Protein Structure and Function - Protein are large.pdf

Protein are large, complex molecules comprised of 200 to 300 amino acids. In humans, there are 20 different amino acids used in the synthesis of proteins. Some of these amino acids can be synthesized by the body, while others must be obtained through the diet. The sequence of amino acids and the interactions that occur between them direct the structure and function of the protein. Proteins perform a broad range of functions throughout the body; therefore, maintaining an appropriate balance of protein synthesis and degradation is essential. This chapter discusses the general properties and functions of amino acids and proteins, the processes involved in protein synthesis and degradation, the clinical significance of select proteins, and test methods used to analyze proteins in the clinical laboratory. Amino Acids Overview Amino acids are simple organic compounds that serve as the building blocks of proteins, but they are also used in the synthesis of nitrogen-containing non-protein compounds such as purines, pyrimidines, porphyrins, creatine, histamine, thyroxine, epinephrine, and coenzyme NAD. This chapter will focus on discussion of proteins. Basic Structure A single amino acid contains at least one amino group and one carboxyl functional group. The N-terminal end of the amino group (–NH2 ) and the C-terminal end of the carboxyl group (– COOH) are bonded to an α-carbon forming an amino acid. 1 The basic structure of an amino acid is depicted in Figure 6.1. Amino acids are amphoteric, meaning they have acidic and basic tendencies. The amino group can accept a proton (basic) forming , while the carboxyl group can donate a proton (acidic) forming –COO–. Since amino acids can have neutral, positive, and negative functional groups, but their net charge is neutral, they are classified as zwitterions. TABLE 6.1 Figure 6.1 General structure of amino acid. © Jones & Bartlett Learning. Description Amino acids differ from one another by the chemical composition of their R group, referred to as side chains. 2 The R groups found on the amino acids used in human protein synthesis are shown in Table 6.1 as well as the three-letter and one-letter codes used to refer to each. The amino group of one amino acid can be covalently linked with the carboxyl group of another amino acid forming a peptide bond (Figure 6.2). When a chain of amino acids is linked by peptide bonds, it is known as a polypeptide, and a large polypeptide constitutes a protein. Proteins found in human plasma range from 100 to 150 amino acids in the length of their polypeptide chains. The content and arrangement of amino acids in a specific protein are determined by the sequence of nucleotide bases in the gene that encodes for that protein. 2 Amino Acids Utilized in Synthesis of Human Proteins Description Figure 6.2 Formation of a peptide bond. © Jones & Bartlett Learning. Description Metabolism About half of the amino acids required by humans for protein synthesis cannot be produced in vivo at a rapid enough rate to support growth. 2 These essential amino acids must be supplied by the diet in the form of proteins. Under normal circumstances, food is mechanically digested in the mouth then transported to the stomach, where chemical digestion occurs. In the stomach, gastrin stimulates the secretion of hydrochloric acid (HCl) and proteolytic enzymes, such as pepsin, to promote denaturation of the proteins and to catalyze their hydrolysis into polypeptides. In the small intestine, HCl is neutralized by sodium bicarbonate secreted from the pancreas under the direction of secretin, a digestive hormone, in order to protect the intestinal lining. Secretin and cholecystokinin, another digestive hormone secreted by the small intestine, stimulate the release of proteolytic enzymes, such as trypsin and chymotrypsin, from the pancreas and bile from the gallbladder. These pancreatic enzymes continue the process of digestion by hydrolyzing polypeptides into tripeptides, dipeptides, and the constituent amino acids. Figure 6.3 illustrates basic processes involved in digestion of dietary proteins. 3 Figure 6.3 Digestion of dietary proteins. © Jones & Bartlett Learning. Description Dipeptides, tripeptides, and amino acids are absorbed from the intestinal lumen through several cotransporters. Carrier proteins on the luminal surface of the epithelial cell bind with an ion, such as hydrogen or sodium, and a dipeptide, tripeptide, or amino acid, which are then transported into the cell. Peptidases within the cell cleave any remaining peptide bonds resulting in free amino acids. Facilitative transporters on the basolateral surface of the epithelial cell allow transport of the amino acids into the bloodstream, where they will be transported to the liver via the hepatic portal vein. In the liver, the amino acids will be used to synthesize new proteins and nonprotein nitrogen compounds or be used to generate energy through gluconeogenesis or ketogenesis. 3 Figure 6.4 illustrates the process for absorbing amino acids in the intestines. Figure 6.4 Intestinal absorption of amino acids. © Jones & Bartlett Learning. Description Classification Essential Amino Acids There are nine essential amino acids: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. 4 As mentioned, these amino acids cannot be synthesized in vivo at a fast enough rate, if at all, and must be acquired through dietary intake. Histidine is needed to help grow and repair body tissues, to maintain the myelin sheaths that protect nerve cells, and to serve as the precursor for several hormones and metabolites essential to renal, gastric, and immune function (e.g., histamine). It also plays an important role in the synthesis of red and white blood cells, in protecting the body from heavy metal toxicity, and serving as a source of carbon atoms in the synthesis of purines for DNA and RNA synthesis. 5 Isoleucine, leucine, and valine are branched-chain amino acids, collectively referred to as the branched-chain amino acid group. Isoleucine is concentrated in muscle tissues and is essential for a number of functions such as wound healing, immune function, glucose homeostasis and hemoglobin formation. 6 Leucine is also important in the regulation of blood glucose and wound healing as well as prevention of muscle protein degradation subsequent to trauma. 7 Valine aids in determining the tertiary structure of proteins and promotes mental health and muscle coordination. 8 Lysine plays a role in the production of antibodies and is required for maintaining healthy tissues. It also helps in the absorption and conservation of calcium and serves an important role in the formation of collagen, a component of cartilage and connective tissue. 9 Methionine helps to initiate translation of messenger RNA, stabilizes protein structure, and is an important cellular antioxidant. It is an important source of sulfur, which is required for normal metabolism and growth. Methionine also assists in the breakdown of fats, helps to detoxify lead and other heavy metals, helps diminish muscle weakness, and prevents brittle hair. Methionine reacts with adenosine triphosphate (ATP) in the synthesis of many important substances, including epinephrine and choline, and is essential for proper absorption of selenium and zinc. 10 Phenylalanine is the metabolic precursor for tyrosine, which, in turn, is the precursor for the neurotransmitters dopamine, norepinephrine, and epinephrine, collectively referred to as catecholamines. Catecholamines perform several important roles in maintaining homeostasis through the autonomic nervous system such as promoting alertness and vitality, elevating mood, decreasing pain, and aiding in memory and learning. When deficient, downstream products such as tyrosine will also be decreased. 11 Threonine is an important component in the formation of collagen, elastin, and tooth enamel. It is also important in the production of neurotransmitters and overall health of the nervous system. Additionally, threonine helps maintain proper protein balance in the body, aids in liver function, and assists in metabolism of porphyrins and fats. 12 Tryptophan is a metabolic precursor for serotonin and melatonin, which regulate appetite, mood, sleep, and pain. As such, tryptophan is a natural relaxant that helps alleviate insomnia by inducing sleep, soothes anxiety, and reduces depression. Tryptophan is also essential for the production of niacin. 13 Nonessential Amino Acids The human body can synthesize adequate amounts of alanine, asparagine, aspartic acid, glutamic acid, selenocysteine, and serine. Since additional dietary intake of these amino acids is unnecessary, they are classified as nonessential. 4 Alanine is a product of the breakdown of DNA, anserine, and carnosine. It is also formed as a result of glycolysis in muscle tissue and the conversion of pyruvate, a pivotal compound in carbohydrate metabolism, into α- ketoglutarate. Alanine plays a major role in the transfer of nitrogen from peripheral tissues to the liver for processing and excretion and strengthens the immune system through production of antibodies. As a ketogenic amine, alanine also serves as a source of energy for the central nervous system, brain, and muscle tissues. It also helps in reducing the buildup of toxic substances released when muscle protein is broken down. 14 Asparagine is derived from aspartic acid and ATP through transamidation, in which an amide group is transferred from one compound to another. It is one of the principal and most abundant amino acids involved in the transport of nitrogen. It is required by the nervous system and plays an important role in the synthesis of ammonia. 15 However, its primary function is in the conversion of one amino acid into another via amination or transamination. Amination is the process by which an amine group is introduced into an organic molecule, and transamination is the transfer of an amino acid to an α-ketoacid. Aspartic acid, or aspartate, is synthesized from oxaloacetate through transamination in the citric acid cycle and the urea cycle. An important role of aspartic acid is to serve as the precursor for several other amino acids, such as asparagine, arginine, lysine, methionine, threonine, and isoleucine, as well as several nucleotides. It also serves as a neurotransmitter and participates in the generation of glucose from non-carbohydrate substrates, a process known as gluconeogenesis. 16 Glutamic acid, or glutamate, is produced from the transamination of amino acids such as alanine and aspartic acid. Glutamic acid serves as a neurotransmitter and has an important role in the metabolism of carbohydrates and fats as well as facilitating amino acid synthesis and degradation. 17 Selenocysteine, unlike other amino acids present in proteins, is not coded for directly in the genetic code. Rather, it is encoded by a UGA codon, which is normally a stop codon; however, like the other amino acids used by cells, selenocysteine has a specialized transfer RNA (tRNA). As its name implies, selenocysteine is the selenium analogue of cysteine, in which a seleniumatom replaces sulfur. Selenocysteine is present in several enzymes, such as formate dehydrogenases, glycine reductases, and some hydrogenases. 