These modifications are not necessary for measuring total protein in serum but are useful in detecting minute amounts of protein that are present after isolation or separation procedures. Modifications of the biuret method are used to enhance sensitivity for proteins at low concentrations, such as those that produce bands on electrophoretic separations.
The Folin-Ciocalteu method involves the addition of molybdenum VI oxide, which is reduced by copper to molybdenum blue; this enhances the sensitivity of the protein assay by 2 or 3 orders of magnitude. Other common staining methods for detecting proteins separated by electrophoresis include the Coomassie Brilliant Blue technique involving a triphenylmethane dye, the amido black napthol black method, and the Ponceau S stain.
Silver also reacts with proteins, producing a dark brown complex; however, the chemical mechanism for this reaction is unknown. Albumin is unique in several respects, 2 of which influence the methods used to measure it in serum: it is not modified with carbohydrate or sialic acid moieties and it is enriched with glutamate and aspartate residues that make it acidic.
On serum protein electrophoresis, albumin migrates toward the anode, just behind the prealbumin proteins transthyretin sometimes called thyroxine-binding prealbumin [TBPA] and retinol-binding protein. Albumin helps to maintain the osmotic balance between intravascular and extravascular fluid, and it is a carrier protein for calcium, bilirubin, and many drugs.
The acid-binding dyes bromocresol green BCG and bromocresol purple BCP selectively bind to albumin to produce chromophores that are used to quantify the protein, based on their reaction with the acidic amino acids. These dyes react with acidic residues on all proteins. As a result, they are not specific for albumin; however, the overwhelming concentration of albumin and its higher content of acidic amino acid residues make their reaction with albumin thermodynamically favorable.
Bias due to reaction of the dyes with other proteins is minimized by careful observation of the reaction kinetics because BCG and BCP react preferentially with albumin; reaction with albumin predominates for approximately 30 seconds. A Michaelis-Menton curve that demonstrates the relationship between reaction rate v ; substrate concentration [S] ; maximal reaction rate V max ; and K m , the Michaelis constant the mathematical relationship is given in the inset.
K m is a thermodynamic quantity that expresses the equilibrium constant between substrate, enzyme, and the substrate-enzyme complex. The maximal rate velocity , V max , is the rate at which the reaction proceeds when the enzyme is fully saturated with substrate. Note that enzyme activity and its concentration are not synonymous, although they are related.
Enzyme activity is measured in the amount of substrate that it can convert to product within a given time interval. In the United States, a more common unit of enzyme activity is the enzyme unit U; 6 defined as the conversion of 1 mmol of substrate to product per minute. In addition, enzyme activity can be influenced by a number of factors, including temperature, pH, the presence of various inhibitors, and genetic polymorphisms.
Several options exist for measuring the rate of an enzyme-catalyzed reaction including the disappearance of substrate or the generation of product. Often, however, it is impractical to measure the substrate or product. In some cases, the product is involved in a secondary linked reaction that is more easily monitored.
Because the secondary reaction depends on the product of the initial reaction, its rate will be proportional to the rate of the initial reaction. Linking enzymatic reactions through their products and substrates is a common strategy to optimize measurement of enzyme-catalyzed reactions.
CK, an enzyme that transfers phosphate groups between creatine and adenosine in muscle, is usually measured by a linked enzyme assay Figure 4. Measurement of creatine kinase CK activity by linking the transfer of a phosphate group from phosphocreatine to adenosine diphosphate ADP to produce adenosine triphosphate ATP with the hexokinase HK reaction that transfers the phosphate group from ATP to glucose.
This produces glucosephosphate, which in turn is oxidized by glucosephosphate dehydrogenase GPDH to form 6-phosphogluconate. Some CK methods measure the reverse reaction, from creatine to phosphocreatine, linking the ADP produced to a pyruvate kinase reaction.
An even more common strategy for monitoring enzymatic reactions involves neither the substrate nor the product; instead, it monitors the conversion of an enzyme cofactor involved in the reaction. Therefore, the rate of the reaction can be monitored by measuring the absorbance at nm Figure 5.
The change in absorbance at that wavelength can be used to monitor the progress of the reaction. The UV absorption maximum at nm in the reduced forms of these coenzymes is due to the resonance structure created in the pyridine ring of nicotinamide or riboflavin moiety.
