Protein electrophoresis is a method for analyzing the proteins in a fluid or an extract. The electrophoresis may be performed in a small volume of sample by a number of alternative ways with or without any supporting media, such as SDS polyacrylamide gel electrophoresis (in short gel electrophoresis, PAGE, or SDS-electrophoresis, free flow electrophoresis, electrofocusing, isotachophoresis, affinity electrophoresis, immunoelectrophoresis, counter electrophoresis, and capillary electrophoresis).
Serum protein electrophoresis is a laboratory examination that is commonly used to identify patients with multiple myeloma and other disorders of serum protein. Many subspecialists include serum protein electrophoresis screening in the initial evaluation for numerous clinical conditions. Sometimes, however, the result of this examination can be confusing or difficult to interpret.
Electrophoresis refers to the migration of charged solutes or particles in a liquid medium under the influence of an electrical field. The first electrophoretic method used in the study of proteins was the free solution or moving boundary method devised by Tiselius in 1937. This technique is still used in research for measurement of electrophoretic mobility and for the study of protein-protein interaction but is not used in clinical laboratories for routine work. In this technique, a complex apparatus is needed, the technique is difficult, and sample of approximately 0.5 mL is required.
The term, zone electrophoresis refers to the migration of charged macromolecules in a porous supporting medium, such as cellulose paper, cellulose acetate sheet, or agarose gel film. Zone electrophoresis generates an electrophoretogram, a display of protein zones, each one sharply separated from neighboring zones on the electrophoretic support material. Solutes analyzed by this technique in clinical chemistry are mainly macromolecular in size and colloidal in nature. They include proteins in serum, cerebrospinal fluid and other biological fluids, as well as in erythrocytes and tissues.
Chemical species carrying an electric charge by virtue of ionization will move either to the cathode or to the anode in an electrophoresis system, depending on the kind of charge on the molecules. In a solution more acidie than the isoelectric point (pI) of the solute, an ampholyte (a solute molecule that can be either positively or negatively charged, also called a zwitterion) takes on a positive charge by binding protons and migrates toward the cathode (i.e. the negatively charged electrode). Conversely, the ampholyte that has lost protons is in the anionic form and migrates toward the anode (positive electrode) where the protein R is shown with terminal amino (–NH2) and carboxyl (–COOH) groups. Since proteins contain many ionizable groups, this equation should be seen as a simplification. The rate of migration is dependent on such factors as (i) net electrical charge of the molecule, (ii) size and shape of the molecule, (iii) strength of the electrical field, (iv) properties of the supporting medium, and (v) temperature of operation. The equation expressing electrophoretic mobility is a general formula that derives from two formulas, one describing the forward motion toward the electrode of charge opposite to that on the molecule and the other describing the retarding force due to frictional resistance:
μ = electrophoretic mobility, cm/(V)(s)
Q = the net charge on the molecule
K = a constant
r = the ionic radius of the migrating particle
η = the viscosity of the solution in which migration is occurring
Clearly, electrophoretic mobility is directly proportional to the net charge, as the protein migrates under the influence of an applied electric current and inversely proportional to the size of the protein ion and the viscosity of the medium (electrophoretic buffer).
This book provides a comprehensive review of serum protein electrophoresis, including a discussion of how the examination is performed, what it measures, and when it is indicated. It provides a simple guide to result interpretation and suggestions on follow-up of abnormal results.
COMPONENTS OF SERUM PROTEIN ELECTROPHORESIS
The pattern of serum protein electrophoresis results depends on the fractions of two major types of proteins—albumin and globulin. Albumin, the major protein component of serum, is produced by the liver under normal physiologic condition. Globulins comprise a much smaller fraction of the total serum protein content. The subsets of these proteins and their relative quantity are the primary focus of the interpretation of serum protein electrophoresis. Albumin, the largest peak, lies closest to the positive electrode. The next five components (globulins) are labeled alpha 1 (α1), alpha 2 (α2), beta 1 (β1), beta 2 (β2), and gamma (γ). The peaks for these components lie toward the negative electrode, with the γ peak being closest to that electrode (Figs 1 and 2).
Prealbumin is the fraction migrating faster than albumin toward the anode. It is same as transthyretin (TTR). Mutations in transthyretin are associated with hereditary amyloidosis. True prealbumin is generally below the limit of detection in serum protein electrophoresis but may be detected in electrophoresis of concentrated cerebrospinal fluid (CSF).
Albumin represents the largest protein component of human serum that is water soluble, is moderately soluble in concentrated salt solutions, and experiences heat denaturation.
Albumins are commonly found in blood plasma and are unique from other blood proteins, as they are not glycosylated. Substances containing albumin, such as egg white, are called albuminoids.
Albumin is the main protein of plasma. It binds water, cations (such as Ca2+, Na+ and K+), fatty acids, hormones, bilirubin, thyroxine (T4) and drugs (including barbiturates). Its main function is to regulate the colloidal osmotic pressure of blood. a- fetoprotein (α-fetoglobulin) is a fetal plasma protein that binds various cations, fatty acids, and bilirubin. Vitamin D-binding protein binds to vitamin D and its metabolites, as well as to fatty acids. The biological role of afamin (α-albumin) has not yet been characterized.
Albumin comprises three homologous domains that assemble to form a heart-shaped molecule. Each domain is a product of two subdomains that possess common structural motifs. The principal regions of ligand binding to human serum albumin are located in hydrophobic cavities in subdomains IIA and IIIA that exhibit similar chemistry. Structurally, the serum albumins are similar, each domain containing five or six internal disulfide bonds as shown in Figure 3.
Serum albumins are important in regulating blood volume by maintaining the oncotic pressure (also known as colloid osmotic pressure) of the blood compartment. They also serve as carriers for molecules of low water solubility this way isolating their hydrophobic nature, including lipid soluble hormones, bile salts, unconjugated bilirubin, free fatty acids (apoprotein), calcium, ions (transferrin) and some drugs like warfarin, phenobutazone, clofibrate and phenytoin. For this reason, it is sometimes referred as a molecular “taxi”. Competition between drugs for albumin binding sites may cause drug interaction by increasing the free fraction of one of the drugs.
Normal range of human serum albumin in adults is 3.5–5 g/dL. For children less than three years of age, the normal range is broader, 2.9–5.5 g/dL.
A decreased albumin level may be indicative of liver failure or diseases such as cirrhosis or chronic hepatitis. Hypoalbuminemia can also present as part of the nephrotic syndrome, in which protein is lost in the urine, because of kidney damage. Low albumin levels can be an indicator of chronic malnutrition or protein losing enteropathy, hormone therapy, and pregnancy. Burns also may result in a low albumin level.
Hypoalbuminemia may cause generalized edema (swelling) via a decrease in oncotic pressure.
Levels below 3.5 g/dL are generally considered low. At the time of hospital admission, 20% of patients have hypoalbuminemia. Serum albumin level is an important prognostic indicator. Among hospitalized patients, lower serum albumin levels correlate with an increased risk of morbidity and mortality.
Absence of albumin, known as analbuminemia, is a benign condition. It is a genetically inherited metabolic defect characterized by an impaired synthesis of serum albumin.