18 Serine is synthesized from 3-phosphoglycerate, which is an intermediate in glycolysis. Serine is needed for the proper metabolism of lipids and fatty acids and plays an important role in the synthetic pathways for pyrimidines, purines, creatine, and porphyrins. It is highly concentrated in all cell membranes, serves as a component of the protective myelin sheaths surrounding nerve fibers, and aids in the production of antibodies. 19 Conditionally Essential Amino Acids Arginine, cysteine, glutamine, glycine, proline, and tyrosine are classified as semi-essential, or conditionally essential, amino acids because adults can synthesize adequate amounts to meet the demands of the body. 4 Infants that are born prematurely, as well as individuals that are in severe catabolic distress, may need supplementation of these amino acids. 2 If a conditionally essential amino acid is produced from an essential amino acid, a deficiency in the essential amino acid will likely require supplementation of both. For example, tyrosine, a conditionally essential amino acid, is produced from phenylalanine, an essential amino acid. If dietary intake of phenylalanine is insufficient, tyrosine will also be deficient and may require dietary supplementation. Arginine is a complex amino acid often found at the catalytic site in proteins and enzymes due to its amine-containing side chain. Arginine plays an important role in cell division, wound healing, stimulation of protein synthesis, immune function, and the release of hormones. Another important role of arginine is in the conversion of ammonia into urea. 20 Cysteine should not be confused with cystine, as they are two different amino acids. Cysteine is potentially toxic, so it is absorbed during digestion as cystine, which is more stable in the gastrointestinal tract and less toxic. Cystine is transported to cells, where it is reduced to two cysteine molecules upon cell entry. Cysteine can also be synthesized in vivo frommethionine through a series of enzymatic reactions. Cysteine is an important structural and functional component of many proteins. It is found in beta-keratin and is important in collagen formation. It also has antioxidant properties and involvement in metabolism of other molecules. 21 Glutamine is synthesized from glutamic acid through the addition of an ammonia group and can donate the ammonia group to form urea, which is excreted by the kidneys. As such, glutamine plays an integral role in regulation of ammonia, which is considered a toxic substance. Additionally, glutamine has many other important functions, including renal maintenance of the acid– base balance, providing fuel for a healthy digestive tract, and acting as the basis of the building blocks for synthesis of RNA and DNA. Glutamine is also a source of cellular energy and aids in immune function. 22 Glycine is synthesized from the amino acid serine. It is essential for synthesis of nucleic acids, bile acids, proteins, peptides, purines, ATP, porphyrins, hemoglobin, glutathione, creatine, bile salts, glucose, glycogen, and other amino acids. Glycine is also an inhibitory neurotransmitter in the central nervous system and a metal-complexing agent. Additionally, glycine limits muscle degeneration, improves glycogen storage, promotes healing, and is utilized by the liver in the detoxification of compounds. 1 Proline is produced from glutamic acid and other amino acids. It serves as the precursor for hydroxyproline, which is manufactured into collagen, tendons, ligaments, and cardiac tissue. Proline is also involved in wound healing, especially that of cartilage, and in the strengthening of joints, tendons, and cardiac tissue. 23 Tyrosine is synthesized from phenylalanine and serves as a precursor for adrenal and thyroid hormones. Tyrosine stimulates metabolism and the nervous system, acts as a mood elevator, and aids in function of the adrenal, thyroid, and pituitary glands. 24 Aminoacidopathies Aminoacidopathies are a class of inborn errors of metabolism in which an enzyme defect inhibits the body’s ability to metabolize certain amino acids. The abnormalities exist either in the activity of a specific enzyme in the metabolic pathway or in the membrane transport system for the amino acid. 25 Aminoacidopathies can cause severe medical complications, such as brain damage, due to the accumulation of toxic amino acids or their by-products in the blood and tissues. Due to the severity of these complications, newborn screening tests are routinely performed to aid in the early diagnosis of numerous inborn errors of metabolism. Refer to Chapter 24, Pregnancy and Prenatal Testing, for additional information on aminoacidopathies as well as maternal and newborn screening. Methods of Analysis Analysis for amino acids may be performed on urine samples to screen for disorders affecting amino acid transport and on plasma samples to diagnose and monitor aminoacidopathies. Amino acid concentrations may also be evaluated in the investigation of conditions involving the liver, endocrine glands, gastrointestinal tract, and kidneys and in patients with severe burns, muscular, neurologic, or neoplastic diseases. 1 Cerebrospinal fluid (CSF) may also be analyzed to aid in the diagnosis of select neurotransmitter disorders. Blood samples for amino acid analysis should be drawn after 6 to 8 hours of fasting to avoid the effect of absorbed amino acids originating from dietary proteins. The sample should be collected in a heparinized tube and the plasma removed from the cells within 2 hours of collection. Care should be taken to avoid aspirating the platelet and white cell layer to prevent contamination with amino acids from these cells. For example, the levels of aspartic acid and glutamic acid in white blood cells are about 100 times higher than plasma levels. Hemolyzed samples are unacceptable for the same reason. 26 For quantitation of urinary amino acids, a well-mixed aliquot from a first morning collection is preferred. 27 For CSF, samples should be immediately centrifuged and the supernatant transferred to a new tube in order to separate it from the cellular material. 28 For all sample types, testing should be performed immediately. If testing is to be delayed, samples can be refrigerated up to 24 hours or frozen for up to 1 month. Techniques, such as high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) may be used to quantitate amino acids and their metabolites in the patient sample. 29 MS/MS methods are considered to have higher specificity and greater sensitivity allowing for detection of lower concentrations of the amino acid(s) and an earlier diagnosis. Genetic assays using DNA analysis may also be performed to aid in the diagnosis and detection of carrier status in families with an inborn error of metabolism. Refer to Chapter 4, Analytic Techniques, for more information on HPLC-MS/MS. Proteins Overview Proteins catalyze almost all reactions in living cells, thereby controlling virtually all cellular processes. Some of their major functions include catalyzing biochemical reactions as enzymes, transporting metals such as iron and copper, acting as receptors for hormones, providing structure and support to cells, and participating in the immune response as antibodies. 4 Basic Structure Proteins consist of the elements carbon, oxygen, hydrogen, nitrogen, and sulfur. The fact that proteins contain nitrogen sets them apart from pure carbohydrates and lipids, which do not contain nitrogen atoms. Proteins are polymers built from one or more unbranched chains of amino acids. A typical protein contains 200 to 300 amino acids, but some are much smaller (peptides) or larger (e.g., titin has 27,000 to 35,000 amino acids). Proteins are considered macromolecules and range from approximately 6000 daltons for insulin to several million daltons for some structural proteins. 3 The four distinct levels of a protein’s structure: primary, secondary, tertiary, and quaternary are depicted in Figure 6.5. A protein’s primary structure refers to the number, type, and sequence of amino acids in the polypeptide chain. 4 In order to function properly, proteins must have the correct sequence of amino acids. For example, when the amino acid valine is substituted for glutamic acid in the β-chain of hemoglobin A, hemoglobin S is formed, resulting in sickle cell anemia. The location of certain amino acids in the primary structure will determine the secondary, tertiary, and quaternary structure of the protein. Figure 6.5 Four Levels of Protein Structure. (A) Primary structure: The sequence of amino acids linked by peptide bonds. (B) Secondary structure: Amino acids near to each other interact through hydrogen bonds. Secondary structure of α helix and β pleated sheet are shown. (C) Tertiary structure: Three-dimensional structure of the protein due to hydrophobic effect, ionic attraction, hydrogen bonds, and disulfide bonds. (D) Quaternary structure: Molecular association of more than one peptide/protein to form a dimer, trimer, tetramer, etc. by noncovalent bonds. © Jones & Bartlett Learning. Description Secondary structure refers to commonly formed arrangements stabilized by hydrogen bonds between nearby amino acids within the protein. Two of the main types of secondary structure are the α-helix and the β-pleated sheet, with most serum proteins forming a helix. Secondary structures add new properties to a protein such as strength and flexibility. 4 Tertiary structure refers to the overall shape, or conformation, of the protein molecule. The conformation is known as the fold, or the spatial relationship of the secondary structures to one another. Tertiary structures are three dimensional and result from the interaction of side chains, which are stabilized through the hydrophobic effect, ionic attraction, hydrogen bonds, and disulfide bonds. The function and physical and chemical properties of a protein are related to its tertiary structure. 4 Quaternary structure is the shape or structure that results from the interaction of more than one protein molecule, or protein subunits, referred to as a multimer, that functions as a single unit. Multimers are held together by noncovalent forces such as hydrogen bonds and electrostatic interactions and occur as dimers (two subunits), trimers (three subunits), tetramers (four subunits), etc. 4 Not all proteins have a quaternary structure. One example of a protein with a quaternary structure is hemoglobin, which has a tetramer globin composed of two α and two β subunits. When the secondary, tertiary, or quaternary structure of a protein is disturbed, the protein may lose its functional and chemical characteristics. This loss of its native, or naturally occurring, folded structure is called denaturation. Denaturation can be caused by heat, hydrolysis by strong acid or alkali, enzymatic action, exposure to urea or other substances, or exposure to ultraviolet light. General Chemical Properties The structure of a protein directly affects its chemical properties as each amino acid side chain has differing properties. 