Enzymes are also used to measure substrate concentrations; in those cases, the reaction conditions ensure that the enzyme is present in abundance and as a result, the rate of the reaction will depend on substrate concentration ie, most of the enzyme is free of substrate. Aside from that difference, most strategies for monitoring enzymatic reactions, such as linked reactions and measuring the conversion of co-factors, are the same as methods used to measure enzyme activity.
Some proteins, particularly peptide hormones, are measured by their physiological effect. Unlike enzymes, these proteins are not catalytic in the sense that an enzyme catalyzes the conversion of a substrate to a product; rather, the proteins produce a physiological response by interacting with tissue receptors.
The approaches to measuring the physiological activity of a protein are in vivo challenge tests and in vitro activity measurements sometimes called bioassays. A challenge test involves a physiological intervention designed to induce a hormonal response; the presence or absence of an appropriate response reflects the integrity of feedback mechanisms and the activity of regulatory intermediates, which often include peptide hormones.
Challenge tests may involve dietary interventions, such as the water-deprivation test to assess the release of antidiuretic hormone ADH; also known as vasopressin or arginine vasopressin from the posterior lobe of the pituitary gland, or the glucose tolerance test, in which glucose is administered orally and the insulin response is measured indirectly by monitoring the change in serum glucose concentration.
Other challenge tests use exogenous agents to assess the integrity of a biochemical feedback loop. An example is the dexamethasone suppression test, in which a cortisol analogue dexamethasone is administered intravenously to determine its effect on corticotropin-releasing hormone CRH and adrenocorticotropic hormone ACTH release by the hypothalamus and anterior pituitary gland, respectively.
Activity tests typically involve tissues, isolated from human or nonhuman sources, that have receptors for the hormone of interest; the tissues produce a biochemical product in proportion to the concentration of the hormone.
Because of their technical complexity and the difficulty in standardizing the reagents and protocol, these are uncommon tests. One activity test that remains relatively common is that which measures plasma renin activity PRA. Renin is an enzyme 7 and can be measured immunochemically see the following paragraphs for further information ; despite this, PRA assays remain available.
Renin assays are complicated by the fact that the enzyme can exist in multiple forms, only some of which are active. In the PRA assay, angiotensinogen isolated from the blood of nephrectomized sheep is mixed with patient serum, along with inhibitors of angiotensinase and angiotensin-converting enzyme ACE to prevent conversion of angiotensin I to degradation products or angiotensin II; the assay measures angiotensin I concentration.
Electrophoretic separations are influenced by a variety of factors, including the strength of the electrical field, the conductivity of the buffer, and the temperature. Each of the fractions includes clinically significant serum proteins, as summarized in Table 4. Protein fractions separated by SPE are stained by one of the chemical methods described previously Commassie brilliant blue, Lowry, or Ponceau S and quantified by densitometry.
A small protein fraction migrates farther toward the anode than albumin peak and is called prealbumin. The 2 proteins in the prealbumin fraction—transthyretin and retinol-binding protein—are not related to albumin.
In electrophoresis, the positive pole is designated as the anode and the negative pole is designated as the cathode. This terminology is the opposite of the conventional designations in electrochemical cells batteries , in which the positive pole is the cathode and the negative pole is the anode. Protein fractions are stained using Lowry, Commassie blue, Ponceau S, and amido black methods and quantitated via densitometry. Immunoglobulins are a necessarily diverse group of proteins that confer immunity against foreign antigens.
These methods can be applied to serum and urine and are useful in the diagnosis of gammopathic diseases. Immunoassays are available for a variety of protein and nonprotein analytes. Proteins express a rich array of epitopes and heterogeneous 2-site sandwich immunoassays for proteins can be highly specific.
For detection and quantitation of specific proteins, immunoassay currently is the most common analytical technique. Many clinically important proteins are commonly measured in serum using immunochemical methods: albumin, immunoglobulins, transferrin, peptide hormones, ceruloplasmin, tumor markers, cardiac markers, coagulation factors, ferritin, myoglobin, haptoglobin, hormone-binding proteins, fibrinogen, and C-reactive protein CRP are examples. Immunoassays can be classified in several ways based on the general approach for using antibodies to detect and measure antigens.
