type of anticogulant has a different color code (e.g Heparin: Green, Blue:Citrate,Purple:EDTA). An appropriate color code is chosen according to the test to be performed in laboratory How to obtain serum from blood? When blood is centrifuged in the absence of anticoagulant, blood cells in fibrin cloth occupy bottom of the blood collection tube. The remainder is a clear, yellowish fluid called serum. How to obtain plasma from blood? When blood is centrifuged in the presence of anticoagulant, the formed elements between ~ 45-46% of the total volume . The remainder is a clear, yellowish fluid called plasma. Overview of plasma proteins • Most plasma proteins are synthesized & destroyed in liver. Hovewer, the γ-globulins are synthesized in plasma cells. • Plasma proteins are generally synthesized on membrane bound polyribosomes • Most plasma proteins are glycoproteins except albumin. Removal of terminal sialic acid residues from certain plasma proteins (e.g. ceruloplasmin) leads to shorten half-lives in plasma. After the removal of sialic acid by endothelial neuraminidases,asialoglycoprotein receptor on the surface of hepatocytes binds it for receptor-mediated endocytosis and lysosomal degradation pathway. • Many plasma proteins exhibit polymorphism and shows Mendelian (monogenic) trait (e.g. Haptoglobin, ceruloplasmin, transferrin) • Plasma protein concentration determines the distribution of fluid between blood & tissues Lower plasma protein concentration leads to the accumulation extracellular fluids (edema) Common methods of analyzing plasma proteins Electrophoresis Radial immunodiffusion Radioimmunoassay (RIA) It is the most common method analyzing plasma proteins. Its use permits resolution, after staining, of plasma proteins into five bands, designated albumin, α1, α2,β, and γ fractions A quantitative immunodiffusion technique used to detect the level of protein (antigen) in a sample by measuring the diameter of the ring of precipitin formed by the complex of the protein (antigen) and the antiserum (antibody). Analysis for e.g. ceruloplasmin,transferrin,α1-antitrypsin A method that uses the competition between radiolabeled and unlabeled substances in an antigenreaction to determine the concentration of the unlabeled substance, which may be an antib ody or a substance against which specific antibodies can be produced. Analysis for e.g. insülin, gastrin,inhibin Enzim An assay that uses an enzyme-bound antibody to detect antigen. immunoassay (EI) The enzyme catalyzes a color reaction when exposed to substrate. Analysis for e.g. Ferritin, IgE, Myoglobin, Troponin I Fluoroimmunoassay An immunoassay that has antigen or antibody labeled with a fluor ophore. Analysis for e.g. alpha-fetoprotein Chemiluminescence Emission of light as a result of a chemical reaction at environmental temperatures. Analysis for e.g.FSH,TSH General functions of plasma proteins Plasma contains over one hundred individual proteins, each with a specific set of functions and subject to specific variations in concentration under different physiologic & pathological conditions Protein Transthyretin Albumin Retinol-binding protein α1-Antiprotease Prealbumin Albumin α1 Thyroxinebinding globulin α1 Transcortin α-Fetoprotein α1 α1 Ceruloplasmin α2-Macroglobulin Haptoglobin Transferrin Hemopexin Fibrinogen C-reactive protein Immunoglobulins α2 α2 α2 β β β γ Electrophoretic Fraction α1 γ The fastest moving band, and normally the most prominent, is the albumin band found closest to the anodic edge of the plate. The band next to this is Alpha1 Globulin, followed by Alpha2 Globulin, Beta, and Gamma Globulins. Transthyretin • Transthyretin, also called prealbumin. • It moves slightly ahead of albumin during electrophoresis • Retinol-binding protein (MW 21000) is a small protein • It binds transthyretin (MW 62000) and prevents its renal excretion • Transthyretin also binds thyroid hormones. • Major transport protein for thyroid hormones is thyroxinebinding globulin (TBG) • TBG binds thyroxine (T4) with higher affinity than does transthyretine General Properties of Serum Albumin • Albumin is the major protein in human plasma and consist of one polypeptide chain which is folded elipsoidal shape. It forms 60% of total protein in serum. • Albumin noncovalently binds many small molecules such as fatty