Levels of albumin are increased (hyperalbuminemia) in patients with a relative reduction in serum water (e.g. dehydration). In some cases of retinol (v itamin A) deficiency of the albumin level can be elevated to high normal values (e.g. 4.9 g/dL). This is because retinol causes cells to swell with water (this is also the reason because excess vitamin A is toxic).
Usually a single band is seen, individuals may produce bisalbuminemia (alloalbminemia), a condition in which two types of albumin exist in an individual. It is considered as an autosomal dominant condition of having serum albumin of a variant type, which differs in mobility on electrophoresis and affects people who are heterozygous or homozygous for one of the alleles for variant albumin types.
Albumin Alpha 1 Interzone
Even staining in this zone is due to alpha 1 (α1) lipoprotein [high density lipoprotein (HDL)]. Decrease occurs in severe inflammation, acute hepatitis and cirrhosis. Also, nephrotic syndrome can lead to a decrease in albumin level due to its loss in the urine through a damaged leaky glomerulus. An increase appears in severe alcoholics and in women during pregnancy and in puberty.
The high levels of alpha fetoprotein (AFP) that may occur in hepatocellular carcinoma may result in a sharp band between the albumin and the α1 zone.
Moving toward the negative portion of the gel (i.e. the negative electrode), the next peaks involve the α1 and α2 components. The α1 protein fraction is comprised of α1 antitrypsin, thyroid binding globulin and transcortin. Malignancy and acute inflammation (resulting from acute phase reactants) can increase the α1 protein band. A decreased α1 protein band may occur because of α1 antitrypsin deficiency or decreased production of the globulin as a result of liver disease. Ceruloplasmin, α2 macroglobulin, and haptoglobin (Hp) contribute to the α2 protein band, and the α2 component is increased as an acute-phase reactant.
Alpha 1 (α1) Zone
Orosomucoid and antitrypsin migrate together but orosomucoid stains poorly so α1 antitrypsin constitutes most of the α1 band. α1 antitrypsin has an SG group and thiol compounds may be bound to the protein altering their mobility. A decreased band is seen in the deficiency state. It is decreased in the nephrotic syndrome, and absence could indicate possible α1 antitrypsin deficiency. This eventually leads to emphsysema from unregulated lung elastase breakdown by neutrophils in the lung tissue. The α1 fraction does not disappear in α1 antitrypsin deficiency; however, because other proteins, including α lipoprotein and α1 acid glycoprotein, also migrate there. As a positive acute phase reactant, α1 antitrypsin is increased in acute inflammation.
Alpha 1 (α1)Antitrypsin
α1 antitrypsin (α1AT) is a protease inhibitor belonging to the serpin superfamily. This was originally named “antitrypsin” because of its ability to covalently bind and irreversibly inactivate the enzyme trypsin in vitro. Trypsin, a type of peptidase, is a digestive enzyme active in the duodenum and elsewhere. The term α1 refers to the protein's behavior on protein electrophoresis. On electrophoresis, the protein component of the blood is separated by electric current. There are several clusters, the first being albumin, the second alpha (α), the third beta (β), and the fourth gamma (γ) (immunoglobulins). The nonalbumin proteins are referred to as globulins. The α region can be further divided into two subregions, termed α1 and α2. α 1 antitrypsin is the main protein of the α globulin 1 region. Another name used is α 1 proteinase inhibitor (α1 PI).
It is generally known as serum trypsin inhibitor. Alpha 1 antitrypsin is also referred to as α1 proteinase inhibitor because it inhibits a wide variety of proteases. It protects tissues from enzymes of inflammatory cells, especially neutrophil elastase and has a reference range in blood of 1.5–3.5 g/L (in US the reference range is generally expressed as mg/dL or micromoles), but the concentration can rise manyfold upon acute inflammation. In its absence, neutrophil elastase is free to break down elastin, which contributes to the elasticity of the lungs, resulting in respiratory complications such as emphysema, or chronic obstructive pulmonary disease (COPD) in adults and cirrhosis in adults or children.
α 1 antitrypsin is a 52 k D serpin, and in medicine, it is considered the most prominent serpin. The terms α1 antitrypsin and protease inhibitor (PI) are often used interchangeably. Most serpins inactivate enzymes by binding to them covalently, requiring very high levels to perform their function. In the acute phase reaction, a further elevation is required to “limit” the damage caused by activated neutrophil granulocytes and their enzyme elastase that breaks down the connective tissue fiber elastin. Like all serine protease inhibitors, α1AT has a characteristic secondary structure of β sheets and α helices. Mutations in these areas can lead to nonfunctional proteins that can polymerize and accumulate in the liver (infantile hepatic cirrhosis).
Disorders of this protein include α1 antitrypsin deficiency, a hereditary disorder in which a deficiency of α1 antitrypsin leads to a chronic uninhibited tissular breakdown. This causes the degradation especially of lung tissue and eventually leads to characteristic manifestations of pulmonary emphysema. Evidence has shown that cigarette smoke can lead to oxidation of methionine 358 of α1 antitrypsin, a residue essential for binding elastase. This is thought to be one of the primary mechanisms by which cigarette smoking (or second hand smoke) can lead to emphysema. Because α1AT is expressed in the liver, certain mutations in the gene encoding the protein can cause misfolding and impaired secretion, which can lead to liver cirrhosis.
Alpha 1 (α1) and Alpha 2 (α2) Interzone
Two faint bands may be seen representing α1 antichymotrypsin and vitamin D binding protein. These bands fuse and intensify in early inflammation, because of an increase in α1 antichymotrypsin, an acute phase protein.
Alpha 2 Zone
This zone consists principally of α2 macroglobulin and Hp. There are typically low levels in hemolytic anemia [Hp is a suicide molecule, which binds with free hemoglobin (Hb) released from red blood cells and these complexes are rapidly removed by phagocytes ]. Haptoglobin is raised as part of the acute phase response, resulting in a typical elevation in the α2 zone during inflammation. A normal α2 and an elevated α1 zone is a typical pattern in hepatic metastasis and cirrhosis.
Haptoglobin/hemoglobin complexes migrate more cathodally than Hp as seen in the α2-β interzone. This is typically seen as a broadening of the α2 zone. Haptoglobin is a protein that in humans is encoded by the HP gene. In blood plasma, Hp binds free Hb released from erythrocytes with high affinity and thereby inhibits its oxidative activity. The Hp Hb complex will then be removed by the reticuloendothelial system (mostly the spleen). In clinical settings, the haptoglobulin assay is used to screen for and monitor intravascular hemolytic anemia. In intravascular hemolysis free Hb will be released into circulation and hence Hp will bind the Hb. This causes a decline in Hp levels. Conversely, in extravascular hemolysis, the reticuloendothelial system, especially splenic monocytes, phagocytose the erythrocytes and Hb is not released into circulation; therefore, serum Hp levels are normal. Haptoglobin is produced mostly by hepatocytes but also by other tissues, e.g. skin, lung and kidney. In addition, the haptoglobin gene is expressed in murine and human adipose tissue and had been shown to be expressed in adipose tissue of cattle as well. Haptoglobin, in its simplest form, consists of two α and two β chains, connected by disulfide bridges. The chains originate from a common precursor protein, which is proteolytically cleaved during protein synthesis. Haptoglobin exists in two allelic forms in the human population, so called Hp1 and Hp2, the latter have arisen because of the partial duplication of Hp1 gene. Three genotypes of Hp, therefore, are found in humans: Hp1-1, Hp2-1 and Hp2-2. Haptoglobin of different genotypes has been shown to bind Hb with different affinities, with Hp2-2 being the weakest binder.
