8) Amino Acids, Peptides and Proteins

SECTION III
Clinical Chemistry-Analytes
CHAPTER 31 Amino Acids, Peptides, and Proteins,* 349
CHAPTER 32 Serum Enzymes,* 350
CHAPTER 33 Tumor Markers,* 351
CHAPTER 34 Kidney Function Tests,* 352
CHAPTER 35 Carbohydrates,* 353
CHAPTER 36 Lipids and Lipoproteins, 354
CHAPTER 37 Electrolytes and Blood Gases,* 415
CHAPTER 38 Hormones,* 416
CHAPTER 39 Vitamins and Trace Elements,* 417
CHAPTER 40 Iron Metabolism,* 418
CHAPTER 41 Porphyrins and Porphyrias,* 419
CHAPTER 42 Therapeutic Drugs and Their Management, 420
CHAPTER 43 Clinical Toxicology,* 454
CHAPTER 44 Toxic Elements,* 455
CHAPTER 45 Body Fluids,* 456
Exam questions, case studies, and additional resources are available on ExpertConsult.com.
*Full versions of these chapters are available electronically on www.ExpertConsult.com.
348
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31
Amino Acids, Peptides, and Proteins*
Dennis J. Dietzen and Maria Alice Vieira Willricha
ABSTRACT
Background genome contains the information to dictate formation of
Amino acids are not only the building blocks of proteins, but approximately 20,000 polypeptides, but the actual diversity
they also play diverse roles in the provision of energy and the of the human proteome and peptidome is manifold more
formation of a number of other important biomolecules, expansive. Proteome diversity arises from linear amino acid
including hormones, neurotransmitters, and signaling mole- sequence and an array of modifications that include acyla-
cules. The polymers of amino acids, peptides and proteins, tion, phosphorylation, glycosylation, and isoprenylation.
orchestrate and control the vast array of human physiologic Systems of short peptides, larger protein monomers, and
and biochemical processes. The catalog of amino acids, pep- multimeric protein complexes are the tools that orchestrate
tides, and proteins in various biological fluids is a target-rich and control human physiologic and biochemical processes.
environment for the detection of pathologic states. Proper synthesis, folding, subcellular targeting, and catabolism
of proteins and peptides are therefore essential for human
Content health. Analytic exploitation of biologic fluids including
This chapter first describes the chemistry, metabolism, trans- blood, urine, and cerebrospinal fluid using chemical, immu-
port, and analysis of amino acids. Polymers of amino acids may nologic, and mass spectrometric methods enables informed
be relatively short (peptides) or long (proteins). The human diagnosis and therapy in a multitude of disease states.
*The full version of this chapter is available electronically on ExpertConsult.com.

a
The authors gratefully acknowledge the preceding foundation for this chapter laid by Glen L. Hortin and A. Myron Johnson, as well as generous
assistance from Carl H. Smith on the topic of amino acid transport.
349
CHAPTER 31 Amino Acids, Peptides, and Proteins 349.e1
specific protein components in healthy and diseased popula-

INTRODUCTION tions, the usual basis for diagnostic applications.
Amino acids, peptides, and proteins are crucial for virtually This chapter begins with a discussion of the chemistry, me-
all biologic processes. Amino acids serve as structural sub- tabolism, and analysis of amino acids. Inherited disorders of
units of peptides and proteins but also play diverse roles in amino acid metabolism are discussed in Chapter 60. A descrip-
metabolism, neurotransmission, and intercellular signaling. tion of the chemistry and biochemistry of the peptide bond is
Peptides serve as autocrine and endocrine signaling mol- then followed by a description of several clinically relevant pep-
ecules that control appetite, vascular tone, and electrolyte tide systems and methods for in vitro assessment. The protein
homeostasis, as well as carbohydrate and mineral metabo- narrative begins with an account of protein structure and cellu-
lism. Proteins, longer peptide chains with molecular mass lar compartmentalization followed by discussion of co- and
typically greater than approximately 6000 Daltons (Da) serve post-translational modifications. Constituents of the proteome
as (1) intracellular and extracellular structural components, in body fluids are also addressed, followed lastly by a description
(2) biologic catalysts, (3) mediators of contractility and mo- of methods for specific and global assessment of the proteome
tility, (4) agents of molecular assembly, (5) ion channels and for clinical purposes. More in-depth treatment of other specific
pumps, (6) molecular transporters, (7) mediators of immu- proteins and protein networks may be found in Chapters 32
nity, and (8) components of intracellular and intercellular (serum enzymes), 78 (enzymes of the red blood cell [RBC]), 33
signaling networks. (tumor markers), 36 (lipoproteins), and 77 (hemoglobin), as
The human genome contains more than 20,000 open read- well as other chapters dedicated to the specific pathophysiology
ing frames that encode proteins. The actual number of pro- of cardiac, liver, renal, bone, pituitary, thyroid, and adrenal dis-
teins is far greater, however, because of alternative splicing of ease. In-depth treatment of measurement modalities for amino
messenger RNA (mRNA), somatic recombination, mutation, acids, peptides, and proteins such as electrophoresis, chromatog-
proteolytic processing, and post-translational modification. raphy, mass spectrometry (MS), and immunoassay may be
The proteome represents the complete set of proteins in an found in Chapters 18, 19, 20, and 26, respectively.
organism or compartment of an organism such as the plasma
space. Efforts to catalog the proteome include those by the AMINO ACIDS
Human Proteome Organization (hupo.org), the National Cen-
ter for Biotechnology Information (ncbi.nlm.nih.gov), the Amino acids were likely among the first organic molecules to
Swiss Institute of Bioinformatics (expasy.org), and the Healthy emerge from the mix of methane, hydrogen, ammonia, and
Human Individual’s Integrated Plasma Proteome Database water in earth’s primordial atmosphere.1 Only 20 of the hun-
(bio.informatics.iupui.edu/HIP2). Most databases were de- dreds of known amino acids account for the vast majority of
signed mainly to assist with peptide and protein identification, residues in human polypeptide chains. Their structure and
but efforts have shifted to characterizing the abundance of molecular properties are summarized in Table 31.1. These
TABLE 31.1 Structure and Chemical Properties of the 20 Proteogenic Amino Acids
Amino Acid MW (Da) Structure (pH 7.0) pK1 pK2 pK3 pI HI
Alanine (ALA, A) 89.09 H O 2.4 9.7 6.0 1.8
H3C C C

NH3 O

Arginine (ARG, R) 174.20 H2N H O 2.2 9.0 12.5 10.8 24.5

C NH CH2 CH2 CH2 C C

H2N NH3 O

Asparagine (ASN, N) 132.12 H2N H O 2.0 8.8 5.4 23.5

C CH2 C C

O NH3 O

Aspartate (ASP, D) 133.10 O H O 2.1 9.8 3.9 2.9 23.5

C CH2 C C

O NH3 O

Cysteine (CYS, C) 121.16 H O 1.7 10.8 8.3 5.1 2.5

HS CH2 C C

NH3 O

Glycine (GLY, G) 75.07 H O 2.3 9.6 6.0 20.4

H C C

NH3 O

Continued
349.e2 SECTION III Clinical Chemistry-Analytes
TABLE 31.1 Structure and Chemical Properties of the 20 Proteogenic Amino Acids—cont’d
Amino Acid MW (Da) Structure (pH 7.0) pK1 pK2 pK3 pI HI
Glutamate (GLU, E) 147.13 O H O 2.2 9.7 4.3 3.2 23.5
C CH2 CH2 C C

O NH3 O

Glutamine (GLN, Q) 146.15 H2N H O 2.2 9.1 5.7 23.5

C CH2 CH2 C C

O NH3 O

Histidine (HIS, H) 155.16 H O 1.8 9.2 6.0 7.6 23.2

HN
CH2 C C

N NH3 O
H
Isoleucine (ILE, I) 131.17 H O 2.4 9.7 6.0 4.5

H3C CH2 CH C C

CH3 NH3 O

Leucine (LEU, L) 131.17 H3C H O 2.4 9.6 6.0 3.8

CH CH2 C C

H3C NH3 O

Lysine (LYS, K) 146.19 H O 2.2 9.0 10.5 9.7 23.9

H3N CH2 CH2 CH2 CH2 C C

NH3 O

Methionine (MET, M) 149.21 H O 2.3 9.2 5.8 1.9

H3C S CH2 CH2 C C

NH3 O

Phenylalanine (PHE, F) 165.19 H O 1.8 9.1 5.5 2.8

CH2 C C

NH3 O

Proline (PRO, P) 115.13 O 2.1 10.6 6.1 1.6

C

NH2 O

Serine (SER, S) 105.09 H O 2.2 9.2 5.7 20.8

HO CH2 C C

NH3 O

Threonine (THR, T) 119.12 HO H O 2.6 10.4 6.5 20.7

CH C C

H3C NH3 O

Tryptophan (TRP, W) 201.22 H O 2.5 9.4 5.9 20.9

CH2 C C

NH3 O
N
Tyrosine (TYR, Y) 181.19 H O 2.2 9.2 10.5 5.7 21.3

HO CH2 C C

NH3 O

Valine (VAL, V) 117.17 H3C H O 2.3 9.6 6.0 4.2

CH C C

H3C NH3 O

HI, Hydropathy index; MW, molecular weight; pk, acid ionization constant; pI, isoelectric point.
20 along with dozens of non–protein-forming amino acids mixture over many years.2,3 Aspartic acid undergoes the most
are critical to the form and function of the human body. Dis- rapid racemization, and this rate can be used to estimate the
rupted amino acid metabolism is not surprisingly associated time of synthesis of proteins with very slow turnover, such as
with a multitude of pathologic processes. ocular lens proteins or intervertebral collagen in which half-
lives may exceed 50 years. Two amino acids, threonine and
Basic Biochemistry isoleucine, have a second asymmetric carbon, and their ste-
Amino acids are organic compounds containing both an reoisomers are referred to as allothreonine and alloisoleucine.
amino group (–NH2) and a carboxyl group (–COOH) or The latter compound has utility in the diagnosis of maple
another acidic group such as a sulfonate group (–SO3). In a syrup urine disease (Chapter 60 and Online Mendelian In-
majority of biologically relevant amino acids, the amine moi- heritance in Man; https://www.omim.org/entry/248600).
ety is primary (–NH2), but some (e.g., sarcosine) are second- In addition to the 20 well-known protein-forming amino
ary (–NH–) amines, and others containing tertiary amines acids, a number of unusual amino acids are recovered from
(e.g., proline) are referred to as imino (5N–) acids. With the protein hydrolysates. For example, 4-hydroxyproline and
exception of proline, the amino acids that occur in protein 5-hydroxylysine are found in collagen lysates, and desmosine
are a-amino acids (below). and isodesmosine are recovered in elastin hydrolysates. Ci-
O OH trulline may also be recovered secondary to deimination of
H C arginine. These amino acids are formed by post-translational
H
mechanisms because no codon is responsible for their incor-
N C
poration into growing polypeptides. Selenocysteine is a special
H R case of an amino acid synthesized on a specific transfer RNA
-Carbon atom and incorporated into a few sites in only about 25 proteins
that include members of the thyroid hormone deiodinase
The R group represents the unique side chains responsible for and glutathione peroxidase families.4 Some of these unusual
the chemical properties of individual amino acids. Not all biologic amino acids are shown in Fig. 31.1.
amino acids are a amino acids. b amino acids such as b-alanine Acid-base properties of amino acids depend on the amino
and taurine, as well as g-amino acids such as g-aminobutyric acid and carboxyl groups attached to the a carbon and on the
(GABA) also play key biochemical roles (Fig. 31.1). basic or acidic groups occurring on some sidechains (R). At a
With the exception of glycine, all a amino acids contain physiologic pH near 7.4, the a-carboxyl group is ionized and
four distinct moieties asymmetrically arranged around the a carries a negative charge, and the a amino group is proton-
carbon. As a consequence, amino acids may exist as mirror ated and carries a positive charge. Molecules existing simul-
images (enantiomers) referred to as the D or L configuration. taneously as cations and anions are referred to as zwitterions
With few exceptions, the biologically relevant amino acids or ampholytes (diagrammed below).
exist in the L configuration. Small quantities of D amino H
acids occur in physiologic fluids but typically do not have H H
specific functions. An exception is D serine, which represents
N O NH3

5 to 20% of total serine in cerebrospinal fluid (CSF) and may R C C or R C COO
serve as a neurotransmitter. Amino acids with the D configu- H O H
ration occur in some bacterial products, foods, and pharma-
ceuticals. D amino acid oxidases in liver and kidney convert The pH at which ionizable groups exist equally as charged
D amino acids to ketoacids, which can be further metabolized. and uncharged forms is referred to as the pK. Amino acids
L amino acids in proteins undergo slow racemization to a DL thus have two or more pKs—one for the carboxyl, one for the
amino group, and an additional one in the presence of an
O H H O H H H ionizable side chain. The isoelectric point (pI) is the pH at
HO S C C NH2 HO C C C C NH2 which an amino acid or other molecule has a net charge of 0.
O H H H H H For a typical neutral amino acid such as glycine, the pI of 5.97
is midway between the pK of 2.34 for the carboxylic acid and
Taurine γ-Aminobutyric acid
the pK of 9.60 for the amino group. The pKs of amino acid
H H side chains in proteins vary somewhat from those in free
H C amino acids because of the influence of neighboring amino
H H O
HO C NH acids. The buffering capacity of ionizable groups is primarily
N2H C C C OH H in a pH range within 61 unit of the pK for the respective
C C C OH
H H
H H
groups. Amino acids and proteins therefore have a limited
buffering capacity near physiologic pH. Glycine, for example,
O
β-Alanine 4-Hydroxyproline is used as a buffer near pH 2.5 or 9.5. The imidazole side
chain of histidine is an exception with a pK near 6.0.
H H O H OH H H H O The structural diversity of side chains permits formation
C C C OH C C C C C
of proteins with a variety of structure and function. Side-
HSe H2N C OH
H H H H H chain diversity is dictated not only by pK but by size and hy-
NH2 NH2 drophobicity as well. Amino acids with longer aliphatic or
Selenocysteine 5-Hydroxylysine aromatic side chains such as isoleucine, leucine, and phenyl-
FIGURE 31.1 Planar structures of rare or unusual, naturally occur- alanine have greater hydrophobicity than shorter side chains
ring amino acids. such as the methyl group found in alanine. Neutral amino
acids with polar groups such as hydroxyl or amide groups in Chapter 46).11 Marasmus results when protein and energy
their side chains are more hydrophilic. Acidic amino acids sources such as carbohydrates are deficient, causing wasting
have side chains with carboxylic acids, and basic amino acids of muscles and subcutaneous tissues. Albumin or prealbumin
have side chains with amino, guanidino, or imidazole groups. concentrations are sometimes used to assess adequacy of the
The thiol side chain (–SH) of cysteine oxidizes easily and amino acid supply. The shorter biologic half-life of prealbu-
may become linked to other molecules via disulfide bonds. min compared with albumin (2 vs. 20 days) makes it a valu-
In plasma, cysteine occurs as cystine (cysteine homodimer able marker for acute dietary assessment.12,13
linked via a disulfide) or as a mixed disulfide with heteroge- Homeostasis of cellular amino acid concentrations is de-
neous thiol compounds, albumin, or other proteins. pendent on supply, catabolism, and excretion. Supply and
With some exceptions, amino acids are water soluble and excretion are regulated by a series of transport systems with
stable in plasma. The most soluble amino acids have small overlapping substrate specificity, strategic tissue expression,
side chains with polar or ionizable moieties such as glycine, and polarized cellular distribution.14,15 Amino acids are de-
alanine, arginine, serine, and threonine. Less soluble amino rived from dietary protein precursors through the action of
acids such as phenylalanine, tyrosine, leucine, and trypto- proteolytic enzymes in the stomach and small intestine that
phan tend to have larger, nonpolar aliphatic or alicyclic side produce shorter oligopeptides and individual amino acids.
chains. Amino acid solubility is rarely limiting in vivo except Enteral absorption of di- and tripeptides is mediated by a single
in some metabolic disorders. Deposition of tyrosine crystals proton-coupled transport system termed peptide transporter
in the eye and skin is common in tyrosinemia, particularly 1 or PEPT1 (encoded by SLC15A1).16 Transport of individual
type II (https://www.omim.org/entry/276600). Likewise, cys- amino acids across the intestinal and renal epithelium and the
tine may crystallize in the renal parenchyma in patients with blood–brain barrier is far more specialized.
cystinuria (https://omim.org/entry/220100). Structural and Early biochemical characterization of amino acid trans-
chemical details for the 20 protein-forming amino acids are port was technologically limited to studies of the plasma
displayed in Table 31.1. membrane. These broad-specificity transport systems func-
tion as co-transporters, exchange transporters, or facilitative
Amino Acid Supply and Transport transporters. Nomenclature was based on substrate specificity,
Amino acids participate in many metabolic pathways in ad- co-transport requirements, and sensitivity to inhibitors. By
dition to serving as substrates for protein synthesis. In the convention, capital letters indicate a requirement for Na1, and
healthy state, women require approximately 46 g/day and lowercase descriptors imply the lack of Na1 dependence. Sys-
men approximately 56 g/day of dietary protein (0.8 g/kg body tems A and ASC are responsible for Na1-mediated symport of
weight), and substantial increases in demand occur during neutral species with small side chains. System L facilitates ex-
growth and in many disease states5 (see Chapter 46). Dietary change of amino acids with large, hydrophobic side chains.
protein is digested by proteases in the stomach (e.g., pepsin) Cationic amino acid transport is mediated by a system termed
and small intestine (e.g., trypsin, chymotrypsin) to yield y1. System y1L facilitates exchange of neutral amino acids
amino acids (see Chapter 52). Endogenous protein turnover with cationic species. System Bo catalyzes Na1 mediated trans-
serves as another source of free amino acids. Eight amino port of branched-chain and aromatic amino acids, and system
acids used for protein synthesis (isoleucine, leucine, lysine, bo enables exchange of bulky neutral and cationic side chains.
methionine, phenylalanine, threonine, tryptophan, and va- Finally, system X2 mediates transport of anionic amino acids
line) are not synthesized by humans and therefore are consid- and system x2c facilitates transmembrane exchange of gluta-
ered “essential” constituents of the diet. Meat, milk, eggs, and mate and cystine. These systems act in a coordinated way to
fish contain a full range of essential amino acids. Gelatin is achieve amino acid homeostasis.17
deficient in tryptophan, and some plant sources of protein Distinct transport systems cooperate to achieve net amino
may be additionally deficient in lysine or methionine. There- acid transport across epithelial barriers. For example, trans-
fore diets based on a single source of plant protein may be cellular transport of cationic amino acids across the renal
deficient in some amino acids. When liver function is com- brush border is achieved by a combination of two exchange
promised, cysteine and tyrosine become essential because they systems, bo and y1L. On the apical surface, positively charged
are not produced from their usual precursors, methionine and amino acids are imported in exchange for an uncharged
phenylalanine, respectively.6 Arginine may be conditionally amino acid via system bo. System bo uses the transmembrane
essential as well because endogenous rates of synthesis may be electrical potential to drive transport. Efflux across the baso-
insufficient to meet requirements in adults under metabolic lateral membrane is also achieved via exchange transport via
stress or in growing children.7 system y1L. System y1L uses the driving force of the trans-
Requirements for dietary protein to maintain nitrogen membrane sodium gradient. Lack of proper polarized expres-
balance increase in infancy and childhood when there are sion in appropriate tissues results in transport disorders such
increased demands for growth.8,9 Daily requirements increase as cystinuria (https://omim.org/entry/220100) and lysinuric
by up to 3.5 to 4 g protein/kg body weight for premature in- protein intolerance (https://omim.org/entry/227100).18
fants, for example.10 Protein demand is also increased in Intracellular amino acid compartments are maintained by
pregnancy, lactation, and states of protein loss or catabolic a distinct set of transport systems. These systems are impor-
states (e.g., burn patients). Persistent negative nitrogen bal- tant for metabolizing, sequestering, and recycling various
ance results in a number of undesirable phenotypic features. amino acids. Substrate concentrations in the urea cycle are
A diet severely deficient in protein and consisting primarily of regulated in part via the mitochondrial ornithine-citrulline
high-starch foods can lead to kwashiorkor, a disorder character- antiporter. Defects in this transport system leads to HHH
ized by decreased serum albumin, immune deficiency, edema, (hyperornithinemia, hyperammonemia, homocitrullinuria)
ascites, growth failure, apathy, and many other symptoms (see syndrome (https://omim.org/entry/238970) (see Chapter 60).
TABLE 31.2 Genetic Basis of Selected Amino Acid Transport Systems

Gene Family System Expression Substrates Disease Link
SLC1 X2, ASC Brain, gut, kidney, liver D,E, A, S, T, C, N, Q Dicarboxylic aminoaciduria
SLC3A1-2 y1, L, y1L, b0 Broad R, K, H, M, L, A, C, L, I, Cystinuria
V, cystine
SLC6 B0 Brain, kidney, gut, liver F, Y, L, I, V, P, C, A, Q, S, Hartnup disorder
H, G, M Iminoglycinuria
SLC7 (A1-14) y1, y1L, b0, ASC, x2 Broad R, K, cystine, ornithine Lysinuric protein intolerance
SLC15 a
Brain, kidney, gut, H None known
immune cells
SLC16 a
Gut, kidney F, Y, W None known
SLC17 a
Neurons E None known
SLC25 ASP/GLU Broad, (mitochondria) D, E, ornithine, citrulline Type II citrullinemia
ORN/CIT HHH syndrome
antiporters
SLC32 a
Neurons G, g-aminobutyrate None known
SLC36 a
Brain, intestine, kidney P, G, W None known
SLC38 A, N Broad Q, A, N, C, H, S, T None known
SLC43 L Liver, kidney, gut, muscle L, I, V None known
CTNS (SLC66) a
Broad Cystine Cystinosis
Transport properties not classified by classical naming convention.

