Which of the following reacts with peptide bonds in the biuret method for measuring total serum protein?

Protein, albumin, enzyme activity, immunoassay, renal disease, liver disease, hyperproteinemia, urine protein, multiple myeloma, nephrotic syndrome, malnutrition, proteomics

Inhaltsverzeichnis Show

Structure and Nomenclature
Synthesis and Function
Measurement of Proteins
Chemical Methods
Activity Assays
Serum Protein Electrophoresis
Immunochemical Methods
Evolving Methods for Protein Analysis
Clinical Significance of Protein and Albumin Measurements
Serum Proteins
Serum Albumin
Urine Proteins
Urine Albumin
Abbreviations
Suggested Reading

Proteins are large polymers of amino acids linked by peptide bonds (Figure 1). The amino acid subunits of proteins are organic molecules that include a carboxylic acid linked through a carbon atom to a primary (or secondary, in proline) amine with the chemical formula H2N-CHR-COOH, in which R is a side group that largely determines the chemical properties of the amino acid. The simplest amino acid is glycine, H2N-CH2-COOH, in which the R side group is a hydrogen atom. Although this chemical template can be modified into an infinite array of molecules based on variations in the R group, only approximately 20 amino acids occur in proteins. The R groups confer acidic, alkaline, polar, or non-polar properties to the various amino acids.

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The peptide bond (or amide bond) is formed when a primary amine reacts with a carboxylic acid. Because a molecule of H2O is lost in the reaction, the reaction constitutes dehydration synthesis, which can be reversed by hydrolytic cleavage. However, the reverse reaction is very slow; as a result, peptide bonds are stable at physiological temperatures and pH. Digestion and metabolism of proteins (proteolysis) are catalyzed by protease enzymes.

Structure and Nomenclature

Amino acid chains vary in size from 2 to thousands of amino acid residues (the term residue refers to an amino acid incorporated into a peptide chain). Small assemblies of amino acids that contain fewer than 50 or so residues are usually referred to as peptides; the smallest peptides have names preceded by a prefix that indicates the number of residues. For instance, dipeptide, tripeptide, and tetrapeptide indicate 2, 3, and 4 amino-acid chains, respectively. Amino acid chains containing more than a dozen or so residues are often referred to as oligopeptides (from the Greek oligos, meaning “few”). Proteins are typically defined as polypeptides with a molecular weight of greater than 5000 Daltons (ie, > 5 kDa). Proteins adopt stable conformations based on chemical interactions between neighboring amino acids within the sequence, between remote domains, and based on interactions with the solvent (in biological systems, the solvent is mostly water).

The primary (1°) structure of a protein is its amino acid sequence; this is determined by the codons in the messenger RNA that serve as a template for synthesis of the protein. Each amino acid has a 3-letter abbreviation (Table 1); hence, the primary structure can be described by a sequence of the amino acids (eg, Asp-Gly-Glu-Lys-Pro-His). A shorthand nomenclature has been developed for peptides that assign single-letter abbreviations to amino acids (for instance, DGQKPH, for the previously-mentioned hexapeptide). It is equally valid to describe the primary structure of a protein by the sequence of nucleic acids that code for that protein.

Table 1

In 1951 Linus Pauling showed that amino acids in the primary structure of proteins interact in predictable ways.1 Some sequences adopt a β-pleated sheet conformation, whereas others coil into α helices. These structural domains, dictated by proximal interactions between amino acids (more precisely, the R groups of amino acids) constitute the secondary (2°) structure of proteins.

Proteins are large molecules that would be insoluble in the aqueous environment of blood if not for their interactions with water. Circulating proteins adopt conformations that expose hydrophilic domains while hiding the hydrophobic domains within their globular structure. The stable 3-dimensional conformation of proteins seems to violate the laws of thermodynamics, which dictate randomness. However, the overall energy of proteins is lowered when they expose dipolar functional groups to the aqueous solvent and while sequestering nonpolar domains that would require energy to solvate. The 3-dimensional conformation of proteins is their tertiary (3°) structure and is very important for their functions because it brings remote regions of the amino acid sequence into close proximity, creating active sites on enzymes and antigen-binding sites on antibodies. John Kendrew and Max Perutz were the first to demonstrate the tertiary structure of a protein, myoglobin, by X-ray crystallography in 1957. They shared the 1962 Nobel Prize in Chemistry for their accomplishment.