Mutations in this gene and/or its regulatory regions cause haptoglobinemia or hypohaptoglobinemia. This gene has also been linked to diabetic nephropathy, the incidence of coronary artery disease in type 1 diabetes mellitus, Crohn's disease, inflammatory disease behavior, primary sclerosing cholangitis, susceptibility to idiopathic Parkinson's disease and a reduced incidence of Plasmodium falciparum malaria.
Since the reticuloendothelial system will remove the haptoglobin-hemoglobin complex from the body, haptoglobin levels will be decreased in hemolytic anemia. In the process of binding Hb, Hp sequesters the iron within Hb, preventing iron utilizing bacteria in benefiting from hemolysis. It is theorized that, because of this, haptoglobin has evolved into an acute phase protein. Haptoglobin has a protective influence on the hemolytic kidney. Some studies associate certain Hp phenotypes with the risk of developing schizophrenia.
Haptoglobin is ordered whenever a patient exhibits symptoms of anemia, such as pallor, fatigue, or shortness of breath, along with physical signs of hemolysis, such as jaundice or dark-colored urine. The test is also commonly ordered as a hemolytic anemia battery, that also includes a reticulocyte count and a peripheral blood smear. It can also be ordered along with a direct antiglobulin test when a patient is suspected of having a transfusion reaction or symptoms of autoimmune hemolytic anemia. Also, it may be ordered in conjunction with a bilirubin.
A decrease in Hp can support a diagnosis of hemolytic anemia, especially when correlated with a decreased red blood cell count, haemoglobin and hematocrit, and also an increased reticulocyte count.
If the reticulocyte count is increased, but the Hp level is normal, this may indicate that cellular destruction is occurring in the spleen and liver, which may indicate a drug-induced hemolysis or a red-cell dysplasia. The spleen and liver recognize an error in the red cells (either drug coating the red cell membrane or a dysfunctional red cell membrane) and destroy the cell. This type of destruction does not release Hb into the peripheral blood, so the Hp cannot bind to it. Thus, the Hp will stay normal, if the hemolysis is not severe. In severe extravascular hemolysis, Hp levels can also be low, when large amount of hemoglobin in the reticuloendothelial system leads to transfer of free Hb into plasma. If there are symptoms of anemia, and both the reticulocyte count and the Hp level are normal, the anemia is most likely not due to hemolysis, but instead some other error in cellular production, such as aplastic anemia. Haptoglobin levels that are decreased but do not accompany signs of anemia may indicate liver damage, as the liver is not producing enough Hp to begin with. As Hp is indeed an acute phase protein, any inflammatory process (infection, extreme stress, burns, major crush injury, allergy, etc.) may increase the levels of plasma Hp.
Alpha 2 (α2) Macroglobulin
α2 macroglobulin is a large plasma protein found in the blood. It is produced by the liver and is a major component of the α2 band in protein electrophoresis. It is the largest major nonimmunoglobulin protein in plasma. The α2 macroglobulin molecule is synthesized mainly in liver, but also locally by macrophages, fibroblasts, and adrenocortical cells.
α2 macroglobulin acts as an antiprotease and is able to inactivate an enormous variety of proteinases. It functions as an inhibitor of fibrinolysis by inhibiting plasmin and kallikrein. It functions as an inhibitor of coagulation by inhibiting thrombin. α2 macroglobulin may act as a carrier protein because it also binds to numerous growth factors and cytokines, such as platelet-derived growth factor, basic fibroblast growth factor, tumor growth factor β (TGF β), insulin, and Interleukin 1 β (IL 1β).
No specific deficiency with associated disease has been recognized, and no disease state is attributed to low concentrations of α2 macroglobulin. The concentration of α2 macroglobulin raises 10-fold or more in the nephrotic syndrome when other lower molecular weight proteins are lost in the urine. The loss of α2 macroglobulin into urine is prevented by its large size. The net result is that α2 macroglobulin reaches serum levels equal to or greater than those of albumin in the nephrotic syndrome, which has the effect of maintaining oncotic pressure.
Human α2 macroglobulin is composed of four identical subunits bound together by S-S bonds. In addition to tetrameric forms of α2 macroglobulin, dimeric and more recently monomeric aM protease inhibitors have been identified. Each monomer of human α2 macroglobulin is composed of many functional domains, including macroglobulin domains, a thiol ester containing domain and a receptor binding domain.
α 2 macroglobulin is able to inactivate an enormous variety of proteinases (including serine, cysteine, aspartic and metalloproteinases). It functions as an inhibitor of fibrinolysis by inhibiting plasmin and kallikrein (Fig. 4). It functions as an inhibitor of coagulation by inhibiting thrombin.
α 2 macroglobulin may be elevated in children and the elderly. This is seen as a sharp front to the α2 band. α2 macroglobin is markedly raised (10-fold increase or greater) in association with glomerular protein loss, as in nephrotic syndrome. Due to its large size, α2 macroglobulin cannot pass through glomeruli, while other lower molecular weight proteins are lost. Enhanced synthesis of α2 macroglobulin accounts for its absolute increase in nephrotic syndrome wherein the kidneys start to leak out some of the smaller blood proteins. Because of its size, α2 macroglobulin is retained in the bloodstream. Increased production of all proteins means α2 macroglobulin concentration increases. This increase has little adverse effect on the health, but is used as a diagnostic clue.
A common variant (29.5%) (polymorphism) of α2 macroglobulin leads to increased risk of Alzheimer's disease and mildly elevated early in the course of diabetic nephropathy.
Ceruloplasmin is the major copper -carrying protein in the blood and in addition plays a role in iron metabolism. It was first described in 1948. The molecular weight of human ceruloplasmin is reported to be 151 kD. Another protein, hephaestin, is noted for its homology to ceruloplasmin and also participates in iron and probably copper metabolism. Ceruloplasmin is an enzyme (EC 22.214.171.124) synthesized in the liver containing six atoms of copper in its structure. Ceruloplasmin carries about 70% of the total copper in human plasma, while albumin carries about 15%. The rest is accounted for by macroglobulins. Albumin may be confused at times to have a greater importance as a copper carrier, because it binds copper less tightly than ceruloplasmin. Ceruloplasmin exhibits a copper-dependent oxidase activity, which is associated with possible oxidation of Fe2+ (ferrous iron) into Fe3+ (ferric iron), therefore assisting in its transport in the plasma in association with transferrin that can carry iron only in the ferric state.
Copper availability does not affect the translation of the nascent protein. However, the apoenzyme without copper is unstable. Apoceruloplasmin is largely degraded intracellularly in the hepatocyte, and the small amount that is released has a short circulation half life of 5 hours as compared to the 5.5 days for the holo-ceruloplasmin.