a
HHH, Hyperornithinemia, hyperammonemia, homocitrullinuria.
Reclamation of amino acids from lysosomal protein diges- intermediates that play a role in energy production. As por-
tion also relies on transport systems. Defects in the CTNS trayed in Fig. 31.2, the transformation of amino acid carbon
gene, for example, inhibits lysosomal cystine transport and to energetic intermediate typically begins with transamination.
results in the clinical disorder known as cystinosis (https:// Excess nitrogen is excreted as urea. Resulting a-ketoacids
omim.org/entry/219750). Finally, neuronal vesicles must may enter the Krebs cycle; undergo conversion to ketone
concentrate synaptic transmitters to achieve interneuronal bodies, fatty acids, or glucose; or be completely oxidized to
communication. Two of these transmitters, glycine and glu- CO2 depending on cellular energy demands. A vast array of
tamate, are actively transported across vesicle membranes. enzyme networks has evolved to orchestrate demand for
The rather coarse biochemical definition of amino acid amino acids. Information regarding the substrates, products,
transport is being redefined as the genetic basis for these sys- kinetics, and inhibitors of these enzymes may be found in
tems is clarified.19,20 Table 31.2 summarizes transporter gene multiple databases, including BRENDA (brenda-enzymes.
families and their connection to functional transport sys- org), ExPASy-enzyme, (enzyme.expasy.org), and ExplorEnz
tems. In some cases, functional characterization of these gene (enzyme-database.org). Pathway databases include KEGG
families is incomplete and many of these transporter families (genome.jp/kegg), GenMAPP (genmapp.org), and BioCyc
have not been linked definitively to human disease. Genes of (biocyc.org).
the SLC (solute carrier) family encode integral transmem- Glucose, fatty acids, and ketones are primary respiratory
brane spanning proteins that catalyze amino acid transport. substrates in humans. These substrates generate adenosine
The SLC1 family, for example, encodes transporters primarily triphosphate (ATP) via the mitochondrial Krebs cycle. Amino
responsible for transport of anionic amino acids that are acids play two key roles in energy provision. First, amino ac-
particularly important in brain and neural tissues.21 Neutral ids are converted to Krebs cycle intermediates to maintain the
amino acid transporters (e.g., System A) are encoded by the activity of the cycle through a process called anaplerosis. Glu-
SLC38 family.22 Mitochondrial transport systems are ex- tamine and glutamate, for example, are converted to a-keto-
pressed by the SLC25 gene family.23 The SLC3 and SLC7 gene glutarate via loss of the epsilon and alpha amino groups.
families encode a wide array of heterodimeric transporters, Fumarate may be derived from asparagine and aspartate, and
including the aforementioned bo and y1L transport sys- succinate is derived from methionine, threonine, and valine.
tems.24 Each of these transporters contains one of two SLC3 Second, amino acids may be mobilized to generate fuels for a
genes that encode membrane-targeting subunits that are di- variety of organ systems. Five amino acids (isoleucine, leu-
sulfide linked to one of at least a dozen SLC7 gene products cine, lysine, phenylalanine, and tyrosine) may be converted to
dictating transport specificity. Metabolic flux of amino acid ketones. All of the amino acids except for leucine may be used
carbon is critically dependent on the proper expression to produce glucose. Therefore in times of high energy de-
and regulation of transport and enzyme-catalyzed chemical mand and limiting fuel sources, flux of amino acid carbon
transformation. through proper pathways becomes an important source of
respiratory fuel.
Amino Acid Metabolism Excess tissue nitrogen is disposed of as urea, which con-
Amino acids serve as scaffolds for the synthesis of many hor- tains two moles of nitrogen per mole (see also Chapter 60).
mones, nucleotides, lipids, signaling molecules, and metabolic Urea production is limited to the liver, so selected amino
Aminotransferases
Amino acids 2-Oxoglutarate Keto acids Glutamate NH4
Pyruvate Glucose Urea

Biosynthesis of liver and (excreted
plasma proteins, purines, by kidney)
pyrimidines, porphyrins,
hormones, and other proteins
Ketone bodies Acetyl-CoA

Tricarboxylic
acid cycle;
Fatty acid O2
oxidative
synthesis phosphorylation
ATP
CO2 H2O
FIGURE 31.2 A generalized scheme of amino acid metabolism in the liver. After transamination,
amino acid carbon may be used in the Krebs cycle directly or transformed into other respiratory fuels such
as glucose and ketones. Waste nitrogen is disposed of via the urea cycle. ATP, Adenosine triphosphate.
acids, primarily glutamine and alanine, serve to shuttle excess The importance of folate and single-carbon metabolism to
nitrogen to the liver. Nitrogen in the form of ammonium ion cell growth and division cannot be overstated. Folate defi-
is first converted to carbamoyl phosphate, which is trans- ciency in a developing embryo can lead to death or severe
ferred to ornithine to form citrulline. Aspartic acid and ci- neurologic birth defects. The use of folate antimetabolites
trulline are condensed to form argininosuccinic acid, which, such as methotrexate has been a mainstay in the treatment of
in turn, is cleaved to arginine and fumaric acid. Arginase hy- cancer for many decades. These pathways are treated in more
drolyzes arginine to urea and ornithine to allow the cycle to detail in Chapter 39.
repeat. Urea is usually viewed simply as waste, but it is also
the primary contributor to the high osmolality in the renal Amino Acid Concentrations
medulla and enables maximal urinary concentrating ability. Plasma amino acid concentrations collectively span four
Amino acids are precursors for many hormones and sig- orders of magnitude from very low micromolar quantities
naling molecules. Tyrosine provides a scaffold for thyroxine, (e.g., b-alanine, cystathionine) to near 1 mmol/L (e.g., gluta-
dopamine, and adrenaline synthesis. Tryptophan is a precur- mine, glycine). With protein intake of 1 to 2 g/kg, daily variation
sor of serotonin and melatonin. The potent vasodilator nitric of approximately 30% in healthy adults has been observed.25
oxide (NO) is produced from arginine. Glycine, aspartate, Concentrations of both essential and nonessential amino
glutamine, and serine contribute atoms to purine and py- acids vary in a coordinated way, suggesting that mechanisms
rimidine synthesis. Glycine and arginine are precursors for beyond diet and enteral extraction are responsible. Amino
creatine synthesis. acid concentrations tend to peak between 12 and 8 pm with
Creatine synthesis and many other biochemical processes a nadir between midnight and 4 am.26,27 After an ingested
rely on a series of single-carbon transfer reactions mediated protein bolus, dietary amino acids rise and tend to return to
by serine, glycine, histidine, and methionine. Transfer of fully preprandial levels in 3 to 6 hours. Therefore determination
oxidized carbonyl carbon (5C5O) to molecules such as pro- of “fasting” amino acid concentrations requires extended
pionyl CoA (to form methylmalonyl CoA) is mediated by bio- periods of dietary abstinence.
tin. Glycine, serine, and histidine contribute less oxidized Most amino acids in blood undergo glomerular filtration
carbon units such as methylidine (5CH–), and methylene but are efficiently reabsorbed in proximal renal tubules by
(–CH2–) groups that enable purine and pyrimidine synthesis previously described saturable transport systems. Increased
via folate derivatives. Folate also mediates transfer of methyl renal excretion of amino acids (aminoaciduria) results from
(–CH3) groups to homocysteine to form methionine. The re- filtration of excessive plasma concentrations, generalized tu-
sulting methionine is, in turn, activated to S-adenosylmethio- bular impairment, or heritable defects in amino acid trans-
nine becoming a methyl donor to a vast array of substrates port systems. Glycine tends to be most abundant in normal
including DNA, RNA, histones, choline, and catecholamines. urine followed by histidine, glutamine, and serine. Increased
concentrations of proteogenic amino acids in plasma tend to Common co-eluting compounds include methionine with
precipitate only mildly elevated excretion because of efficient homocitrulline, phenylalanine with aminoglycosides, and his-
reabsorption. Other amino acids that accumulate in plasma tidine with gabapentin.
secondary to metabolic errors (e.g., argininosuccinate, homo MS is increasingly being adopted as the method of choice
citrulline) demonstrate pronounced excretion because of the for amino acid profiling. Newborn screening programs
absence of specific tubular mechanisms enabling reclamation quantitate amino acid butyl esters derived from dried blood
from the filtrate. spots using flow-injection MS protocols. These methods
With the exception of glutamine, CSF amino acid concen- do not use chromatographic separation and so cannot
trations are typically less than 10% of those found in plasma. distinguish between isomeric or isobaric amino acids such
This high plasma to CSF gradient suggests active net brain to as leucine, isoleucine, alloisoleucine, and hydroxyproline.
blood transport across the blood–brain barrier.28,29 Glutamine Liquid chromatography–tandem MS (LC-MS/MS) methods
concentrations in CSF are generally equal to those in plasma, for detection of amino acids in plasma and other body fluids
suggesting a bidirectional facilitative transport process. Insofar that use liquid chromatography have also been developed
as CSF concentrations reflect synaptic concentrations, regula- (see Chapters 19 and 20).38–40 Some of these use amine-
tion of neurotransmitter amino acid concentrations is critical targeted derivatives, others target the carboxyl group, and
for normal neural action potential propagation. Glutamate some others use no chemical derivatization. Advantages of
is the most abundant amino acid in the brain and is the MS-based techniques include improved analytic specificity, a
primary excitatory transmitter. Glycine and GABA are the three- to four-order-of-magnitude dynamic range, and rapid
predominant inhibitory transmitters. Lumbar puncture to (20-minute) throughput. MS methods may also be optimized
access the CSF amino acid pool must be done with great care for profiling multiple molecular species in addition to amino
to avoid overestimation of central amino acid concentrations acids. Such approaches promise to improve the scope of
secondary to contamination with peripheral blood. metabolic disorders detectable with a single patient specimen
Assessment of amino acid concentrations in blood, urine, in a single analytic run.
and spinal fluid has been historically applied to the detection
of inborn errors of metabolism. These are comprehensively PEPTIDES
covered in Chapter 60. Aside from the measurement of
homocysteine as a marker of vitamin B12 and folate status, This section describes the basic biochemistry of peptides. In
clinical applications of amino acid measurement beyond general, the term peptide applies to relatively short polymers
metabolic diseases are limited. Future applications may include of amino acids with molecular weights less than 6000 Da
assessment of immunity using tryptophan and its metabo- (,~50 amino acid residues). The chemistry of the peptide
lites such as kynurenine.30,31 Increased plasma concentrations bond and the physical characteristics of the peptide backbone
of a-aminobutyric acid may be useful in detecting liver re- are discussed in this section along with a number of clinically
generation.32 Branched-chain amino acid concentrations relevant peptide systems.
may be early indicators of diabetes,33 while a combination of
phenylalanine, glutamate, and alanine has some value to Peptide Bond
predict the onset of preeclampsia.34 Finally, quantitation of A peptide bond, also referred to as an amide bond, is formed
arginine and its dimethylated derivatives (asymmetric and between the a-nitrogen atom of one amino acid and the car-
symmetric dimethylarginine) may have utility in assessing bonyl carbon of a second (diagrammed below).
endothelial function.35,36 These applications require further
H H O OH H2O O OH
clinical validation. N C H2N C
O H O
Analysis of Amino Acids R1 C C N C H R1 C C N C H
For decades, the standard method of amino acid analysis was H OH H R2 H H R2
cation-exchange chromatography with postcolumn spectro- First Second
photometric or fluorescent detection of various primary amine amino acid amino acid Dipeptide
derivatives. Derivatizing agents have included dansyl chloride,
o-phthalaldehyde, and ninhydrin. The ninhydrin approach So-called isopeptide bonds refer to amide bonds between
developed by Stein and Moore in the 1950s was initially sidechain amines or carbonyl carbons on the side chain rather
applied to determination of amino acid content of protein than a-amine or a-carbonyl. In glutathione, for example, the
hydrolysates and then adapted for profiling of free amino acids g-carboxyl group of glutamic acid is linked to the a-amino
in deproteinized body fluids.37 Other systems commercialized group of cysteine. During translation, peptide bonds are formed
by Beckman (Brea, CA) and Hitachi (Tokyo, Japan) were large from the amino (N) to the carboxyl (C) terminus by removal of
floor models and required as long as 2 to 3 hours to quantitate water (also referred to as dehydration or condensation) and
30 to 50 physiologic amino acids in a single patient specimen. catalyzed by RNA (referred to as a ribozyme) that forms part of
These systems have given way to smaller bench-top systems the ribosome.41 Peptides are also synthesized in vitro for thera-
using ninhydrin (Biochrom, Cambridge, United Kingdom) or peutic and experimental purposes. Such chemical peptide syn-
fluorescent (quinolyl-N-hydroxysuccinimidyl) amine deriva- thesis proceeds from C to N terminus using N-protected amino
tives (Waters, Milford, MA) that still require 90 to 120 minutes acids and catalyzed by N,N9-dicylohexylcarbodiimide.42,43 In
for full sample analysis. In addition to long cycle times, these this scheme, the nucleophilic amine group reacts with a car-
methods are subject to interference from co-eluting amines, bodiimide : carbonyl intermediate, resulting in the formation of
leading to overestimation of some amino acid concentrations. a new peptide bond and dicyclohexylurea. Dicyclohexylurea is
insoluble in most solvents and can be easily removed from the develop in vitro. Thus narrowly targeted MS assays may ex-
maturing peptide. Cleavage of peptide bonds may be nonspe- clude active peptide species and run the risk of underesti-
cifically achieved by acid hydrolysis or accomplished specifically mating bioactive peptide. Cross-reactive immunoassays, on
by a host of proteolytic enzymes with affinity for bonds be- the other hand, may stoichiometrically detect both active
tween specific amino acid residues. These protease systems are and inactive peptide, thus running the risk of overestimating
described later in this chapter. bioactive peptide concentrations. Examples of several im-
Electron sharing in the amide bond (also known as the v portant biologic peptide systems and their analytic consider-
bond) is delocalized, effectively preventing rotation about ations follow.
this bond. This bond is fixed in one plane. Conformational
flexibility of the peptide backbone results entirely from rota- Selected Clinically Relevant Peptide Systems
tion about the axes of the two bonds to the a-carbon. Angles Pro-Opiomelanocortin System
of rotation about these bonds are referred to as Ramachan- The pro-opiomelanocortin (POMC) gene on chromosome
dran angles.44 The nitrogen to a-carbon bond angle is re- 2 is expressed primarily in the pituitary gland, arcuate nu-
ferred to as the F angle, and the a-carbon to carbonyl bond cleus of the hypothalamus, and skin melanocytes. The gene
is referred to as the C angle (below). produces a 241–amino acid prohormone that can yield as
many as 10 distinct biologically active peptides depending
on patterns of cleavage in specific tissue types. The POMC
peptides have diverse effects on glucose and electrolyte ho-
meostasis (via adrenocorticotropic hormone [ACTH]),
N φ ψ C body mass and appetite (via lipotropins and melanocor-
ω
tins), pigmentation (also via melanocortins), and pain (via
Cα N endorphins).46–48
R
Clinical exploitation of this complex peptide system is
currently limited to the impact of ACTH on the adrenal
gland and subsequent feedback by cortisol. Measurement of
Theoretically, free rotation about these bonds allows an- circulating ACTH concentrations may be used to clarify the
gles ranging from 2180 to 180 degrees. In reality, steric and mechanism of adrenal disease. For example, Cushing syn-
energetic factors limit the possible combinations. These drome may result from autonomous adrenal function (ACTH
bond angles play a key role in dictating the secondary struc- low) or may be fueled by ectopic ACTH production (ACTH
ture of proteins. For example, values of F and C in a-helices high). Likewise, Addison disease may result from adrenal
are approximately 260 and 245 degrees, respectively. Sec- failure (ACTH high) or pituitary failure (ACTH low). The
ondary structure is addressed more extensively later in this pathophysiology of this axis is treated more extensively in
chapter. Chapters 55 and 56. Full-length ACTH consists of 39 amino
acid residues and circulates with a half-life ranging from 10
Peptide Heterogeneity and Analysis to 15 minutes. The biologic activity of ACTH is contained in
Assessment of circulating peptide concentrations has a num- residues 1 to 18, and the length of the C-terminus mediates
ber of limitations. In the absence of enzymatic activity, peptide circulating half-life. Two-site immunoassays typically target
measurements have been historically limited to immunologic the extreme N and C termini to avoid detection of shorter,
techniques (discussed in more detail in Chapter 26). Antibod- inactive circulating species. This approach, however, can lead
ies may recognize linear sequence epitopes or discontinuous, to cross-reactivity with longer precursor forms of ACTH
conformational epitopes. These epitopes typically involve 10 such as pro-ACTH and the full POMC gene product.49 These
to 20 amino acids binding exposed areas of 600 to 1000 Å.45 precursors typically circulate at concentrations that are five
Measurement of short peptides (,20 to 30 amino acids) times greater (5 to 50 pmol/L) than ACTH (1 to 10 pmol/L).
are therefore limited to single-site, competitive assays that lack ACTH assays are poorly standardized.
the analytic specificity of two-site (sandwich) immunoassays.
The molecular specificity issues may be addressed using MS Natriuretic Peptides
as an alternative. Small peptides may be ionized via electro- This peptide family consists of atrial natriuretic peptide
spray (ESI) or matrix-assisted laser desorption (MALDI) (ANP), B-type natriuretic peptide (BNP, formerly brain na-
and interfaced to tandem quadrupole mass analyzers (see triuretic peptide), and C-type natriuretic peptide (CNP).50
Chapter 20). ANP and BNP are highly expressed in cardiac tissue relative
Absolute analytic specificity is not always ideal when ap- to CNP, which is expressed at low concentrations in a broad
plied to biologic peptide systems. Peptide populations may variety of tissue types. The mature forms of these peptides
consist of species with a variable number of amino acid contain a 17–amino acid loop stabilized by an intramolecular
residues possessing sometimes unknown biologic potency. disulfide bond. Each peptide acts via a specific guanylate
Hepcidin, for example, is an iron transport regulatory pep- cyclase-coupled receptor to promote sodium and water ex-
tide that circulates principally as a 25–amino acid peptide cretion, blunt activation of the renin–angiotensin system,
but also as shorter peptides of 22 and 20 amino acids with and decrease vascular resistance. Circulating concentrations
diminished biologic activity (see Chapter 40). Likewise, doz- of ANP and BNP (but not CNP) increase rapidly in response
ens of truncated forms of the mature 32–amino acid B-type to increased cardiac filling pressures that are characteristic of
natriuretic peptide ranging from 24 to 31 amino acids are heart failure. Clinical measurement of BNP has become
detectable in heart failure patients (see Chapter 48). Some of a widely used tool to detect heart failure and monitor its
these truncated forms are present in vivo, and others likely progression.
B-type natriuretic peptide is synthesized as a 108–amino the renal juxtaglomerular apparatus.64 Renin acts on circulat-
acid precursor (pro-BNP) that is cleaved upon cellular release ing angiotensinogen to initiate the formation of vasoactive
to the active 32–amino acid peptide and an N-terminal frag- peptides that act to reestablish glomerular flow. The N-terminal
ment (NT-proBNP), which lacks biologic activity.51 The decapeptide cleaved from the 452–amino acid angiotensinogen
diagnostic and prognostic role of these peptides is treated molecule by renin is referred to as angiotensin I. Angiotensin-
extensively in Chapter 48. Two-site immunoassays for both converting enzyme (ACE) cleaves 2 C-terminal residues from
BNP and NT-proBNP are commercially available and widely angiotensin I to form the octapeptide, angiotensin II, which
used. NT-proBNP circulates at higher concentration than promotes contraction of vascular smooth muscle and stimu-
BNP by virtue of its longer biologic half-life (~60 vs. lates proximal tubular sodium reabsorption to increase blood
~20 minutes). Recent evidence suggests that the BNP detected pressure. ACE inhibitors (e.g., captopril, enalapril, quinapril)
immunologically in heart failure is not the bioactive form of are important pharmacologic tools used to treat hyperten-
BNP. Using MS, almost no mature 32–amino acid peptide was sion (see also Chapter 56).
detected in heart failure patients despite the significant pres- Although the renin–angiotensin–aldosterone axis is an
ence of immunoreactive BNP.52,53 Further studies suggest that important therapeutic target, it is a far more infrequent target
the immunoreactive BNP in the plasma of heart failure pa- for diagnostic laboratory studies. Plasma renin activity is
tients may be attributed to higher molecular weight forms assessed to explore the possibility of renovascular hyperten-
such as proBNP that exhibit a fraction of the bioactivity of the sion. In this condition, unilateral restriction of renal blood
mature peptide.54,55 This new analytic information may flow results in inappropriate release of renin and severe
prompt a reevaluation of the role of BNP in the pathophysiol- hypertension. In nonpathologic states, renin activity in the
ogy, diagnosis, and treatment of heart failure. plasma is normally very low (,10 ng/mL/h) and is typically
measured by assessing the production of angiotensin I from
Hepcidin endogenous angiotensinogen after a long (.12 hours) incu-
Hepcidin was initially described as an antimicrobial pep- bation period. Angiotensin I generation is most commonly
tide.56 The role of hepcidin in iron metabolism was noted by monitored via a competitive immunoassay with the potential
Nicolas et al. in 2001.57 The mature 25–amino acid molecule to cross-react with shorter peptides. MS approaches that
is derived from a 60–amino acid precursor expressed from address peptide specificity and stability may mitigate these
3 exons of the HAMP gene on chromosome 19. The tightly analytic limitations.65,66
looped structure of hepcidin is stabilized by four intramo-
lecular disulfide bonds. The biologic activity of hepcidin is Endothelins
mediated via its interaction with ferroportin, the transport Endothelins (ETs) are peptides with 21 amino acids derived
protein that mediates iron transport from duodenal entero- from the vascular endothelium (ET-1), intestinal and renal
cytes and macrophages. Hepcidin binding promotes the in- tissue (ET-2), and neural tissue (ET-3).67 ET-1 is produced
ternalization and degradation of ferroportin, thus inhibiting from a 203–amino acid precursor (preproendothelin) and
mobilization of iron stores.58,59 Physiologic states such as smaller, 30– to 40–amino acid “big” ET molecules that
chronic inflammation are characterized by microcytic ane- are inactive. ET-1 is a potent vasoconstrictor and may medi-
mia with paradoxically adequate iron stores known as anemia ate pathology associated with diabetic nephropathy and
of chronic disease. Increased hepcidin expression is at the hypertension.68 Increased circulating concentrations of ET
pathologic root of this condition. In addition to its diagnostic after myocardial infarction suggest a negative survival prog-
role in differentiating iron deficiency anemia from the ane- nosis.69 The reliability of these observations using currently
mia of chronic disease, hepcidin measurement may aid in the available immunoassays and other potential clinical applica-
treatment of hemochromatosis, transfusion-associated iron tions for ET measurement remain unclear.
overload, and anemia associated with chronic renal failure.60
Despite the important role of hepcidin in iron metabo- Vasopressin
lism, its clinical use remains infrequent partly because of Vasopressin (arginine vasopressin [AVP]), also known as
difficulties associated with its measurement. Antibodies to- antidiuretic hormone (ADH), is a nonapeptide stored in
ward hepcidin have been difficult to develop because of its and secreted from the posterior pituitary gland (see also
small, compact size and because it is highly conserved across Chapter 55). Its primary target organ is the distal convoluted
multiple species. Immunoassays have largely been limited to tubule and collecting duct, where it acts to promote water
single-site, competitive formats that cross-react significantly reabsorption. ADH circulates at very low concentrations
with shorter versions of the molecule (22-, 20-mers).61 This (,40 pmol/L) and has a very short half-life (15 to 20 min-
can lead to overestimation of circulating hepcidin compared utes), making routine diagnostic measurement impractical.
with MS techniques62 when applied to patients with renal Copeptin, a prohormone form of ADH, exhibits a longer
failure in whom shorter hepcidin peptides tend to accumu- half-life and is an attractive alternative target for measure-
late in the plasma.63 Improvements in the molecular specific- ment.70 Diabetes insipidus (DI) may result from faulty secre-
ity and harmonization of hepcidin determination will further tion (central DI) or from end-organ resistance (nephrogenic
clarify its role in both normal and pathologic physiology and DI). Head injury, tumors, and some medications may also
also promise to enhance its clinical utility. For additional induce pathologic secretion of ADH, resulting in fluid over-
discussion on hepcidin, see Chapter 40. load referred to as the syndrome of inappropriate antidiuretic
hormone secretion (SIADH). A synthetic analog referred to as
Angiotensins DDAVP (1-desamino, 8-D-AVP) is used therapeutically to
Renin is secreted by the afferent arterioles of the kidney in treat DI and some forms of coagulopathy. DDAVP stimulates
response to decreased flow, pressure, and sodium delivery to release of von Willebrand factor from endothelial cells
and extends the half-life of circulating factor VIII, thereby (DTNB). Reaction of DTNB with thiols results in the for-
mediating improvements of circulating hemostatic factors mation of 2-thio-5-thiobenzoate, which absorbs with high
associated with various forms of von Willebrand disease and extinction (~14,000 L cm21 mol21) at 410 nm. Other tech-
hemophilia A. niques use derivatization and stabilization of GSH followed
by high-performance liquid chromatography or MS. Inborn
Glutathione errors of glutathione metabolism such as glutathione synthe-
Glutathione consists of a glutamate residue linked to cysteine tase deficiency are detected via the accumulation of compo-
via its g-carboxyl rather than the a-carboxyl group and fol- nents of the g-glutamyl cycle such as pyroglutamic acid
lowed by a conventional peptide bond between cysteine and (5-oxoproline).
glycine.71 This ubiquitous tripeptide is the most abundant in-
tracellular thiol (1 to 10 mmol/L) and circulates in the blood at GASTROINTESTINAL PEPTIDE HORMONES
micromolar concentrations. The cellular ratio of reduced glu-
tathione (GSH) to oxidized glutathione (GSSG) ranges from Enteral organs produce a number of peptides that mediate
10 to 100. Intracellular glutathione performs a variety of im- important digestive processes. Assessment of these peptides is
portant functions. It plays an important role in maintaining indicated in rare digestive disorders. In addition to enteral
the proper ratio of oxidized to reduced forms of metabolically effects, many of these peptides also impact the central ner-
important thiols such as coenzyme A. It also provides reducing vous system. Clarification of their function in the interplay
equivalents that detoxify reactive oxygen species such as perox- between brain, gut, and microbiome may clarify the patho-
ides (catalyzed by glutathione peroxidase). Through the activity logic basis of multiple disorders including obesity, anxiety,
of glutathione-S-transferase, glutathione also serves to detoxify depression, bowel disease, and various forms of neurodegen-
other xenobiotic compounds via formation of a thioether de- eration.73,74 These peptides are still largely assessed by immu-
rivative, which can then be excreted. Amines and peptides are nologic techniques but improvements to analytic specificity
transported across the plasma membrane via the g-glutamyl would likely be achieved using MS. Select peptides are con-
moiety of glutathione, a reaction catalyzed by g-glutamyl- sidered briefly here but treated in detail in Chapter 52.
transpeptidase (see also Chapter 32). The tripeptide is then Gastrin is produced as a larger precursor (preprogastrin,
regenerated through the concerted action of enzymes in the progastrin) by G cells in the pyloric antrum of the stomach.
so-called g-glutamyl cycle (Fig. 31.3). Precursor forms are cleaved to peptides of multiple lengths
Determination of circulating GSH and GSSG is not rou- including the most potent 17–amino acid version. Gastrin
tinely called for in clinical practice as the site of action is in- stimulates acid production by the parietal cells of the stom-
tracellular. Nonetheless, a variety of techniques to measure ach and is overproduced in Zollinger-Ellison syndrome (gas-
glutathione have been used.72 Measurement of total glutathione trinoma) with a worldwide incidence of 0.5 to 3 cases per
requires prior reduction of the sample to release all oxidized million population annually.
forms. The simplest techniques employ the colorimetric de- Cholecystokinin (CCK) is expressed as a 115 amino acid
tection of free thiol using 5,59-dithio-bis-2-nitrobenzoic acid precursor and cleaved into progressively shorter fragments of
AA
Glutathione
γ -GLU-CYS-GLY
ADP
6
1
ATP
GLY
ADP
CYS-GLY 3
CYS 5
γ -GLU-AA ATP
GLU
2 Pyro-GLU
AA
(5-oxoproline) 4 ADP
ATP
FIGURE 31.3 Transmembrane transport of amino acids (AAs) using the g-glutamyl cycle. Three-letter
AA abbreviations are used. Extracellular AA is transferred to glutathione via activity of membrane-
bound g-glutamyl transpeptidase (1). AA is released in the cytoplasm via the activity of g-glutamyl cy-
clotransferase (2), which also results in the formation of pyroglutamate (5-oxoproline). Cysteine and
glycine generated via dipeptidase activity (3) are recycled with pyroglutamate to reform glutathione via
successive activities of 5-oxoprolinase (4), g-glutamyl-cysteine synthetase (5), and glutathione synthe-
tase (6). ADP, Adenosine diphosphate; ATP, adenosine triphosphate; CYS, cysteine; GLU, glutamate;
GLY, glycine.
39, 33, 22, 12, and 8 amino acids that also possess biological multimeric unit. Examples range from the relatively sim-
activity (see also Chapter 52). CCK is secreted by the duode- ple creatine kinase, a heterodimer of M and B subunits, to
nal mucosa and stimulates gall bladder contraction and branched chain a-ketoacid dehydrogenase, which is a het-
secretion of pancreatic enzymes. eromeric complex of 12 E1, 24 E2, and 6 E3 subunits.
Secretin is synthesized as a 120 amino acid precursor by 5. Ligands and prosthetic groups provide additional func-
the duodenal mucosa. The mature 27 amino acid peptide is tional and structural elements, such as metals in metalloen-
released in response to acid and acts to stimulate pancreatic zymes, heme in hemoglobin and cytochromes, and lipids in
bicarbonate release and neutralize the proximal intestinal lipoproteins. Proteins without their associated ligands are
environment and ensure proper pancreatic enzyme function. often referred to as apoproteins (e.g., apotransferrin with-
Recombinant secretin is used to stimulate and assess pancre- out iron, apolipoproteins without lipid).
atic secretory function.
Vasoactive intestinal peptide (VIP) is a 28 amino acid Physical Properties of Proteins
peptide secreted by enteric neurons throughout the gut. It The diverse structural features of proteins result in unique
shares structural similarity with glucagon and secretin. In the physical properties that can be exploited for analysis. For ex-
gut, VIP acts to increase pancreatic water and electrolyte se- ample, tyrosine and tryptophan residues absorb light at 280
cretion, gut motility, as well as hepatic bile flow. VIP also nm, and the abundance of these amino acids determines the
impacts coronary vasodilation and myocardial contractility. extinction coefficient of a peptide or protein. A pure protein,
In the brain, VIP is an important mediator of circadian therefore may be quantitated using A280. Some prosthetic
rhythms. groups such as heme also possess intrinsic absorbance that
Ghrelin is a 28 amino acid peptide that requires covalent may be monitored to assess the presence of specific proteins.
modification by octanoic acid for full activity. Ghrelin is pro- Automated clinical analyzers assess the presence of hemoglo-
duced in the stomach and is best known as a potent stimulus bin at 540 to 570 nm, for example, to detect hemolyzed
of growth hormone production. Ghrelin itself is metabolized plasma or serum specimens. Ionizable groups exert a strong
to obestatin, a 24 amino acid peptide that antagonizes the effect on physical properties depending on the pH of the sur-
function of ghrelin. rounding solution. Differing physical properties serve as the
basis of methods to separate proteins. Some important char-
acteristics include the following:
PROTEINS 1. Differential solubility. The solubility of proteins is affected
The structural diversity of proteins may be described using by pH, ionic strength, temperature, and the characteristics
the following features: of the solvent. Changing solvent pH affects the net charge
1. Primary structure is the linear sequence of amino acids in of a protein. Changing ionic strength affects the hydration
a peptide or protein. Post-translational modifications of and solubility of proteins. “Salting-in” and “salting-out”
amino acids contribute to increased diversity. procedures were early methods for separating and charac-
2. Secondary structure describes the nature of the peptide terizing protein. Albumin, for example, stays in solution at
backbone dictated by the peptide bond angles described high concentrations of ammonium sulfate that precipitate
earlier and stabilized by hydrogen bonds. Examples of globulins. Addition of organic solvents and polyethylene
secondary structure include a-helix, b-sheet, and b-turn. glycol is also useful for differential precipitation. Frac-
An a-helix has about 3.6 residues per turn and is stabilized tional precipitation of plasma with ethanol, using proto-
by hydrogen bonds between the N–H and C5O group of cols developed by Cohn and coworkers,75 enables isolation
the fourth following amino acid. A b-sheet involves hy- of plasma fractions that are enriched in immunoglobu-
drogen bonds between the peptide bonds of adjacent lins, a- and b-globulins, or albumin (fraction V). Polyeth-
peptide chains arranged in parallel or antiparallel configu- ylene glycols induce precipitation by steric exclusion and
rations. Random coils refer to segments of peptide that therefore preferentially precipitate large proteins or com-
lack defined secondary structure. plexes.
3. Tertiary structure refers to the folding of the polypeptide 2. Molecular size. Separation of small and large molecules is
chain and elements of secondary structure into a compact commonly achieved by differential migration through
three-dimensional (3D) shape. Folding is a complex pro- molecular filters. Examples are size exclusion chromatog-
cess driven by energy minimization of intramolecular and raphy (also known as gel filtration), ultracentrifugation,
solvent interactions. Hydrophobic groups tend to fold into and electrophoresis. These techniques may be used under
the interior with less exposure to solvent, while charged conditions when proteins and peptides are in native
and polar sidechains tend to be located on the surface. The globular states or under denaturing conditions. Addition
3D structure is stabilized by intramolecular hydrogen of reducing agents allows separation of disulfide-linked
bonds, van der Waals forces, and hydrophobic interactions. components.
Disulfide bonds between cysteine residues also stabilize 3D 3. Molecular mass. Advances in MS allow the determination
structure. Denaturation of protein refers to unfolding that of masses of peptides and proteins with increasing accu-
occurs with temperature change or in the presence of or- racy. Peptides and proteins can be ionized by MALDI or
ganic solvents, detergents, or reagents that disrupt hydro- by ESI (see Chapter 24).
gen bonds. Limited denaturation can be reversible, but 4. Electrical charge. Ion-exchange chromatography, isoelec-
extensive unfolding and denaturation of proteins often tric focusing (IEF), and electrophoresis separate peptides
lead to irreversible aggregation and precipitation. and proteins based on charge (see Chapters 18 and 19).
4. Quaternary structure refers to the incorporation of two 5. Surface adsorption. The affinity of peptides and proteins
or more polypeptide chains or subunits into a larger for a variety of physical surfaces may also be used as the
basis for separation. Reverse-phase chromatography, for in response to a variety of stresses are molecular chaperones.
example, exploits the interaction of hydrophobic molecular Increased accumulation of misfolded proteins induces an adap-
moieties with hydrophobic surfaces (C8 or C18 alkyl chains) tive mechanism—the unfolded protein response.81 This re-
when the ratio of water to organic solvent is high but not sponse increases production of chaperones and slows general
when organic content is increased (see Chapter 19). protein synthesis to allow more time to fold new proteins.
6. Affinity chromatography. Specific ligands, antibodies, and Despite these protective mechanisms, several families of
other recognition molecules have been used to separate age-related, genetic, and infectious diseases appear connected
peptides or proteins selectively (see Chapter 19). to disorders of protein folding and protein aggregation. Prion
diseases are infectious diseases in which the transmissible
Protein Formation protein agent may catalyze misfolding of endogenous proteins.
Folding In Alzheimer disease, deposits of amyloid may contribute to
Proteins are synthesized by ribosomes reading from the 59- pathogenesis. Polyglutamine diseases result from genetic expan-
end of mRNA. Triplet codons in mRNA are matched with sion of repeat units encoding glutamine and are associated with
complementary sequence in transfer RNA carrying specific Huntington disease and other neurodegenerative disorders.82
amino acids. Protein synthesis begins with an AUG codon These expanded polyglutamine sequences tend to aggregate as
encoding methionine, and the polypeptide chain is synthe- b-sheets. TDP-43 proteinopathies include amyotrophic lateral
sized from the N terminus. During translation, the initiator sclerosis (Lou Gehrig disease), resulting from aggregation of
methionine is typically cleaved and the resulting N-terminal transactive DNA-binding proteins.83 Several inherited disorders
residue commonly acetylated. Although 80 to 90% of pro- related to mutations in specific proteins probably result from
teins carry an N-terminal acetyl group, the function of this problems in protein folding. In AAT deficiency, hepatic injury
modification is not entirely clear, but it may play a role in results from aggregation and accumulation of misfolded
stabilizing the growing peptide chain. protein.84,85 The most common cause of cystic fibrosis results
Instructions for folding are largely contained in the primary from a single amino acid deletion (DF508), which results in
amino acid sequence of the growing polypeptide chain. The rapid degradation of the cystic fibrosis transmembrane conduc-
rate of elongation (typically 5 to 10 amino acids per second in tance regulator (CFTR). Accumulation of misfolded proteins
eukaryotes) may have a significant impact on folding. The use has been suggested as a pathogenic mechanism contributing
of rare codons, secondary structural elements of the mRNA, to vascular, cardiac, and b-cell failure in diabetes.81 Small
and polybasic stretches may dictate pauses in translation and molecule therapeutics capable of modulating protein folding
enhance formation of secondary structural elements.76,77 Fold- have shown some promise in mitigating disease caused by
ing begins as the chain is elongated and still associated with the abnormal protein aggregation.86
ribosome, assisted by a family of proteins referred to as chap-
erones.78 The function of chaperones was originally ascribed to Targeting
a group of proteins called “heat shock proteins” that prevented As originally outlined by Lingappa and Blobel,87 proteins that
protein denaturation and aggregation in response to heat and are secreted, located in vesicular compartments, or oriented
other extreme environmental conditions. on the external surface of cell membranes usually contain a
Many gene products that share common 3D features hydrophobic N-terminal signal peptide about 15 to 30 amino
have arisen from common ancestral genes. The serpin (serine acids in length. Signal peptides interact with signal recogni-
proteinase inhibitor) superfamily consists of more than tion particles (SRPs) and mediate interaction with the endo-
1000 related proteins in different organisms.79 Humans have plasmic reticulum (ER). Nascent peptide chains are inserted
36 serpins, 29 of which are protease inhibitors and 7 of which through the membrane of the ER as the protein is synthe-
lack protease inhibitor function. Serpins that act as protease sized. Signal peptides of most secretory proteins are removed
inhibitors in plasma include a1-antitrypsin (AAT), a1-anti- even before synthesis of the entire protein chain is completed.
chymotrypsin, a2-antiplasmin, antithrombin, C1 inhibitor, Co-translational membrane retention may be achieved via an
heparin cofactor II, protein C inhibitor, and plasminogen uncleaved signal sequence, by one or more hydrophobic
activator inhibitor-1 (PAI-1). Serpins without known prote- transmembrane domains, or by lipid modifications such as
ase inhibitor function are cortisol-binding globulin, thyrox- N-myristoylation.88
ine-binding globulin, angiotensinogen, intracellular proteins, Newly synthesized proteins ultimately reside in a number
heat shock protein 47, and the tumor suppressor maspin. of membranous or soluble compartments, including the nu-
Serpins illustrate how a common structure motif may be cleus, lysosome, peroxisome, mitochondrion, or plasma
adapted to multiple functions. Other examples of plasma membrane. Plasma membrane sorting is further complicated
protein families are the albumin and lipocalin families. The in polarized epithelial cells where proteins may be targeted to
albumin family includes albumin, a-fetoprotein, and afamin. basolateral or apical environments. In the so-called “secre-
The lipocalin family includes several plasma proteins such as tory” pathway, proteins are shuttled via membrane-bound
a1-acid glycoprotein (AAG), retinol-binding protein, apoli- vesicles bearing COP II (coat protein II) from the ER through
poprotein D, a1-microglobulin, prostaglandin D synthase the Golgi apparatus.89,90 Intra-Golgi transport and retrograde
(b-trace), b-lactoglobulin, neutrophil gelatinase-associated Golgi to ER transport is mediated by COP I vesicles. Upon
lipocalin (NGAL), inter-a-trypsin inhibitor, and C8 g-chain. fusion of vesicle with specific membranes, soluble compo-
Lipocalins generally have a barrel-shaped structure that is nents are extruded, and lipid-associated components take up
well suited to serve as a carrier for small molecules. residence as stable membrane components. Sorting in polar-
Protein folding is an error-prone process, and many molecu- ized epithelia is mediated by association of proteins with
lar chaperones work to refold, prevent aggregation, or degrade unique membrane domains. For example, proteins anchored
misfolded proteins.80 Several heat shock proteins that increase to the membrane via a glycosylphosphatidylinositol (GPI)
anchor tend to cluster in cholesterol- and sphingolipid-rich Prenylation