30 Proteins contain many ionizable groups on the side chains of their amino acids as well as on their N- and C-terminal ends. As a result, proteins can be positively and negatively charged. For example, the side chains of lysine, arginine, and histidine include basic groups (proton acceptors), whereas acidic groups (proton donors) are found on the side chains of glutamic acid and aspartic acid. The acid or base groups that are not involved in the peptide bond can exist in different charged forms depending on the pH of the surrounding environment (Figure 6.6). The pH of the solution, the pKa of the side chain, and the side chain’s environment influence the charge on each side chain. The relationship between pH, pKa , and charge for individual amino acids can be described by the Henderson-Hasselbalch equation: (Eq. 6.1) Figure 6.6 Charged states of amino acids. The charge of the protein depends upon the pH of its environment. © Wolters Kluwer. Description In general terms, as the pH of a solution increases, becoming more alkaline, deprotonation of the acidic and basic groups occurs; carboxyl groups are converted to carboxylate anions (R– COOH to R–COO– ) and ammonium groups are converted to amino groups (R–NH3+ to R–NH2 ). The pH at which an amino acid or protein has no net charge is known as its isoelectric point (pI). When the pH is greater than the pI, the protein has a net negative charge, and when the pH is less than the pI, the protein has a net positive charge. As such, the pI is the point at which the number of positively charged groups equals the number of negatively charged groups in a protein. If a protein is placed in a solution that has a pH greater than the pI, the protein will become negatively charged. If a protein is placed in a solution that has a pH less than the pI, the protein will become positively charged. Proteins differ in their pI values, but most occur in the pH range of 5.5 to 8.0. Because proteins carry different net charges depending on the pHof their environment, the difference in net charge at a given pH is the basis for several procedures used to separate and quantify proteins. A protein’s solubility is also dependent on the charge on its surface, with its lowest solubility at its pI, where the positive and negative charges balance resulting in a net charge of zero. If there is a charge at the protein surface, the protein is referred to as hydrophilic and prefers to interact with water rather than with other protein molecules, making it more soluble. Without a net surface charge, the protein is less soluble and protein–protein interactions and precipitation are more likely. Proteins in the blood require a pH in the range of 7.35 to 7.45 to remain soluble. In the laboratory, the relative solubilities of proteins may be used to separate and quantify them. Synthesis Plasma proteins are predominantly synthesized in the liver and secreted into circulation. Immunoglobulins are an exception, as they are synthesized by B lymphocytes. It is the information encoded in genes that provides each protein with its own unique amino acid sequence. The amino acid sequence of a polypeptide chain is determined by a corresponding sequence of bases (guanine, cytosine, adenine, and thymine) in the DNA contained in the specific gene. This genetic code is a set of three nucleotides known as codons, with each three-nucleotide combination standing for a specific amino acid. Because DNA contains four nucleotides, the total number of possible codons is 64; therefore, some redundancy in the genetic code allows for some amino acids to be specified by more than one codon. Doublestranded DNA unfolds in the nucleus, and one strand is used as a template for the formation of a complementary strand of mRNA in a process known as transcription. The mRNA is manufactured in the cell nucleus and then translocated across the nuclear membrane into the cytoplasm for translation. 30 In the process of translation, the mRNA strand is used as a template for protein synthesis by the ribosome. The mRNA is surrounded by the ribosome and is read three nucleotides at a time by matching each codon to its base pairing anticodon located on a tRNA molecule. tRNA is a short chain of RNA that occurs freely in the cytoplasm and brings amino acids to the ribosome. Each amino acid has a specific tRNA that contains three bases (anticodon) corresponding to the three bases in the mRNA (codon). The tRNA carries its particular amino acid to the ribosome and attaches to the mRNA in accordance with the matching codon. As each new amino acid is added, the preceding amino acid is transferred onto the amino group of the new amino acid and enzymes located in the ribosome form a peptide bond. The tRNA is released into the cytoplasm, where it can pick up another amino acid, and the cycle repeats. In this manner, the amino acids are aligned in sequence and linked by peptide bonds to form a polypeptide. This process continues until a stop codon is reached signaling that all amino acids have been joined in the specific sequence to form the polypeptide chain. When the terminal codon is reached, the peptide chain is released and the ribosome and mRNA dissociate. 30 Figure 6.7 illustrates basic protein synthesis through transcription and translation. Figure 6.7 Protein synthesis through transcription and translation. © Jones & Bartlett Learning. Description Protein targeting is the process by which proteins are directed to the location they are needed (translocated) during or after translation. This occurs under the direction of signal peptides, known as signal recognition particles (SRPs), that occur at their N-terminus. In the absence of an SRP, the protein will be synthesized and remain in the cytosol. Proteins with an SRP that signals post-translational translocation are synthesized in the cytosol then moved to a nonendomembane location such as the nucleus or peroxisomes. Intracellular proteins are generally synthesized in this fashion, whereas proteins destined for the endomembrane systemor secretion from the liver are made on ribosomes attached to the rough endoplasmic reticulum. Translation for these proteins also begins in the cytosol, but is paused by SRPs signaling cotranslational translocation. After attaching to the endoplasmic reticulum, the SRP is cleaved, allowing translation to resume. The polypeptide is delivered through a protein channel into the lumen of the endoplasmic reticulum. Additional SRPs can direct the polypeptide to organelles in the endomembrane system or to the cell membrane for secretion into circulation. 31 Catabolism and Nitrogen Balance Unlike fats and carbohydrates, nitrogen- containing compounds, such as proteins, have no designated storage. Most proteins in the body are repetitively synthesized (anabolism) and then degraded (catabolism) allowing for efficient recycling of amino acids. These processes result in a turnover of about 125 to 220 g of protein each day, with the rate of individual proteins varying widely. For example, plasma proteins and most intracellular proteins are rapidly degraded, having half-lives of hours or days, whereas some of the structural proteins, such as collagen, are metabolically stable and have half-lives of years. In health, nitrogen balance is maintained by equal intake and excretion of amino acids. Pregnant women, growing children, and adults recovering from major illness are often in positive nitrogen balance because their nitrogen intake exceeds their loss. When more nitrogen is excreted than incorporated, an individual is in negative nitrogen balance, which may occur in conditions associated with excessive tissue destruction, such as burns, wasting diseases, continual high fevers, or starvation. When in excess, amino acids can be converted to urea for excretion by the kidneys and into glucose or ketones to be used as an alternative source of energy. These catabolic processes typically involve transamination and oxidative deamination of the amino acid, which primarily occurs in the liver. Transamination involves transferring an amino group from the α-amino acid to the keto-carbon of an α-ketoglutarate resulting in the production of glutamate and an αketoacid. These reversible reactions are catalyzed by a group of intracellular enzymes known as aminotransferases or transaminases. Glutamate is then deaminated, catalyzed by glutamate dehydrogenase, forming ammonia and α- ketoglutarate. For some amino acids, such as serine and threonine, only deamination is required to produce ammonia and the αketoglutarate. In the hepatocytes, ammonia is then converted to urea by the urea cycle, which is less toxic and can be excreted in urine. 32 The resultant ketoacids enter into the Krebs cycle (citric acid cycle) to release stored energy derived from carbohydrates, fats, and proteins. Glucogenic amino acids generate precursors of glucose, such as pyruvate or citric acid cycle intermediates. Examples include alanine, which can be deaminated to pyruvate; arginine, which is converted to α- ketoglutarate; and aspartic acid, which is converted to oxaloacetate. Ketogenic amino acids, such as leucine and lysine, are degraded to acetyl-CoA or acetoacetyl-CoA and form ketone bodies. Isoleucine, TABLE 6.2 phenylalanine, tryptophan, tyrosine, and threonine are both ketogenic and glucogenic. 32 Hormonal Regulation of Protein Metabolism In some tissues, protein metabolism is regulated through interaction of several hormones including insulin, growth hormone (GH), insulin-like growth factor I (IGF-1), sex hormones (testosterone/estrogen), glucocorticoids (e.g. cortisol), glucagon, catecholamines, and thyroxine. 33 The effects of each of these hormones on protein synthesis and/or degradation are listed in Table 6.2. Hormones Regulating Protein Metabolism Hormone Primary Influence on Protein Metabolism Insulin Suppresses proteolysis Growth Hormone Stimulates protein synthesis Insulin-like Growth Factor-1 Inhibits proteolysis in fasting state, but stimulates protein synthesis in fed state Glucocorticoids Stimulates protein degradation in muscles Glucagon Promotes uptake of amino acids in the liver Sex Hormones Increases protein deposition in tissues Catecholamines Increases production of gluconeogenic amino acids Thyroxine Increases the basal metabolic rate © Jones & Bartlett Learning. Classification Proteins are often discussed or classified based on their structure, composition, and function. Simple proteins contain peptide chains composed of only amino acids and may be globular or fibrous in shape. Globular proteins are globe-like, have symmetrical proteins that are soluble in water, and commonly function as transporters, enzymes, and messengers. Examples of globular proteins are albumin, hemoglobin, and the immunoglobulins, IgG, IgA, and IgM. Fibrous proteins form long protein filaments or subunits, are asymmetrical and usually inert, and are generally water- insoluble due to their hydrophobic R groups. They provide structure to cells, such as connective tissues, tendons, bone, and muscle. Examples of fibrous proteins include troponin and collagen. Conjugated or complex proteins consist of a protein and a nonprotein. The non-amino part of a conjugated protein is generally referred to as the prosthetic group, which may be a lipid, carbohydrate, porphyrin, metal, etc. In a conjugated protein, it is the prosthetic group that defines the characteristics of the protein. Examples of conjugated proteins include metalloproteins, glycoproteins, lipoproteins, and nucleoproteins. Metalloproteins have a metal ion attached to the protein, either directly, as in ferritin, which contains iron, and ceruloplasmin, which contains copper, or as a complex metal such as hemoglobin. Lipoproteins, including highdensity lipoproteins (HDLs) and very low-density lipoproteins (VLDLs), are composed of a protein and a lipid, such as cholesterol or triglyceride. There are several terms used to describe conjugated proteins that are bound to carbohydrates. In general, molecules, such as haptoglobin and α1 - antitrypsin, that have a composition of 10% to 40% carbohydrate are