Mutations in the ceruloplasmin gene (CP), which are very rare, can lead to the genetic disease aceruloplasminemia, characterized by hyperferritinemia with iron overload. In the brain, this iron overload may lead to characteristic neurologic signs and symptoms, such as cerebellar ataxia, progressive dementia, and extrapyramidal signs. Excess iron may also deposit in the liver, pancreas and retina, leading to cirrhosis, endocrine abnormalities and loss of vision, respectively.
Like any other plasma protein, levels drop in patients with hepatic disease because of reduced synthesizing capabilities and the low levels may indicates Wilson disease, Menkes disease, overdose of v itamin C, c opper deficiency, a ceruloplasminemia. On the other hand, greater levels than normal may indicate pregnancy, oral contraceptive pill, lymphoma, rheumatoid arthritis, angina, Alzheimer's disease, schizophrenia, obsessive-compulsive disorder, and acute and chronic inflammation.
Alpha 2-(α2) Beta Interzone
Cold insoluble globulin forms a band here which is not seen in plasma because it is precipitated by heparin. There are low levels in inflammation and high levels in pregnancy. β lipoprotein forms an irregular crenated band in this zone. High levels are seen in type II hypercholesterolemia, hypertriglyceridemia, and in the nephrotic syndrome.
The β fraction has two peaks labeled β1 and β2. b1 is composed mostly of transferrin, and β2 contains β lipoprotein. Immunoglobulin A (IgA), IgM, and sometimes IgG, along with complement proteins, also can be identified in the β fraction. Transferrin and β lipoprotein [low-density lipoprotein (LDL )] comprises β1. Increased β1 protein due to the increased level of free transferrin is typical of iron deficiency anemia, pregnancy, and estrogen therapy. Increased β1 protein due to LDL elevation occurs in hypercholesterolemia.
β2 comprises c omplement protein 3 (C3). It is raised in the acute phase response. Depression of C3 occurs in autoimmune disorders as the complement system is activated and the C3 becomes bound to immune complexes and removed from plasma. Fibrinogen, a β2 protein, is found in normal plasma but also absent from normal serum. Occasionally, blood drawn from heparinized patients, does not fully clot, resulting in a visible fibrinogen band between the β and γ globulins.
Complement Component 3
Complement component 3 is a protein of the immune system. It plays a central role in the complement system and contributes to innate immunity. In humans, it is encoded on chromosome 19 by a gene called C3. C3 plays a central role in the activation of complement system. Its activation is required for both classical and alternative complement activation pathways. People with C3 deficiency are susceptible to bacterial infection.
One form of C3 convertase, also known as C4b2a, is formed by a heterodimer of activated forms of C4 and C2. It catalyzes the proteolytic cleavage of C3 into C3a and C3b, generated during activation through the classical pathway as well as the mannan binding lectin pathway. C3a is an anaphylotoxin and the precursor of some cytokines, such as ASP and C3b, serves as an opsonizing agent. Factor I can cleave C3b into C3c and C3d in which the latter plays a role in enhancing B cell responses. In the alternative complement pathway, C3 is cleaved by C3bBb, another form of C3 convertase composed of activated forms of C3 (C3b) and factor B (Bb). Once C3 is activated to C3b, it exposes a reactive thioester that allows the peptide to covalently attach to any surface, which can provide a nucleophile, such as a primary amine or a hydroxyl group. Activated C3 can then interact with factor B. Factor B is then activated by factor D to form Bb. The resultant complex, C3bBb, is called the alternative pathway (AP) C3 convertase.
C3bBb is deactivated in steps. First, the proteolytic component of the convertase, Bb, is removed by complement regulatory proteins having decay accelerating factor (DAF) activity. Next, C3b is broken down progressively to first iC3b, then C3c + C3dg, and then finally C3d. Factor I is the protease that performs these cuts but it requires the help of another protein to supply what is termed cofactor activity. Several crystallographic structures of C3 have been determined and reveal that this protein contains 13 domains. Levels of C3 in the blood may be measured to support or refute a particular medical diagnosis. For example, low C3 levels are associated with some types of kidney disease, such as postinfectious glomerulonephritis and shunt nephritis.
Gamma (γ) Zone
Much of the clinical interest is focused on the γ region of the serum protein spectrum, because immunoglobulins migrate to this region. It should be noted that immunoglobulins often can be found throughout the electrophoretic spectrum. C reactive protein (CRP) is located in the area between the β and γ components. The immunoglobulins (IgA, IgM, IgG, IgE and IgD) are the only proteins present in the normal γ region, but note that immunoglobulins may be found in the α and β zones. If the γ zone shows an increase (or spike), the first step in interpreting the graph is to establish if the region is narrow or wide. If it is elevated it could be elevated in a single narrow “spike like” manner indicating monoclonal gammopathy or a broad «swell like» manner (wide) indicating polyclonal gammopathy.
A narrow spike indicates a monoclonal gammopathy, also known as an “M spike”. Typically, a monoclonal gammopathy is malignant or clonal in origin, myeloma being the most common cause of IgA and IgG spikes. Chronic lymphatic leukemia and lymphosarcoma are not uncommon and usually give rise to IgM paraproteins. Note that up to 8% of healthy geriatric patients may have a monoclonal spike. Waldenström macroglobulinemia (WM) IgM, monoclonal gammopathy of undetermined significance (MGUS), amyloidosis, plasma cell leukemia and solitary plasmacytomas also produce an M spike.
A “swell like” elevation in the γ zone indicates a polyclonal gammopathy, and this typically points to a nonmalignant condition (although is not exclusive to nonmalignant conditions). The most common causes of polyclonal hypergammaglobulinemia detected by electrophoresis are severe infection, chronic liver disease, rheumatoid arthritis, systemic lupus erythematosus and other connective tissue diseases. Lysozyme may be seen as a band cathodal to the slowest γ in myelomonocytic leukemia in which it is released from tumor cells. Fibrinogen from plasma samples will be seen in the fast γ region.
This is easily identifiable as a “slump” or decrease in the γ zone. It is normal in infants. It is found in patients with X-linked agammaglobulinemia. IgA deficiency occurs in 1:500 of the population, as is suggested by pallor in the γ zone.
Immunoglobulin A (IgA) antibodies are found in areas of the body, such as the nose, breathing passages, digestive tract, ears, eyes and vagina. IgA antibodies protect body surfaces that are exposed to outside foreign substances. This type of antibody is also found in saliva, tears, and blood. About 10–15% of the antibodies present in the body are IgA antibodies. A small number of people do not make IgA antibodies. It plays a critical role in mucosal immunity, more IgA is produced in mucosal linings than all other types of antibodies combined: between 3 g and 5 g are secreted into the intestinal lumen each day. This accumulates to 75% of the total immunoglobulin produced in the entire body. IgA has two subclasses (IgA1 and IgA2) and can exist in a dimeric form called secretory IgA (sIgA).
In its secretory form (Fig. 5), IgA is the main immunoglobulin found in mucous secretions, including tears, saliva, colostrum, and secretions from the genitourinary tract, gastrointestinal tract, prostate, and respiratory epithelium. It is also found in small amounts in blood. The secretory component of sIgA protects the immunoglobulin from being degraded by proteolytic enzymes; thus, sIgA can survive in the harsh gastrointestinal tract environment and provide protection against microbes, which multiply in body secretions.