domains called lipid rafts that are selectively sorted to apical Isoprenoid compounds such as farnesyl pyrophosphate
surfaces.91 Proteins destined for mitochondria contain a (15 carbons) and geranylgeranyl pyrophosphate (20 carbons)
unique N-terminal targeting sequence that mediates their are hydrophobic moieties formed from 3-hydroxy-3-methyl-
interaction and import into the proper submitochondrial glutaryl CoA (HMGCoA) and mevalonate via HMG CoA
location (e.g., outer membrane, inner membrane, matrix, reductase. These groups modify more than 300 members of
intermembrane space).92 the human proteome via enzymatic attachment to a cysteine
residue in a so-called “CaaX” motif where C is cysteine, aa
Post-translational Modifications represents aliphatic amino acids such as glycine or alanine,
Acetylation and X is typically serine, methionine, glutamine, alanine, or
Eighty to 90% of eukaryotic cellular proteins are acetylated. threonine.104,105 Isoprenylation regulates membrane and mo-
Acetyl-CoA is the typical substrate for a variety of acetyl lecular association of a number of proteins important for
transferase enzymes localized to the nucleus, cytoplasm, and signal transduction (H-Ras, K-Ras), vesicular trafficking
mitochondria. Most acetylation occurs co-translationally on (Rab2, Rab3a), cytoskeletal function (RhoA, RhoB), and the
the N-terminal a-amino group of methionine or another integrity of the nuclear membrane (lamin A).106
exposed N-terminal residue after excision of the initiator
methionine. Acetylation targeting the e-amino group of ly- Glycosylphosphatidylinositol Anchor
sine is post-translational and reversible. Common targets of The GPI anchor is a glycoglycero-phospholipid construct
reversible lysine acetylation include histones, cytoskeletal that mediates membrane attachment for a variety of proteins.
proteins, mitochondrial proteins, and proteins controlling The anchor is presynthesized in the ER and then transferred
cell growth including the tumor suppressor, p53. Acetylation, to the target protein via a C-terminal hydrophobic signal
therefore contributes to control of gene expression, cell mo- sequence. After modification, this hydrophobic sequence
tility and division, metabolism, and oncogenesis.93 is removed, leaving a protein that is uniquely membrane
anchored via interdigitation of two fatty acyl chains with a
Fatty Acylation single membrane leaflet.107,108 The purpose of the GPI anchor
The activity and localization of a variety of proteins may be remains unclear, although it has been proposed that such
modulated by covalent attachment of fatty acyl chains. lipid-anchored proteins diffuse in the lateral plane of biologic
Co-translational attachment of myristate via a glycine residue membranes more rapidly than transmembrane proteins.109
has been previously mentioned as a mechanism for membrane GPI-anchored proteins also uniquely associate with choles-
association. The most common acylation of eukaryotic proteins terol- and glycosphingolipid-rich plasma membrane domains
involves thioester linkage of palmitate to membrane proximal referred to as lipid rafts and caveolae.91 Notable examples of
cysteine residues.94 S-palmitoylation reversibly controls local- GPI-anchored proteins include decay accelerating factor
ization to membrane microdomains such as lipid rafts and thus (DAF, CD55), membrane inhibitor of reactive lysis (MIRL,
regulates interaction of proteins with signaling and other effec- CD59), alkaline phosphatase, 59-nucleotidase, and glypican
tor molecules. Examples of palmitoylated proteins include family members. Defects in the PIG-A gene product that me-
caveolin-1, some members of the SRC protein kinase family, diates GPI synthesis are responsible for paroxysmal nocturnal
NO synthase (NOS), b-adrenergic receptor, and transferrin hemoglobinuria (PNH). PNH is characterized by abnormal
receptor. Ghrelin, a potent growth hormone secretagogue, complement-mediated lysis of erythrocytes deficient in CD55
is modified by covalent attachment of an octanoyl moiety and CD59.
at serine 3 of the polypeptide.95,96 Only octanoylated ghrelin
promotes growth hormone release. g-Carboxylation
Glutamic acid is a five-carbon a-amino dicarboxylic acid.
Phosphorylation Vitamin K acts as a cofactor in the enzymatic addition of
Reversible phosphorylation may impact as many as one third a second carboxyl group to the g-carbon.110 A cluster of
of all human cellular proteins.97 O-phosphorylation occurs at g-carboxylated glutamyl residues is referred to as a “gla” domain.
serine, threonine, and tyrosine residues. The human genome This modification mediates calcium-dependent membrane
encodes approximately 1000 kinases, enzymes responsible for association and is required for full functional activity. Many
phosphorylation, and about 500 phosphatases responsible proteins of the coagulation cascade contain the gla domain,
for removal of covalent phosphate groups. Detailed treatment including thrombin, factors VII, IX, and X, and protein C, S, and
of reversible phosphorylation is beyond the scope of this Z. Warfarin exerts its anticoagulant effect by abrogating the
chapter but, in general, serine and threonine phosphorylation activities of the vitamin K–dependent factors.
acutely modifies enzyme activity (e.g., glycogen phosphory-
lase)98 and subcellular localization (e.g., cAMP-dependent Glycosylation
protein kinase).99 Tyrosine phosphorylation, on the other Secreted proteins and the extracellular domains of transmem-
hand, regulates a plethora of signaling pathways (e.g., mitogen brane proteins are commonly modified with carbohydrate.
activated protein kinase, Janus kinase pathways) in part by Carbohydrate plays an important role in folding, secretion,
providing docking site proteins that propagate a transmem- and stability of the modified polypeptide. Sugar chains are
brane signal such as those of the SRC kinase family (lyn, lck, added in the ER and Golgi apparatus via O-linkage to serine
fyn).100 Mitochondria contain members of a primitive kinase and threonine residues or N-linkage to the amide nitrogen of
family that modulate flux through the pyruvate dehydroge- asparagine residues.111 O-linked sugar modifications are typi-
nase and branched chain a-ketoacid dehydrogenase via a cally simple and consist of one to four residues. The sugars
unique phosphohistidine intermediate.101–103 are transferred to the nascent polypeptide via the energy of
the phosphoester bonds of nucleotide sugars such as uridine (S–N5O) derivatives with free cysteine thiol groups.122,123
diphosphate–galactose and guanosine diphosphate–mannose. Thousands of proteins with a diverse array of functions are
N-linked sugars are far more complex. A core glycan consisting reversibly modified in this way.124 Although no rigorous deter-
of 2 N-acetylglucosamine, 9 mannose, and 2 glucose moieties minants of nitrosylation sites have been defined, solvent ex-
is preassembled on a membrane embedded isoprenoid posed cysteine residues in alpha helices within 6 Å of charged
molecule, dolichol, and then transferred en bloc to the newly residues seem to be preferred targets.122 Nitrosylation may oc-
synthesized peptide chain. The core glycan structure may then cur directly with the possible aid of metal catalysis via transfer
be lengthened and trimmed by a host of enzymatic processes. of SNO groups from low molecular weight thiols such as glu-
Approximately 200 human gene products are involved in tathione, or through exchange mediated by disulfide reducing
glycosylation, and more than 100 genetic defects in this enzymes such as thioredoxin.125,126 Effects of nitrosylation are
process have now been documented.112,113 pleiotropic. Whereas nitrosylation of the N-methyl-d-aspar-
tate receptor in neurons blunts its activity,127 nitrosylation of
Sulfation matrix metalloprotease-9 (MMP-9) stimulates its activity.128
Sulfation of proteins on tyrosine residues was originally de- The role of protein nitrosylation in health and disease remains
scribed in 1954.114,115 Nearly 50 secreted and transmembrane to be fully clarified.
proteins carry this irreversible modification that occurs in the
Golgi apparatus. Examples of sulfated proteins include coagu- Ubiquitination
lation factors V, VIII, and IX, fibrinogen, thyroglobulin, and Ubiquitin is a highly conserved 76–amino acid polypeptide
a-fetoprotein. The sulfation reaction is catalyzed by two containing seven lysine residues encoded by four human
widely expressed isoforms of tyrosylprotein phosphotransferase genes.129 Intracellular polypeptide chains may be modified with
(TPST) and uses 39-phosphoadenosine-59-phosphosulfate as ubiquitin through the concerted action of three ubiquitin ligase
the sulfonic acid donor.116 The consensus peptide sulfation enzymes (E1 to E3). In the presence of ATP, ubiquitin is first
site is poorly understood aside from the fact that acidic resi- activated via formation of a thioester bond between E1–cysteine
dues surrounding the target tyrosine appear to promote this residue and the C-terminal carboxyl of ubiquitin. Activated
modification. The function of sulfation is likewise not well ubiquitin is then trans-esterified to the E2 ligase, which cooper-
understood beyond its capacity to enhance the affinity of ates with E3 to effect the formation of an isopeptide bond be-
molecular recognition events. A naturally occurring Tyr n Phe tween the C-terminal glycine of ubiquitin and a lysine residue
at position 1680 of factor VIII, for example, prevents sulfation on the target protein.130 The target protein may be modified by
and weakens its interaction with von Willebrand factor, caus- a single ubiquitin moiety, or multiple molecules may be added
ing a mild form of hemophilia.117 to the original ubiquitin at one or more of the seven ubiquitin
lysine residues. Limited ubiquitination of target proteins gener-
Hydroxylation ally modifies their subcellular location or intermolecular interac-
Hydroxylation is the most prevalent post-translational modifi- tions, and polyubiquitination targets the protein for destruction.
cation of human proteins. Hydroxylation most commonly Proteolysis occurs in the 26S proteasome, consisting of a 20S
occurs at the 4-position of the proline ring. Approximately catalytic and 19S regulatory subunit constructed from greater
30% of proline residues in collagen are modified in this way. than 60 polypeptides.131 After recognition of the protein target by
Therefore hydroxyproline residues are more common in pro- the 19S subunit, the unfolded polypeptide is threaded into the
teins than many other common amino acid residues, including 20S subunit, where peptide bond fission occurs via a threonine-
cysteine, histidine, methionine, phenylalanine, tryptophan, mediated nucleophilic attack.
and tyrosine.118 Hydroxylation of proline residues is thought to
alter the flexibility (“pucker”) of the pyrrolidine ring, thereby Sumoylation
stabilizing the triple helical structure of collagen119 and other Small Ubiquitin-like MOdifier (SUMO) proteins consist of
connective tissue proteins (e.g., elastin). approximately 100 amino acids (~12 kDa) and exhibit 15 to
Proline hydroxylation also plays a unique role in mediat- 20% homology with ubiquitin.132 There are four human
ing the cellular response to oxygen through hypoxia induc- SUMO proteins dubbed SUMO 1, 2, 3, and 4 and at least
ible factor-1 (HIF-1). Under hypoxic conditions, HIF-1a/ 2500 sumoylated proteins have been identified.133 SUMO is
HIF-1b heterodimers induce expression of genes that medi- attached via C-terminal glycine and forms an isopeptide
ate angiogenesis, erythropoiesis, vascular tone, citric acid bond with lysine in the target protein. SUMO targets are
cycle activity, and iron metabolism.120 During normoxia, modified on the e-amino of lysine residues, most within a
proline residues at position 402 or 564 are targets for enzy- CKxD/E motif in which C is a hydrophobic residue, K is the
matic hydroxylation, which mediates ubiquitination by a target lysine, x is any amino acid followed by an acidic residue
protein complex containing von Hippel-Lindau (VHL) pro- (aspartate or glutamate). Covalent SUMO attachment is
tein.121 Ubiquitination of HIF-1a mediates its degradation ATP-dependent and achieved in a coordinated way by the
via the proteasome (see section on ubiquitination) and sub- action of E1, E2, and E3 enzymes. SUMO proteins themselves
sequent blunting of the genetic response to hypoxia. may be multiply sumoylated. The modification is reversible
via a series of SUMO-specific proteases. Mice that lack the
Nitrosylation ability to conjugate SUMO to proteins die early in embryonic
NO is a volatile free radical produced from arginine via three life. Sumoylation has a significant impact on protein survival
human NOS enzyme systems: neuronal (nNOS), endothelial as it antagonizes protein modification by ubiquitin. SUMO-
(eNOS), and an inducible form (iNOS). NO exerts its primary modification affects a vast array of cellular functions including
biologic effects (e.g., vasodilation) via guanylate cyclase– gene expression, DNA repair, and protein translocation.134
coupled receptors, but it also forms covalent nitrosothiol Altered sumoylation has been implicated in numerous
pathologic states including cancer and Parkinson, Alzheimer, inactivates a broad variety of protease enzymes via forma-
and Huntington disease.135 tion of a covalent complex with the active site serine. Mem-
bers of the serpin superfamily with antiprotease activity
Protein Catabolism include AAT, antithrombin, PAI-1, and protein C. Some
The steady-state concentration of any specific intra- or extra- members of the serpin family, including angiotensinogen
cellular protein reflects not only its rate of synthesis but also and thyroxine-binding globulin, do not possess known ac-
its rate of degradation. The degradative process is much more tivity against specific proteases. Serine protease activity in
than a passive, nonspecific mechanism for disposing of un- vitro can be mitigated with a number of inhibitors, including
wanted cellular material. It is highly specific and tightly con- phenylmethanesulfonylfluoride (PMSF), [4-(2-aminoethyl)
trolled. As supporting evidence, consider that the human benzenesulfonyl fluoride] (AEBSF), aprotinin, and leupeptin.
genome contains more than 500 proteases. These proteases Cysteinyl proteases. This group of proteases uses a cysteine
belong to one of four families based on the catalytic mecha- thiol in nucleophilic attack on the peptide bond.137 A thioester
nism for hydrolyzing peptide bonds. Representative members intermediate with the carbonyl carbon of the peptide bond is
of the four protease families are detailed in Table 31.3. formed before hydrolysis and formation of two peptides with
new C and N termini. In humans, cysteine proteases mediate
Protease Families apoptosis via a series of enzymes referred to as caspases.138
Serine proteases. The serine proteases are the most Other notable cysteine proteases include some in the cathep-
abundant family in humans and are so named because serine sin family and interleukin 1b converting enzyme (ICE).
serves as the nucleophilic residue at the active site of the Cystatins are endogenous inhibitors of cysteine protease
enzyme.136 Peptide bond hydrolysis is achieved via a con- activities. Some chemical inhibitors of serine proteases such
served “catalytic triad” of spatially adjacent histidine, serine, as PMSF and leupeptin also exhibit activity toward cysteine
and aspartate residues. These enzymes enable a vast array of proteases. Unique in vitro inhibitors of cysteine proteases in-
physiologic processes, including protein digestion, comple- clude L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane
ment activation, and blood coagulation. As indicated earlier, (E-64) and N-[N-(N-acetyl-L-leucyl)-L-leucyl]-L-norleucine
a family of endogenous serine protease inhibitors (serpins) (ALLN).
TABLE 31.3 Selected Members of the Four Protease Families