Some proteins assemble with other proteins to form superstructures essential to their functions. An example is hemoglobin, which contains 4 protein subunits, each incorporating an oxygen-binding heme moiety. The lactate dehydrogenase (LD) enzyme also has 4 protein subunits; creatine kinase (CK) has 2. Immunoglobulin M (IgM), a pentamer is one of the largest circulating proteins in humans. Multiprotein complexes constitute the quaternary (4°) structure of proteins.

Synthesis and Function

Proteins have a vast array of functions in biological systems (Table 2). The biochemical properties of proteins are modified by the post-translational addition of various constituents to the side groups (R groups) on the amino acid backbone. Some of them incorporate metal ions (metalloproteins), some are modified with carbohydrates (glycoproteins), some are phosphorylated (phosphoproteins), and some bind to lipids (lipoproteins). Proteins can be catalytic (enzymes), can promote the structural integrity of cellular membranes (lipoproteins and phosphoproteins), can transduce chemical signals (cell-surface receptors), can transport essential ions (eg, transferrin and ceruloplasmin), can have endocrine and exocrine functions (eg, insulin, thyrotropin, and gonadotropins), can bind to foreign antigens and activate humoral immune attacks (immunoglobulins), or can maintain osmotic balance between intravascular and interstitial fluids (albumin).

Table 2

Biological Functions of Proteins

The chemical properties of proteins are the basis of life. With few exceptions, proteins are synthesized in the liver; hence, hepatic failure is usually accompanied by a deficit in the production of proteins. However, not all amino acids can be synthesized by mammals; therefore, dietary intake of the essential amino acids (namely, histidine, leucine, isoleucine, lysine, phenylalanine, methionine, threonine, tryptophan, and valine2) is necessary for protein synthesis. Most calorically adequate diets provide sufficient amounts of the essential amino acids; however, malnutrition is a cause of deficient protein synthesis.

Genetic diseases are caused by DNA mutations that result in the absence, deficiency, or dysfunction of enzymes that regulate metabolism. An amino acid substitution due to a single nucleotide polymorphism (SNP; pronounced “snip”) can have a profound effect on the functional integrity of a protein. A familiar example is sickle cell disease, which is caused by an SNP in the β-globulin gene located in the short arm of chromosome 11. In the gene, an adenosine residue is replaced by a threonine, resulting in the substitution of valine for glutamic acid at position 6 in the hemoglobin protein.3 The mutation causes hemoglobin to polymerize in oxygen-deficient conditions, distorting the red blood cells into the characteristic sickle shape.

Many genetic mutations affect proteins involved in metabolism. Some of the mutations are incompatible with life and result in fetal demise; others cause profound physical and neurological abnormalities. However, a few are relatively benign, causing only mild or episodic symptoms; these can be treated medically or with dietary and other lifestyle modifications.

Measurement of Proteins

There are several ways to measure proteins. Chemical methods rely on the properties of amino acids or peptide bonds that are common to all proteins. Activity assays measure the biological function of proteins (eg, enzymes and hormones). Immunoassays use antibodies to recognize unique structural domains (epitopes) on proteins. All of these methods are applied in clinical laboratories to measure proteins and to detect abnormalities that cause or result from disease. Measurement of total protein concentration in serum has limited clinical value; measurement of specific proteins by their activity or via immunoassay provides more specific diagnostic information. Serum protein electrophoresis (SPE) separates serum proteins into general classifications based on size and charge; immunoelectrophoresis (IEP) and immunofixation electrophores is (IFE) are modifications of SPE that provide greater specificity based on the immunoreactivity of proteins, primarily immunoglobulins. Proteomics is a relatively new approach to the diagnostic use of proteins; it involves high-resolution chromatographic methods to separate and to map many proteins, with the goal of detecting global variations in protein expression that are associated with disease.

Chemical Methods

Amino acids have characteristic chemical properties that are used to quantify proteins by densitometric or spectrophotometric methods. Chemical methods for detecting proteins are summarized in Table 3.

Table 3

Chemical Methods for Measuring Proteins

Although it is not the most sensitive method to detect proteins, the biuret reaction is universally used to measure total protein concentration in serum on automated chemistry instruments.4 The biuret reaction is illustrated in Figure 2. In alkaline solution, copper II (cupric ion) forms a complex with secondary or tertiary amino groups separated by a carbonyl group and, in the case of peptide bonds, another tertiary carbon atom. The method derives its name from the reaction of cupric ion with biuret, a combination of 2 urea molecules (H2NCONH2). The complex absorbs visible radiation at 540 nm, giving it a blue color. Potassium sodium tartrate is added to the reagent to stabilize the cupric ions because copper I (cuprous) ions do not form the complex. The biuret method does not distinguish between proteins because the reaction is specific for peptide bonds, which are common to all proteins. Therefore, the method is used to quantify the total protein content of serum.