IgA is a poor activator of the complement system and opsonies only weakly. Its heavy chains are of the type α. This immunoglobulin has the most anodal mobility and migrates in the region between the β and γ zones also causing a β/γ fusion in patients with cirrhosis, respiratory infection, skin disease or rheumatoid arthritis (increased IgA).
Immunoglobulin A exists in two isotypes : IgA1 and IgA2. While IgA1 predominates in serum (~80%), IgA2 percentages are higher in secretions than in serum (~35% in secretions). The ratio of IgA1 and IgA2 secreting cells varies in the different lymphoid tissues of the human body:
- IgA1 is the predominant IgA subclass found in serum. Most lymphoid tissues have a predominance of IgA producing cells
- In IgA2, the heavy and light chains are not linked with disulfide, but with noncovalent bonds. In secretory lymphoid tissues [e.g. gut associated lymphoid tissue (GALT)], the share of IgA2 production is larger than in the nonsecretory lymphoid organs (e.g. spleen, peripheral lymph nodes).
Both IgA1 and IgA2 have been found in external secretions like colostrum, maternal milk, tears and saliva, where IgA2 is more prominent than in the blood. Polysaccharide antigens tend to induce more IgA2 than protein antigens.
It is also possible to distinguish forms of IgA based upon their location serum IgA vs secretory IgA.
In secretory IgA, the form found in secretions, polymers of two to four IgA monomers are linked by two additional chains; as such sIgA holds a molecular weight of 385,000. One of these is the joining chain (J chain), which is a polypeptide of molecular mass 15 kD, rich with cysteine and structurally completely different from other immunoglobulin chains. This chain is formed in the IgA secreting cells.
The oligomeric forms of IgA in the external (mucosal) secretions also contain a polypeptide of a much larger molecular mass (70 kD) called the secretory component, which is produced by epithelial cells. This molecule originates from the poly Ig receptor (130 kD) that is responsible for the uptake and transcellular transport of oligomeric (but not monomeric) IgA across the epithelial cells and into secretions, such as tears, saliva, sweat, and gut fluid.
The high prevalence of IgA in mucosal areas is a result of cooperation between plasma cells that produce polymeric IgA (pIgA) and mucosal epithelial cells that express an immunoglobulin receptor called the polymeric Ig receptor (pIgR). Polymeric IgA is released from the nearby activated plasma cells and binds to pIgR. This results in transportation of IgA across mucosal epithelial cells and its cleavage from pIgR for release into external secretions.
In the blood, IgA interacts with an Fc receptor called FcαRI (or CD89), which is expressed on immune effector cells, to initiate inflammatory reactions. Ligation of FcαRI by IgA containing immune complexes cause antibody dependent cell-mediated cytotoxicity (ADCC), degranulation of eosinophils and basophils, phagocytosis by monocytes, macrophages and neutrophils, and triggering of respiratory burst activity by polymorph nuclear leukocytes.
Polymeric IgA (mainly the secretory dimer) is produced by plasma cells in the lamina propria, adjacent to mucosal surfaces. It binds to the polymeric immunoglobulin receptor on the basolateral surface of epithelial cells and is taken up into the cell via endocytosis. The receptor IgA complex passes through the cellular compartments before being secreted on the luminal surface of the epithelial cells, still attached to the receptor. Proteolysis of the receptor occurs, and the dimeric IgA molecule, along with a portion of the receptor known as the secretory component, are free to diffuse throughout the lumen. In the gut, it can bind to the mucous layer on top of the epithelial cells to form a barrier capable of neutralizing threats before they reach the cells. Decreased or absent IgA, termed selective IgA deficiency, can be a clinically significant immunodeficiency.
Neisseria gonorrhoeae (that causes gonorrhea), Streptococcus pneumoniae, and Haemophilus influenzae type B all release a protease which, destroys IgA.
Immunoglobulin A nephropathy is caused by IgA deposits in the kidneys. It is not yet known why IgA deposits occur in this chronic disease. Some theories suggest an abnormality of the immune system results in these deposits.
Celiac disease involves IgA pathology due to the presence of IgA antiendomysial antibodies.
The IgA fixes the complement by the alternative path: polyclonal increases of IgA can be present in chronic inflammatory panels of the respiratory tract and the gastroenteric tract, including the liver. About one-fourth of the patients with IgA deficit have anti-IgA antibodies and are at risk for serious anaphylactic reactions in case of blood/plasma transfusions.
Increased IgA levels—chronic hepatopathy, cirrhosis, chronic respiratory infections, neoplasia of the final section of the intestine, gastroenteric apparatus diseases (Crohn's disease, ulcerative colitis, etc.), rheumatoid arthritis, ankylosing spondylitis, neuropathies, Wiskott Aldrich syndrome; IgA myeloma, MGUS (monoclonal).
Decreased IgA levels—infancy, selective deficiency of IgA (approximately 1/700), protide dispersion syndrome, macroglobulinemia, or non-IgA myeloma.
Immunoglobulin G (IgG) antibodies are found in all body fluids. They are the smallest but most common antibody (75–80%) of all the antibodies in the body. IgG antibodies are very important in fighting bacterial and viral infections. IgG antibodies are the only antibody that can cross the placenta in a pregnant woman to help protect her baby (fetus).
IgG is composed of four peptide chains two heavy chains γ and two light chains (Fig. 6). Each IgG has two antigen -binding sites. Other immunoglobulins may be described in terms of polymers with the IgG structure considered the monomer.
Ig G constitutes 75% of serum immunoglobulins in humans. IgG molecules are synthesized and secreted by plasma B cells. IgG antibodies are involved predominantly in the secondary immune response. The presence of specific IgG, in general, corresponds to maturation of the antibody response.
Human Immunoglobulin G Subclasses
Immunoglobulin G is the only isotype that can pass through the human placenta, thereby, providing protection to the fetus in utero. Along with IgA secreted in the breast milk, residual IgG absorbed through the placenta provides the neonate with humoral immunity before its own immune system develops. Colostrum contains a high percentage of IgG, especially bovine colostrum.
Immunoglobulin G can bind to many kinds of pathogens, (for example, viruses, bacteria and fungi) and protects the body against them by agglutination and immobilization, complement activation (classical pathway), opsonization for phagocytosis, and neutralization of their toxins. It also plays an important role in ADCC and intracellular antibody mediated proteolysis, in which it binds to TRIM21 (the receptor with greatest affinity to IgG in humans) in order to direct marked virions to the proteasome in the cytosol, IgG is also associated with type II and type III hypersensitivity.
Immunoglobulin G antibodies are large molecules of about 150 k D composed of four peptide chains. It contains two identical class γ heavy chains of about 50 kD and two identical light chains of about 25 kD, thus a tetrameric quaternary structure. The two heavy chains are linked to each other and to a light chain each by disulfide bonds. The resulting tetramer has two identical halves, which together form a Y like shape. Each end of the fork contains an identical antigen binding site. The Fc regions of IgGs bear a highly conserved N glycosylation site. The N glycans attached to this site are predominantly core fucosylated diantennary structures of the complex type. In addition, small amounts of these N glycans also bear bisecting GlcNAc and α 2, 6 linked sialic acid residues.