Protease/Family Gene Loci Tissue Expression Subcellular Localization Substrate, Function, Pathology
Serine
Corin 4p13 Broad Plasma membrane Natriuretic peptides (heart failure)
Trypsin 7q34 Pancreas Secreted Promiscuous (cleavage of LYS-X, ARG-X)
Chymotrypsin 16q23 Pancreas Secreted Promiscuous (cleavage of TRP-X, PHE-X,
TYR-X)
Neutrophil elastase 19p13 Myeloid cells Cytoplasm, secreted Promiscuous (cleavage of VAL-X, ALA-X)
Factor IX Xq27 Liver Secreted, plasma Conversion of factor X to Xa (hemophilia B)
membrane
Activated protein C 2q14 Liver Factor Va, factor VIIIa (coagulopathy)
PSA (kallikrein) 19q13 Prostate Secreted Semen liquefaction; marker of prostate
mass and cancer
C1s 12p13 Broad Secreted Complement C1r, C2, and C4 (angioedema)
Cysteinyl
Caspase-3 4q34 Broad Cytosol, nucleus, ASP-X-X-ASP (apoptosis)
mitochondria
Cathepsin C 8p23 Broad Lysosome Amyloid precursor (Alzheimer disease)
Aspartyl
Renin 1q32 Ovary, broad Secreted Angiotensinogen (hypertension)
Pepsin 6p21 GI tract, lung Secreted Cleavage at adjacent hydrophobic residues
(PHE-VAL, ALA-LEU, LEU-TYR)
Presenilin 14q24 Broad Endoplasmic reticulum/ Amyloid precursor (Alzheimer disease)
Golgi
Metallo
ADAMTS13 9q34 Liver, erythroid Secreted von Willebrand factor (thrombotic
precursors, broad thrombocytopenic purpura)
MMP-1 11q22 Muscle Secreted Collagen (tissue remodeling,
embryogenesis, metastasis)
Angiotensin I 17q23 Testes, broad Secreted Angiotensin I (hypertension)
converting enzyme
Aspartyl proteases. In contrast to the serine and cysteinyl four proteins referred to as tissue inhibitors of matrix metal-
proteases, aspartyl proteases do not act via a covalent acyl loproteases (TIMPs). TIMPs act via formation of an equimo-
enzyme intermediate. Instead, peptide bond lysis is achieved lar complex with target MMPs. Chemical inhibition of MMP
in a single step through the coordination of a water molecule activity may be achieved by metal chelation and hydroxamic
between two highly conserved aspartate residues. One aspar- acid derivatives.
tate residue abstracts a proton from water, which then be-
comes a nucleophile, attacking the carbonyl carbon of the Proteins in Human Serum and Plasma
peptide bond.139 Human aspartyl protease enzymes include The circulating proteome is a complex mixture of thousands
members of the pepsin, cathepsin, and renin families. The of gene products. The most abundant products are proteins
target for HIV protease inhibitors (e.g., indinavir, ritonavir) secreted directly into the circulation primarily by the liver
is also an aspartyl protease. Pepstatin is a potent hexa-peptide and immunoglobulins contributed by lymphatic tissue. Clas-
inhibitor of aspartyl protease activity. sical methods for protein fractionation and purification over
Metalloproteases. Members of this protease family, com- several decades led to isolation and characterization of about
monly referred to as MMPs, include approximately 25 Zn21 100 of the most abundant proteins.144 The 12 most abundant
dependent enzymes sub-classified by their reactivity to col- proteins represent more than 95% of total protein mass.
lagen, gelatin, and other extracellular matrix proteins.140–142 Albumin alone represents more than 50% of the total mass of
MMPs contain a conserved motif in which a Zn atom is coprotein and an even higher proportion of the number of
ordinated by three histidine residues and a glutamate residue. molecules so that albumin is the main contributor to colloid
MMPs achieve peptide bond fission through nucleophilic osmotic pressure (oncotic pressure). The distinction between
activation of a water molecule bound between Zn and the mass and molar concentrations of circulating proteins may
g-carboxyl of glutamate.143 MMPs play a multitude of roles be significant in considering oncotic pressure, protease inhi-
in wound healing and repair, pathogen defense, cancer me- bition, and the binding capacity for ions, drugs, or small
tastasis, and rheumatoid arthritis. Endogenous control of molecules. Table 31.4 lists the 30 most abundant proteins by
MMP activity is achieved through an endogenous group of mass and molecular abundance. An exhaustive list of the
TABLE 31.4 High-Abundance Plasma Proteins