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Biuret (top) and peptide bonds (bottom) that react with cupric ion (Cu2+) to form a blue adduct that absorbs at a wavelength (λ) of 540 nm. The reaction only occurs under alkaline conditions. The biuret reagent also includes sodium potassium tartrate to stabilize the cupric ions that would otherwise be susceptible to reduction to cuprous ion (Cu+). The biuret reaction is the most common technique for measuring total protein using automated chemistry analyzers. Modifications of the biuret method include the Lowry and Folin-Ciocalteu techniques, which greatly enhance the sensitivity. 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 Lowry method for protein quantification uses a phosphomolybdic/phosphotungstic acid solution to react with the copper-protein complex, producing a chromophore that absorbs at 650 to 750 nm. 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 the most abundant protein (constituting approximately 50% of the total proteins) in serum and is measured to detect and monitor a variety of diseases. 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.5

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.

Activity Assays

Enzymes are usually measured by their activity. An enzymatic reaction can be expressed as: where E = enzyme, S = substrate, ES = the enzyme-substrate complex, and P = product. When measuring the activity of an enzyme, sufficient substrate is added to ensure that the enzyme is completely saturated with substrate and that the substrate concentration, [S], does not change significantly during the time the reaction is being monitored. Under those circumstances, the rate of the reaction is exclusively dependent on enzyme concentration, which equals [ES] because it is saturated with substrate. The activity of an enzyme is related to its affinity for substrate (as expressed by the Michaelis constant, Km) and the efficiency with which it converts substrate to product (expressed as Vmax, which is the rate at which the reaction occurs when the enzyme is completely saturated with substrate). Graphical and mathematical representations of the relationship between [S], Vmax, and Km appear in Figure 3.

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A Michaelis-Menton curve that demonstrates the relationship between reaction rate (v); substrate concentration ([S]); maximal reaction rate (Vmax); and Km, the Michaelis constant (the mathematical relationship is given in the inset). Km is a thermodynamic quantity that expresses the equilibrium constant between substrate, enzyme, and the substrate-enzyme complex. The maximal rate (velocity), Vmax, is the rate at which the reaction proceeds when the enzyme is fully saturated with substrate. The Lineweaver-Burk derivation of the Michaelis-Menton equation produces a linear plot of 1/[S] vs 1/v with a slope of Km/Vmax and a y-intercept of −1/Km.

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. The Système International (SI) unit for catalytic activity is the katal (abbreviated as kat), which corresponds to 1 mol of substrate converted to product per second. 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).

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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 glucose-6-phosphate, which in turn is oxidized by glucose-6-phosphate dehydrogenase (G-6-PDH) to form 6-phosphogluconate. The reaction is monitored by the change in absorbance at 340 nm when the G-6-PDH cofactor of the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+) is converted to the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH). 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. Nicotinamide adenine dinucleotide (NADH; a coenzyme), for example, is required for most dehydrogenase enzymes because it has oxidized (NAD+) and reduced (NADH) forms, which gives it the ability to accept or to donate a proton. NAD+ and NADH have different ultraviolet (UV) absorption profiles; NADH has an absorption maximum at λ = 340 nm that is absent in NAD+, the oxidized cofactor. Therefore, the rate of the reaction can be monitored by measuring the absorbance at 340 nm (Figure 5).

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The ultraviolet (UV) absorption spectra of reduced and oxidized forms of nicotinamide adenine dinucleotide (NADH and NAD+, respectively), which differs most widely at the wavelength (λ) of 340 nm. The change in absorbance at that wavelength can be used to monitor the progress of the reaction. Some dehydrogenase enzymes prefer the phosphorylated form of the coenzyme (NADPH) or a coenzyme with riboflavin (vitamin B6) substituted for nicotinamide (flavin adenine dinucleotide; FAD+ and FADH). The UV absorption maximum at 340 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.