referred to as glycoproteins. When the percentage of carbohydrate is greater than 40%, the protein conjugate is referred to as a mucoprotein or proteoglycan. An example of a mucoprotein is mucin, which is a lubricant that protects body surfaces from friction or erosion. Nucleoproteins are proteins that are combined with nucleic acids. Chromatin is an example of a nucleoprotein as it is a complex of DNA and protein. Enzymes are proteins that catalyze biochemical reactions. They are normally found intracellularly and are released into the bloodstream when tissue damage occurs, making enzyme measurements an important diagnostic tool. Aminotransferases, dehydrogenases, and phosphatases are just a few examples of enzyme groups routinely analyzed in the clinical laboratory to evaluate possible tissue damage. Hormones are chemical messenger proteins that control the action(s) of specific cells or organs. Hormones directly affect growth and development, metabolism, sexual function, reproduction, and behavior. Examples of hormones commonly measured in the clinical laboratory include insulin, testosterone, growth hormone, thyroid stimulating hormone, and cortisol. Many proteins serve as transporters for molecules such as hormones, vitamins, minerals, and lipids, across a biologic membrane. Hemoglobin, albumin, ceruloplasmin, haptoglobin, and transferrin are examples of transport proteins. Immunoglobulins, or antibodies, are proteins that are produced by B cells (lymphocytes) in the bone marrow. Immunoglobulins mediate the humoral immune response to identify and neutralize foreign antigens. Examples of immunoglobulins of clinical importance are IgG, IgM, IgE, IgD, and IgA. Structural proteins are fibrous proteins that provide structure to many cells and tissues throughout the body, such as muscle, tendons, and bone matrix. Examples include collagen, elastin, and keratin. Storage proteins serve as reservoirs for metal ions and amino acids so they can be stored without causing harm to the cell and released when needed. An example is ferritin, which stores iron in the hepatocyte until it is needed in the synthesis of hemoglobin. Some proteins serve as an energy source for tissues and muscle. Creatine is one example of an energy source protein, as it helps to supply energy to cells throughout the body but is primarily found in muscle tissue. Proteins provide up to 20% of the total energy required daily by the body. Maintaining water distribution throughout the compartments of the body is another important function of proteins. Due to their size, plasma proteins cannot cross the capillary membrane. As a result of colloid osmotic pressure, water is absorbed from the tissue into the capillary. When the concentration of plasma proteins is significantly decreased, the decrease in osmotic pressure results in increased levels of interstitial fluid and edema in the tissues. This often occurs in renal disease when proteins are inappropriately excreted in urine and plasma protein concentrations are decreased. Proteins also play an important role in maintaining the acid– base balance by serving as buffers to maintain pH. They are also involved in hemostasis by participating in the formation and dissolution of blood clots. TABLE 6.3 Plasma Proteins Plasma proteins are commonly analyzed in the clinical laboratory and can be divided into two major groups: albumin and globulins. Routine analysis of blood specimens will typically include measurement of total protein and albumin and calculation of the albumin-to-globulin (A/G) ratio; however, there are many other clinically important proteins that may be measured when indicated by clinical presentation. Some of the more significant plasma proteins and their function, structure, and relation to disease states are discussed below. The characteristics of select plasma proteins are listed in Table 6.3. Characteristics of Select Plasma Proteins Description Prealbumin Prealbumin, or transthyretin, is so named because it migrates before albumin in classic serumprotein electrophoresis (SPE). It can also be separated using high-resolution electrophoresis (HRE) or immunoelectrophoresis techniques. Prealbumin is a transport protein for the thyroid hormones, thyroxine (T4 ) and triiodothyronine (T3 ). It also forms a complex with retinol- binding protein to transport retinol (vitamin A) and is rich in the amino acid tryptophan. Serum and plasma concentrations may be decreased in hepatic damage due to decreased protein synthesis, during an acute-phase inflammatory response, or as a result of tissue necrosis. A low concentration may also indicate poor nutritional status. Diets deficient in protein may not provide sufficient amino acids for protein synthesis by the liver, resulting in decreased plasma concentrations of prealbumin, albumin, and globulins. Prealbumin has a half-life of approximately 2 days, so blood concentrations decrease rapidly when protein synthesis is inhibited. Blood concentrations may be increased in patients receiving steroid therapy, who have issues with alcohol abuse, or who are in chronic renal failure. 34 Albumin Albumin is synthesized in the liver at a rate of 9 to 12 g/day. Because it is decreased during an acute-phase response, it is classified as a negative acute-phase reactant, meaning it decreases in the presence of acute disease states, such as inflammation, infection or injury. It is the most abundant plasma protein and is also found in significant amounts in the extravascular (interstitial) space. Interestingly, the amount of extravascular albumin exceeds the intravascular amount by about 30%; however, the concentration of albumin in plasma (albumin mass/plasma volume) is much greater. Albumin leaves the bloodstream at a rate of 4% to 5%of the total intravascular concentration per hour. This rate of movement, known as the transcapillary escape rate, measures the systemic capillary efflux of albumin. Due to its high presence in the plasma, albumin is responsible for nearly 80% of the colloid osmotic pressure, meaning it is the primary protein involved in maintaining the fluid balance between the intravascular and extravascular spaces. Albumin also plays an important role in maintaining a homeostatic pH by serving as a buffer in the circulation. 35 Another key function of albumin is its ability to bind and transport a large variety of substances throughout the body. There are four binding sites on albumin, each with varying specificities for the different substances requiring transport in circulation. For example, albumin is involved in the transport of thyroid hormones, unconjugated bilirubin, fat-soluble hormones, iron, fatty acids, calcium (Ca 2+ ), magnesium (Mg 2+ ), and many drugs such as salicylic acid (aspirin). Albumin also binds well with certain dyes, which is the basis for several methods used to quantitate it in fluids such as serum, plasma, and urine. 35 Decreased blood concentrations of albumin are most commonly associated with an acute inflammatory response as albumin is a negative acute-phase reactant. Liver and kidney disease may also result in low blood albumin concentrations. The liver is a primary site for protein synthesis; damage to hepatocytes, such as in liver cirrhosis, may result in decreased protein synthesis. Note that an increase in globulins occurs in early liver cirrhosis, which balances the loss in albumin to give a total protein concentration within acceptable limits. 36 Albumin is normally excreted in very small amounts by the kidneys; however, increased renal loss of albumin, termed albuminuria, commonly occurs in renal disease. This increased excretion occurs when the glomerulus no longer restricts the passage of proteins from the blood into the ultrafiltrate, as occurs in nephrotic syndrome and glomerular damage associated with diabetes mellitus. 37 Low albumin concentrations may also be the result of malnutrition and malabsorption in which an inadequate ingestion of proteins or amino acid-rich foods results in decreased protein synthesis by the liver. Less commonly, low blood albumin concentrations occur as a result of hypothyroidism, burns or exfoliative dermatitis, dilution by excessive intake (polydipsia), or infusion of intravenous liquids. 36 Albumin may be redistributed by hemodilution, increased capillary permeability, or decreased lymphatic clearance. In sepsis, there is a profound reduction in plasma albumin associated with marked fluid shifts. Mutations resulting from an autosomal recessive trait can cause an absence of albumin, known as analbuminemia. Bisalbuminemia, which is inherited or acquired, is characterized by the presence of two albumin bands during serum protein electrophoresis instead of the single band usually observed. Elevated albumin concentrations are generally considered clinically insignificant as they most likely indicate dehydration or excessive albumin infusion. Another important clinical application for measuring albumin is to determine the percent glycated albumin as a means to monitor short-term efficacy of therapies for diabetes mellitus. 38 Typically, glycated hemoglobin is measured to assess glucose concentrations over the past 2 to 3 months for patients with diabetes mellitus, as the lifespan of a red blood cell is approximately 120 days. While this is the preferred test for monitoring long-term therapy, it may not be appropriate for patients with certain red blood cell disorders. For this patient population, glycated albumin or fructosamine determinations may be monitored instead. Fructosamine is a measure of the non-enzymatic glycation of circulating plasma proteins, including albumin, globulins, and lipoproteins. The half-life of serum albumin is approximately 20 days, making it a better indicator of short-term glycemic control. 39 Methods used to determine the concentration of glycated albumin in serum often use affinity chromatography based on specific interaction of boronic acids with the glycated proteins. Globulins There are four major types, or fractions, of globulins designated as α1 , α2 , β, and γ based on their electrophoretic mobilities. Each fraction consists of a number of different proteins with individual functions. Select globulins from each fraction are discussed below. α1 - Globulins α1 -Antitrypsin (AAT) is a glycoprotein synthesized predominantly in the liver. Its main function is to inhibit neutrophil elastase, a protease released from neutrophils and macrophages during an infection. Mutations in the SERPINA1 gene can lead to a deficiency of α1 -antitrypsin or an abnormal form of the protein that does not properly control neutrophil elastase. As a result, damage to the alveoli can lead to emphysema. The abnormal form of α1 -antitrypsin can also accumulate in the liver, causing cirrhosis. α1 - Antitrypsin is a positive acute-phase reactant, meaning it increases in the presence of an acute onset of disease states, such as inflammation, infections, or injuries; therefore, increased levels are seen in inflammatory reactions, as well as in pregnancy and contraceptive use. Decreased α1 -antitrypsin levels are most often identified by the absence of an α1 -globulin band on serum protein electrophoresis because α1 -antitrypsin accounts for approximately 90% of the α1 -globulin fraction. 40 α1 -Fetoprotein (AFP) is synthesized in utero by the developing embryo and then by the fetal liver and gastrointestinal tract. AFP concentrations decrease gradually after birth, reaching adult concentrations by 8 to 12 months of age. As a positive acute-phase reactant, increases of AFP are seen in inflammatory conditions. 