Immunoglobulin G has a low frequency wave number of 28 cm−1 in the Raman spectra. This emission has been assigned to the breathing motion in the β barrel of nine β strands in its VH domain. The dynamic mechanism of the “chelate effect” and “trigger effect” of IgG has been analyzed from the angle of low frequency resonance among the 12 β barrels of an IgG molecule.
There are four IgG subclasses (IgG1, IgG2, IgG3, and IgG4) in humans, named in order of their abundance in serum (IgG1 being the most abundant).
Immunoglobulin G affinity to Fc receptors on phagocytic cells is specific to individual species from which the antibody comes as well as the class. The structure of the hinge regions gives each of the four IgG classes its unique biological profile. Even though there is about 95% similarity between their Fc regions, the structure of the hinge regions is relatively different.
In a model of autoantibody mediated anemia using IgG isotype switch variants of an antierythrocytes autoantibody, it was found that IgG2a was superior to IgG1 in activating complement. Moreover, it was found that the IgG2a isotype was able to interact very efficiently with Fc-γ receptors (FcγR). As a result, 20 times higher doses of IgG1 in relationship to IgG2a autoantibodies, were required to induce autoantibody mediated pathology.
The IgG represent from 75 to 80% of the total immunoglobulins, fix the complement through the classic path (IgG1, IgG2, IgG3) and alternative path (IgG4) and constitute the specific immune response. They are the only immunoglobulin, which passes the placenta. Deficiency of IgG is associated with repeated serious infections.
Increased IgG levels—autoimmune diseases [Lambert-Eaton syndrome (LES), AR, systemic sclerosis, Sjogren syndrome, etc.), chronic hepatopathy, recurrent or chronic infections, sarcoidosis, intrauterine contraceptive devices.
Oligoclonal: lymphoid and nonlymphoidal neoplasia, viral infections, autoimmune diseases
Monoclonal: IgG myeloma, MGUS, lymphomas
Decreased IgG levels—infancy, pregnancy (moderate), hypogammaglobulinemia, agammaglobulinemia, nephrotic syndrome, non-IgG myelomas.
Immunoglobulin M antibodies are the largest antibody. They are found in blood and lymph fluid and are the first type of antibody made in response to an infection. They also cause other immune system cells to destroy foreign substances. IgM antibodies are about 5–10% of all the antibodies in the body.
Immunoglobulin M is produced by B cells ; it is by far the physically largest antibody in the human circulatory system. It is the first antibody to appear in response to initial exposure to antigen. The spleen is the major site of specific IgM production. IgM forms polymers where multiple immunoglobulins are covalently linked together with disulfide bonds, mostly as a pentamer but also as a hexamer.
IgM has a molecular mass of approximately 900 k D (in its pentamer form). Because each monomer has two antigen binding sites, a pentameric IgM has 10 binding sites (Fig. 7). Typically, however, IgM cannot bind 10 antigens at the same time because the large size of most antigens hinders binding to nearby sites.
The J chain is found in pentameric IgM but not in the hexameric form, perhaps due to space constraints in the hexameric complex. Pentameric IgM can also be made in the absence of J chain. At present, it is still uncertain what fraction of normal pentamer contains J chain, and to this extent, it is also uncertain whether a J chain containing pentamer contains one or more than one J chain. Because IgM is a large molecule, it cannot diffuse well and is found in the interstitium only in very low quantities. IgM is primarily found in serum ; however, because of the J chain, it is also important as a secretory immunoglobulin. Due to its polymeric nature, IgM possesses high avidity and is particularly effective at complement activation. By itself, IgM is an ineffective opsonin ; however, it contributes greatly to opsonization by activating complement and causing C3b to bind to the antigen. In germline cells, the gene segment encoding the μ constant region of the heavy chain is positioned first among other constant region gene segments. For this reason, IgM is the first immunoglobulin expressed by mature B cells. It is also the first immunoglobulin expressed in the fetus (around 20 weeks) and also phylogenetically the earliest antibody to develop.
Immunoglobulin M antibodies appear early in the course of an infection and usually reappear, to a lesser extent, after further exposure. IgM antibodies do not pass across the human placenta (only isotype IgG).
These two biological properties of IgM make it useful in the diagnosis of infectious diseases. Demonstrating IgM antibodies in a patient's serum indicates recent infection, or in a neonate's serum indicates intrauterine infection (e.g. congenital rubella).
The IgM fixes the complement through the classic path and constitutes the primary response to the infections. Viral IgM specific antibodies in newborns are due to congenital infections (they do not pass through the placenta filter).
Increased IgM levels: Viral infections, parasitosis, chronic hepatopathy, primitive biliary cirrhosis, primary sclerosing cholangitis.
Monoclonal: Waldenstrom macroglobulinemia, malignant lymphoma, reticulosis, cryoglobulins, cryoagglutinins.
Decreased IgM levels: Infancy, immunodeficiency conditions (Wiskott-Aldrich syndrome), non-IgM myeloma.
Discovered by Gillett and Francis in 1930, it was initially thought that CRP might be a pathogenic secretion, as it was elevated in people with a variety of illnesses, including cancer, however, discovery of hepatic synthesis demonstrated that it was a native protein, CRP was so named because it was first discovered as a substance in the serum of patients with acute inflammation that reacted with the C (capsular) polysaccharide of P neumococcus.
C-reactive protein is a protein found in the blood, the levels of which rise in response to inflammation (i.e. C-reactive protein is an acute-phase protein). Its physiological role is to bind to phosphocholine expressed on the surface of dead or dying cells (and some types of bacteria) in order to activate the complement system via the C1Q complex.
C-reactive protein is synthesized by the liver in response to factors released by macrophages and fat cells (adipocytes). It is a member of the pentraxin family of proteins, and it is not related to C peptide or protein C. C-reactive protein was the first pattern recognition receptor (PRR) to be identified. The CRP gene is located on the first chromosome (1q21-q23). CRP is a 224 residue protein with a monomer molar mass of 25106 Da. The protein is an annular pentameric disk in shape and a member of the small pentraxins family.
The acute phase response develops in a wide range of acute and chronic inflammatory conditions like bacterial, viral or fungal infections; rheumatic and other inflammatory diseases; malignancy; and tissue injury or necrosis. These conditions cause release of IL 6 and other cytokines, which trigger the synthesis of CRP and fibrinogen by the liver. During the acute phase response, levels of CRP rapidly increase within 2 hours of acute insult, reaching a peak at 48 hours. With resolution of the acute phase response, CRP declines with a relatively short half-life of 18 hours. Measuring CRP level is a screen for infectious and inflammatory diseases. Rapid, marked increases in CRP occur with inflammation, infection, trauma and tissue necrosis, malignancies, and autoimmune disorders. Because there are a large number of disparate conditions, which can increase CRP production, an elevated CRP level does not diagnose a specific disease. An elevated CRP level can provide support for the presence of an inflammatory disease, such as rheumatoid arthritis, polymyalgia rheumatica, or giant cell arteritis.