acids, thyroxine, cortisol, heme, bilirubine, and various drugs (e.g.aspirin,dicumarol,sulfonamides and penicilin). Only free,unbound drugs are pharmacologically active in human plasma. • Albumin also functioning as an powerful antioxidant in human plasma. • Albumin provides osmotic/oncotic pressure of plasma. Lower albumin concentration leads to the accumulation extracellular fluids (edema). Preparations of human albumin have been widely used the treatment of hemorrhagic shock and of burns Albumin & Protein Electrophoresis • The diminished synthesis of albumin is seen in various diseases (e.g.liver diseases, neoplasm, including multiple myeloma). The plasma of patients with liver disease often shows decreased albumin-globulin ratio • The lack of albumin in serum protein electrophoresis is named as analbuminemia.The cause of analbuminemia is a mutation that affects splicing. • Bisalbuminemia or alloalbuminemia is an inherited or acquired serum protein abnormality characterized by the presence of 2 different albumin fractions, in equal or unequal amounts. Inherited bisalbuminemia has no pathological effects Bisalbuminemia • α1-Antitrypsin (α1-Antiprotease, α1-Protease inhibitor) • It exhibits genetic genetic polymorphism • α1-Antitrypsin inhibits serine proteases (e.g. leucocyte derived elastase) • α1-Antitrypsin deficiency leads to excessive proteolitic activity & tissue damage in chronic bronchitis. Alveolar macrophages,neutrophils secrete lysosomal proteases during phagocytosis. • α1-Antitrypsin can be inactivated by smoking. α1-antitrypsin deficiency appears as either a decrease in or complete absence of the α1 band. Thyroxine-binding globulin • Thyroxine binding globulin (TBG) binds thyroxine with higher affinity than does transthyretin • Individuals with congenital absence of TBG show no any clinical complication, because the level of free (unbound) hormone is kept in physiological range by homeostatic mechanisms. Transcortin • Steroid hormones have to binding proteins: transcortin for glucocorticoids and sex-hormone-binding globulin (SHBG) for androgens and estrogens. • Only the unbound fraction of the hormone determines the biological response. α-Fetoprotein • Abnormalities in the concentration of minor plasma proteins cannot be observed from the electrophoretic patern and have to be determined immunological methods. • α-fetoprotein is synthesized in the fetal liver but occurs in only trace amounts in normal adult blood. It levels are increased in most patients with hepatocellular carcinoma. α-fetoprotein is also used for the prenatal diagnosis of the open neural tube defects. Ceruloplasmin • Ceruloplasmin is an α2-globulin and carries 90% of the copper in plasma. • It binds six atoms of copper very tightly and copper is not readly exchangeable. • Albumin carries the other ~10% of plasma copper but binds copper less tightly than does ceruloplasmin and donates its copper to more easily to the tissues • Ceruloplasmin exhibits a copper-dependent ferro oxidase activity. Ceruloplasmin & Wilson Disease • Copper is carried to the liver bound to albumin taken up by liver cells, and part of it is excreted in the bile. • Copper is found in the liver attached to ceruloplasmin. • The amount of plasma ceruloplasmin is decreased in liver disease and Wilson disease (hepatolenticular degeneration). • A variety of mutations in gene encoding a copper-binding Ptype ATPase (ATP7B protein) were responsible the occurence of Wilson disease. This ATPase is responsible for the efflux of copper from liver cells into the bile. • Copper accumulates in liver and brain • Administration penicillamine which chelates copper is used to treatment Clinical Findings of Wilson Disease • A frequent clinical finding is the Kayser-Fleischer ring. This is a green or golden pigment ring around the cornea due to deposition of copper • Hemolytic anemia • Chronic liver disease • Neurologic syndromes Menkes Disease (kinky or steely hair disease), another condition involving abnormal copper metabolism • Menkes disease was mutations in the gene (ATP7A) for a copper binding P-type ATPase. • Copper is not mobilized from intestine • X-linked affects only male infants • Normal liver expresses very little of ATPases (No hepatic involvement) α2-Macroglobulin • α2-macroglobulin is a large plasma glycoprotein and