41 Maternal serum with an elevated AFP concentration is associated with increased likelihood of spina bifida, neural tube defects, abdominal wall defects, anencephaly, general fetal distress, and the presence of multiple gestation. Low levels of maternal AFP indicate an increased risk for trisomy 21 (Down syndrome) and trisomy 18 (Edwards syndrome). 42 Refer to Chapter 24, Pregnancy and Prenatal Testing, for additional information on maternal screening of AFP. AFP may also be used as a tumor marker, as elevated concentrations are associated with hepatocellular carcinoma, metastatic liver disease, and nonseminomatous germ cell tumors. AFP’s utility as a tumor marker will be discussed further in Chapter 28, Tumor Markers. α1 -Acid glycoprotein (AGP), or orosomucoid, is a positive acute-phase reactant produced primarily by the liver. Its physiological functions include maintaining the barrier function of capillaries, regulating immunity, mediating sphingomyelin metabolism, and acting as a transport protein. AGP elevates as a result of stress, inflammation, tissue damage, acute myocardial infarction (AMI), trauma, pregnancy, cancer, pneumonia, rheumatoid arthritis, and surgery. 43 α2 -Globulins Haptoglobin (Hp) is an α2 -glycoprotein synthesized in the liver as a tetramer consisting of two α and two β chains. It is a positive acute-phase reactant that increases in many inflammatory diseases, such as ulcerative colitis, acute rheumatic disease, acute myocardial infarction, and severe infection. The primary function of haptoglobin is to bind free hemoglobin to prevent the loss of its constituent, iron, into the urine. When haptoglobin and hemoglobin attach, mononuclear phagocytic cells, predominantly in the spleen, remove the haptoglobin–hemoglobin complex from circulation. The hemoglobin constituents, iron and amino acids, can be recycled, but haptoglobin is destroyed in the process. For this reason, haptoglobin concentrations are primarily used to evaluate possible hemolytic anemias and to aid in distinguishing intravascular hemolysis from extravascular hemolysis. Patients with a hemolytic anemia have a decreased haptoglobin concentration due to intravascular hemolysis. In contrast, the haptoglobin concentration would be normal if the anemia is related to extravascular destruction of red blood cells in organs such as the spleen and liver. In extravascular hemolysis, the hemoglobin is not released into the bloodstream, and the haptoglobin therefore stays intact. In the case of in vitro hemolysis, the haptoglobin concentration would appear normal, indicating hemolysis occurred during or after the collection. If haptoglobin concentrations are decreased without any sign of hemolytic anemia, it is possible the liver is not producing adequate amounts of haptoglobin. 44 Ceruloplasmin is a copper-containing, α2 -glycoprotein synthesized in the liver. As a positive acute-phase reactant, it is frequently elevated in inflammation, severe infection, and tissue damage and may be increased with some cancers. Ceruloplasmin may also be increased during pregnancy and in patients who are taking estrogen, oral contraceptives, and medications such as carbamazepine, phenobarbital, and valproic acid. The primary function of ceruloplasmin is to serve as a transport protein for copper. Approximately 90% of copper in circulation is bound to ceruloplasmin, with the remaining 10% bound to albumin. Ceruloplasmin is primarily measured along with blood and urine copper concentrations to aid in the diagnosis of Wilson’s disease, an inherited autosomal recessive disorder. Patients with Wilson’s disease generally have decreased concentrations of ceruloplasmin, increased serum concentrations of free copper, and increased urinary excretion of copper. 45 Due to the limited availability of ceruloplasmin, free copper is deposited in the liver, brain, and other organs, which results in hepatic cirrhosis and neurologic damage. Copper is also deposited in the cornea producing the characteristic Kayser-Fleischer rings. Low ceruloplasmin is also seen in malnutrition, malabsorption, severe liver disease, nephrotic syndrome, and Menkes’ syndrome, in which a decreased absorption of copper results in a decrease in ceruloplasmin. 46 α2 -Macroglobulin, a major component of the α2 -globulin fraction, is a tetramer of four identical subunits synthesized by the liver. Alpha2 - macroglobulin is a protease inhibitor. It inhibits proteases such as pepsin, trypsin, thrombin, and plasmin, which reduces the accessibility of the protease functional sites but does not completely inactivate them. After binding with proteases, α2 -macroglobulin is removed by the mononuclear phagocytic system. Additional functions of α2 - macroglobulin include regulation of growth factors and cytokines as well as chaperoning of misfolded proteins. In nephrotic syndrome, serum concentrations of α2 -macroglobulin may appear increased relative to other proteins. Its large size inhibits filtration at the renal glomeruli, making measurement of α2 -macroglobulin useful in evaluation of renal disease and damage to the glomeruli. Increased concentrations may be seen during pregnancy, while using hormonebased contraceptives, and in patients with renal disease secondary to diabetes mellitus or hepatorenal syndromes. 47 β-Globulins Transferrin, a negative acute- phase glycoprotein synthesized by the liver, is the major component of the β-globulin fraction on protein electrophoresis. Transferrin’s primary function is to bind and transport iron to and from storage sites, such as the liver and bone marrow, and to prevent iron from being inappropriately deposited in other tissues. Because transferrin is made in the liver, it may be decreased in patients with liver disease or when dietary intake of proteins is insufficient. A low transferrin concentration may also be due to excessive loss through the kidneys, as occurs in protein-losing disorders such as nephrotic syndrome. Many conditions, including infection, inflammation, and malignancy, lead to decreased transferrin levels, as it is a negative acute-phase reactant. Low transferrin concentrations can lead to anemia due to decreased delivery of iron to the bone marrow and subsequent impairment of hemoglobin synthesis. A deficiency of plasma transferrin may also result in the inappropriate accumulation and precipitation of iron in tissues as hemosiderin. An increase in transferrin is generally associated with iron-deficiency anemia as a compensatory mechanism to promote iron absorption and mobilization. Similarly, transferrin concentrations decrease in patients with iron overload. As such, transferrin concentrations are routinely measured to aid in determining the cause of an anemia, to evaluate processes associated with iron metabolism, and to determine the total iron- binding capacity of the blood. 48 Additional information on iron metabolism and iron status studies can be found in Chapter 27, Trace Elements, Toxic Elements, and Vitamins. Hemopexin is an acute-phase β-globulin, synthesized in the parenchymal cells of the liver, whose main function is to bind with free heme. When heme is released during the breakdown of hemoglobin, myoglobin, or catalase, it binds to hemopexin. The heme– hemopexin complex is carried to the liver, where it is destroyed, releasing the heme components to be recycled. In this manner, hemopexin preserves the body’s iron and amino acid stores and prevents oxidative damage by free heme. Because hemopexin is destroyed during this process, low concentrations are associated with intravascular hemolytic conditions such as hemolytic anemias. Increased concentrations of hemopexin are found in inflammation, diabetes mellitus, Duchenne-type muscular dystrophy, and some malignancies, especially melanomas. 49 Lipoproteins are complexes of proteins and lipids whose function is to transport cholesterol, triglycerides, and phospholipids in the bloodstream. Lipoproteins are subclassified according to their apolipoprotein and lipid content into chylomicrons, very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). In high-resolution electrophoresis, HDL migrates between the albumin and α1 -globulin bands, VLDL migrates at the beginning of the β-globulin band (pre-β), and LDL appears as a separate band in the β-globulin region. 50 A detailed discussion of the structure, function, and laboratory methods used to measure lipoproteins can be found in Chapter 10, Lipids and Lipoproteins. β2 -Microglobulin (B2M) is the light chain component of the major histocompatibility complex or human leukocyte antigen (HLA). This protein is found on the surface of most nucleated cells and is present in high concentrations on lymphocytes. Because of its small size (molecular weight: 11,800 kDa), B2M is filtered by the renal glomeruli, but is almost completely reabsorbed and catabolized in the proximal tubules. Elevated plasma concentrations reflect impaired renal clearance or overproduction, which occurs in a number of inflammatory diseases, such as rheumatoid arthritis and systemic lupus erythematosus (SLE). In patients with human immunodeficiency virus (HIV), a high B2M level in the absence of renal failure indicates a large lymphocyte turnover rate, suggesting the virus is killing lymphocytes. Elevated urine concentrations of B2M are associated with renal tubular damage or disease due to decreased reabsorption. 51 The complement system is a natural defense mechanism against infections. These proteins are synthesized in the liver as single polypeptide chains and circulate in the blood as nonfunctional precursors. Complement C3 is the most abundant complement protein in human plasma, with complement C4 being the second most abundant. In the classic pathway, activation of these proteins begins when the first complement factor, C1q, binds to an antigen– antibody complex. Each complement protein (C2–C9) is then activated sequentially and can bind to the membrane of the cell to which the antigen–antibody complex is bound, eventually leading to cell lysis. An alternate pathway for complement activation, known as the properdin pathway, bypasses early components of the cascade allowing the process to begin with C3. This pathway is triggered by different substances and does not require the presence of an antibody; however, the lytic attack on the cell membrane follows the same sequence of C5 to C9. Increased levels of both C3 and C4 are linked to acute inflammatory disease and tissue inflammation. In contrast, decreased levels of C3 are typically associated with autoimmune disease, neonatal respiratory distress syndrome, bacteremia, tissue injury, and chronic hepatitis. Complement C3 has been found to be important in the pathogenesis of age-related macular degeneration, and this finding further underscores the influence of the complement pathway in the pathogenesis of this disease. Decreased levels of C4 are seen in disseminated intravascular coagulation (DIC), acute glomerulonephritis, chronic hepatitis, and SLE. Inherited deficiencies of individual complement proteins have also been described. 