RANKED BY MASS ABUNDANCE (mg/L) RANKED BY MOLECULAR ABUNDANCE (mmol/L)
Rank Protein Concentration Protein Concentration
1 Albumin 35,000–52,000 Albumin 500–800
2 Immunoglobulin G 7,000–16,000 Immunoglobulin G 40–120
3 Transferrin 2,000–3,600 Apolipoprotein A-I 30–70
4 Immunoglobulin A 700–4,000 Apolipoprotein A-II 30–60
5 a2-Macroglobulin 1300–3,000 Transferrin 25–45
6 Fibrinogen 2,000–4,000 a1-Antitrypsin 18–40
7 a1-Antitrypsin 900–2,000 Haptoglobin 6–40
8 Apolipoprotein A-I 910–1,940 a1-Acid glycoprotein 15–30
9 C3 900–1,800 a2HS-glycoprotein 9–30
10 IgM 400–2,300 Immunoglobulin A 5–30
11 Haptoglobin 300–2,000 Hemopexin 9–20
12 Apolipoprotein B 600–1,550 Apolipoprotein C-III 6–20
13 a1-Acid glycoprotein 500–1,200 Fibrinogen 5–18
14 a2HS-glycoprotein 400–1,300 Gc-globulin 8–14
15 Hemopexin 500–1,150 Apolipoprotein C-I 6–12
16 Gc-globulin (vitamin D–BP) 400–700 C3 5–10
17 Factor H 240–740 a1-Antichymotrypsin 4–9
18 a1-Antichymotrypsin 300–600 Apolipoprotein D 2–10
19 Inter-a-trypsin inhibitor 200–700 Prealbumin 4–8
20 Apolipoprotein A-II 260–510 b2-Glycoprotein I 3–6
21 C4b-binding protein 200–530 Apolipoprotein A-IV 3–6
22 Ceruloplasmin 200–500 Apolipoprotein C-II 2–7
23 Factor B 180–460 Serum amyloid A4 3–6
24 Prealbumin 200–400 Inter-a-trypsin inhibitor 3–5
25 Gelsolin 200–400 Antithrombin III 3–5
26 Fibronectin 300 a1B-glycoprotein 3–5
27 C1 inhibitor 190–370 Gelsolin 3–5
28 C4 100–400 Ceruloplasmin 2–5
29 Plasminogen 150–350 Factor H 2–5
30 Antithrombin III 170–300 Factor B 2–5
Data from in Hortin GL, Sviridov D, Anderson L. High-abundance polypeptides of the human plasma proteome comprising the top 4 logs of
polypeptide abundance. Clin Chem 2008;54:1608–1616.
contents of the circulating proteome would exceed 12,000 component anodal to albumin on routine serum electropho-
entries.145 resis and is not routinely observed by most methods. It is a
The range of protein concentrations measured in clinical proportionately greater component of CSF. Prealbumin is
assays spans more than 10 orders of magnitude and thus poses most commonly assessed using immunonephelometric or
a significant analytic challenge. In decreasing order of abun- immunoturbidimetric methods.
dance, the source of circulating proteins include (1) proteins
secreted directly into plasma, (2) proteins associated with the Albumin
cell membrane and shed into the circulation, (3) secretory The name albumin (L. albus, meaning white) originated from
proteins in exocrine secretions, (4) high-abundance cytoplasmic the white precipitate formed during the boiling of acidic
proteins, (5) low-abundance cytoplasmic proteins, (6) trans- urine from patients with proteinuria. Normally, albumin is
membrane proteins, and (7) organellar proteins that must the most abundant plasma protein from the fetal period on-
traverse more than one membrane to exit cells. Many of these ward, accounting for about half of the plasma protein mass.
serve as useful markers of physiology and disease. It is a major component of most body fluids, including inter-
Circulating concentrations of proteins depend not only on stitial fluid, CSF, urine, and amniotic fluid. More than half of
rates of production and efficiency of entry to the circulation the total pool of albumin is in the extravascular space.
but also on rates of clearance. Proteins and peptides substan- Albumin has a nonglycosylated polypeptide chain of 585
tially smaller than albumin are cleared from the circulation by amino acids and a calculated molecular weight of 66,438 Da.
glomerular filtration unless they are bound to larger carrier It is synthesized in the liver and has a 3D structure stabilized
molecules. Peptides and small proteins not bound to carriers by 17 intrachain disulfide bonds.150 It is both chemically and
are cleared with half-lives of about 2 hours under conditions of biologically stable because it resists denaturation at higher
normal kidney function and accumulate in kidney failure. Ex- temperatures than most plasma proteins and circulates with
amples of proteins and peptides that increase dramatically in a half-life of 15 to 19 days. Albumin has a high abundance
renal failure include b2-microglobulin (BMG), cystatin C, im- of charged amino acids that contribute to high solubility, and
munoglobulin light chains, parathyroid hormone fragments, it has a net negative charge of about 212 at neutral pH.151
complement factor D, ANP, and interleukins.146,147 Other pro- Albumin therefore contributes about 6 to 10 mmol/L to
teins and bioactive peptides such as insulin, intact parathyroid the anion gap at normal albumin concentrations of 0.5 to
hormone, and growth hormone have much shorter circulating 0.8 mmol/L (3.5 to 5.2 g/dL) and lesser amounts at lower albu-
half-lives of only a few minutes, indicating receptor-mediated min concentrations. At a pH of 8.6 for alkaline electrophoresis,
uptake or degradation by exopeptidases or endopeptidases.148 albumin has a net charge of about 225, resulting in high
For all circulating proteins, the choice of measurement mobility toward the anode. One unpaired cysteine at position
using plasma or serum is not without consequence. Plasma 34 occurs partially in reduced form and partially in ex-
refers to the fluid portion of blood in the presence of an an- changeable disulfide bonds with other free thiols.
ticoagulant after the cells are removed by centrifugation. Albumin has two critical biologic functions. First, it serves
Common anticoagulants include dry heparin or ethylenedi- as the major component of colloid osmotic pressure. Patients
aminetetraacetic acid (EDTA) or solutions containing citrate. with hypoalbuminemia caused by nephrotic syndrome, for
Small molecular weight additives such as EDTA may intro- example, develop edema. Second, it serves as a transporter
duce osmotic effects on plasma volume, and citrate solutions for a diverse range of substances, including fatty acids and
introduce some specimen dilution. Variation in platelet con- other lipids, bilirubin, foreign substances such as drugs, thiol-
centration is noted, depending on the time of centrifugation containing amino acids, tryptophan, calcium, and metals.
and the force applied. Preparation of platelet-poor plasma Some of these substances, such as fatty acids and unconju-
commonly requires spinning specimens twice to ensure gated bilirubin, have very low solubility in water in the ab-
platelet removal and remove their contribution to plasma sence of a carrier molecule.
proteins. Serum is the fluid component of blood after blood Most clinical laboratories assay albumin in plasma or se-
is allowed to clot. It differs from plasma in several respects. rum samples by dye-binding methods, which rely on a shift
An approximate 4% decrease in total protein content is related in the absorption spectrum of dyes such as bromocresol
mainly to removal of fibrinogen during coagulation. Because green (BCG) or purple (BCP) upon albumin binding. The
of the absence of fibrinogen, serum has a lower viscosity than affinity of these dyes is higher for albumin than for other
plasma. Intact clotting factors are consumed during the clot- proteins, providing some specificity for albumin. BCP gener-
ting process and in their place proteolytic fragments and the ally is slightly more specific for albumin and yields lower
contents of platelet granules produced by the clotting process values than BCG, particularly for patients with kidney fail-
may be recovered in serum.149 ure.152 Heparin in collection tubes is reported to affect some
dye-binding methods.153 Dye-binding assays also tend to be
Abundant Components of the Circulating Proteome less accurate when the serum or plasma protein composition
Prealbumin (Transthyretin) is abnormal. The many ligands of albumin do not typically
Prealbumin (molecular weight, 35 kDa) is composed of four affect dye-binding assays of serum or plasma significantly
identical noncovalently bound subunits with the capacity to unless their concentrations are very high.
bind and transport 10% of circulating triiodothyronine (T3)
and thyroxine (T4). Prealbumin concentrations are often a1-Antitrypsin
used as an indicator of adequacy of protein nutrition because Schultze et al.154 described the inhibition of trypsin in the
of its relatively short half-life (;2 days), high proportion of 1950s. However, AAT inhibits a variety of serine proteinases,
essential amino acids, and small pool size.8,12,13 Concentra- leading to the term a1-proteinase inhibitor. AAT is the term
tions also fall in cirrhosis of the liver and protein-losing dis- commonly used by clinicians and clinical laboratorians,
eases of the gut or kidneys. Prealbumin migrates as a minor but phenotypes are commonly abbreviated as Pi (protease
inhibitor). AAT is synthesized by hepatocytes as a single poly- single peptide chain by hepatocytes and cleaved into a- and
peptide chain with 394 amino acid residues and 3 N-linked b-chains. Variable numbers of a- and b-chains combine and
oligosaccharides, yielding a total molecular weight of ap- become covalently linked by disulfides to form Hp.159 Hp
proximately 51 kDa. It belongs to the serpin superfamily, a scavenges hemoglobin in the vascular space. Hp–hemoglobin
group of suicide inhibitors of serine proteases, which abort- complexes are bound by CD163 receptors and rapidly cleared
ively cleave the inhibitor at the reactive site residue but re- by the reticuloendothelial system, which degrades protein
main covalently linked to the reactive site. Serpins usually and recycles heme and iron. This process prevents renal clear-
occur in a “stressed” conformation, and cleavage leads to a ance of hemoglobin until Hp-binding capacity is exceeded.
dramatic conformational shift to a “relaxed” form. AAT and Because Hp is degraded after complexing with hemoglobin,
other serpins serve as important models for conformational its concentration drops severely in the event of intravascular
change in protein function and aggregation.84 hemolysis. The normal plasma half-life of Hp is about
More than 75 genetic variants of AAT have been noted. 5.5 days. Hemolysis of specimens after blood collection does
Clinical AAT deficiency is inherited in an autosomal codomi- not decrease Hp. Hp has a capacity to bind only about 1% of
nant fashion with a prevalence of 1 in 3000 to 5000 in the the hemoglobin in RBCs at usual hematocrits, so minimal
United States. AAT alleles are designated B to Z in order of intravascular lysis completely depletes plasma Hp. When
decreasing electrophoretic mobility.155 The allele designated Hp capacity is exceeded, free hemoglobin in the circulation
M is the wild type. Clinically important variants are the S allele increases. Free hemoglobin can oxidize to methemoglobin
characterized by a Glu n Val substitution at position p264 (Fe31) followed by dissociation of metheme from globin. Met-
and the Z allele characterized by a Lys n Glu missense muta- heme binds to hemopexin (high affinity) or albumin (low af-
tion at position p340. Whereas individuals homozygous for finity), keeping it in solution. Hemopexin–heme complexes
the S allele have serum concentrations approximately 60% of are removed by the reticuloendothelial system. Hp depletion
normal, those homozygous for the Z allele possess about 15% is usually a sensitive biochemical indicator of intravascular
of normal levels.85 The lung disease of AAT deficiency is hemolysis followed by hemopexin depletion and finally by the
thought to be associated with unchecked elastase activity. presence of methemalbuminemia, hemoglobinuria, or both.
Pulmonary disease is characterized by onset of emphysema in Hp measurement, typically by immunoturbidimetry or im-
the third to fifth decades of life.156 The onset is particularly munonephelometry, is therefore part of the assessment of
early in smokers. Liver manifestations include neonatal cho- possible transfusion reactions or other causes of hemolysis.
lestasis or hepatitis, cirrhosis, and hepatocellular carcinoma
(see also Chapter 51). Transferrin
Assessment of circulating AAT concentrations is usually Transferrin (originally named siderophilin) is the principal
performed by immunoturbidimetry or immunonephelometry. plasma transport protein for iron (Fe31). Chapter 40 de-
Normal concentrations range from 70 to 200 mg/dL (14 to scribes iron metabolism. Transferrin has a molecular weight
50 mmol/L). Concentrations are higher in patients with in- of 79.6 kDa, including 5.5% carbohydrate. It is a single poly-
flammatory disorders, malignancy, or trauma and in women peptide chain, with two N-linked oligosaccharides and two
who are pregnant, on estrogen therapy, or taking oral contra- homologous domains, each with an Fe31-binding site. It is
ceptives. Neonates also have increased concentrations, possibly synthesized mainly in the liver and circulates with a half-life of
secondary to maternal estrogen. Individuals with decreased 8 to 10 days. Transferrin reversibly binds two ferric (Fe31) ions
AAT may be phenotyped using electrophoresis or MS or with high affinity at physiologic pH but lower affinity at de-
alternatively, genotyped. creased pH, which allows release of iron in intracellular com-
partments. After cellular delivery of iron via receptor-mediated
Ceruloplasmin endocytosis, apotransferrin is recycled back into the circula-
Ceruloplasmin (Cp) is an a2-globulin that contains about 95% tion. Clinical indications for direct measurement of transferrin
of serum copper. Each molecule of Cp contains six to eight tightly are few. Indirect assessment of transferrin concentration may
bound copper atoms. A solution of Cp is blue (L. caeruleus, be inferred by total iron-binding capacity (TIBC).
meaning blue), and increased concentrations of Cp may lend Under normal circumstances, transferrin oligosaccharides
plasma a green tint. Cp is a polypeptide chain with 1046 terminate in four sialic acid residues, but this pattern is al-
amino acids and three asparagine-linked oligosaccharides tered under both normal and pathologic conditions. Trans-
yielding a molecular weight of approximately 132 kDa. Cp is ferrin glycan structure is widely used to detect congenital
synthesized primarily by hepatic parenchymal cells, with disorders of glycosylation.160 These forms are generically re-
small amounts from macrophages and lymphocytes. The ferred to as carbohydrate-deficient transferrin (CDT).161 A
peptide chain is formed first and then copper is added via the desialated version of transferrin, termed tau-transferrin or
activity of an intracellular ATPase (ATP7B). ATP7B is com- b2-transferrin, is a substantial component of CSF but not se-
monly mutated in Wilson disease.157 Cp synthesized in the rum. It has been used as an indicator of CSF leakage in fluid
absence of copper or the ATPase is degraded intracellularly, from the ear or nasal passages (see later section on CSF pro-
but some apoCp reaches the circulation, where it has a short- teins).162,163 Increased CDT in plasma also has been used as an
ened half-life of a few hours (normal, 4 to 5 days). Conse- indicator of alcohol abuse.164 The decreased amount of sialic
quently, serum Cp is low in those with Wilson disease (see acid and the reduced negative charge of CDT may be detected
Chapter 51).158 by electrophoresis, IEF, ion-exchange chromatography, or MS.
Haptoglobin b2-Microglobulin
Haptoglobin (Hp) is an a2-glycoprotein that binds hemoglo- BMG is a small, nonglycosylated 99 residue protein with a
bin (G. haptein, meaning to bind). Hp is synthesized as a molecular weight of 11.8 kDa. It is the noncovalently bound
light chain subunit of class I major histocompatibility com- the complement cascade are to recruit effector phagocytes for
plex molecules present on the surface of all nucleated cells. opsonization and clearance of foreign pathogens and trigger
BMG is shed into the blood, particularly by B lymphocytes, direct destruction of foreign organisms. Activation of the
and some tumor cells. Its small size allows efficient glomeru- cascade proceeds by three different recognition molecule
lar filtration, resulting in a plasma half-life of approximately systems responsive to different threats. The classical pathway
100 minutes. In addition to renal failure, high plasma concen- (CP) is activated primarily by immune-complex formation. A
trations occur in inflammation and neoplasms, especially single immunoglobulin M (IgM) or two immunoglobulin G
those associated with B lymphocytes. In patients with chronic (IgG) molecules binding to antigens are sufficient to activate a
lymphocytic leukemia, high BMG concentrations are a nega- complex consisting of C1q, C1r, and C1s. CP components
tive prognostic marker for decreased survival. Plasma BMG are numbered consecutively from C1 to C9. The alternative
concentrations are used as a staging criterion in multiple pathway (AP), described in 1981, is activated in the absence of
myeloma.165 BMG concentrations are also increased in states antibodies. It is triggered by foreign pathogenic surfaces
of immune activation and have been applied as an indicator such as lipopolysaccharides from microbes and it can also be
of transplant rejection. spontaneously activated in a surveillance role. Spontaneous
hydrolysis of C3 generates C3b that can bind a variety of mi-
C-Reactive Protein crobial surfaces and trigger C5 hydrolysis in the presence of
In 1930, Tillet and Francis described a substance in the sera factor B and factor D. AP components are commonly identi-
of acutely ill patients that bound cell wall C-polysaccharide fied with letters and include Factors B, D, H, I, and P. The lectin
of Streptococcus pneumoniae and agglutinated the organ- pathway (LP) is activated after recognition of danger-associated
isms.166 In 1941, the substance was shown to be a protein and molecular patterns (DAMPs) and pathogen-associated molec-
named C-reactive protein (CRP).167 CRP consists of five ular patterns (PAMPs), by binding of mannan-binding lectin
identical, nonglycosylated 23 kDa subunits noncovalently as- (MBL), ficolins, or collectins to mannose-rich oligosaccharides
sociated to form a disk-shaped structure with radial symme- that are present in the cell walls of many microorganisms.170
try and total mass of approximately 115 kDa. CRP aids in This event triggers activation of MBL-associated serine prote-
nonspecific host defense against infectious organisms by ac- ases termed MASP-1 and MASP-2, which are homologous to
tivating the classical complement pathway. C1r and C1s in the CP. The nomenclature of the complement
CRP is one of the strongest acute-phase reactants, with system was revised in 2014.171 During activation of the cas-
plasma concentrations rising up to 1000-fold after myocardial cades, many complement components are enzymatically
infarction, stress, trauma, infection, inflammation, surgery, cleaved into two or more fragments. The larger fragments were
or neoplastic proliferation.168 Concentrations greater than 5 designated by a lowercase b and the smaller ones by a lowercase
to 10 mg/L suggest the presence of an infection or inflamma- a. The larger fragments usually contain a binding site for mem-
tory process. Concentrations are generally higher in bacterial branes, immune complexes, and protein association, or in some
than viral infection, although concentrations greater than cases, yield new protease activities that activate subsequent
100 mg/L (10 mg/dL) may be seen in uncomplicated influ- component(s) of the cascade. Smaller fragments typically serve
enza and infectious mononucleosis. The increase with in- as anaphylatoxic or chemotactic peptides. The exception to that
flammation occurs within 6 to 12 hours and peaks at about rule is C2. C2a, produced in the C3 and C5 convertases of the
48 hours and is generally proportional to the extent of tissue CP, turned out to be the larger fragment of C2.171 Inactivated
damage. Because the increase is nonspecific, however, it can- fragments are designated by the letter i (e.g., C3bi). Activated
not be interpreted without other clinical information. complexes used to be indicated by a bar over the components
CRP is normally present in plasma at a concentration below (e.g., C567), but the bar was removed in the new 2014 consen-
5 mg/L. High concentrations in inflammatory states are mea- sus and currently there is no definitive designation to distin-
sured with direct immunoturbidimetric or immunonephelo- guish native from activated complement components.
metric assays. Epidemiologic studies demonstrate that mildly Via coordinated proteolysis, all three pathways converge at
increased CRP concentrations are associated with risk of car- C3 convertase. The CP and LP C3 convertase consists of C4b
diovascular disease (see Chapters 36 and 48).169 Increased and C2a, while the AP C3 convertase consists of C3b and an
concentrations may reflect low-grade, chronic intimal inflam- activated fragment of Factor B, Bb. C3 convertase has high-
mation. The use of CRP for these purposes requires assays with affinity for C3 and catalyzes cleavage and subsequent formation
detection limits below 0.3 mg/L that generally are referred to as of C3b and C3a. Surface-bound C3b serves as a docking site for
high-sensitivity CRP assays. High-sensitivity assays require phagocyte receptors. The C3 convertase eventually saturates
particle-enhanced (also termed latex-enhanced) light scatter- the cleavage of C3 when there are sufficient circulating C3b
ing assays or sandwich-type immunoassay formats. fragments for opsonization of targets. The C3b fragments in-
teract with the C3 convertase, changing its conformation and
Complement affinity. The C5 convertase is formed with this change. The CP
The complement system consists of more than 50 soluble pro- and LP C5 convertase is formed by C4bC2aC3b and the AP C5
teins synthesized primarily by the liver, as well as cell-bound convertase is C3bBbC3b. The C5 convertase targets C5, and
receptors and control proteins. A much smaller, local produc- with C5 cleavage, the release of C5a results in the most potent
tion of complement components can also happen in the organ anaphylatoxin of the complement cascade, and the availability
level, such as the gastrointestinal (GI) and urogenital tracts, of C5b ultimately leads to the formation of a membrane attack
where threats from pathogens are frequent, although it is un- complex (MAC) consisting of components C5b, C6, C7, C8,
likely that the local production of complement proteins ends and several C9 molecules. The MAC forms an ion channel in
up in systemic circulation. A basic schematic of the comple- the membrane of the foreign pathogen triggering lysis and
ment cascade is presented in Fig. 31.4. The basic functions of destruction of the target organism.172
FIGURE 31.4 Complement initiation, amplification, regulation, and formation of the terminal comple-
ment complex (TCC). Initiation, top: Classical pathway (CP) initiation via C1q and the C1 complex to
form the C3 convertase (C4bC2a). Initiation, middle: Lectin pathway (LP) initiation via ficolins, mannan-
binding lectin (MBL), and collectins to form the C3 convertase (C4bC2a). The roles of C1-INH, C4BP,
and FI in regulating CP and LP initiation are highlighted. Initiation, bottom: Alternative pathway (AP)
initiation via the “tick-over” mechanism (spontaneous hydrolysis of C3) to form the AP C3 convertase
C3H2OBb. Amplification: The amplification loop shows the role of properdin in complement initiation and
amplification. The roles if FI, FH, CFHR1 (HR-1) in regulation of complement amplification are high-
lighted along with the role of FD and FB in complement cascade amplification. Inflammation and cell
lysis: The role of the amplification loop in generating the C5 convertases is shown along with the C5
convertase formation of the TCC. Reproduced with permission of the Mayo Clinic Foundation.
The constant slow, chronic activation of complement com- 50% of the added erythrocytes. Many new versions of the test
ponents would have devastating consequences were it not for a have become available in the past decade, including ones
host of regulatory proteins designed to limit complement ac- amenable to automation, which utilize liposomes coated
tivity. A few of these factors include decay-accelerating factor with immune complexes instead of RBCs, a feature that stan-
(DAF or CD55), MIRL (CD59), membrane cofactor protein dardizes testing across reagent lots and different institutions
(MCP), C1 inhibitor, and factor H. Deficiency of DAF and (Fig. 31.5). Enzyme-linked immunosorbent assay (ELISA)
CD59 can lead to abnormal lysis of RBCs, such as observed in versions of the test, which can assess CP, AP, or LP function,
PNH.173 Defects in C1 inhibitor are linked to hereditary angio- depending on the type of trigger coated on the ELISA plate
edema.174 Factor H plays a role in age-related macular degen- (Fig. 31.6), made the functional tests relatively simple to adopt,
eration,175 atypical hemolytic uremic syndrome,176 and C3 if laboratories would shy away from the hemolytic method for
glomerulopathies.177 Proper function of complement regula- CH50. All the different methods employed for functional assays
tory factors prevents destruction of endogenous cells at the (hemolytic, liposome or ELISA) evaluate the progression of
same time that foreign cells are destroyed by complement. the complement cascade from the recognition molecule until
Laboratory analyses of the complement system can be di- the formation of the MAC, and require all components to
vided into five different types of testing. The oldest and most be available for a successful assay measurement, hence sample
widely available type of testing is the assessment of (1) total collection is important to preserve the complement components
complement function or activity. Total serum complement intact function. Serum samples should be frozen at 220 °C or
activity is a screening test that demonstrates an intact CP lower temperatures immediately after blood collection.
from activation to formation of the MAC. The classic version The second most common type of complement test is
of this assay tests the capacity of patient sera to lyse sheep (2) measurement of individual component concentrations,
RBCs coated with rabbit anti-sheep antibodies. Progress of such as the assessment of circulating C3 and C4. C3 and C4 are
the reaction is monitored using hemoglobin release from the typically determined with immunoturbidimetry or immu-
RBCs. CH50 refers to the amount of serum required to lyse nonephelometry. Measurement of C3 may be complicated
sample type as it chelates calcium, a required metal for the

progression of the cascade. Measurement of the more stable
activation fragments such as C4d, Bb, sC5b-9 (the soluble
MAC) using ELISA are part of complex evaluations per-
formed as large test panels by few reference laboratories and
allow for a full picture of the complement cascade. Clinical
disorders of inherited complement deficiency or dysregula-
tion are listed in Table 31.5.
The detection of (4) autoantibodies against complement
components has gained importance in the last decade as the
evidence of antibodies against the C3 convertases, called C3
and C4 nephritic factors (for the AP and CP C3 convertases,
respectively). These autoantibodies are present in cases of
kidney diseases such as the C3 glomerulopathies, a rare type
of membranoproliferative glomerulonephritis where the AP
damages kidney structures. Antibodies to Factor H have also
been described in acquired causes of atypical hemolytic ure-
mic syndrome and C3 glomerulopathies.
Genetic studies of complement genes (5) allow for dif-
ferentiation between hereditary or acquired causes of defi-
ciency or disease, and testing frequently helps clinicians in
determining prognosis, course of therapy, and potential
risk of relapses. Acquired causes of complement dysregula-
tion, such as the conditions where autoantibodies to the
complement system are present, may be treated with im-
munosuppressants and plasma exchange. Known patho-
genic genetic variants, on the other hand, may require
specific treatment with complement inhibitors such as ecu-
lizumab that targets the terminal pathway component C5
and prevents formation of the MAC and the subsequent
lytic effects of complement.
Immunoglobulins
Immunoglobulins (antibodies) are generated against foreign
immunogens and initiate clearance of the foreign molecule
FIGURE 31.5 Liposome enzymatic assay for measurement of or organism. Human immunoglobulin molecules consist of
total complement activity (CH50). The total complement lytic one or more basic units built of two identical heavy (H)
activity is measured by mixing patient serum in the presence chains and two identical light (L) chains. L chains have one
of immune complexes created by the reagents. Liposomes con- variable and one constant domain, whereas H chains have
taining glucose-6-phosphate (G6P) will be lysed if the classical one variable and three to four constant domains, with the
and terminal complement pathways are activated. G6P is then variable region involved in antigen recognition and binding
exposed and an enzymatic reaction using nicotinamide adenine (see Chapter 26). Extensive diversity in the variable domains
dinucleotide (NAD) can be measured on several automated plat- is generated by somatic recombination and mutation of the
forms, with absorbance measured at 340 nm. Reproduced with
immunoglobulin genes. Individual plasma cells or clonally
permission of the Mayo Clinic Foundation.
expanded cells are committed to synthesis of a single variable
domain sequence for heavy and light chains. The amino acid
by in vitro conversion of C3 to C3c. Because of differences in sequences of the variable domains at the N-terminal ends of
antibody reactivity toward C3 and C3c, C3 concentrations the four chains form two antigen-binding sites with a high
measured on fresh samples may be lower than those deter- degree of variation in binding specificity. The constant do-
mined after long-term storage. Measurement of C3 and C4 mains are the same for every immunoglobulin molecule of a
are used to assess activation of the alternative and CPs, re- given subclass and carry sites for binding to complement re-
spectively. Low concentrations are observed in complement ceptors and activation of complement.
deficiency, glomerulonephritis, lupus erythematosus, and The variable domains contain the antigen-binding regions
sepsis. and the constant domains of the heavy chains contain sites
More recently, the availability of assays to measure (3) acti- for complement activation and receptor binding. Cleavage of
vation fragments of complement components has helped immunoglobulins with pepsin or papain can yield antigen-
to differentiate complement deficiencies from complement binding fragments (Fab) and constant region fragments (Fc).
consumption in which the parent molecules are being Variations in the constant domains of heavy chains (Fc region)
cleaved constantly due to an auto-immune or infectious result in the classes and subclasses into which immunoglobu-
process. Measurement of these activation fragments re- lins are grouped: IgM, IgG (four subclasses), immunoglobu-
quires a stable sample where the cascade can no longer lin A (IgA) (two subclasses), immunoglobulin D (IgD), and
progress in vitro. EDTA plasma is the most appropriate immunoglobulin E (IgE), respectively. Light chains, which
FIGURE 31.6 ELISA assays are available to measure function of classical, lectin, and alternative path-
ways of complement as described in Mollnes et al., Mol Immunol. 2007;44:3838–3849. For the clas-
sical pathway (CP) function assessment, the plate is coated with IgM. For the lectin pathway (LP)
function the plate has mannose, and for the alternative pathway (AP) function there are lipopolysaccha-
rides from Gram negative bacteria coated on a solid-phase support ELISA plate. The CP and LP assays
are run with diluted serum at 1:101, and the AP assay uses 1:18 dilution. In addition, the buffer used for
the LP uses antibodies to C1q to block initiation of CP. The high dilution makes it difficult to trigger the
AP. The buffer used in the AP assay uses magnesium EGTA to chelate calcium, necessary for the activity
of CP and LP but not the AP. Function is tested when the complement system in patient’s serum is acti-
vated by the plate triggers, and its cascades amplified to create C3 and C5 convertases, and ultimately
the membrane attack complex (MAC). The conjugate antibody for all assays targets neoepitopes gener-
ated after the formation of the MAC. (Reproduced with permission of the Mayo Clinic Foundation.)
TABLE 31.5 Inherited Deficiencies of Complement Components