Serum Protein Electrophoresis

Electrophoresis is an analytical technique that separates components of a mixture based on their mobility in a viscous medium to which an electrical field has been applied. Electrophoretic mobility is influenced by 2 factors, namely, the charge on the molecule and its resistance to movement (drag) through a viscous medium, which for globular proteins is roughly proportional to size. In an electrophoretic separation, proteins experience 2 counterbalancing forces: the electromotive force that attracts their positively or negatively charged residues to the oppositely-charged electrode and the resistance to movement through a viscous medium. Electrophoretic mobility is defined by the following equations:

Femf=EQ=V·QdFdrag=6πrηvwhenFemf=Fdrag,velocity is constant

where Femf is the electromotive force, E is the electrical field strength, Q is the charge on the molecule, V is the voltage applied to the electrophoretic gel, d is the distance over which the voltage is applied, Fdrag is the force that resists movement, r is the Stokes radius of the molecule, η is the viscosity of the medium, and v is the velocity of the molecule through the medium. Proteins accelerate towards the oppositely-charged electrode until the drag force equals the electromotive force and then migrate at a constant velocity until the electrical field is turned off.8

Electrophoretic separations are influenced by a variety of factors, including the strength of the electrical field, the conductivity of the buffer, and the temperature. In SPE, serum proteins are separated into fractions designated as prealbumin, albumin, α-1, α-2, β, and γ (Figure 6). 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.9

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Serum proteins that are separated by electrophoresis into the general categories of albumin, α1, α2, β, and γ fractions. 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.

Table 4

Serum Protein Electrophoresis Fractions

Immunoglobulins are a necessarily diverse group of proteins that confer immunity against foreign antigens. The γ region of an SPE gel ordinarily is a broad band that includes the vast polyclonal array of immunoglobulins in serum. Certain hematological disorders express abnormal amounts of monoclonal antibodies that appear as a spike in the γ region. IEP and IFE are modifications of SPE that use immunospecific reagents to identify immunoglobulin heavy and light chains. These methods can be applied to serum and urine and are useful in the diagnosis of gammopathic diseases.

Immunochemical Methods

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).

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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. In the absence of free antigen (•), the antibody binds to the labeled antigen in the reagent, making it possible to measure a chemical property of the label (eg, fluorescence polarization, enzyme activity, and cross-linking of microparticles). 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.10 In noncompetitive immunoassays, the antibodies are in excess and therefore capture all the target antigens present. 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. The prototype for 2-site noncompetitive immunoassays is the enzyme-linked immunosorbent assay (ELISA; Figure 8).

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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. Many modifications of the ELISA approach exist. 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). One-site (Figure 9) and 2-site (Figure 10) immunoassay configurations have been adapted to POC devices. Most of these assays are qualitative; however, quantitative methods involving densitometric measurement of the labeled antibody with portable instruments have been designed.

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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).

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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).

Evolving Methods for Protein Analysis

Proteome is a term coined in 1994 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 1997 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. When liquid chromatography–mass spectrometry (LC-MS) techniques that use low energy ionization methods to detect proteins became available, LC-MS (and LC-tandem MS [LC–MS/MS]) were applied to identify and to quantitate multiple proteins with high specificity and sensitivity.

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.

Clinical Significance of Protein and Albumin Measurements

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.

Serum 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. In healthy kidneys, the glomerular membrane excludes most proteins from crossing into the Bowman’s capsule, and the small amount of protein that is filtered is mostly reabsorbed by the renal tubules. This issue is discussed in greater depth in the section “Urine Proteins.” Damage to the glomerular membrane from toxins or inflammation causes it to become leaky and the greater concentration of proteins in the filtrate overwhelms the capacity of the renal tubules to reabsorb protein. 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. CRP is an example of an acute phase reactant; others include ferritin, ceruloplasmin, haptoglobin, and α-1 antitrypsin. 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]).

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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. Immunofixation electrophoresis of serum and urine from patients with multiple myeloma will typically reveal a monoclonal gammopathy in the serum and the presence of a κ or a λ light chain in the urine (Bence-Jones protein).

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.

Table 5

Clinically Important Serum Proteins

Serum Albumin

In addition to being the most abundant protein in serum (3.5–5.0 g/dL, which constitutes approximately half of all serum proteins), albumin has several important characteristics. 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. Albumin also functions as a transport protein for calcium (approximately half of circulating calcium ions are bound to albumin), unconjugated bilirubin (which can be covalently or noncovalently bound to albumin), thyroid hormones (approximately 20% of T4 and 10% of T3 is bound to albumin), and many drugs.