52 Fibrinogen is one of the largest proteins in blood plasma. It is synthesized in the liver and classified as a glycoprotein due to its considerable carbohydrate content. The function of fibrinogen is to form a fibrin clot when activated by thrombin; therefore, all fibrinogen is virtually removed in the clotting process and should not be present in serum specimens. Fibrinogen is a positive acute-phase reactant and increases significantly during an inflammatory process. Fibrinogen levels also rise with pregnancy and the use of oral contraceptives. Decreased values generally reflect extensive coagulation during which fibrinogen is consumed. On electrophoresis, fibrinogen can be seen as a small, distinct band between the β- and γ-globulin regions and indicates use of plasma instead of serum. 53 C-reactive protein (CRP) is synthesized in the liver and is one of the first acute-phase reactants to rise in response to inflammatory disease. CRP rises sharply whenever there is tissue inflammation and has been demonstrated through many studies to be important in the development of atherosclerosis. Atherosclerosis, in addition to being a disease of lipid accumulation, also represents a chronic inflammatory process, which leads to an elevated CRP concentration. Elevated levels of CRP stimulate the production of tissue factor, which initiates coagulation, activates complement, and binds to LDL in the atherosclerotic plaque. As a result, high-sensitivity CRP (hsCRP) concentrations are measured during the evaluation of arteriosclerosis and as a risk indicator for cardiovascular disease. CRP is not tissue-specific; however, it does have value as a general indicator of inflammation. Normally, there are minimal amounts of CRP in blood. A high or increasing amount suggests an acute infection or inflammation. Results above 1 mg/dL are considered high for CRP, and most infections and inflammatory processes result in levels above 10 mg/dL. In cases of inflammatory rheumatic diseases, such as rheumatoid arthritis and SLE, CRP is used to assess the effectiveness of treatment and monitor periods of disease eruption. However, even in known cases of inflammatory disease, a low CRP concentration is possible and is not indicative of the absence of inflammation. CRP is also significantly elevated in acute rheumatic fever, bacterial infections, myocardial infarctions, carcinomatosis, gout, and viral infections. 54 High-sensitivity CRP (hsCRP) refers to a monoclonal antibody–based test method that can detect CRP at levels below 1 mg/L. This test is most commonly used to determine risk of cardiovascular disease (CVD) as high levels of CRP consistently predict recurrent coronary events in patients with unstable angina and AMI. 54 A detailed discussion of cardiovascular risk factors and markers of cardiac damage can be found in Chapter 20, Cardiac Function. γ-Globulins Immunoglobulins (Igs), or antibodies, are glycoproteins composed predominately of protein with a small percentage of carbohydrate (14–18%). Immunoglobulins are produced by white blood cells, known as B lymphocytes, that confer humoral immunity. These proteins consist of two identical heavy (H) chains and two identical light (L) chains linked by two disulfide bonds. They can be in the form of a monomer (one unit), dimer (two units), or a pentamer (five units). There are five classes, or isotypes, of immunoglobulins: IgG, IgA, IgM, IgD, and IgE. This classification is based on the type of heavy chain the immunoglobin possesses, which include γ, α, μ, δ, and ε, respectively. There are two types of light chains, kappa (κ) and lambda (λ). For example, IgG has two γ-type heavy chains and two identical light chains, either κ or λ. Each heavy chain has two regions; the constant region and the variable region. The constant region is identical in all antibodies of the same isotype but differs in antibodies of different isotypes. The variable region of the heavy chain differs in antibodies produced by different B lymphocytes but is the same for all antibodies produced by a single B lymphocyte or B-lymphocyte clone. The ratio of κ to λ chains is 2:1, which is sometimes used as a marker of immune abnormalities. 55 The N-terminal regions of the heavy and light chains exhibit highly variable amino acid composition referred to as VH and VL, respectively. This variable region is involved in antigen binding. Similar to the variable region, the constant domains of light and heavy chains are referred to as CL and CH, respectively. The constant regions are involved in complement binding, placental passage, and binding to cell membranes. 55 Figure 6.8 illustrates the structure and forms of an immunoglobulin. Figure 6.8 Immunoglobulin structure of IgG and IgM. © Jones & Bartlett Learning. Description Genes for the variable regions contain three distinct types of segments encoded in the human genome. For example, the immunoglobulin heavy chain region contains 65 Variable (V) genes plus 27 Diversity (D) genes and six functional Joining (J) genes. The light chains also possess numerous V and J genes, but do not have D genes. By the mechanism of DNA rearrangement of these regional genes, it is possible to generate an antibody repertoire of more than 10 7 possible combinations. V(D)J recombination is a genetic mechanism that randomly selects and assembles segments of genes encoding specific proteins, which generates a diverse repertoire of T-cell receptor and immunoglobulin molecules. These molecules are necessary for the recognition of diverse antigens from bacterial, viral, and parasitic pathogens and fromdysfunctional cells such as tumor cells. 55 Immunoglobulin class switching, or isotype switching, is a biologic mechanism that changes an antibody from one class to another. 56 For example, an isotype IgM could be changed to an isotype IgG. This process occurs after activation of the B lymphocyte, which allows the cell to produce different classes of antibody. Only the constant region of the antibody heavy chain changes during class switching. Because the variable region does not change, class switching does not affect the antigens that are bound by the antibody. Instead, the antibody retains affinity for the same antigens but can interact with different effector molecules, which are regulatory molecules that bind to a protein and alter their activity. The antibody molecule has a “Y” shape, with the top being the site that binds antigen, and, therefore, recognizes specific foreign objects. This region of the antibody is called the Fab (fragment, antigen binding) region. It is composed of one constant and one variable domain from each heavy and light chain of the antibody. The base of the Y is called the fragment crystallizable (Fc) region and is composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response for a given antigen. The Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins. By doing this, it mediates different physiologic effects including opsonization, cell lysis, and degranulation of mast cells, basophils, and eosinophils. 55 Immunoglobulin G (IgG) is the most abundant class of antibodies found in blood plasma and lymph. IgG antibodies act on bacteria, fungi, viruses, and foreign particles by agglutination, opsonization, and complement activation and by neutralizing toxins. IgG is increased in liver disease, infections, IgG myeloma, parasitic disease, and many rheumatic diseases. Decreased IgG levels are associated with acquired immunodeficiency, an increased susceptibility to infections, hereditary deficiency, protein-losing states, and non- IgG myeloma. Immunoglobulins are not synthesized to any extent by the developing fetus; however, IgG can cross the placenta, so any IgG present in a newborn’s serum was synthesized by the mother. 55 Immunoglobulin A (IgA) is the main immunoglobulin found in mucous secretions, including tears, saliva, colostrum, vaginal fluid, and secretions from the respiratory and gastrointestinal mucosa, and is also found in small amounts in blood. It is made by B lymphocytes. IgA exists in two isotypes, IgA1 and IgA2. While IgA1 and IgA2 are both found in serum and mucosa, IgA1 predominates in serum, while IgA1 and IgA2 tend to be evenly distributed in the mucosa. IgA is also classified, based upon location, as serum IgA or secretory IgA. Secretory IgAs are polymers of two to four IgA monomers linked by a joining protein (J chain), which is a polypeptide-containing cysteine and a secretory component. It is different structurally fromother immunoglobulins. Secretory IgA is resistant to enzyme degradation and remains active in the digestive and respiratory tracts to provide antibody protection in body secretions. Increases in serum IgA are found in liver disease, infections, and autoimmune diseases, while decreases are found in impaired protein synthesis and immunodeficiency. 57 Immunglobulin M (IgM) is the first antibody to appear in response to antigenic stimulation and is present in B lymphocytes. IgM is a pentamer and contains a J chain. Both anti-A and CASE STUDY TABLE 6.1 anti-B are naturally occurring IgM antibodies as part of the ABO blood group system. An increased IgM concentration is found in bacterial infections, toxoplasmosis, primary biliary cirrhosis, cytomegalovirus, rubella, herpes, and various fungal diseases. A monoclonal increase in IgM antibodies is seen in Waldenström’s macroglobulinemia as a spike in the vicinity of the late β zone on protein electrophoresis. Decreases are seen in protein-losing conditions and hereditary immunodeficiencies. IgM cannot cross the placenta, and it is the only immunoglobulin synthesized by the neonate. 58 Immunoglobulin D (IgD) molecules are present on the surface of most, but not all, B lymphocytes early in their development, though little IgD is ever released into the circulation. IgD may help regulate B lymphocyte function; however, the specific function of circulating IgD is largely unknown. Its concentration is typically increased in infections, liver disease, and connective tissue disorders. 55 Immunoglobulin E (IgE) is produced by B lymphocytes; increased concentrations are associated with allergic and anaphylactic reactions, autoimmune processes, and parasitic infections. In contrast to other immunoglobulins, the concentration of IgE in the circulation is very low. An elevated concentration is not diagnostic of any single condition but is observed in many inflammatory and infectious diseases, including asthma and hay fever. Monoclonal increases are seen in IgE myeloma, but these are rare. 59 CASE STUDY 6.1, PART 2 Remember Sean, the 3-week-old infant who was seen in the emergency department. Sean is now admitted to the hospital with pneumonia. The physician orders a number of laboratory tests to evaluate the child’s immune system. Hematology results show that the child does not have anemia and that his white blood cell count is slightly elevated compared to the reference range. Additional testing reveals normal levels of B lymphocytes and T lymphocytes. Chemistry results for total protein, albumin, and immunoglobulin levels are listed in Case Study Table 6.1. Laboratory Results Test Result Reference Range Total protein 8.7 g/dL 1 y: 5.4–7.5 g/dL Albumin 3.8 g/dL 1–3 y: 3.4–4.2 g/dL IgG 153 mg/dL 1–3 y: 507–1407 mg/dL IgM 576 mg/dL 1–3 y: 18–171 mg/dL IgA 11 mg/dL 1–3 y: 63–298 mg/dL IgD 0 mg/dL Newborn to adult: 0–8 mg/dL IgE 1 kU/L 0–5 mos. 1 month 14–45 mg/dL ALBUMIN—CSF 0–35 mg/dL CSF—SERUM ALBUMIN RATIO 2.7–7.3 IgG INDEX 0.26–0.70 © Jones & Bartlett Learning. An abnormally increased CSF total protein is generally associated with increased permeability of the capillary endothelial barrier through which ultrafiltration occurs. Examples of such conditions include meningitis, traumatic lumbar puncture, multiple sclerosis, obstruction, neoplasm, disk herniation, and cerebral infarction. The degree of permeability of the blood– brain barrier can be evaluated by measuring the CSF albumin and comparing it with simultaneous measurement of serum albumin. Albumin is used as the reference protein for permeability because it is not synthesized to any degree in the central nervous system. The reference range for the CSF-to-serum albumin ratio is 2.7 to 7.3; a value above the reference range may indicate damage to the blood–brain barrier. Low CSF protein values are found in hyperthyroidism and when fluid is leaking from the central nervous system. 