Component Frequency of Deficiency Associated Disorders
Ficolin 3 (H-ficolin) Rare, less than 10 cases described Respiratory infections, necrotizing enterocolitis
MBL 5% in Caucasians Mostly asymptomatic, increased susceptibility to bacte-
rial and fungal infections; infection in infancy; less ef-
fect on adults, SLE-like
MASP-2 Rare, less than 10 cases described Respiratory infections
C1qa Relatively rare, over 40 cases described SLE or SLE-like, GN, infections
C1r, C1s Rare, less than 20 cases described SLE or SLE-like, infections
C1 inhibitor 1:50,000 Hereditary angioedema (autosomal dominant)
C2 1:10,000–1:20,000 Recurrent infection, vasculitis, SLE, DLE (no antinuclear
antibody); half of affected individuals are asymptomatic
C3 Rare, less than 30 cases described Severe and recurrent bacterial infection, especially with
encapsulated, pyogenic bacteria, pneumococci, Hae-
mophilus influenzae and streptococci.
C4 (C4A/C4B) Less than 30 homozygous cases described; SLE-like, RA, IgA nephropathy, infections. Often asymp-
13% heterozygosity for C4A or C4B tomatic in heterozygous deficiency.
C4 binding protein Rare, less than 10 cases described Atypical Morbus Behçet, angioedema
C5 Rare, 40 cases described Severe or recurrent infection with Neisseria species, SLE
C6 Relatively rare, over 50 cases described Severe or recurrent infection with Neisseria species
C7 Relatively rare, over 50 cases described Severe or recurrent infection with Neisseria species
C8 a-g/C8b Relatively rare, over 50 cases described, Severe or recurrent infection with Neisseria species
majority is deficiency of C8 b subunit
C9 1:1000 in Japan, otherwise rare Neisserial infections, mostly asymptomatic
Properdin Over 100 cases described X-linked; Neisserial infection
Factor B Rare, two cases described for deficiency, Neisserial infections (deficiency), aHUS (gain-of-function)
less than 10 cases described for gain-
of-function mutation
Factor D Rare, less than 10 cases described Recurrent infection
Factor H Over 100 cases described Hypercatabolism of C3, recurrent bacterial infections,
aHUS, C3G, DDD, AMD
FHR1 (FHR3) 5% in Caucasians aHUS, C3G, AMD, RA, SLE
Factor I Over 50 cases described Hypercatabolism of C3, severe infections, aHUS, C3G
Thrombomodulin (CD141) Rare, over 10 cases described aHUS
Decay accelerating Rare PNH related to decreased DAF and CD59 on cell
factor (DAF, CD55) surfaces
CD46/MCP Relatively rare, over 50 cases described aHUS, C3G
CD59 PNH
CR2 (CD21) Rare Infections, associated with CVID
CR3 (CD18/CD11b) and 1:1 million Leukocyte adhesion deficiency with severe bacterial
CR4 (CD18/CD11c, LFA-1) infection
a
Both quantitative and qualitative (functional) deficiencies reported.
aHUS, Atypical HUS; AMD, age-related macular degeneration; DLE, discoid lupus erythematosus; GN, glomerulonephritis; HUS, hemolytic-
uremic syndrome; MBL, mannan-binding lectin; PNH, paroxysmal nocturnal hematuria; SLE, systemic lupus erythematosus (or SLE-like
disease). C3G, C3 glomerulopathy; DDD, dense deposit disease; RA, rheumatoid arthritis.
Data from Colten HR, Rosen FS. Complement deficiencies. Annu Rev Immunol 1992;10:809–834; Unsworth DJ. Complement deficiency and
disease. J Clin Pathol 2008;61:1013–1017 and Grumach AS, Kirschfink M. Are complement deficiencies really rare? Overview on prevalence,
clinical importance and modern diagnostic approach. Mol Immunol 2014;61:110–117.
are produced independently and in slight excess of heavy producing antibody to the target antigen. Somatic mutation
chains, are of two types—kappa (k) and lambda (l). The of immunoglobulins leads to further diversity of immuno-
heavy-chain genes are located on chromosome 14, whereas k globulin variable region and antibody maturation, generally
light chains are encoded by a gene on chromosome 2, the l- leading to antibodies with higher affinity. B lymphocytes at
chain gene is on chromosome 22. first have IgM surface receptors and secrete IgM as the first or
Immunoglobulins are synthesized by plasma cells, the “primary” response to an antigen. Membrane and secreted
progeny of B-lymphocyte stem cells in bone marrow. More forms of the antibody arise from differential splicing of the
mature B lymphocytes, found mainly in lymph nodes and in mRNA for heavy chains, which adds a transmembrane seg-
blood, develop receptor immunoglobulins on their surface ment to the membrane-bound form. Heavy chains of the
membranes. Upon binding a target antigen, these B lympho- IgM surface receptor molecules undergo class switching to
cytes proliferate and develop into a clone of plasma cells, produce immunoglobulins with g- or a-heavy chains (IgG or
IgA), but the variable regions remain unchanged; as the cells (70 kDa) and a J chain (15.6 kDa). It is synthesized mainly by
change into plasma cells, second exposure to the same anti- plasma cells in the mucous membranes of the gut and bron-
gen causes a larger secondary or anamnestic response of IgG chi and in the ductules of the lactating breast. The secretory
secretion. component assists with transport of secretory IgA across
Individual immunoglobulins and light chains mucosal epithelium and into secretions. Secretory IgA in
Immunoglobulin G. IgG accounts for 70 to 75% of the colostrum and milk is more abundant than IgG and may aid
total immunoglobulins in plasma. Only 35% is found in the in protection of neonates from intestinal infection. IgA can
plasma space, and 65% is extravascular. IgG consists of two activate complement by the AP, but the exact role of IgA in
g-heavy and two light chains, linked by disulfide bonds (see serum is not clear.
Chapter 26). The molecular weight of IgG is approximately Immunoglobulin D. IgD accounts for less than 1% of
150 kDa, including one N-linked oligosaccharide on each serum immunoglobulin. It is monomeric, contains about
heavy chain. The oligosaccharide structure may change in 12% carbohydrate, and has a molecular weight of 184 kDa.
inflammatory states and affect interactions with receptors.178 Its structure is similar to that of IgG. Similar to IgM, IgD is a
On agarose gel electrophoresis, IgG migrates broadly in the surface receptor for antigen on B lymphocytes, but its pri-
g- and slow b-regions as a result of its heterogeneity of mary function is unknown. A condition named hyperimmu-
charge from sequence variation. noglobulin D syndrome is a rare, autosomal recessive genetic
Immunoglobulin G has four subclasses: IgG1, IgG2, IgG3, disorder characterized by recurrent episodes of fever with
and IgG4. The circulating half-life of IgG1, IgG2, and IgG4 is lymphadenopathy, abdominal pain, and increased serum IgD
about 22 days. IgG3 has a half-life of 7 days. IgG1 and IgG3 concentration above 14 mg/dL or 100 IU/mL. It usually pres-
strongly activate complement via the CP, IgG2 is weakly ents during the first year of life and is equally prevalent in
complement fixing, and IgG4 does not activate complement. males and females. The syndrome is a result of compound
Clustering of multiple IgG molecules is required to activate heterozygous or homozygous mutation in the mevalonate
complement. Both IgG1 and IgG3 bind Fc receptors on phago- kinase gene (MVK). Initially it was thought that the increase
cytic cells, activate killer monocytes, and cross the placenta via of IgD was etiologically linked to the disease, and some now
receptor-mediated active transport. IgG1 is the principal IgG consider the IgD increase to be a secondary phenomenon to
to cross the placenta, and neonatal concentrations are similar the inflammatory processes. In hyper-IgD syndrome, the IgD
to maternal concentrations. Neonates have low production of increase is polyclonal.181,182 However, monoclonal gammopa-
IgG as the result of immaturity of their immune systems, and thies caused by IgD have worse prognosis than non-IgD M-
IgG concentrations fall through infancy as the maternally proteins and usually mean a malignancy such as multiple
acquired antibody repertoire is cleared. myeloma requiring therapy.
Immunoglobulin M. IgM is produced at earlier stages of Immunoglobulin E. Immunoglobulin E (IgE) contains
B-cell development. In the immature immune systems of 15% carbohydrate and has a molecular weight of 188 kDa.
neonates, IgM is the major immunoglobulin synthesized. In IgE is so rapidly and firmly bound to specific IgE receptors on
adult serum, it is the third most abundant immunoglobulin, mast cells that only trace amounts are normally present in
usually accounting for 5 to 10% of total circulating immuno- serum. IgE binds to mast cells via sites on its Fc region. When
globulins. IgM as a membrane receptor molecule is mono- the antigen (allergen) cross-links two of the attached IgE
meric, but most of the serum IgM is a pentamer containing molecules, the mast cell is stimulated to release histamine and
five monomers linked via disulfide bonds to the small J (join- other vasoactive amines that increase vascular permeability
ing) chain. Plasma cell malignancies may secrete monomeric and smooth muscle contraction, mediating type 1 hypersen-
IgM in addition to, or instead of, pentamers. The high mo- sitivity reactions such as hay fever, asthma, urticaria, and ec-
lecular weight of IgM (970 kDa; 10% carbohydrate) pre- zema. Rare regulatory disorders with hyperproduction of IgE
vents its ready passage into extravascular spaces. IgM is not lead to a primary immunodeficiency disorder, Job syndrome,
transported across the placenta and therefore is not involved with eczema, recurrent infection, and markedly increased
in hemolytic disease of neonates. It activates complement IgE.183 IgE molecules specific for particular allergens are
even more efficiently than IgG. Binding of one IgM molecule commonly assessed to identify the specificity of allergies. The
may be adequate to activate the complement CP. In rare total serum concentration of IgE may be increased in indi-
hyper-IgM syndromes, class switching to IgG and IgA is defi- viduals with allergic disorders.
cient. Affected patients have deficiency of IgG and IgA and Free immunoglobulin light chains. Light chains are
increased susceptibility to infection.179 usually synthesized in slight excess of heavy chains beyond
Immunoglobulin A. IgA has a molecular weight of 160 quantities required for intact immunoglobulins. Conse-
kDa, including about 10% carbohydrate derived from both N- quently, small amounts of free light chains (FLC), represent-
and O-linked oligosaccharide chains. IgA accounts for about 10 ing only about 0.1% of total immunoglobulin, are present in
to 15% of serum immunoglobulin and has a half-life of serum or plasma. Amounts in plasma are kept low by renal
6 days. In its monomeric form, its structure is similar to that clearance. Free k-light chains (23 kDa) are cleared about
of IgG, but 10 to 15% of IgA in serum is dimeric, particularly two to three times faster than free l-light chains (a disulfide-
IgA2, which is more resistant to destruction by pathogenic linked dimer of 46 kDa), which have a half-life of 4 to
bacteria than IgA1. On electrophoresis, IgA migrates in the 6 hours. Consequently, even though production of k-light
b-g region, anodal to most IgG. IgA is an important compo- chains is about twice as great as that of l-light chains, the
nent of mucosal immunity.180 Secretory IgA is found in tears, plasma concentration of free l-light chains is usually higher,
sweat, saliva, milk, colostrum, and GI and bronchial secre- except in renal failure.184 FLC usually are not functional, but
tions. Secretory IgA has a molecular weight of 380 kDa immunoassays specific for FLC are applied to detect plasma
and consists of two molecules of IgA: a secretory component cell disorders.185
Quantitative FLC assays use antisera directed against epit- monoclonal antibody, which may appear as a sharp, narrow
opes that are exposed only when the light chains are free band on protein electrophoresis. Unbalanced production of
(unbound to heavy chain) in solution. These cryptic sites are FLCs might also lead to a second band representing FLCs. If
involved in the very tight, noncovalent binding of light chains a few clones proliferate, several sharp bands may be seen.
to heavy chains. FLC immunoassays can be used to specifi- Therefore disease may be associated with a decrease or an
cally quantitate FLC even in the presence of large concentra- increase in normal polyclonal immunoglobulins or an in-
tions of polyclonal serum immunoglobulins. The approach is crease in one or more monoclonal immunoglobulins. These
to quantitate the k FLC and the l FLC and use the ratio of disease states are detailed below.
k/l FLC to detect unbalanced light chain synthesis. This ap- Immunoglobulin deficiency. Immunodeficiency states
proach has proven surprisingly sensitive for detecting clonal may be the result of deficiency of a single factor or combina-
free light chain diseases. Abnormal serum FLC ratios have tions affecting multiple systems of immune defense. Severe
been detected in 100% of patients with light chain multiple combined immune deficiency (SCID) is a disorder of B-cell
myeloma, 90 to 95% of primary light chain (AL) amyloidosis development or activation affecting 1 in 100,000 newborns
patients, and 60 to 70% of patients with nonsecretory and resulting in broad-spectrum immunoglobulin deficiency.
multiple myeloma. Despite the sensitivity in free light chain The more common primary deficiencies188 involve only one
diseases, abnormal serum FLC ratios have only been detected or two immunoglobulin classes (IgA) or subclasses (IgA or
in 90% to 95% of intact immunoglobulin multiple myeloma IgG subclasses) or ability to generate antibodies against poly-
and 40% of monoclonal gammopathy of undetermined saccharide antigens. IgA deficiency may lead to false-negative
significance (MGUS). Since these two groups have easily assays for celiac disease detection, and some affected indi-
detectable serum M-proteins by protein electrophoresis and viduals are at risk for anaphylaxis if they receive blood prod-
immunofixation, it is clear that not all monoclonal gam- ucts containing IgA.189 Selective deficiency of IgG subclasses
mopathies secrete excess FLC and that a combination of is not rare, but it is unclear whether it is an important risk for
tests is needed.186 FLC testing was included in the diagnostic infection. Deficiency of IgG2 may be related to poorer re-
criteria for multiple myeloma requiring treatment in 2014. sponses to polysaccharide antigens and increased risk of in-
When the involved free light chain concentration is greater fection with encapsulated organisms.188
than 100 mg/L and the involved FLC ratio is greater than 100, The diagnosis of major deficiencies in immunoglobulin
this qualifies as a biomarker of malignancy, and patients may production is clinically important to avoid infection, particu-
benefit from treatment even in the absence of end-organ larly in neonates, as their maternally acquired antibodies de-
damage for the disease (CRAB: hypercalcemia, renal damage, cline. Infants have transient physiologic deficiency of IgG,
anemia, bone lesions).187 The criteria will need to be updated with a nadir at about 3 months of age (Fig. 31.7). Prolonged
again once different assays for FLC gain market share. Several or severe physiologic deficiency may be associated with in-
diagnostic companies are now producing assays for FLC and creased infection rates, especially with encapsulated bacteria.
the tests are not comparable quantitatively. The assays have Concentrations of maternal IgG, transferred across the pla-
different calibrators and reference intervals and likely differ- centa, rise rapidly in the fetus during the last half of preg-
ent clinical decision points. nancy but then drop over a few months after birth. Two
Clinical utility of immunoglobulin measurement. groups of neonates are at risk for clinically significant IgG
Serum normally contains a diverse, polyclonal mixture of deficiency: premature infants who begin life with less mater-
antibodies with varying amino acid sequences, which repre- nal IgG and infants with delayed initiation of IgG synthesis.
sent multiple “idiotypes” (i.e., the products of many different Monitoring of IgG concentrations can identify this problem.
clones of plasma cells, each producing a specific immuno- Rising IgM and normal salivary IgA concentrations at 6 weeks
globulin molecule). Benign or malignant proliferation of of age suggest a favorable prognosis. Contact of the neonate
one such clone produces a high concentration of a single with environmental antigens normally causes B lymphocytes
100
Maternally transferred
IgG IgM
Total IgG
Mean adult levels (%)
IgG
50
IgA
5 7.5 Birth l
Gestation (months) Age (years)
FIGURE 31.7 Serum immunoglobulin concentrations as percent of mean adult concentrations before
birth and for the first year of life. IgA, Immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M.
to begin to multiply and IgM concentrations to start to rise, However, from the laboratory standpoint, paraproteins are
followed weeks to months later by IgA and IgG. For a full also significant as a potentially unpredictable source of inter-
description of primary and secondary immunodeficiencies, ference with many assays. Paraproteins may aggregate or
refer to Chapter 100. precipitate, causing interference in a variety of photometric
Polyclonal hyperimmunoglobulinemia. Polyclonal in- reactions and in light-scattering hematology analyzers.
creases in plasma immunoglobulins are the normal response to Many patients with paraproteins have nonspecific presenta-
infection. IgG predominates in autoimmune responses. IgA is tions such as anemia or infection. Identification of paraproteins
increased in skin, gut, respiratory, and renal infections, and IgM in serum usually is based on serum protein electrophoresis
is increased in primary viral infections and bloodstream infec- and immunofixation electrophoresis (IFE) (described in a later
tion with parasites such as malaria (see Chapter 88). Chronic section), along with measurement of FLCs. Urine protein
bacterial infection may cause increased concentrations of all electrophoresis and urine IFE are helpful mainly in identifying
immunoglobulins. Measurements of total IgE are used in the patients with free immunoglobulin light chains and amyloidosis.
management of asthma and other allergic conditions, especially Urinary FLCs, as described by Bence Jones in the 1850s, were
in children. Measurements of allergen-specific IgE assist in the first tumor marker. FLC are often referred to as Bence Jones
identifying the stimulus for hypersensitivity responses. proteins.191
Monoclonal immunoglobulins (paraproteins). A single
clone of plasma cells produces immunoglobulin molecules Acute Phase Response
with a single defined amino acid sequence. If the clone ex- Systemic inflammation in response to infection, tissue injury,
pands greatly, it may produce a discrete band on electropho- or inflammatory disease triggers changes in hepatic produc-
resis, often referred to as an M-spike, or M-protein. These tion of multiple plasma proteins, as indicated in Box 31.1.
monoclonal immunoglobulins, termed paraproteins, may be This process, mediated by the action of interleukin-6 (IL-6) and
polymers, monomers, individual immunoglobulin chains other cytokines has been termed the acute-phase response
such as FLC or heavy chains, or fragments of immunoglobulins. (APR).168 It is a nonspecific reaction to inflammation, analo-
Clinical, epidemiologic, and biochemical characteristics of gous to the increase in temperature or leukocyte count. In the
monoclonal paraprotein diseases are summarized in Table 31.6. APR, synthesis of a few proteins, including albumin, transferrin,
About 60% of paraproteins are associated with plasma cell and prealbumin, is downregulated. These proteins are termed
malignancies (light chain amyloidosis, multiple myeloma, or negative acute-phase reactants. Albumin concentrations fall be-
solitary plasmacytoma), and approximately 15% are caused cause of acutely decreased synthesis and from redistribution to
by overproduction by B lymphocytes, mainly in lymph nodes extracellular fluids. Production of a number of proteins, includ-
(lymphomas, chronic lymphocytic leukemia, Waldenström ing AAT, AAG, Hp, Cp, C4, C3, fibrinogen, and CRP, increases
macroglobulinemia, or heavy-chain disease). Virtually every several-fold. Plasma concentrations of these individual positive
multiple myeloma is preceded by the premalignant condition acute-phase reactants rise at different rates, and all reach max-
MGUS.190 The incidence of MGUS increases with age, with 1% ima within 2 to 5 days after an acute insult. Qualitative changes
incidence for people 50 to 70 years of age and 3% incidence for in proteins, such as altered glycosylation, are also observed sec-
people older than 70. The occurrence of MGUS is associated ondary to the APR. Changes to glycosylation of immunoglobu-
with increased risk of progression to multiple myeloma that lins may have an immunomodulatory effect.
should be monitored after the initial diagnosis is made. MGUS
is the most commonly diagnosed monoclonal gammopathy, Methods for Analyzing Proteins
followed by multiple myeloma and AL amyloidosis (Fig. 31.8). Determination of Total Protein
The primary clinical interest in identifying paraproteins Plasma normally contains about 6.5 to 8.5 g/dL protein
is to detect or monitor proliferative disorders of B cells. and serum about 4% less. Determination of total protein in
TABLE 31.6 Monoclonal Immunoglobulins (Paraproteins) in Multiple Myeloma

Plasma Incidencea Mean Age of Incidence of Free Light
Paraprotein (%) Occurrencea (yr) Chain in Urine (%) Comments
IgG 50 65 60 Patients more susceptible to infection; parapro-
teins reach highest concentrations
IgA 25 65 70 Tend to have hypercalcemia and amyloidosis
Free light chain 20 56 100 Often renal failure; bone lesions; amyloidosis;
only poor prognosis
IgD 2 57 100 90% l type; often have extraosseous lesion, amy-
loidosis, renal failure; poor prognosis
IgM 1 — 100 May or may not have hyperviscosity syndrome
IgE 0.1 — Most Rare, less than 50 cases described
Biclonal 1 — — —
None detected ,1 — 0 Usually found with reduction of normal immuno-
globulins (hypogammaglobulinemia on PEP);
increased plasma cells in bone marrow biopsy
a
Approximate.
Ig, Immunoglobulin. PEP, protein electrophoresis.
n = 58,327
SMM
4% (2,188)
Lymphoproliferative
Solitary or
2.5% (1,508)
extramedullary
2% (1,026)
AL amyloidosis Macro**
9.5% (5,454) 3% (1,690)
Other
5% (2,670)
Multiple
myeloma
17% (10,518)
MGUS†
57% (33,273)
† Includes LC MGUS (n = 2435)