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 T4 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.13 However, an extremely rare autosomal recessive genetic defect impairs albumin synthesis and produces analbuminemia, or the absence of albumin in the blood. This condition is benign; it produces only mild edema. Elevations in serum albumin are uncommon and not clinically significant.

Urine Proteins

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.14 Minute amounts of some serum proteins normally appear in the urine, including albumin (<100 mg/d) and certain enzymes such as CK and amylase; proteinuria refers to amounts that exceed the amount of naturally excreted proteins. The varying degrees of severity of proteinuria and their most common causes are summarized in Table 6.

Table 6

Classification and Causes of Proteinuria

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. Clinically, NS is characterized by proteinuria (usually >3.5 g/d); hypoalbuminemia (because albumin is the most abundant protein in blood, its loss in NS is most pronounced); edema due to loss of albumin-regulated maintenance of intravascular osmolality; and hyperlipidemia, the causes of which are multifactorial. In children aged 1 to 7 years, approximately 90% of NS cases are caused by minimal change disease (lipoid nephrosis), the etiology of which has not been established. 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). Most cases of MM produce immunoglobulin G, although clonal proliferation of all the other heavy chain immunoglobulin isotypes (IgA, IgE, IgM, and IgD) have been observed in the disease. In MM, the plasma cells express not only intact immunoglobulins but also free heavy and light (κ and λ) chain fragments. Ig-heavy chains are approximately 50 kDa in size, whereas light chains are approximately 25 kDa; intact IgG is usually 150 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.

Other gammopathies, such as Waldenstrom’s macroglobulinemia and certain lymphoproliferative disorders, can cause overproduction of immunoglobulins and the appearance of light chains in the urine; however, MM is the most common. 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. Approximately 99% of these filtered proteins are normally reabsorbed by the renal proximal tubular epithelium. 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. Whereas glomerular causes of proteinuria generally spill out large quantities of heavier proteins such as albumin that are not reabsorbed by the renal tubules, tubular proteinuria is mostly characterized by the appearance of small proteins in the urine. The most commonly used protein marker for tubular proteinuria is the 11-kDa beta-2 microglobulin, a serum protein associated with the major histocompatibility complex class I heavy chain found on the surface of all nucleated human cells. This protein is filtered in the glomerulus but is normally reabsorbed in the proximal renal tubules. Thus, the appearance of elevated concentrations of beta-2 microglobulin in the urine is associated with renal tubular disease.

Urine protein concentrations ideally are measured in 24-hour specimens and expressed as mg (or g) of protein per day. In spot (untimed) urine specimens, the protein concentration is often expressed as a ratio to creatinine concentration, which partially compensates for the variability of urine volume and concentration.

Urine Albumin

When renal function is normal, less than 100 mg of albumin is filtered in the glomerulus per day and is not reabsorbed. Nephrotic syndrome results in larger amounts of all proteins in the urine; however, albumin, by virtue of its abundance in serum, is the primary protein that will be observed in the urine. Because NS is the most common presentation of renal failure, an increased urinary albumin level is a sensitive indicator of most renal diseases. The dry reagent technology incorporated into urine dipsticks that semiquantitatively detect protein in urine is therefore designed to react most intensely with albumin; other causes of proteinuria such as overflow or tubular disease may not produce a positive result by the dipstick methodology. An older technique for the qualitative detection of proteins in urine that is not selective for albumin is the sulfosalicylic-acid precipitation method; this method is still used in some clinical laboratories.

Routine quantitative methods for urinary albumin are not sufficiently sensitive to measure mg quantities of the protein; however, optimized techniques can distinguish between normal (<100 mg/d) and slightly elevated (usually 100–300 mg/d) amounts of albumin in urine. These methods have been given the unfortunate blanket term of urinary microalbumin methods, in reference to the minute amounts of albumin they can detect. The term is unfortunate because it falsely implies that the analyte is a micro form of albumin. Measurement of urinary albumin is used to detect early renal disease, particularly in diabetes.

Summary

Proteins are multifunctional biomolecules that perform and regulate most metabolic processes in living organisms. Measurement of proteins in serum and urine is an essential component of medical diagnosis and treatment. A wide array of analytical techniques has been applied to measuring proteins in biological specimens; most of these methods are routinely available in clinical laboratories. Yet, on the horizon are powerful analytical methods that promise to greatly expand the clinical applications of protein measurements in health and disease. Measurement of proteins is certain to remain a central element of laboratory medicine for many years to come.