Heterogeneous immunoassays require physical isolation of the antibody-bound antigen fraction, whereas in homogeneous methods the bound antigen can be chemically distinguished from free antigen so separation is not required.
Most homogeneous immunoassays involve small antigens because detection of the bound fraction in the presence of unbound antigens requires that a chemical property of the antigen or, more specifically, its label is changed when it binds to the comparatively large antibody molecule.
Rotational frequency influenced by the mass of the antibody fluorescence polarization immunoassay [FPIA] , enzyme activity influenced by the antibody obscuring the active site on the enzyme enzyme-multiplied immunoassay technique [EMIT] , the ability of enzyme subunits to spontaneously associate cloned enzyme-donor immunoassay [CEDIA] , and cross-linking of antigen-labeled microparticles that produce turbidity kinetic interaction of microparticles in solution [KIMS] all have been used in homogeneous immunoassays Figure 7.
Homogeneous immunoassay results that measure a chemical property of the label green attached to an antigen blue when the labeled antigen is bound to an antibody. When endogenous unlabeled antigen cells are present, the labeled antigen is displaced from the antibody; its chemical properties are then unaffected by antibody binding. Virtually all immunoassays for proteins are heterogeneous.
Immunoassays can be classified as competitive or noncompetitive. In competitive immunoassays, antigens are in excess and labeled antigens compete with endogenous antigens for binding sites on a limited number of antibodies. Although both competitive and noncompetitive methods exist for measuring proteins, the noncompetitive methods are more common and have the advantage of greater sensitivity because all the target antigens are captured and available for measurement. Another distinction exists between 1-site and 2-site immunoassays.
In the former, a single antibody preparation polyclonal or monoclonal is used to recognize and bind with an epitope on the target antigen. In the latter, two antibody preparations are used both may be monoclonal or polyclonal that recognize 2 different epitopes on the target antigen; 2-site methods are often called sandwich immunoassays because the antigen is sandwiched between 2 antibodies.
Although exceptions exist, competitive homogeneous and heterogeneous immunoassays are almost always of the 1-site type, whereas noncompetitive assays are of the 2-site type and involve a labeled second antibody that reveals the antigens adsorbed by the capturing antibodies.
Enzyme-linked immunosorbent assay ELISA results showing the capture antibody white covalently linked to a microtiter well. This process captures all of the antigen cells in the specimen, which had been added first. After washing, a second, enzyme-labeled antibody yellow is added to create a veritable sandwich consisting of antibody-antigen-antibody.
After a second wash, the enzyme activity remaining in the well is measured by addition of substrate that is converted by the enzyme into product. In the microparticle enzyme immunoassay MEIA method, the capturing antibodies are attached to microparticles that are mixed with serum and then separated from unbound components via filtration through a glass fiber matrix.
Most contemporary 2-site sandwich immunoassays use a soluble capture antibody with a paramagnetic particle attached; the captured antibody-antigen complexes are immobilized after equilibrium is reached by activating a magnet.
These modifications improve the kinetics of antigen capture and reduce the amount of time required for the assay. Also, chemiluminescent labels have largely replaced enzymes as the labels on the second antibodies. The key element in many immunoassays, competitive and noncompetitive, is the label used to detect the competing antigen in single-site competitive methods, or the secondary antibody in 2-site noncompetitive methods.
Immunoassays at least partially derive their names from the type of label used. Radioimmunoassay RIA uses a radioactive isotope as the label, enzyme immunoassays EIA use an enzyme, and the fluorescent immunoassay uses a fluorophore.
Many contemporary immunoassays use a chemiluminescent label that creates a burst of light when a reactant usually a oxidizing or reducing agent is added. Chemiluminescent labels have advantages over the enzymes used for labels. They are small and therefore do not interfere with antigenicity; also, the signal that chemiluminescent agents produce is similar to radioactivity, in that the signal is measured against a blank background.
Enzyme activity is usually measured by changes in UV absorbance and the background signal can be significant. Point-of-care POC technologies are available for qualitative and quantitative measurement of several proteins eg, urine pregnancy tests that measure human chorionic gonadotropin [hCG]. Almost all of these devices use a technique usually called immunochromatographic lateral flow ILF.