**Includes SWM (n = 253)
FIGURE 31.8 Monoclonal gammopathies diagnosed at the Mayo Clinic (Rochester, MN) from 1960 to
2018. There have been over 58,300 cases diagnosed. AL, Primary light chain amyloidosis; LC MGUS,
light chain only MGUS; Macro, Waldenström macroglobulinemia; MGUS, monoclonal gammopathy of
undetermined significance; SMM, smoldering multiple myeloma; SWM, smoldering Waldenström
macroglobulinemia. Other includes cases of light chain deposition disease, heavy chain deposition
disease, acquired Fanconi syndrome, Castleman disease, cold agglutinin disease, plasma cell leukemia,
POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy/edema, monoclonal protein and
skin abnormalities). (Figure courtesy Dr. Robert A. Kyle, MD, Professor of Laboratory Medicine and
Pathology and Medicine, College of Medicine, Mayo Clinic, Rochester, MN.)
BOX 31.1 The Acute Phase Response: Changes in Plasma Protein Concentrations
Positive Acute Phase Response Lipopolysaccharide-binding protein
C-reactive protein (extreme) Mannan-binding protein (lectin)
Serum amyloid A (extreme) Plasminogen
a1-Acid glycoprotein Procalcitonin
a1-Antitrypsin
a1-Antichymotrypsin Negative Acute Phase Response
Antithrombin III Albumin
C3, C4, and C9 Apolipoprotein A-I
C1 inhibitor Apolipoprotein B
C4b-binding protein a2-HS glycoprotein
Ceruloplasmin Insulin-like growth factor I
Factor B Prealbumin
Ferritin Retinol-binding protein
Fibrinogen Thyroxine-binding globulin
Haptoglobin Transferrin
Hemopexin
Data from Craig WY, Ledue TB, Ritchie RF. Plasma proteins: clinical utility and interpretation. Scarborough, ME: Foundation for Blood Research;
2001; Gabay C, Kushner I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med 1999;340:448–454; and Vollmer T,
Piper C, Kleesiek K, Dreier J. Lipopolysaccharide-binding protein: a new biomarker for infectious endocarditis? Clin Chem 2009;55:295–302.
biologic fluids in some respects represents a greater challenge and proteins. Absorbance at 280 nm depends primarily on
than analysis of a specific protein because variable protein the tryptophan and tyrosine content of a protein. This tech-
composition of biologic fluids leads to variable carbohydrate nique works best for purified proteins with known absorptiv-
composition, charge, and physical characteristics of proteins ity. For complex mixtures, accuracy and specificity suffer
in the mixture. Many methods of protein analysis respond from variable content of tryptophan and tyrosine and from
differentially to different proteins and present problems absorbance of low molecular weight compounds such as free
when applied to specimens of varying protein composition. amino acids, uric acid, and bilirubin. From 200 to 225 nm,
Most methods other than the biuret method have not been peptide bonds are chiefly responsible for UV absorbance.
thoroughly examined for interactions with small peptide Absorptivity by proteins at these shorter wavelengths is 10 to
components and intact proteins, and this may become a 30 times greater than at 280 nm. Many low-molecular-weight
significant issue in renal failure patients with increased compounds such as urea also have absorbance at wavelengths
accumulation of peptides and small proteins. Many methods below 220 nm. Accurate measurement of proteins by this
have been developed to measure the total protein content of method may require removal of low molecular weight mol-
biologic fluids. Several are reviewed here. ecules before absorbance measurements are performed. Sev-
Kjeldahl method. This method is rarely used in clinical eral other optical methods using infrared or Raman analysis
laboratories but is of historical importance and is sometimes of specimens offer methods for total protein determination
used as a reference method. Protein nitrogen is converted to based on complex spectral analysis.
ammonium ion by heating with sulfuric acid in the presence Dye-binding methods. Dye-binding methods depend on
of a catalyst. Ammonium ion is measured by alkalinization, shifts in the absorbance spectra of dyes when they bind to
distillation, and acid titration or by Nessler’s reagent. Protein proteins. Variable binding of dyes to different proteins is a limi-
is estimated to contain 16% nitrogen. Errors in protein esti- tation. Calibration with a protein mixture is particularly diffi-
mation occur if the amino acid composition is unusual and cult to define consistently. Calibration with a pure protein such
if nitrogen content differs from 16%. Nonprotein nitrogen, as albumin may not simulate binding to the complex mixture
such as from urea and amino acids, also is measured, so a of proteins in serum or plasma. Using Coomassie blue, for ex-
protein precipitation step may be required. The Kjeldahl ample, immunoglobulins often give only 60% of the response
method was one of the first methods used for reproducible of an equivalent concentration of albumin or transferrin. This
total protein measurement, but it is time consuming and dye binds to polypeptide chains under acidic conditions, result-
impractical for routine use. This method has been used to ing in decreased absorbance at 465 nm and increased absor-
assign values to reference materials for the biuret method. bance at 595 nm. Some dyes offer sensitivity at low protein
Biuret method. Under strongly alkaline conditions, Cu21 concentrations. Pyrogallol red, for instance, has become one of
ions form multivalent complexes with peptide bonds in pro- the most commonly used dyes for analysis of fluids with lower
teins. Binding shifts the absorption spectrum of Cu21 ions to protein concentrations such as urine and CSF.
shorter wavelengths, leading to a color change from blue to Lowry (folin-ciocalteu) method. The detection limit of the
violet that has been termed the biuret reaction. Absorbance Lowry method is about 100 times lower than that of the biuret
attributable to protein is measured spectrophotometrically at method.193 In this technique, specimens are mixed with an alka-
540 nm. Absorbance changes at 540 nm also result from line copper solution followed by addition of the Folin-Ciocalteu
binding of Cu21 ions by many compounds that can form reagent. Phosphotungstic acid and phosphomolybdic acid are
chelates with five- or six-member rings where amino, amide, reduced to tungsten blue and molybdenum blue by copper com-
or hydroxyl groups bind to Cu21 ions. Such compounds in- plexed with peptide and by tyrosine and tryptophan residues.
clude serine, asparagine, ethanolamine, and TRIS buffer, Absorbance of products is measured between 650 and 750 nm.
among others.192 Small compounds lead to a smaller spectral Reactivity of proteins varies with the content of tyrosine
shift than proteins. Historically, the biuret method was not and tryptophan. Low-molecular-weight compounds, including
thought to be sensitive to free amino acids and dipeptides, tryptophan and tyrosine, as well as drugs such as salicylates,
but absorbance changes do occur with some amino acids, chlorpromazine, tetracyclines, and some sulfa drugs, also inter-
amino acid amides, and with dipeptides. The biuret reaction fere. Analysis of a fluid such as urine with high concentrations of
is also commonly thought to react equally with all proteins phenolic compounds requires removal of low-molecular-weight
and peptides longer than two amino acids, but peptides substances before protein is measured.
with high proline content have reduced reactivity. As long as Refractometry. Refractometry is a method used to rapidly
proteins are not extremely proline rich or do not have a very estimate protein at high concentrations. Accuracy decreases
unusual composition, different proteins probably have similar at protein concentrations below 3.5 g/dL, where salts, glucose,
reactivities as long as the biuret reaction is performed in the and other low molecular weight compounds have a larger
typical endpoint manner. Biuret rate assays are also available proportional effect on refractive index. Refractometry is used
but should be considered as a separate category from end- more often in clinical laboratories to assess the concentration of
point assays. Cu21 ions complex with small molecules and solutes in urine specimens than for determining total protein.
accessible sites in proteins almost instantaneously while ad- Light-scattering methods. Many different reagents have
ditional absorbance change depends on the rate of unfolding been used to aggregate protein for turbidimetric or nephelo-
of a protein and exposure of additional binding sites for Cu21 metric assays, including trichloroacetic acid, sulfosalicylic acid,
ions under prescribed alkaline conditions. benzethonium chloride, and benzalkonium salts under alka-
Direct optical methods. Absorbance of ultraviolet (UV) line conditions. Precipitation methods for total protein assay
light between 200 to 225 nm and 270 to 290 nm has been depend on forming a suspension of uniform, insoluble protein
used to measure protein concentrations and is commonly particles, which scatter incident light. Albumin and globulins
applied to monitor chromatographic separations of peptides often give different reactivities in precipitation methods.
Variables affecting measured protein concentrations. Cali- with low ionic strength buffers (0.05 mol/L) at slightly basic
bration of biuret methods commonly uses bovine or human pH (~8.6). For agarose gels, the usual sample is approximately
albumin. Protein mixtures with specific albumin-to-globulin 15 mL. Much smaller volumes are injected for CE. Separation
ratios have been recommended for calibration of other meth- and processing times are typically about 1 hour for agarose
ods. Using these calibration schemes, the total protein con- gels and a few minutes for CE. A variety of stains are used to
centration of plasma obtained from healthy ambulatory visualize proteins in gels, including amido black, Ponceau S,
adults is typically 6.5 to 8.5 g/dL. Serum usually contains a acid violet, and Coomassie blue. Intensity of staining and
protein concentration about 0.3 g/dL less because fibrinogen linearity of protein detection vary with different dyes. Only a
and other proteins are removed during clotting to form se- few of the most abundant proteins are visualized, and inten-
rum. Hemoconcentration and relative hyperproteinemia, sities of bands with protein stains usually relate to the mass
with increased concentrations of all plasma proteins, occur of peptide; oligosaccharides and lipids may reduce rather
with inadequate water intake or excessive water loss as in se- than contribute to band intensities. Therefore glycoproteins
vere vomiting, diarrhea, Addison disease, or diabetic ketoaci- with a high proportion of carbohydrate (e.g., a1 acid glyco-
dosis. Some hemoconcentration also occurs with standing protein) have lower detection responses than a nonglyco-
(reduced intravascular volume) or prolonged tourniquet slyated protein such as albumin. Quantitative analysis relies
time during blood collection. Hemodilution and relative hy- on densitometry, which provides relative proportions of
poproteinemia, with decreased concentrations of all plasma different components rather than absolute amounts. Quanti-
proteins, occur with water intoxication or salt retention tation of individual components relies on calculations derived
syndromes, during massive intravenous infusions, and physi- from total protein concentration. Lipophilic stains such as
ologically when a recumbent position is assumed. A recum- Sudan black are needed to visualize lipoproteins such as high-
bent position decreases total protein concentration by 0.3 to density lipoprotein (a1-lipoprotein), very-low-density lipo-
0.5 g/dL and many individual proteins, including albumin, by protein (LDL) (pre–b-region), (b-lipoprotein), or chylomicrons
up to 10%. This reflects the redistribution of extracellular (origin) (see also Chapter 36). CE detects proteins passing
fluid from the extravascular space to the intravascular space through a flow cell by their light absorbance at wavelengths
and therefore dilution of a constant amount of plasma pro- below 220 nm. This offers a more unbiased assessment of
tein in a larger volume. See Chapter 5. protein concentration than staining intensity because absor-
bance of proteins in the low UV region is more consistently
Immunochemical Techniques for Specific Proteins related to mass. Small molecules at high concentrations, such
Nephelometric and turbidimetric methods are performed as as metabolites, radiocontrast dyes, or drugs, may also yield
equilibrium or rate methods for measuring the amount of light absorbance peaks, and this presents problems with analysis
scattering by antigen–antibody complexes (see Chapter 16). of urine specimens.
Limits of detection of approximately 10 mg/L are attained The major clinical application of serum protein electro-
with routine nephelometric and turbidimetric methods us- phoresis is the detection of monoclonal immunoglobulins
ing antibodies in solution. Binding antibodies to particles of (paraproteins) to assist in the diagnosis and monitoring of
latex or other materials enhances light scattering and can multiple myeloma and related disorders. Most monoclonal
lower limits of detection by 10- to 100-fold. Such assays immunoglobulins are observed in the b-region or g-region.
may be described as latex-enhanced or as particle-enhanced Quantitation of monoclonal components serves as a means
assays. Nephelometric and turbidimetric assays commonly of monitoring disease progression and response to therapy.
offer within-run coefficients of variation (CVs) of less than Identification of paraproteins requires distinction from a
5% except as limits of detection are approached. Turbidi- variety of other sources of additional bands or pseudopara-
metric methods can be applied on most automated chemis- proteins by means such as IFE as described later. Incom-
try analyzers capable of performing photometric methods. pletely clotted specimens contain fibrinogen. Genetic or
Nephelometry requires instrumentation capable of measur- post-translational variants of proteins such as transferrin, Hp,
ing light scattering at an angle to the incident light. and C3 may migrate in different positions than usual. Large
increases in CRP may yield a detectable band in the b- or
Electrophoresis g-region. Increased lysozyme in monocytic leukemia may
Electrophoresis is used to separate proteins by charge and produce a band in the post–g-region. Hemoglobin will yield
thereby to assess protein variants or the concentrations of a band in hemolyzed specimens.
specific components in serum or other fluids. Electrophoretic Protein electrophoresis may also aid in the diagnosis of
techniques commonly performed in clinical laboratories other conditions unrelated to monoclonal gammopathies.
include nondenaturing electrophoresis on cellulose acetate Changes in the a1-region are typically related to AAT. De-
strips or agarose gels, capillary electrophoresis (CE), immu- creases are associated with AAT deficiency or protein-losing
nofixation, and “Western blotting.” Protein separation in one disorders. Increases are related to inflammation. Changes
or two dimensions with polyacrylamide gel is a powerful in the a2-region usually relate to changes in Hp and a2 mac-
separation technique frequently used for research, but has roglobulin (AMG). Migration of Hp varies with genotype.
been largely replaced by MS. Hp decreases with in vivo hemolysis and increases with
Serum protein electrophoresis. Generally, serum rather inflammation. AMG and high-molecular-weight forms of
than plasma is used for electrophoresis of proteins on agarose Hp increase in nephrotic syndrome, but most other protein
gels to avoid the fibrinogen band at the b-g interface. components decrease. Bands in the b-region are related to
The principles of electrophoresis are described in Chapter 18. transferrin, C3, and LDL. Migration of transferrin may
Fig. 31.9 provides examples of normal and a variety of patho- change from the b1-region to the b2-region when significant
logic electrophoretic patterns. Analysis usually is performed carbohydrate-deficient forms are present. An increase
Normal (Adult) Normal (Pediatric) Chronic Renal Disease

Pattern Protein Concentration Pattern Protein Concentration Pattern Protein Concentration
(mg/dL) (mg/dL) (mg/dL)
TP 6800-8300 TP 6900 TP 2300
Alb 3500-5000 Alb 4390 Alb 1110
AAT 100-200 AAT 240 AAT 260
AAG 50-150 AAG 59 AAG 72
Hp 30-215 Hp 65 Hp 101
AMG 125-140 AMG 490 AMG 180
TRF 200-350 TRF 300 TRF 81
C3 70-150 C3 127 C3 71
C4 10-40 C4 27 C4 14
IgA 40-390 IgA 180 IgA 67
IgM 25-210 IgM 140 IgM 47
IgG 525-1650 IgG 870 IgG 200
CRP 2 CRP 1 CRP 1
IgG Monoclonal Gammopathy IgA Monoclonal Gammopathy
(Multiple Myeloma) Nephrotic Syndrome
TP 6900 TP 9100 TP 2900
Alb 4380 Alb 2170 Alb 680
AAT 200 AAT 250 AAT 160
AAG 50 AAG 63 AAG 35
Hp 75 Hp 97 Hp 370
AMG 220 AMG 170 AMG 460
TRF 270 TRF 150 TRF 101
C3 122 C3 90 C3 125
C4 24 C4 20 C4 22
IgA 70 IgA 5800 IgA 250
IgM 170 IgM 24 IgM 93
IgG 1330 IgG 200 IgG 440
CRP 1 CRP 1 CRP 1
Inflammation (Acute) Systemic Lupus Erythematosus Rheumatoid Arthritis (Adult)
TP 5700 TP 7800 TP 6300
Alb 2470 Alb 3390 Alb 2840
AAT 400 AAT 230 AAT 400
Hp 340 Hp 111 Hp 290
AMG 210 AMG 240 AMG 148
TRF 71 TRF 310 TRF 220
C3 120 C3 94 C3 90
C4 17 C4 12 C4 13
IgA 270 IgA 650 IgA 260
IgM 137 IgM 170 IgM 880
IgG 1440 IgG 2480 IgG 930
CRP 9.8 CRP 7.8 CRP 6.1
Iron Deficiency Chronic Hepatic Disease Chronic Hemolysis and Iron Deficiency
TP 6800 TP 6300 TP 6300
Alb 4770 Alb 2240 Alb 4010
AAT 280 AAT 97 AAT 190
Hp 101 Hp 1 Hp 1
AMG 220 AMG 290 AMG 400
TRF 530 TRF 129 TRF 390
C3 136 C3 53 C3 134
C4 22 C4 4 C4 14
IgA 150 IgA 480 IgA 180
IgM 82 IgM 620 IgM 170
IgG 880 IgG 2370 IgG 700
CRP 1 CRP 1 CRP 1
FIGURE 31.9 Electrophoretic patterns typical of normal conditions and of some pathologic conditions
(agarose gel). The upward- and downward-pointing arrows indicate increase and decrease from the
reference interval, respectively. Right- and left-slanting arrows indicate variation from normal to an in-
crease or from normal to a decrease from the reference interval, respectively. AAG, Alpha-1 acid gly-
coprotein; AAT, alpha-1 antitrypsin; Alb, albumin; AMG, alpha-2 macroglobulin; C3, complement com-
ponent 3; C4, complement component 4; CRP, C-reactive protein; Hp, haptoglobin; Ig, immunoglobulin;
TP, total protein concentration; TRF, transferrin.

1 1 1 1
2 2 2 2
3 3 3 3
4 4 4 4
5 5 5 5
6 6 6 6
7 7 7 7
SPE lgG lgA lgM SPE lgG lgA lgM
FIGURE 31.10 Immunofixation electrophoresis (IFE). Left, Patient specimen with an immunoglobulin
G (IgG; k) monoclonal protein. Right, Patient specimen with an immunoglobulin A (IgA; l) monoclonal
protein. The arrow indicates the position of monoclonal protein.
between b- and g-bands, so-called bridging of b- and g- challenges is to avoid adsorption of proteins to the surface of
bands, suggests an increase in IgA as seen with cirrhosis, re- the capillary. CE is suitable for automation and offers rapid
spiratory tract or skin infection, and rheumatoid arthritis. analysis with no need for gel handling or staining. Direct UV
Finally, increases or decreases in the g-region suggest changes detection offers slightly different specificity than protein
in immunoglobulin concentrations. Increases result from staining and offers better reproducibility of quantitation than
chronic infection or paraproteins. Decreases occur with many densitometry. Immunofixation cannot be performed with
immunodeficiency states including immunosuppression CE. Immunosubtraction with specific antisera is used as an
therapies. Multiple myeloma may suppress global production alternative procedure to identify paraproteins.194
of immunoglobulins other than from the expanded clone. A Western blotting. For Western blotting, proteins separated
decrease in the g-region may suggest the need for additional on a gel after electrophoresis are transferred by diffusion or
studies such as IFE to detect paraproteins. electroblotting onto a membrane made of nitrocellulose or
Immunofixation electrophoresis. IFE is complementary to polyvinylidene fluoride. Proteins bound on the membrane
serum protein electrophoresis and employs antisera targeted are identified with specific antibodies conjugated to enzymes
to specific proteins rather than nonspecific dyes. Examples of such as peroxidase or alkaline phosphatase that act on photo-
IFE from two patients with monoclonal gammopathies are metric, fluorescent, or chemiluminescent signaling molecules.
shown in Fig. 31.10. Specific lanes of the gel are overlaid with Mass spectrometry. Multiple types of MS instrumentation
antisera against k and l light chains or g, m, and a-immuno- provide qualitative or quantitative information about pro-
globulin heavy chains. Antibody dilutions are adjusted to teins (see Chapter 24). An advantage of MS is the ability to
provide approximate equivalence with immunoglobulins analyze a large number of components in a single analysis,
separated in the gel so as to form immune complexes pre- including rapid-sequence analysis of peptides. Therefore MS
cipitated in the gel. Proteins not precipitated are washed from has been an enabling technology in proteomics, defined as the
the gel to lower background, and precipitated proteins are effort to study the complete set of proteins in an organism or
stained. Paraproteins characteristically yield sharper precipi- in subcompartments of an organism such as plasma. Ioniza-
tin bands than the heterogeneous polyclonal immunoglobu- tion of peptides and proteins has been accomplished by elec-
lins. IFE provides more sensitive detection of paraproteins trospray or MALDI sources. Electrospray is better suited to
because of the lower background and signal amplification analyzing small peptides than larger intact proteins. After
from immune complex formation. Additionally, immuno- ionization, proteins can be separated by quadrupoles, ion
fixation helps identify the immunoglobulin type of the para- traps, time-of-flight, and other types of mass analyzers. Use
protein. Sometimes more than one clone may be expanded, of tandem MS with an intermediate fragmentation step be-
or a monoclonal free light chain may occur concurrently with tween two stages of MS separation offers high sensitivity and
a monoclonal intact immunoglobulin. Uncommonly, para- specificity for the quantitative analysis of peptides, as it does
proteins may be of the IgD or IgE class. These paraproteins for most small molecules.
should be detected by standard antisera directed against k Advantages of using MS for quantitative analysis include
and l light chains but require d or e heavy chain–specific the ability to analyze components without developing
antisera to distinguish them from FLC. High concentrations specific antibodies and the ability to multiplex a large num-
of paraproteins may interfere with fixation (prozone effect) ber of measurements. MS can provide information about
requiring specimen dilution for optimal studies. For para- post-translational modifications that is difficult to assess by
proteins present in high concentrations, quantitative analysis immunoassays and chromatographic or electrophoretic tech-
of immunoglobulins can confirm the unbalanced production niques. Examples of clinical applications include identification
of a specific class of immunoglobulin and may assist in of genetic variants of prealbumin and CDT. The use of MS
proper dilution of specimens for immunofixation. is likely to increase for accurate determination of protein
Capillary electrophoresis. CE of proteins relies on zone concentrations as recently applied for standardization of he-
electrophoresis in small-bore (10 to 100 mm), fused silica moglobin A1c, insulin, and C-peptide.195,196 Likewise, MS is
capillary tubes 20 to 200 cm in length (see Chapter 18). Elec- also able to distinguish peptides differing in length by one or
trokinetic or hydrostatic injection introduces a small amount of two amino acids or by a post-translational modification. For
protein that is resolved rapidly under high voltage. One of the these reasons, MS is likely to find increased use for clinical
laboratory analysis of bioactive peptides and other compo- specimens are usually collected), the protein concentration
nents of the peptidome. increases.
In the setting of proteomics, several MS assays have been Low to intermediate molecular weight plasma proteins
implemented in the past decade for large proteins. For immu- such as prealbumin, albumin, and transferrin normally pre-
noglobulins, MALDI-TOF has been applied to the detection dominate because CSF is mainly an ultrafiltrate of plasma.
and characterization of monoclonal proteins.197,198 The methods No protein with a molecular weight greater than that of IgG
combine nanobody immunoenrichment coupled to MALDI- is present in sufficient concentration to be visible on electro-
TOF MS to identify, isotype, and quantitate M-proteins. In phoresis. The normal plasma/CSF gradient is about 14 for
this method, the mass-to-charge distributions of immuno- prealbumin, 240 for albumin, 140 for transferrin, 800 for IgG,
globulin light chains are examined for the presence of an and more than 1000 for larger proteins such as IgA, AMG,
overexpressed clone, in a similar fashion to protein electro- fibrinogen, IgM, and b-lipoprotein.201
phoresis. This method has equal or slightly improved limits The electrophoretic pattern of normal CSF after concen-
of detection in comparison to immunofixation. It is capable tration of the fluid has two striking features—a prealbumin
of isotyping and quantitating M-proteins when coupled to band and two transferrin bands—one at b2 in addition to the
nephelometric quantitation.198 Given the automation poten- usual b1 position. As mentioned previously, b2-transferrin
tial, rapid analysis time (,1 minute per patient), improved has decreased charge because its glycosyl chains lack terminal
lower limit of quantitation and the adoption of MALDI-TOF sialic acid residues. Both b2-transferrin and another relatively
MS in clinical microbiology, this method could be a cost- abundant CSF protein, prostaglandin D synthase or b-trace,
competitive replacement for electrophoresis and immuno- have been used to determine whether clear fluids from nasal
fixation in the near future. or ear passages represent leakage of CSF, so-called CSF rhi-
norrhea and otorrhea.162 The utility of b2-transferrin in this
Proteins in Other Body Fluids context is compromised in patients with congenital disorders
Complex mixtures of proteins are present in all biologic flu- of glycosylation. Prostaglandin D synthase is a protein of
ids; analysis of a variety of other specimens is diagnostically about 30 kDa that is relatively enriched in CSF relative to
useful, including analyses of urine (see Chapter 34), CSF, serum or plasma, where it is typically cleared rapidly by glo-
pleural and peritoneal fluids (see Chapter 45), amniotic fluid merular filtration. Apolipoprotein E (Apo E) is also abundant
(see Chapter 59), saliva, and feces (see Chapter 52). in CSF because it plays a primary role in lipid transport as
opposed to apo AI– and apo B100–containing lipoproteins
Saliva which serve this purpose in the periphery but are relatively
Saliva has a very different protein composition than plasma.199,200 scarce in CSF.
Protein composition varies with the site and method of sam- Concentrations of other proteins may increase in CSF sec-
pling of saliva. In addition to the well-known presence of ondary to brain injury or other pathologic processes. Proteins
amylase, proteomic approaches have detected sequences from such as calcium-binding protein B (S-100B), neuron-specific
hundreds of different proteins in saliva. Proteins involved in enolase (NSE), glial fibrillary acidic protein, visinin-like protein-1
host defense against pathogens such as immunoglobulins, (VLP-1), and ubiquitin carboxyl hydrolase-L1 (UCHL1) have
lysozyme, and lactoferrin are particularly abundant. Efforts been associated with traumatic or ischemic brain injury.202-204
are under way to exploit the salivary proteome to detect In acute leukemia and lymphoma with central nervous system
and characterize susceptibility to dental caries, periodontal involvement, the concentration of BMG is increased in CSF.
disease, head and neck cancers, diabetes, and cystic fibrosis. Concentrations of tau and b-amyloid isoforms may be useful
Patients with Sjögren syndrome exhibit increased concentra- in the diagnosis and prognosis of Alzheimer disease.205 Detection
tions of BMG and other inflammatory proteins compared of these CSF changes in the peripheral circulation is limited by
with unaffected patients. Interrogation of saliva for the pres- the blood-brain barrier.
ence of secretory IgA is common in the diagnostic workup of The blood–brain or blood–CSF barrier limits exchange of
hypogammaglobulinemia. many compounds, particularly large compounds such as
proteins. Analyses of total protein and specific proteins in
Cerebrospinal Fluid CSF are used primarily to detect increased permeability of
CSF is the extracellular fluid around the brain and spinal the blood–CSF barrier, increased intrathecal synthesis, or
column. CSF usually has total protein concentrations about increased release of proteins from neural and glial tissue.
100-fold lower than plasma and a different protein composi- Conditions such as viral meningitis, encephalitis, increased
tion because most proteins have limited passage across the intracranial pressure, trauma, and hemorrhage may all com-
blood–brain barrier. CSF for testing is most frequently ob- promise the blood–brain barrier, resulting in increased CSF
tained by lumbar spinal tap. CSF is secreted by the choroid protein. Protein concentrations associated with these condi-
plexus, around the cerebral vessels, and along the walls of the tions are displayed in Table 31.7. Increased intrathecal
ventricles of the brain. It fills the ventricles and cisternae, synthesis of immunoglobulins, particularly IgG, occurs in
bathes the spinal cord, and is reabsorbed into the blood demyelinating diseases of the CNS, especially multiple sclero-
through the arachnoid villi. CSF turnover is rapid, exchang- sis. Methods used to assess potential abnormalities in CSF
ing totally about four times daily. More than 80% of CSF protein content are detailed next.
protein content originates from plasma by ultrafiltration and Total protein in cerebrospinal fluid. The total protein con-
pinocytosis; the remainder is derived from intrathecal syn- centration in CSF is an indicator of blood–CSF permeability.
thesis. The lowest protein concentration and the smallest The protein concentration of CSF usually is more than 100-
proportion of larger protein molecules are in the ventricular fold lower than for plasma, and methods with greater sensitiv-
fluid. As the CSF passes down to the lumbar spine (where ity or increased specimen volume are required for measuring
TABLE 31.7 Cerebrospinal Fluid Total Protein in Various Diseases