The physiological role of CRP is to bind to phosphocholine expressed on the surface of dead or dying cells (and some types of bacteria) in order to activate the complement system. CRP binds to phosphocholine on microbes and damaged cells and enhances phagocytosis by macrophages. Thus, CRP participates in the clearance of necrotic and apoptotic cells.
CRP is a member of the class of acute phase reactants, as its levels rise dramatically during inflammatory processes occurring in the body. This increment is due to a rise in the plasma concentration of IL 6, which is produced predominantly by macrophages as well as adipocytes. CRP binds to phosphocholine on microbes. It is thought to assist in complement binding to foreign and damaged cells and enhances phagocytosis by macrophages (opsonin mediated phagocytosis), which express a receptor for CRP. It is also believed to play another important role in innate immunity as an early defense system against infections.
CRP rises up to 50,000-fold in acute inflammation, such as infection. It rises above normal limits within 6 hours and peaks at 48 hours. Its half-life is constant and therefore, its level is mainly determined by the rate of production (hence the severity of the precipitating cause).
CRP levels also tend not to be elevated in systemic lupus erythematosus (SLE) unless serositis or synovitis is present. Elevations of CRP in the absence of clinically significant inflammation can occur in renal failure. CRP level is an independent risk factor for atherosclerotic disease. Patients with high CRP concentrations are more likely to develop stroke, myocardial infarction, and severe peripheral vascular disease.
Recent research suggests that patients with elevated basal levels of CRP are at an increased risk of diabetes, hypertension, and cardiovascular disease. A study of over 700 nurses showed that those in the highest quartile of trans fat consumption had blood levels of CRP that were 73% higher than those in the lowest quartile. Although one group of researchers indicated that CRP may be only a moderate risk factor for cardiovascular disease, this study (known as the Reykjavik Study) was found to have some problems for this type of analysis related to the characteristics of the population studied, and there was an extremely long follow-up time, which may have attenuated the association between CRP and future outcomes. Others have shown that CRP can exacerbate ischemic necrosis in a complement -dependent fashion, and that CRP inhibition can be a safe and effective therapy for myocardial and cerebral infarcts ; so far, this has been demonstrated in animal models only.
CRP protein is used mainly as a marker of inflammation. Apart from liver failure, there are few known factors that interfere with CRP production. Measuring and charting CRP values can prove useful in determining disease progress or the effectiveness of treatments. Blood, usually collected in a serum separating tube, is analyzed in a medical laboratory or at the point of care. Various analytical methods are available for CRP determination, such as ELISA, immunoturbidimetry, rapid immunodiffusion and visual agglutination.
A high sensitivity CRP (hs CRP) test measures low levels of CRP using laser nephelometry. The test gives results in 25 minutes with sensitivity down to 0.04 mg/L. Normal concentration in healthy human serum is usually lower than 10 mg/L, slightly increasing with aging. Higher levels are found in late pregnant women, mild inflammation and viral infections (10–40 mg/L), active inflammation, and bacterial infection (40–200 mg/L); and severe bacterial infections and burns (> 200 mg/L).
CRP is a more sensitive and accurate reflection of the acute phase response than the erythrocyte sedimentation rate (ESR). The half-life of CRP is constant. Therefore, CRP level is mainly determined by the rate of production (and hence the severity of the precipitating cause). In the first 24 hours, ESR may be normal and CRP elevated. CRP returns to normal more quickly than ESR in response to therapy.
Arterial damage results from white blood cell invasion and inflammation within the wall. CRP is a general marker for inflammation and infection, so it can be used as a very rough proxy for heart disease risk. Since many things can cause elevated CRP, this is not a very specific prognostic indicator. Nevertheless, a level above 2.4 mg/L has been associated with a doubled risk of a coronary event compared to levels below 1 mg/L. However, in the study group in this case consisted of patients who had been diagnosed with unstable angina pectoris, whether elevated CRP has any predictive value of acute coronary events in the general population of all age ranges remains unclear.
Immunoglobulin Light Chain
Light chains are of two types, kappa (κ) and lambda (λ), and in any given antibody molecule only one type occurs. Approximately twice as many κ as λ molecules are produced in humans, but in other mammals, this ratio can vary. Each free light chain (FLC) molecule contains approximately 220 amino acids in a single polypeptide chain that is folded to form the constant and variable region domains (Fig. 8).
Domains are constructed from two β sheets. These are elements of protein structure made up of strands of the polypeptide chain (β strands) packed together in a particular shape. The sheets are linked to a disulfide bridge and together form a roughly barrel-shaped structure known as a β barrel.
Fig. 8: An antibody molecule showing the heavy and light chain structure, together with kappa and lambda free light chains
Genes, which encode antibodies are assembled in B lymphocytes by joining multiple gene segments, which are far apart in germline DNA. The light chain variable domain is constructed from V and J gene segments, whilst the constant domain is encoded by a separate C gene segment.
There are multiple copies of the gene segments in germline DNA, and a random selection of individual V, J and C genes contributes to the diversity of immunoglobulin light chains. The constant domains of light chains show little amino acid sequence variation. The human genome contains a single κ constant gene for which three serologically defined allotypes have been defined, and designated Km1, Km2 and Km3. These allotypes define three Km alleles that differ in two amino acids. The genome also contains a variable number of λ constant genes, giving rise to multiple λ chain isotypes. These λ isotypes can be distinguished serologically through the expression of Mcg, Kern, and Oz markers. For example, the protein product of the IGLC1 gene is Mcg+Kern+Oz-. There is no evidence that these variants affect FLC measurements using free light assays, which are based on polyclonal antisera. In contrast, the variable domains of light chains exhibit huge structural diversity, particularly in association with the antigen binding amino acids. In addition, the first 23 amino acids of the first variable domain framework region have a limited number of variations known as subgroups. Using monoclonal antibodies, four κ (Vκ1–Vκ4) and six λ subgroups (Vλ1–Vλ6) can be identified. The specific subgroup structures influence the potential of the FLCs to polymerize such that amyloid light chain (AL) amyloidosis is associated with Vλ6 and light chain deposition disease (LCDD) with Vκ1 and Vκ4.
Kappa FLC molecules (chromosome 2) are constructed from approximately 40 functional Vκ gene segments, five Jκ gene segments and a single Cκ gene. Lambda FLC molecules (chromosome 22) are constructed from approximately 30 Vλ gene segments and four (or more) pairs of functional Jλ gene segments and Cλ genes. FLCs are incorporated into immunoglobulin molecules during B lymphocyte development and are expressed initially on the surface of pre-B cells. Production of FLCs occurs throughout the rest of B cell development and in plasma cells, where secretion is highest. Tumors associated with the different stages of B cell maturation will secrete monoclonal FLCs into the serum where they may be detected by FLC immunoassays (Fig. 9).