Most of these assays are qualitative; however, quantitative methods involving densitometric measurement of the labeled antibody with portable instruments have been designed. A single-site design using immunochromatographic lateral flow, in which antigen is covalently attached to a solid support.
A , Negative result in the absence of endogenous antigen. The antibodies are captured on the solid support structure, and the label [star] can be detected.
B , Positive result. When the specimen contains free endogenous antigen, the antibodies are saturated and do not react with immobilized antigens on the solid support structure. Note that the signal is present when the specimen does not contain antigen, which is opposite from most qualitative assays, in which the signal represents a positive result.
Most of the devices contain an internal control that verifies the presence of antibodies by capturing them on another area of the solid support structure with immobilized anti—immunoglobulin IgG antibodies not shown.
Two-site immunochromatographic lateral flow design, in which the captured antibody is covalently attached to a solid support structure. Antigens that have adsorbed to the surface are detected with a second, labeled star antibody. In the absence of endogenous antigen, no signal is detected A. If antigens are present in the specimen, an antibody-antigen-antibody complex is formed, and the labeled antibody is detected B.
In this configuration, a positive result produces a signal, and a negative result means the absence of a signal. As with 1-site methods, an internal control line produces a signal when the second labeled antibody is captured by anti—immunoglobulin IgG antibodies immobilized to another region of the test pad known as the control line ; not shown. Proteome is a term coined in by Marc R. Wilkins, a PhD student at Macquarie University in Sydney, Australia, in reference to the entire complement of proteins expressed by a genome.
The term proteomics followed in combining protein and genomics. Using high-resolution 2-dimensional electrophoresis, serum proteins can be mapped to detect changes in protein expression related to various diseases. Proteomics has been used as a research tool to study protein expression and function; however, the technique may become commonplace in clinical laboratory practice when the vast amounts of information produced by these methods are characterized, classified, and correlated with various diseases.
One recently developed application of mass spectrometry involves the identification of microbes, which display unique protein signatures. A technique known as matrix-assisted laser desorption ionization MALDI combined with use of a time-of-flight TOF mass spectrometer has been applied to the identification of microbial proteins. This analytical method is much faster than growing and identifying microbes in culture and is likely to become more economical when MALDI-TOF instruments configured for this application become widely available.
Many diagnostically important proteins can be quantified using the analytical methods described in the previous section; a complete discussion of enzymes and all the diseases associated with protein abnormalities is beyond the scope of this article. The production and function of hormones, some of which are proteins, are topics that span the discipline of endocrinology. Genetic polymorphisms that affect the expression, function, or activity of proteins are within the realm of molecular diagnostics.
This review focuses on the clinical significance of total protein and albumin in serum and urine, as well as SPE and IFE applications and selected individual proteins. Measurement of total serum protein concentration via automated methods such as the biuret reaction is used to assess the synthesis and maintenance of proteins in circulation.
Because albumin accounts for half the serum protein content, a decrease in albumin is often associated with a decrease in total protein, even if most other proteins are present in normal concentrations. Variations in serum albumin are discussed later in this article. A decrease in serum total protein may reflect decreased protein synthesis or increased protein loss. Nearly all proteins are synthesized in the liver; hence, hepatic failure is a cause of decreased serum protein.
However, serum total protein is not a sensitive measure of hepatic failure because most proteins have biological half-lives of days to weeks. Therefore, inadequate production of proteins by a failing liver may not be reflected in low serum protein until after other symptoms of hepatic failure are already present, such as jaundice due to decreased hepatic conjugation of bilirubin , hyperammonemia due to urea cycle failure , 11 and coagulopathy due to a deficiency in the short-lived coagulation factors produced in the liver parenchymal cells.
Liver failure is ordinarily diagnosed by hyperbilirubinemia particularly the unconjugated, indirect fraction , increased serum ammonia levels, and prolonged prothrombin time, along with elevations in the serum activities of hepatic enzymes such as alanine aminotransferase ALT , aspartate aminotransferase AST , LD, and other proteins such as ferritin that are released by damaged hepatocytes.
Protein synthesis requires dietary amino acids that cannot be synthesized ie, the essential amino acids.