85 The concentration of total protein in CSF may be determined by the same methods referred to earlier in the discussion on urinary proteins. Although total protein concentrations in the CSF are informative, electrophoresis may be performed to characterize individual protein fractions. The pattern of proteins present can be visualized best by using a concentrated CSF specimen. A normal CSF pattern shows prealbumin (transthyretin), a prominent albumin band, an α1 - globulin band composed predominantly of α1 -antitrypsin, an α2 - globulin band consisting primarily of haptoglobin and ceruloplasmin, a β1 band composed principally of transferrin, and a CSFspecific transferrin that is deficient in carbohydrate, referred to as τ protein, in the β2 zone. The globulin present in the γ band is typically IgG with a small amount of IgA. 85 Electrophoretic patterns of CSF from patients with multiple sclerosis demonstrate multiple, distinct oligoclonal bands in the γ region (Figure 6.11B). The presence of discrete bands in the γ region of CSF that are not also present in the serum is consistent with production of IgG in the CSF. These bands cannot be seen on routine cellulose acetate electrophoresis, but require a high-resolution technique using agarose. More than 90% of patients with multiple sclerosis demonstrate the presence of oligoclonal bands, although oligoclonal bands have also been found in inflammatory conditions and infectious neurologic diseases, such as Guillain-Barré syndrome, bacterial meningitis, viral encephalitis, subacute sclerosing panencephalitis, and neurosyphilis. 86 To differentiate elevated CSF IgG concentrations due to local CNS production from leakage of plasma into the CSF, the laboratory may compare CSF and serum IgG concentrations with reference to albumin in a value known as the IgG index. The CSF albumin concentration corrects for increased permeability of the blood–brain barrier. To identify the source of an elevated CSF IgG level, the IgG index can be calculated as follows: The reference range for the IgG index is 0.26 to 0.70. A high IgG index is indicative of local CNS production of IgG, whereas a low IgG index is suggestive of hypergammaglobulinemia or low serum albumin. 85 In the investigation of multiple sclerosis, myelin basic proteins present in the CSF may also be assayed because these proteins can provide an index of active demyelination. Myelin basic proteins are constituents of myelin, the sheath that surrounds many of the CNS axons. In very active demyelination, concentrations of myelin basic proteins of 17 to 100 ng/mL are found using an enzyme-linked immunosorbent assay (ELISA). In slow demyelination, values of 6 to 16 ng/mL occur, and in remission, the values are less than 4 ng/mL. In addition to multiple sclerosis, other conditions that induce CNS demyelination and elevated concentrations of myelin basic protein include meningoencephalitis, SLE, diabetes mellitus, and chronic renal failure. 85 CASE STUDY 6.3, PART 2 Remember Fiona, the 36- year-old woman, was seen by her physician with complaints of intermittent blurred vision. CSF was collected via lumbar puncture and sent to the laboratory along with a paired serum sample. The CSF specimen was clear and colorless with a normal cell count. The CSF total protein concentration was 49 mg/dL with an IgG of 8.1 mg/dL. Electrophoresis of Fiona’s serum and CSF revealed more than two oligoclonal bands in the CSF electropherogram (seen in Figure 6.11) and a polyclonal pattern on SPE. 1. What is the significance of the CSF protein bands indicated by the arrows? 2. What conditions would produce this type of CSF protein electrophoresis pattern? 3. What other tests would be helpful in the investigation of this patient’s diagnosis? 4. What laboratory test can be useful for monitoring the course of this patient’s condition? (Eq. 6.4) © Westend61/Getty Images. Amyloids are insoluble fibrous protein aggregates formed due to an alteration in their secondary structure known as β-pleated sheets. Amyloidosis refers to conditions in which amyloids are abnormally deposited in organs and tissues, including the heart, blood vessels, brain, peripheral nerves, kidneys, liver, spleen, and intestines causing localized or widespread organ failure. Deposition of amyloid in the brain is associated with the development of Alzheimer’s. As such, measurements of cerebrospinal fluid for amyloid β42 (Aβ42) may aid in differentiating Alzheimer’s disease from other forms of dementia. CSF concentrations are decreased in patients with Alzheimer’s due to deposition in the brain. Hemoglobin Hemoglobin is classified as a transport protein. Its role is to transport oxygen from the lungs to the tissues, and transport carbon dioxide back to the lungs. It is one of the major buffering systems in the body. Hemoglobin is produced in red blood cells during their development in the bone marrow. Hemoglobin has a mass of 64,500 daltons, making it similar in mass to albumin. Hemoglobin is a tetramer, having two α-like and two β-like globin chains. Each chain holds one heme molecule, which reversibly binds oxygen, dependent on the PO2 of the surrounding tissue. The major secondary structure of the globin chains is α helix. The helices surround the heme to form the tertiary structure. The globin chains first form homodimers, then come together as a tetramer to form the quaternary structure (Figure 6.13A). Figure 6.13 Molecular structure of hemoglobin compared to myoglobin. (A) Hemoglobin. The four different colors are four proteins that have formed a tetramer. (B) Myoglobin. Myoglobin is a monomer. The secondary structure is predominantly α helix. Wang J, Youkharibache P, Zhang D, Lanczycki CJ, Geer RC, Madej T, Phan L, Ward M, Lu S, Marchler GH, Wang Y, Bryant SH, Geer LY, Marchler-Bauer A. iCn3D, a Web-based 3D Viewer for Sharing 1D/2D/3D Representations of Biomolecular Structures. Bioinformatics. 2020 Jan 1;36(1):131-135. (Epub 2019 June 20.) doi: 10.1093/bioinformatics/btz502 Different globin chains are expressed over the course of development from separate genes on chromosomes 11 and 16. The relative proportion of the different chains varies in embryos, fetuses, newborns, and adults. The major hemoglobin in fetuses is HbF, which consists of two α and two γ chains. In adults the major hemoglobin is HbA, which consists of two α and two β chains. HbA accounts for up to 97% of the total hemoglobin. The remainder is comprised of HbA2 and a small amount of HbF. HbA2 consists of two α and δ chains. Hemoglobin concentration is measured in peripheral blood as part of a complete blood count, along with a red blood cell (RBC) count. A decreased hemoglobin concentration is indicative of anemia. Hemoglobinopathies are qualitative defects caused by mutations in the DNA of the globin chain that produce a structurally different form leading to a decrease in red blood cell survival. For example, HbS is a mutated form of HbA, causing sickle cell anemia. Thalassemias are quantitative defects due to mutations that reduce the synthesis of normal hemoglobin. This chapter will focus on discussion of disorders in the production of heme classified as porphyrias. Synthesis and Degradation of Hemoglobin Hemoglobin synthesis occurs in the immature RBCs located in the bone marrow. Newly synthesized heme exits the mitochondria and complexes with four globin molecules in the cell cytoplasm. Iron is transported from storage sites to the developing RBCs, where it is inserted into heme and is available to bind oxygen. Red blood cells may lyse in the blood vessels (intravascular hemolysis) or be degraded outside of the circulatory system within the phagocytic cells of the spleen, liver, and bone marrow (extravascular hemolysis) releasing hemoglobin. In intravascular hemolysis, circulating haptoglobin and hemopexin will bind with free hemoglobin and free heme, respectively, and transport them to the liver so their constituent parts can be recycled. Iron is reclaimed and stored until it is needed (see Chapter 27, Trace Elements, Toxic Elements, and Vitamins), and the globin chains are broken down so the amino acids can be recycled. Heme is converted to bilirubin and urobilinogen through several steps (see Chapter 19, Liver Function), and laboratory measurement of these degradation products can help determine the underlying cause for increased RBC destruction. TABLE 6.11 Myoglobin Myoglobin is the primary oxygen-carrying protein found in striated skeletal and cardiac muscle, accounting for approximately 2% of total muscle protein. It can reversibly bind oxygen and requires a very low oxygen tension to release the bound oxygen. Myoglobin transports oxygen from the muscle cell membrane to the mitochondria, and it serves as an extra reserve of oxygen to help exercising muscle maintain activity longer. Myoglobin is a monomer, a singlechain globular protein with a mass of 16,700 daltons. Its secondary and tertiary structure is similar to hemoglobin. Much of the secondary structure is α helix, surrounding a heme prosthetic group, shown in Figure 6.13B. When striated muscle is damaged, myoglobin is released into the bloodstream resulting in elevated blood and urine concentrations. Elevations are found shortly after an acute myocardial infarction (see Chapter 20, Cardiac Function). Elevations are also seen in conditions in which skeletal muscle is damaged, such as progressive muscular dystrophy, 87 crush injuries, and rhabdomyolysis. 88 Myoglobin is a nephrotoxin, and in severe muscle injury, concentrations of myoglobin can rise very quickly causing damage to the kidneys. Renal failure can also elevate blood concentrations of serum myoglobin due to impaired filtration at the glomerulus. 89 Table 6.11 lists some causes of serum myoglobin elevation. Myoglobin levels in urine are normally very low or not detected. Increased levels of myoglobin in urine will make the urine appear reddish-brown. Myoglobin will cross-react with the hemoglobin pad on the urine dipstick and cause a positive reaction. Myoglobin is measured in urine and blood by immunometric methods. Causes of Myoglobin Elevations Acute myocardial infarction Angina without infarction Rhabdomyolysis Multiple fractures; muscle trauma Renal failure Myopathies Vigorous exercise Intramuscular injections Open heart surgery Tonic–clonic seizures Electric shock Arterial thrombosis Certain toxins Malignant hyperthermia Muscular dystrophy Systemic lupus erythematosus © Jones & Bartlett Learning. Heme Heme is not a protein; it is a prosthetic group found in hemoglobin, myoglobin, chlorophyll, cytochromes, and several other enzymes. Heme is a flat cyclic tetrapyrrole, with a central ferrous iron atom (Fe 2+ ) that reversibly binds oxygen (Figure 6.14). Figure 6.14. Structure of heme. © Jones & Bartlett Learning. Description Heme is produced by a series of eight enzymatic reactions within the mitochondria and cytosol of all cells. It is most concentrated in RBCs and liver cells. Intermediates in the enzymatic pathway are collectively referred to as porphyrins. A mutation in any of the enzymes in this pathway leads to an accumulation of specific porphyrins. The symptoms of porphyrias that manifest are related to which porphyrins are elevated. Therefore, porphyrias are classified as either erythropoietic or hepatic. 