In normal individuals, approximately 500 mg of FLCs are produced each day from bone marrow and lymph node cells. The molecules enter the blood and are rapidly partitioned between the intravascular and extravascular compartments. The normal plasma cell content of the bone marrow is about 1%, whereas in multiple myeloma this can rise to over 90%. The bone marrow may contain 5–10% plasma cells in chronic infections and autoimmune diseases, and this is associated with hypergammaglobulinemia and corresponding increases in polyclonal serum free light chain (sFLC) concentrations. Bone marrow identification of monoclonal plasma cells by histology or flow cytometery is an essential part of multiple myeloma (MM) diagnosis and is frequently based on identifying intracellular κ and λ by direct immunofluorescence techniques.
Plasma cells produce one of five heavy chain types, together with κ or λ molecules. There is approximately 40% excess FLC production over heavy chain synthesis, to allow proper conformation of the intact immunoglobulin molecules. As mentioned previously, there are twice as many κ producing plasma cells as λ producing plasma cells. Kappa FLCs are normally monomeric, while λ FLCs tend to be dimeric, joined by disulfide bonds; however higher polymeric forms of both FLCs may occur. In normal individuals, sFLCs are rapidly cleared and metabolized by the kidneys depending upon their molecular size. Monomeric FLCs, characteristically κ, are cleared in 2–4 hours at 40% of the glomerular filtration rate. Dimeric FLCs, typically λ, are cleared in 3–6 hours at 20% of the glomerular filtration rate, while larger polymers are cleared more slowly. Removal may be prolonged to 2–3 days in MM patients in complete renal failure. In contrast, IgG has a half- life of 21 days with minimal renal clearance.
Bence Jones Protein
Considered as the first tumor marker, it is a monoclonal globulin protein or immunoglobulin light chain found in the blood or urine, with a molecular weight of 22–24 k D. It is excreted in the urine of myeloma patients. The presence of protein is a sign that there are higher than normal levels of protein in the blood, as in the case of myeloma where there is an overproduction of immunoglobulin protein. When Bence Jones proteins are in the urine, they can accumulate and cause kidney damage.
Detection of Bence Jones protein may be suggestive of MM or WM. Bence Jones proteins are particularly diagnostic of multiple myeloma in the context of end-organ manifestations, such as renal failure, lytic (or “punched out”) bone lesions, anemia, or large numbers of plasma cells in the bone marrow of patients. Bence Jones proteins are present in two-third of MM cases.
The proteins are immunoglobulin light chains (paraproteins) and are produced by neoplastic plasma cells. They can be κ (most of the time) or λ. The light chains can be immunoglobulin fragments or single homogeneous immunoglobulins. They are found in urine due to the kidneys’ decreased filtration capabilities due to renal failure, sometimes induced by hypercalcemia from the calcium released as the bones are destroyed or from the light chains themselves. The light chains have historically been detected by heating and now by electrophoresis of concentrated urine. More recently serum free light chain assays have been utilized in a number of published studies, which have indicated superiority over the urine tests, particularly for patients producing low levels of monoclonal FLCs, as seen in nonsecretory MM and AL amyloidosis. This is primarily because of the reabsorption of FLCs in the kidneys, creating a “threshold” of light chain production which must be exceeded before measurable quantities overflow into the urine. As such, urinalysis is a fickle witness to changing FLC production.
URINARY PROTEIN ELECTROPHORESIS
The association of the proteinuria with abnormality of the kidney has been clearly demonstrated by Richard Bright around 150 years ago. The role of the kidney in the storage and metabolism of the plasma proteins has been studied many times in the past, but it is in the last two decades that the subject has been the focus of numerous studies.
The interest in the study of proteinuria is derived by the frequency of appearance of this sign, present in almost all kidney diseases, by the development of biochemical methods for the qualitative and quantitative study of urinary proteins, and a more precise knowledge of the physiological processes of glomerular filtration and tubular reabsorption of the proteins in normal and pathological conditions (Fig. 10).
The presence of proteinuria does not necessarily imply the existence of a renal pathology, even normal subjects have “physiological” proteinuria (Box 1), and instead, there are the qualified electrophoretic studies to define a proteinuria as “pathological”, highlighting the presence of plasma originating proteins in the urine that under normal conditions do not exceed the glomerular barrier of proteins present only when the tubule does not carry out its function of reabsorption of abnormal plasmatic proteins that pass in the urine, and of proteins secreted by the kidney and urinary tract.
Four main factors intervene in the determination of the glomerular filtration of the proteins:
- The selective permeability: molecular weight and form
- The presence of anionic structure with the function of electrostatic repulsion
- The plasmatic concentration
- The local hemodynamic conditions.
Mechanisms of Reabsorption
All proteins with a molecular weight less that 40 D do not encounter, almost, difficulty in being filtered at the glomerular level, and they are over 90% reabsorbed in the proximal tubule.
From the semiological viewpoint, the proteinuria may be:
It consists of the exhibition of occasional proteinuria with pathological values, usually moderate that do not repeat over time in subsequent checks and has a benign type character. With the end of functional proteinuria, meaning the proteinuria that independently from pathological kidney states accompanies several clinical conditions characterized by states of fever to intense and prolonged exercise to conditions of physical and emotional stress, such as surgical interventions or the exposure to cold. This proteinuria is defined as transitory type and resolves together at the recovery of the physical condition altering it was not the origin.
These proteinurias are characterized, as their definition indicates, by the appearance of pathological proteins over time intervals and in episodes in which the proteinuria re-enters into the range of physiological values. These proteinurias may have completely random and irregular courses, or show a regular recurrence such as in orthostatic type proteinuria, in addition to normal values in the samples taken in the morning, following a period of supine rest, which presents pathological values in serotin samples, after the common orthostatic activity. The intermittent proteinurias are of glomerular type, although usually quantitatively small, they deserve attention because they can be indexes of incipient renal suffering. The verification of finding an intermittent proteinuria requires the doctor to deepen the investigations in order to be able to resolve the potential doubt of a renal lesion that, as we have said, may be the cause. The typology of the intermittent proteinuria is often a prerogative of the child and adolescent age, where it is fundamental to exclude the possibility of silent Streptococcus infections.
It can be distinguished in the prerenal, renal, and postrenal forms. The prerenal proteinurias are the once from “overflow”, which are not the expression of renal disease, but proteins physiologically filtered by glomerulus that are present in an increased concentration in the plasma for various pathological contingencies, exceeding the normal threshold of tubular reabsorption paradigmatic expression of this type of proteinuria and the Bence Jones proteinuria. The renal proteinurias are those already described in the etiopathogenetic classification as glomerulary, tubulary, and mixed. The postrenal proteinurias are due to lesions, which often refer to an inflammatory nature of the tissues of the kidney pathways, such as renal pelvis, ureter, bladder, prostate, and genital organs. Sometimes, these proteinurias have a composition, which allows the identification of the presence, of specific proteins such as the presence of enzymes or the Tamm Horsfall protein.
Pathogenetic Classification of the Proteinurias
The complexity of the mechanisms underlying the determinism of proteinuria is reflected in the complexity of the interpretative diagnostic process of the proteinuria frameworks found in clinical practice on the basis of the probable origin of urinary proteins (Tables 1 to 5).
Proteinurias of Plasmatic Derivation
Proteinurias of Tubular Derivation
- Nephrogenetic (Table 4)
Post renal Proteinurias (Table 5)