Thus, decreased serum protein levels may also result from malnutrition; nevertheless, more sensitive tests exist for adequate dietary protein levels see the discussion of prealbumin in the following paragraphs. As in hepatic failure, the decrease in total protein resulting from malnutrition does not appear until existing proteins are degraded, which may take several weeks.
A group of malabsorptive disorders, such as celiac disease, Crohn syndrome, and short-bowel syndrome cause hypoproteinemias, often known as protein-losing enteropathy. This is a misnomer because the protein is not lost; rather, the inability to absorb proteins causes a deficiency in essential amino acids, resulting in deficient protein synthesis even with adequate protein intake.
A decrease in total protein is also observed when hepatic function is normal but the proteins are lost in the urine. Loss of proteins in the urine results in a decrease in total serum protein. Whether hypoproteinemia results from deficient synthesis due to hepatic failure, malnutrition, or from renal loss due to increased glomerular membrane permeability, the concentrations of all proteins do not diminish at the same rate.
When protein synthesis is deficient, proteins with the shortest biological half-lives disappear first. The persistence of proteins in serum varies from minutes to weeks, with an average half-life of 10 days; certain proteins degrade very rapidly. Prealbumin proteins transthyretin and retinol-binding protein and coagulation factors are examples of proteins that have short biological half-lives; their concentrations fall below normal levels quickly when protein synthesis is deficient.
Protein loss in the urine due to renal disease typically begins with smaller proteins, which are the first to leak across a deteriorating glomerular membrane, and albumin, which is favored for filtration by virtue of its high concentration in serum. Elevation of serum protein concentration has 2 principal causes: dehydration, in which there is less water in the body and the blood volume decreases, thereby concentrating the proteins, and overproduction of specific proteins, which is more common.
The most commonly overproduced proteins are immunoglobulins, the levels of which can be elevated in infections and in hematological neoplasms. A variety of proteins are classified as acute-phase reactants because their concentrations increase rapidly in response to inflammation. Specific methods are available to measure each of these proteins. Because most of the protein in serum is albumin, however, the increase in these proteins does not significantly affect the total serum protein concentration.
Changes in the concentrations of these proteins can be detected via SPE; however, direct measurement via immunoassay is the most sensitive and specific way to assess the acute-phase response. Multiple myeloma is a hematological malignant neoplasm characterized by unregulated proliferation of antibody-producing plasma cells.
Plasma cells originate in the bone marrow as B lymphocytes and mature into plasma cells in lymph nodes. In multiple myeloma, B cells accumulate in the bone marrow and inhibit the production of erythrocytes along the normal myeloid pathway, producing a normocytic and normochromic form of anemia.
The unchecked proliferation of a plasma-cell clone results in the overproduction of a single immunoglobulin clone, called a paraprotein , that can be detected by an increase in serum total protein and a single spike in the gamma region of an SPE gel known as an M spike [ Figure 11 ].
Serum protein electrophoresis pattern in multiple myeloma. Note the M spike in the gamma region of the gel, as reflected in the densitometric tracing at the top of the figure. In rare cases, the disease may produce a biclonal peak. Disorders associated with high or low serum concentrations of specific proteins other than albumin are numerous. Transferrin and ferritin are used to assess iron status; ceruloplasmin reflects copper transport and storage; cardiac troponins reveal myocardial damage; tumor markers such as prostate-specific antigen PSA , alpha fetoprotein AFP , carbohydrate antigen CA markers, carcinoembryonic antigen CEA , and so forth are used to detect, and monitor treatment of, cancer; fibrinogen and coagulation factors are used to assess hemostatic function; and various enzymes reveal tissue damage and necrosis.
Table 5 lists some of the clinically important serum proteins. In addition to being the most abundant protein in serum 3. It is an anionic protein, containing an abundance of aspartate and glutamate residues; it is not functionally modified with carbohydrates; 12 among all serum proteins, it has a midrange molecular weight 67 kDa ; and it has a longer-than-average half-life of approximately 20 days.
Albumin helps maintain osmotic balance between intravascular and interstitial spaces; therefore, a deficiency in albumin ordinarily results in edema as water is redistributed to tissues. Because albumin has a longer half-life relative to many other proteins, its concentration in serum is a poor indicator of nutritional deficiency or impaired synthesis; prealbumin proteins and coagulation factors are more sensitive measures of impaired protein synthesis because their half-lives are much shorter.