90 Porphyrins are photoactive due to extensive conjugation of the tetrapyrrole ring, which is highlighted in the diagram of heme shown in Figure 6.14. Their dark red color is due to a strong absorbance in the visible region of the spectrum. The compounds absorb light of 400 nmwavelength and emit a characteristic orange-red fluorescence between 600 and 650 nm. 91 This property produces some of the manifestations of disease, such as photosensitivity, and provides a means for detection of the compounds in body fluids tested in the laboratory. Aqueous solubility of porphyrins varies with the number of carboxylic acid substituents present in the porphyrin compound. Because of the variable solubilities of the porphyrins, selection of an appropriate sample type for analysis is essential. Uroporphyrin (Uro) has eight carboxylic acid groups. It is most soluble in water and is excreted by the kidneys. Coproporphyrin (Copro) has four carboxylic acid substituents and intermediate solubility and is found in blood, urine, and feces. Protoorphyrins (Proto) with only two carboxyl groups are the least water soluble and are not found in urine. They are present in the blood and are excreted in the feces. Synthesis of Heme Heme biosynthesis is diagrammed in Figure 6.15. In the first step in the pathway, glycine and succinyl coenzyme A are condensed to form δ-aminolevulinic acid (ALA) in the mitochondrion. In the next reaction, the porphobilinogen synthase catalyzes condensation of two molecules of ALA to form porphobilinogen (PBG). Polymerization of four molecules of PBG creates the linear tetrapyrrole, hydroxymethylbilane (HMB). This process is catalyzed by the enzyme hydroxymethylbilane synthase, or PBG deaminase. Uroporphyrinogen III synthase catalyzes intramolecular rearrangement and ring closure resulting in the cyclic isomer, uroporphyrinogen III. Sequential removal of one carboxyl group from each pyrrole ring in the cyclic tetrapyrrole is catalyzed by uroporphyrinogen decarboxylase. The hepta-, hexa-, penta-, and tetra-carboxyl intermediates are formed in succession. In subsequent reactions, coproporphyrinogen oxidase catalyzes the oxidation of two additional carboxylic acid substituents to formprotoporphyrinogen IX, which is oxidized to protoporphyrin in a reaction catalyzed by protoporphyrinogen oxidase. In the final step of biosynthesis, ferrochelatase catalyzes the insertion of ferrous iron into the porphyrin ring to form heme. The process of heme biosynthesis is regulated by negative feedback of heme itself. TABLE 6.12 Figure 6.15. Heme biosynthesis. © Wolters Kluwer. Description Disorders of Heme Biosynthesis The porphyrias are a group of rare inherited or acquired metabolic disorders caused by loss or gain of function mutations in the enzymes responsible for heme biosynthesis. The defective enzyme results in overproduction, accumulation, and excretion of toxic precursor compounds and porphyrins. Conditions corresponding to enzyme abnormalities have been identified in every step of heme synthesis. Table 6.12 lists the porphyrias, their associated enzyme deficiencies, the intermediates that accumulate, and the body fluids that might be collected for analysis. Heme Biosynthesis: Metabolites and Diseases Description The porphyrias can be categorized as either erythropoietic (CP and EPP) or hepatic (AIP, ADP, VP, HCP, PCT, and HEP). Porphyrias may also be classified based on their associated symptoms as acute or cutaneous (non-acute). Acute porphyrias, which include ADP, AIP, HCP, and VP, are associated with neurological symptoms and acute attacks of abdominal pain. The cutaneous porphyrias, CEP, PCT, EPP, and XLPP, present as chronic conditions. Exposure to sunlight results in photosensitization of the skin due to accumulation of excess porphyrins in tissues. The porphyrins absorb light and react, producing blistering skin lesions on sun- exposed areas that are characteristic of cutaneous porphyrias. Chronic photosensitivity is also a symptom in VP and HCP. Many of the porphyrias have an autosomal dominant inheritance pattern, meaning that a mutation of one gene causes disease, which leads to a 50% reduction in activity of the affected enzyme. Exceptions are X-linked protoporphyria (XLPP) and CEP, both are autosomal recessive. Some individuals demonstrate an enzyme deficiency but do not experience clinical or biochemical manifestations of porphyria indicating that other factors, such as an increased demand for heme biosynthesis, must be present to cause disease expression. 90 Other Conditions Associated with Increased Porphyrins Secondary porphyrinurias are acquired conditions associated with increased excretion of urinary porphyrins. 92 These disorders are not the result of an inherited biochemical defect in heme synthesis, but are secondary to another disorder or toxin that interferes with heme formation or metabolism. Impaired heme synthesis may result from functional changes due to liver diseases such as hepatitis, cirrhosis, hereditary tyrosinemia, and obstructive jaundice. 93,94 Urinary porphyrins are also increased in inherited disorders of bilirubin metabolism such as Dubin Johnson, Rotor, and Gilbert syndromes. 95 Heavy metal poisoning, such as lead toxicity, can also lead to excess urinary porphyrin excretion in the absence of porphyria. Zinc Protoporphyrin When iron stores are insufficient to meet the demands of heme synthesis, erythrocyte protoporphyrin can assimilate zinc instead of iron to become zinc protoporphyrin (ZPP). As such, ZPP has been suggested as a biomarker to screen for iron deficiency in the absence of lead toxicity. 96,97 Lead inhibits enzymatic formation of PBG, which increases concentrations of ALA and coproporphyrinogen oxidase function leading to an accumulation of Copro III. Lead also interferes with insertion of ferrous iron into heme, causing ZPP to be formed by incorporation of zinc ions into the protoporphyrin ring instead. Some symptoms of lead intoxication are similar to those found in acute porphyria, so identification of the underlying cause is essential. If lead poisoning is suspected, measurement of whole blood lead concentrations is reommended. 98 Clinical Application Testing for porphyrins and porphyrin precursors is used to diagnose and monitor porphyrias and other disorders affecting heme metabolism. Methods available for identification of porphyrias include genetic assays to detect DNA mutations, functional and quantitative assays for the enzymes responsible for heme biosynthesis, and biochemical assays to identify and quantify porphyrins and precursor compounds. Clinical diagnostic tests for porphyrin disorders are based most often on identification of characteristic patterns of excess porphyrin precursors and intermediate compounds in body fluids (Table 6.11). The specimen to be tested (urine, blood, or feces) depends on the solubility of the metabolic intermediates associated with particular disease symptoms. Testing for Porphyrin Disorders When an acute porphyria is suspected, initial testing will likely include measurement of a random or timed urine collection for PBG. If the symptoms are due to an acute porphyria, increased PBG concentrations will be detected in the urine specimen. The urine may actually have a deep red color, referred to as port wine. 99 Follow-up testing for a positive PBG test may include measurement of the deficient enzyme, hydroxymethylbilane synthase (PBG deaminase) to identify AIP, and analysis of fecal porphyrins to differentiate among the acute porphyrias (AIP, VP, and HCP). PBG may not be increased in asymptomatic individuals; therefore negative results do not exclude porphyria completely, and testing should be repeated on a 24-hour urine specimen or when acute symptoms recur. Initial testing for cutaneous disease requires measurement of porphyrins and PBG in a random or timed urine collection. The suspicion for CEP in infants may begin when the caregiver notices red urine in the diaper. 100 Porphyrins can also be detected on a wet diaper by characteristic fluorescence of the urine when exposed to long wave UV light. 90 Increased excretion of porphyrins in a characteristic pattern particular for each disease is diagnostic for PCT and CEP. Testing for PBG is negative in these conditions. Suspected EPP or XLPP is evaluated by assessing erythrocyte porphyrins in whole blood. 101 Porphyrin analysis is typically limited to large reference laboratories or “porphyrin centers.” Important considerations for porphyrin analyses include appropriate selection of which test to perform and careful attention to specimen collection, storage, and transport requirements. Analytical Methods Testing for the precursor compounds, PBG and ALA, utilizes ion exchange chromatography to remove interferences and isolate each compound. Detection is accomplished by the addition of 4-dimethylaminobenzaldehyde (Ehrlich’s reagent) to produce a characteristic rose-red color that is detected spectrophotometrically. The principle is that of the Watson-Schwartz assay. 102 The absorbance spectrum has a maximum at approximately 555 nm and a shoulder at 525 nm. Current assays allow quantitative measurement of analyte concentrations. Porphyrin intermediates may be evaluated using HPLC with fluorescence detection. 92 Urine is acidified to enhance the natural fluorescence of the porphyrins, and an aliquot is injected into the HPLC system. Measured porphyrin concentrations can be normalized for random urine collections by expressing them as a ratio to creatinine. Results for 24-hour collections are expressed as excretion per day. Fecal porphyrin analysis using HPLC with fluorescence detection allows separation and quantification of coproporphyrin isomers and protoporphyrin to differentiate the acute porphyrias: AIP, HCP, and VP. An aliquot of the fecal specimen is lyophilized and reconstituted in acidic solution to enhance the natural porphyrin fluorescence before it is subjected to chromatographic analysis. Testing for protoporphyrin to determine presence of EPP or XLPP is performed by scanning fluorescence of a plasma specimen and by analysis of whole blood. CASE STUDY 6.4, PART 2 Remember Mateo, the 2-month-old infant with blisters. His mother explains that their current visit is because Mateo developed multiple blisters on his exposed skin when they had only been outside for a short period of time, and his urine-soaked diaper appears reddish in color. The pediatrician checks his current diaper with long-wave UV light and notes fluorescence. 1. What porphyria should be suspected? 2. How is this porphyria classified? 3. What is the first analyte that should be measured, and in which body fluid? 4. Mateo’s mother was instructed to collect a urine and a stool sample and bring them to the laboratory for analysis for Uro I and Copro I. Ablood sample was also collected and sent at the same time. Only the blood sample was positive. What preanalytical error could lead to this false result?

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