transports 10% of zinc in plasma. • Monocytes, hepatocytes, astrocytes are the main sources of α2-macroglobulin • α2-macroglobulin contains internal cyclic thiol ester bond formed between a glutamine and a cysteine. This reactive bond is responsible for the biological actions of α2macroglobulin. • α2-macroglobulin binds many proteinases and considered as panproteinase inhibitor for cytokines Haptoglobin • Haptoglobin (Hp) is a plasma glycoprotein that binds extracorpuscular hemoglobin (Hb) in a tight noncovalent complex. • Hp exists in three polymorphic forms (Hp 1-1, Hp 2-1, Hp 2-2) • The Hb (MW:65kDa)- Hp (MW:90kDa) has a molecular mass of approximately 155 kDa. Free hemoglobin pases through the glomerulus of the kidney, enters the tubules, and tends to precipitate and leads to tubular damage (e.g. Incompatible blood transfusion). However, Hb-Hp complex is too large to pass through the glomerulus and prevent to loss free hemoglobin into the kidney • Low levels of Hp are found in patients with hemolytic anemia. Transferrin shuttles iron to sites where it is needed • • Free iron is extremely toxic because of its ability to form reactive oxygen species (ROS) In biological systems, iron is always bound to proteins in order to limit ROS formation. • In the plasma, iron is tightly bound β1-globulin called transferrin (Tf) which plays a central role in iron (Fe+++) transport. It is a glycoprotein and synthesized in liver. Tf transports iron in circulation to sites where iron required (e.g.the bone marrow) • Tf has two high-affinity binding sites for Fe+++ (holotransferrin (Tf-Fe). The concentration of Tf in plasma is approximately 300 μg/dL. This amount of can bind a total of approximately 300 μg of iron (per deciliter of plasma). This reprecents the total iron-binding capacity (TIBC) of plasma • Glycosylation of transferrin is impaired in congenital disorders of glycosylation and chronic alcoholism Ferritin stores iron in cells • Ferritin stores excess iron in various tissues • It is composed of 24 subunits which surround 3000-4500 ferric atoms. • Plasma ferritin levels are considered to be an indicator of body iron stores. Hemopexin • Certain other plasma proteins bind heme but not hemoglobin.Hemopexin is a β1-globulin that binds free heme. • Albumin binds some metheme (ferric heme) to form methalbumin which then transfers the metheme to hemopexin. Three major components of the immune system • B lymphocytes: B lymphocytes are mainly derived from bone marrow cells. They are responsible for the synthesis of circulating humoral antibodies, also known as immunoglobulins. • T lymphocytes: The T lymphocytes are of thymic origin. These cells are involved in cell-mediated immunological process such as graft rejection, hypersensitivity reactions, and defense against malignant cells and many viruses • The innate immune system defends against the infection in a nonspecific manner and unlike B and T cells is not adaptive. It contains a variety of cells such as phagocytes, neutrophils, natural killer cells and others. Blood components are used for transfusions Levels of plasma proteins are affected by many diseases • Acute-phase reactants whose levels change within 1 or 2 days after acute trauma or surgery, and especially during infections and inflammation. Acute-phase pattern : the typical acute inflammatory pattern is characterized by a decrease in the albumin fraction with an increase in both α1 and α2 fractions. Polyclonal gammopathy (seen in several conditions that enlist antibody production); a diffuse broad gamma band suggests a polyclonal increase in immunoglobulin fractions as seen in chronic inflammatory disorders, liver disease, and infectious disorders Amyloidosis Occurs by the Deposition of Protein fragments in various tissues • Amyloidosis is the accumulation of various insoluble fibrillar proteins between cells of tissues to an extent that affects function. • The accumulation is generally due to either increased production of certain proteins or accumulation of mutated forms of other proteins (deposition of amyloid fibrils). Fibrils possess a β-pleated sheet structure Primary & Secondary Amyloidosis • Primary amyloidosis a usually due to monoclonal plasma cell disorder in which the protein that accumulates is a fragment of light chain of an immunglobulin. • Secondary