Clinical Condition Appearance and Cells 3 106/L Total Protein, mg/dL
Normal Clear, colorless; 0–5 lymphocytes 15–45a
INCREASED ADMIXTURE OF PROTEINS FROM BLOOD

Increased capillary permeability
• Bacterial meningitis Turbid, opalescent, purulent, usually .500 polymorphs 80–500
• Cryptococcal meningitis Clear or turbid; 50–150 polymorphs or lymphocytes 25–200
• Leptospiral meningitis Clear to slight haze; polymorphs early, then 5–100 50–100
lymphocytes
• Viral meningitis Clear or slight haze, colorless; usually #500 lymphocytes 30–100
• Encephalitis Clear or slight haze, colorless; usually #500 lymphocytes 15–100
• Poliomyelitis Clear, colorless; #500 lymphocytes 10–300
• Brain tumor Usually clear; 0–80 lymphocytes 15–200 (usually normal)
Mechanical obstruction:
• Spinal cord tumorb Clear, colorless, or yellow 100–2000
Hemorrhage
Cerebral hemorrhage Colorless, yellow, or bloody; blood cells 30–150
Local immunoglobulin production
• Neurosyphilis Clear, colorless; 10–100 lymphocytes 50–150
• Multiple sclerosisc Clear, colorless; 0–10 lymphocytes 25–50
Both increased capillary permeability and
local immunoglobulin production:
• Tuberculous meningitis Colorless, fibrin clot, or slightly turbid; 50–500 lymphocytes 50–300 (occasionally
#1000)
• Brain abscess Clear or slightly turbid 20–120
After myelography (inflammatory reaction) Slight increase
a
Premature infants: #400 mg/dL; children: 30–100 mg/dL; older adults: #60 mg/dL.
b
Froin syndrome: Lumbar fluid values are much higher than cisternal fluid values.
c
Similar values may occur in certain other chronic inflammatory conditions of the nervous system.
INTRATHECAL PROTEIN SYNTHESIS

total serum or plasma protein. In practice, methods com-
monly used by clinical laboratories to measure total CSF Measurement of the CSF–serum immunoglobulin ratio and
protein include (1) pyrogallol red, (2) benzethonium chlo- assessment of oligoclonal immunoglobulin bands on electro-
ride, (3) reverse-biuret, and (4) biuret. The reverse-biuret phoretic separations of CSF serve as assays for intrathecal
method measures free copper remaining after formation of immunoglobulin synthesis. At least 90% of cases of multiple
biuret complexes by reduction and complexation with a che- sclerosis give positive findings, but increased immunoglobu-
lating dye. Analyzers often use the same method, possibly lins and oligoclonal immunoglobulins may be found in other
with some adjustment of specimen volume, for CSF and inflammatory diseases of the central nervous system such as
urine protein. The usual reference interval for CSF total pro- bacterial, viral, or parasitic infections, as well as neurosyphilis,
tein is 15 to 45 mg/dL. Total protein concentrations are con- subacute sclerosing panencephalitis, and Guillain-Barré
siderably higher in neonates and in healthy elderly adults. In syndrome. Multiple sclerosis is less likely if CSF total protein
CSF from premature and full-term neonates, concentrations exceeds 100 mg/dL or if the CSF leukocyte count is greater
up to 400 mg/dL are observed. In term newborns, a progres- than 50/mL. The CSF albumin concentration in 70% of cases
sive decline in the reference interval CSF protein is seen over of multiple sclerosis is within the reference interval. Increases
the first few weeks of life, with values approaching adult con- in CSF IgG concentration or in the CSF–serum IgG ratio may
centrations after 4 months of age.206 result from increased permeability of the blood–CSF barrier,
Permeability of the blood–brain barrier. A more specific increased local production of IgG, or both. To identify intra-
measure of the permeability of the blood–CSF barrier in- thecal production specifically, correction for increased per-
volves determination of the ratio of albumin concentration meability is necessary. These corrections use CSF and serum
in CSF versus plasma. This ratio, the CSF/serum albumin albumin and IgG concentrations in one of several ways:
index, is usually calculated for CSF albumin in milligrams 1. Concentrations in CSF of IgG and albumin are measured,
per deciliter and for serum protein in grams per liter, effec- and the IgG–albumin ratio is calculated. A ratio greater
tively multiplying values by 1000. A CSF–serum albumin than 0.27 is considered indicative of increased synthesis;
index less than 9 indicates an intact blood–CSF barrier. in about 70% of cases of multiple sclerosis, the ratio
Values of 9 to 14 represent slight impairment, 14 to 30 mod- exceeds 0.27.
erate impairment, and greater than 30 severe impairment. IgGCSF (mg/dL)
The index helps correct for variation in serum albumin Ratio =
concentration. AlbuminCSF (mg/dL)
2. Concentrations in CSF and serum of IgG and albumin Peritoneal and Pleural Fluids
are measured, and the CSF immunoglobulin index is Pathologic accumulations of fluid in the peritoneal and pleu-
calculated. ral cavities or elsewhere vary greatly in protein content. These
The reference interval for the index is 0.30 to 0.70. Values fluids may be ultrafiltrates with low-protein concentrations
greater than 0.70 indicate increased IgG synthesis; in relative to plasma and scant amounts of large proteins (tran-
more than 80% of cases of multiple sclerosis, this index sudates). Alternatively, fluids may have protein concentrations
exceeds 0.70. This CSF immunoglobulin index is now approaching those of plasma and significant amounts of large
frequently used. proteins such as immunoglobulins and AMG (exudates) in
IgGCSF (mg/dL) × Albuminserum (g/dL) response to local inflammation and increased vascular perme-
Index = ability. Distinction between transudates and exudates assists
AlbuminCSF (mg/dL) × IgGserum (g/dL) in diagnosing the cause of fluid accumulation. The major
cause of pleural transudates is congestive heart failure. Exu-
3. The rate of intrathecal IgG synthesis is estimated by Tour- dates occur with infection, pleuritis, pulmonary embolism,
tellotte’s formula.207 The rate of synthesis of IgG in milli- and cancer. Criteria for identifying exudates in pleural fluid
grams per day is equal to: were proposed by Light.214 According to these criteria ratios
of (1) pleural fluid protein to serum protein greater than
{
5 dL/d  IgGCSF −

IgGserum 
369 }
−  AlbuminCSF −
Albuminserum 
230 
0.5, (2) pleural lactate dehydrogenase (LDH) to serum LDH
activity greater than 0.6, and (3) total pleural fluid LDH
0.43 (IgGserum )  greater than 200 IU/L likely result from an exudative process.215
× 
Albuminserum 
Pleural fluid may be turbid from large numbers of white
blood cells (WBCs), fibrin particles, or chylomicrons. Chylous
effusions (containing chylomicrons) result from lymphatic
where protein concentrations are in mg/dL. The 5 dL/day obstruction related to cancer, surgery, trauma, sarcoidosis, or
term is daily CSF production. The first bracketed term other causes.216 Lymphatics, particularly the major trunk, the
represents the difference between IgG found in CSF and thoracic duct, serve as the major route for chylomicrons from
the IgG expected if the blood–brain barrier is intact. The the intestines to the blood circulation. Therefore entry of chy-
second bracketed term represents the same for albumin lomicrons into the pleural space is related in part to dietary fat
but is corrected by a factor of 0.43, corresponding to the intake to generate chylomicrons, and fasting lowers the fat
ratio of molecular weights of albumin to IgG, assuming content of chylous effusions. Chylomicrons in fluids may be
that 1 mole of IgG accompanies every mole of albumin identified by separation of a cream layer upon standing or by
that passes the blood-brain barrier. The constants 369 triglyceride analysis. For peritoneal fluid, the serum to ascites
and 230 originate from the normal serum–CSF ratios for albumin gradient (i.e., the difference between serum and
IgG and albumin. The reference interval for the synthesis peritoneal fluid albumin) helps distinguish transudates
rate is 0 to 3.3 mg/day. Values exceeding 8 mg/day are (mainly in portal hypertension from cirrhosis) from infec-
found in most cases of multiple sclerosis. This estimator tion and other causes of ascites. Usually, the serum to ascites
provides no more clinical information than the IgG in- albumin gradient is greater than 1.1 g/dL in portal hyperten-
dex; the complex formula merely rearranges the results sion and is lower than 1.1 g/dL for other causes that generate
for serum and CSF IgG and albumin and then factors in exudates.217
several constants.
The current, most reliably accepted method for detection Fecal Material
of intrathecal immunoglobulin is the detection of CSF The use of fecal material for protein analysis is relatively infre-
specific oligoclonal bands through IEF coupled with IgG- quent but indicated in some specific clinical circumstances.
specific immunoblotting. IEF is a manual technique with an Assessment of protein content in feces is fraught with a num-
average turn-around time of 3 hours. It requires a paired ber of limitations. Results of fecal protein content are often
serum sample to be analyzed with the CSF, as is the case normalized to fecal weight. Thus watery stool specimens
with most of the index calculations described above. Detec- generally provide decreased estimates of protein concentra-
tion of at least two CSF-specific oligoclonal bands that are tion. Extraction is likewise dependent on stool consistency
not present in serum is a positive result, according to the and homogeneity, leading to considerable within-subject and
revised 2017 McDonald criteria. The presence of oligoclonal between-subject variability.
bands may substitute for dissemination in time for diagnosis Protein loss in the GI tract may be assessed using an assay
of multiple sclerosis.208 Even though the McDonald criteria of fecal AAT. The amount of fecal AAT excreted over time is
considers the detection of two or more CSF-exclusive bands determined as a function of the serum AAT concentration.
to be positive, this definition is characterized differently Correction for serum AAT concentration is necessary because
by various institutions with positivity being defined by any- of variation in serum AAT from severe enteric losses or from
thing ranging from one to four unique CSF bands.209 Since APRs. AAT is relatively stable in the lower digestive tract but is
2008, several studies have attempted to use automated, digested in the stomach at acid pH. Therefore suppression of
quantitative markers to replace oligoclonal banding assays. acid secretion is necessary to use AAT excretion as a measure
Measurement of kappa FLC in CSF has been shown to po- of gastric protein-losing enteropathy.218 An alternative measure
tentially reflect intrathecal immunoglobulin synthesis and of increased GI loss of protein has been the assessment of stool
have comparable sensitivity and specificity to oligoclonal radioactivity following the injection of radiolabeled albumin.
bands.210–212 Indexes replacing the total immunoglobulins Inflammatory bowel disease (IBD) includes Crohn dis-
for kappa FLC have also been developed and adopted, ease and ulcerative colitis. Although GI histology remains
mostly in Europe.211,213 the gold standard for diagnosis, a number of fecal protein
markers have been employed as tools for screening and Exocrine pancreatic disease occurs in the setting of multi-
response to therapy. In the setting of IBD, it may be more ple pathologic states, including chronic alcoholism, diabetes,
useful to have indicators of inflammation rather than leak- HIV, celiac disease, and cystic fibrosis. Determination of fecal
age of plasma proteins. Fecal products secreted by WBCs, proteins such as elastase that are derived from pancreatic se-
such as lactoferrin and calprotectin, have been used as a cretions provides an alternative for the diagnosis of pancreatic
measure of disease activity in IBD.219 Lactoferrin has diag- insufficiency compared with gold standard duodenal sam-
nostic sensitivity and specificity ranging from 47 to 92% pling after secretin administration.221,222 For example, fecal
and 60 to 100%, respectively, for the diagnosis of IBD. The elastase concentrations of less than 100 mg/mg stool suggest
sensitivity and specificity of calprotectin is similar and severe functional deficiency with sensitivity ranging from 72
range from 61 to 100% and 72 to 100%, respectively.220 to 100% and specificity ranging from 29 to 96%.220
POINTS TO REMEMBER
• Amino acid homeostasis depends on enteral extraction, • Protein diversity is derived from linear amino acid sequence
broad-specificity transport systems, and catabolism. and an array of cotranslational and post-translational modifi-
• Amino acids are the building blocks of protein but also serve cations, including acylation, prenylation, phosphorylation, and
as substrates for energy generation and other important glycosylation.
biomolecules. • More than 100 congenital disorders of glycosylation have
• Amino acids such as glycine, glutamate, and g-aminobutyric been documented among the nearly 200 human gene prod-
acid are important neurotransmitters. ucts involved in protein glycosylation.
• An array of small peptide systems derived from larger pro- • Current catalogs of the circulating human proteome contain
tein precursors dictate control of numerous physiologic pro- more than 12,000 components.
cesses, including glucose and electrolyte homeostasis • Complement activation is triggered by direct detection of li-
(ACTH), fluid retention (natriuretic peptides, vasopressin), popolysaccharide or oligosaccharide components of foreign
iron metabolism (hepcidin), and vascular tone (angiotensins, organisms (alternative and lectin pathways) or via antibody
endothelins). recognition of microbes (classical pathway).
• Broad specificity immunoassays may detect both active • The human proteome includes more than 500 different pro-
and inactive peptides, overestimating active peptide concen- teases, classified into four families according to the catalytic
trations. mechanism employed to cleave peptide bonds.
• Narrowly targeted mass spectrometry assays may underes- • Infants typically manifest a transient IgG deficiency with a
timate the concentration of active peptides by excluding nadir at about 3 months of age coinciding with the decline of
detection of some molecules with substantial biologic maternally acquired antibodies.
potency. • Proteins in CSF are available in concentrations approximately
• Pathologic conditions such as cystic fibrosis, sickle cell dis- 100-fold lower than plasma. The primary utility of CSF protein
ease, Alzheimer disease, and amyloidosis result largely from testing is to evaluate the integrity of the blood–brain barrier
protein folding defects. and the intrathecal synthesis of proteins.
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M U LT I P L E CHOICE QUESTIONS 6. Which of the following is the second most abundant

circulating immunoglobulin in adults?
1. Which of the following techniques is used to determine a. IgG
total protein concentration? b. IgM
a. Jaffe Method c. IgA
b. Watson-Schwartz Method d. IgD
c. Jendrassik-Grof Method e. IgE
d. Biuret Method 7. Which two of the following proteins are positive acute-
e. Berthelot Method phase reactants?
2. a1-Antitrypsin inhibits which of the following classes of a. C-reactive protein
proteases? b. Alanine aminotransferase
a. Phosphoryl Proteases c. Creatine phosphokinase
b. Serine Proteases d. a1-Antitrypsin
c. Aspartyl Proteases e. Prealbumin
d. Metalloproteases 8. The terminal lytic membrane attack complex of the
e. Cysteinyl Proteases complement cascade consists of which protein factors:
3. Which of the following amino acids is nonproteogenic. a. C1q,C1r,C1s
a. Threonine b. C4b,C2a,C3b
b. Ornithine c. C5b,C6,C7,C8,C9
c. Glutamate d. B,D,H,I,P
d. Methionine 9. Which of the following proteins exhibits the most anodic
e. Selenocysteine migration during electrophoresis at pH 8.6?
4. Which of the following is the most abundant protein in a. Transferrin
plasma? b. IgG
a. Prealbumin c. C3
b. Ceruloplasmin d. C-reactive protein
c. Albumin e. Haptoglobin
d. Haptoglobin 10. Which two of the following amino acids may be reversibly
e. b2-Microglobulin phosphorylated?
5. Ubiquitin is covalently attached to which of the following a. Threonine
amino acids? b. Tyrosine
a. Cysteine c. Alanine
b. Glutamine d. Lysine
c. Methionine e. Arginine
d. Lysine
e. Arginine

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SECTION III

*The full version of this chapter is available electronically on ExpertConsult.com.

specific protein components in healthy and diseased popula-

Arginine (ARG, R) 174.20 H2N H O 2.2 9.0 12.5 10.8 24.5

Asparagine (ASN, N) 132.12 H2N H O 2.0 8.8 5.4 23.5

Aspartate (ASP, D) 133.10 O H O 2.1 9.8 3.9 2.9 23.5

Cysteine (CYS, C) 121.16 H O 1.7 10.8 8.3 5.1 2.5

Glycine (GLY, G) 75.07 H O 2.3 9.6 6.0 20.4

Glutamine (GLN, Q) 146.15 H2N H O 2.2 9.1 5.7 23.5

Histidine (HIS, H) 155.16  H O 1.8 9.2 6.0 7.6 23.2

Isoleucine (ILE, I) 131.17 H O 2.4 9.7 6.0 4.5

Leucine (LEU, L) 131.17 H3C H O 2.4 9.6 6.0 3.8

Lysine (LYS, K) 146.19 H O 2.2 9.0 10.5 9.7 23.9

Methionine (MET, M) 149.21 H O 2.3 9.2 5.8 1.9

Phenylalanine (PHE, F) 165.19 H O 1.8 9.1 5.5 2.8

Proline (PRO, P) 115.13 O 2.1 10.6 6.1 1.6

Serine (SER, S) 105.09 H O 2.2 9.2 5.7 20.8

Threonine (THR, T) 119.12 HO H O 2.6 10.4 6.5 20.7

Tryptophan (TRP, W) 201.22 H O 2.5 9.4 5.9 20.9

Tyrosine (TYR, Y) 181.19 H O 2.2 9.2 10.5 5.7 21.3

Valine (VAL, V) 117.17 H3C H O 2.3 9.6 6.0 4.2

TABLE 31.2 Genetic Basis of Selected Amino Acid Transport Systems

Transport properties not classified by classical naming convention.

HHH, Hyperornithinemia, hyperammonemia, homocitrullinuria.

Pyruvate Glucose Urea

Ketone bodies Acetyl-CoA

anchor tend to cluster in cholesterol- and sphingolipid-rich Prenylation

TABLE 31.3 Selected Members of the Four Protease Families

TABLE 31.4 High-Abundance Plasma Proteins

sample type as it chelates calcium, a required metal for the

TABLE 31.5 Inherited Deficiencies of Complement Components

TABLE 31.6 Monoclonal Immunoglobulins (Paraproteins) in Multiple Myeloma

† Includes LC MGUS (n = 2435)

Normal (Adult) Normal (Pediatric) Chronic Renal Disease

SPE lgG lgA lgM   SPE lgG lgA lgM  

TABLE 31.7 Cerebrospinal Fluid Total Protein in Various Diseases

INCREASED ADMIXTURE OF PROTEINS FROM BLOOD

INTRATHECAL PROTEIN SYNTHESIS

REFERENCES 23. Palmieri, F., The mitochondrial transporter family SLC25:

M U LT I P L E CHOICE QUESTIONS 6. Which of the following is the second most abundant

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Histidine (HIS, H) 155.16 H O 1.8 9.2 6.0 7.6 23.2

SPE lgG lgA lgM SPE lgG lgA lgM