The reason for decreased serum albumin is usually renal loss. Glomerular membrane permeability is partially a function of size but also is related to charge; the negative charge on albumin inhibits its filtration because the membrane likewise is negatively charged.
Diseases that cause damage to the glomerular membrane increase its permeability to all proteins; however, its permeability to albumin may be particularly affected if the negatively charged groups on the membrane surface are neutralized. This appears to be the principal mechanism of albuminuria associated with diabetic nephropathy. Although albumin is highly conserved across many species, there exist mostly benign polymorphisms in the genes that code for this protein.
For example, there are forms of albumin that have higher-than-normal affinity for thyroid hormones; these do not produce clinical manifestations but may cause errors in immunochemical methods that measure free hormone concentrations because the methods are based on competition between antibodies against thyroid hormones and endogenous hormone-binding proteins. Albumin with increased affinity for thyroxine will also result in elevated total T 4 concentrations in patients with healthy thyroid-gland function because only the free fraction of thyroid hormones is biologically active.
Another albumin variant results in bisalbuminemia, a benign disorder in which 2 distinct albumin peaks appear on an SPE gel. A fascinating paradox surrounds albumin: it is a protein that is highly conserved across many species and has unique properties that seem functionally indispensable, such as buffering the serum ionized-calcium concentration, osmoregulation of plasma volume, solubilizing unconjugated bilirubin, and binding cationic drugs.
The logic of evolutionary design would argue that such a protein must be essential for life. This condition is benign; it produces only mild edema. Elevations in serum albumin are uncommon and not clinically significant. Most circulating proteins are conserved in the kidneys by exclusion from the glomerular filtrate or reabsorption from the renal tubules; failure of either mechanism causes excess protein levels in the urine.
Proteinuria may also occur when renal function is adequate if the concentration of a circulating protein is so high that filtration and reabsorption mechanisms are overwhelmed overflow proteinuria.
Finally, protein secreted by the renal tubular epithelium appears in the urine Tamm-Horsfall protein; also known as uromodulin ; however, its clinical significance is not well established. The varying degrees of severity of proteinuria and their most common causes are summarized in Table 6.
The methods commonly used to measure urine proteins are urine total protein measured via the biuret method, urine albumin measured via bromocresol green or purple methods, and IFE used to identify immunoglobulin light chains in urine.
The urine dipstick method for measuring protein in urine involves dye that produces a change in pH in the presence of protein. Because the dye is most reactive with albumin, dipstick urine protein methods are poor indicators of overflow and tubular proteinurias.
The most common cause of proteinuria is nephrotic syndrome NS , a nonspecific term that refers to increased permeability of the glomerular membrane. NS typically results from glomerulonephritis , or inflammation of the glomeruli.
Glomerulonephritis may occur because the disease affects only the kidney primary glomerulonephritis or it may result from systemic illness secondary glomerulonephritis ; the latter is more common.
In adults, NS is usually the result of secondary glomerulonephritis due most often to diabetes or lupus. Milder proteinuria can have many causes, including pyelonephritis, drug toxicity, nephrosclerosis usually due to hypertension , and overflow proteinuria. Overflow proteinuria is most often caused by overproduction of immunoglobulins in multiple myeloma MM. Ig-heavy chains are approximately 50 kDa in size, whereas light chains are approximately 25 kDa; intact IgG is usually kDa.
Because of their small size, light chains can cross the glomerular membrane; when their concentration is high enough, they can overwhelm the reabsorptive capacity of renal tubules. Thus, immunoglobulin light chains appear in the urine in MM, and collectively are called Bence-Jones protein.
Urine IFE testing can confirm the monoclonality of a gammopathic entity when a single spot appears in the kappa- or gamma-stained lane of the gel. Immunoassays are available to quantify free light chains in serum, in lieu of urine IFE.
Small proteins, of less than 40 to 50 kDa, pass across the glomerular membrane into the Bowman capsule as part of the filtrate. Failure of the reabsorptive mechanism results in minimal or mild proteinuria and can be caused by genetic deficiencies in the transport components or renal tubular damage from drugs or persistent exposure to high glucose concentrations; diabetes is the most common cause of renal failure.
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