amyloidosis usually occurs secondary to chronic infections or cancer and is due to accumulation of degredation products of serum amyloid A (SAA). SAA production in liver is stimulated by cytokins. Classification of Amyloidosis Type Protein implicated Primary Principally light chains of immunoglobulins Secondary Serum amyloid A (SAA) Familial Mutated forms of Transthyretin, apolipoprotein A1, Cystatin C, fibrinogen Alzheimer disease Amyloid β-peptide Dialysis-related β2-microglobulin All immunoglobulins contain a minimum of two light & two heavy chains • • Immunglobulins contain a minimum two identical light (L) chains and two identical heavy (H) chains, held together as a tetramer (L2H2) by disulfide bonds . Both tips of Y shaped immunoglobulin molecules have antigen binding affinity Each chain can be divided into specific domains from amino terminal to carboxy terminal (VL,CL; VH,CH1,CH2,CH3). Regions between the CH1,CH2 domains is named as hinge region. Hinge region confers the flexibility Digestion of a hinge region of immunoglobulin by the enzyme papain produces two antigen-binding fragments (Fab) and one crystallizable fragments. Since there are two Fab regions, IgG molecules bind two molecules of antigen (divalent). The site on the antigen to which an antibody binds is termed an antigenic determinant or epitope. Fc and hinge regions differ in the different class of immunoglobulins All light chains are either kappa or lambda in type • There are two general type of light chains kappa (κ) and lambda (λ). • They can be distinguished on the basis of structural differences in their constant light regions • A given immunglobulin molecule always contains two kappa or to lambda light chains. Never a mixture of kappa and lambda. • In humans the kappa chains are more frequent than lambda chains in immunglobulin molecules Some important properties of immunoglobulins • The five types of heavy chain (γ, α, μ, δ, ε) determine immunoglobulin class. They can be distinguished by differences in their CH regions • The constant regions determine class-specific effector functions (e.g. Complement fixation, transplacental passage) • Both light & heavy chains are products of multiple genes. • Antibody difference depends on gene rearrangements by recombination • Class (ısotype) switching occurs during immune response (e.g. IgM normally precede molecules of the IgG class. Main antibody in the secondary response. Opsonizes the bacteria, making them easier to phagocytose. Fixes complement which enhances bacterial killing. Neutralizes bacterial toxins and viruses. Crosses the placenta Secretory IgA prevents attachment of bacteria and viruses to mucous membranes. Does not fix complement Produces in the primary response to an antigen. Fixes complement. Does not cross the placenta. Antigen receptors on the surface of B cells Found on the surface of B cells where it acts as a receptor for antigen Mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure of allergen (antigen) Does not fix complement. Main host defense against helmintic infections. Monoclonal gammopathies are neoplastic diseases of plasma cells • Abnormal proliferation of a single plasma cell leads to overproduction of a single antibody. • The resulting sharp peak seen on plasma protein electrophoresis is called a paraprotein, and the disorder is called a monoclonal gammopathy. • The evaluation of monoclonal gammopathies is one of the most common indications for plasma protein electrophoresis. • Paraproteins are common in the geriatric age group. They are diagnosed as benign monoclonal gammopathy (MGUS) as long as no signs of malignant disease are present. Multiple myeloma Multiple myeloma is a malignant disease associated with an overproduced IgG, IgA, or IgD antibody. In some patients, the malignant cells overproduce not a complete immunoglobulin but loose κ or λ light chains that eventually are excreted in the urine. These overproduced light chains are known as Bence Jones protein. The malignant plasma cells thrive in the bone marrow, where they cause bone pain and abnormal fractures. Waldenstrom macroglobulinemia • Waldenstrom macroglobulinemia is a malignant disease with overproduction of an IgM antibody. • Because of its high MW of 900,000 D, this IgM antibody causes a dangerous increase of blood viscosity.