Chapter 1:Amino acids and proteins

D. Ceruloplasmin
• It is a copper-containing α2-glycoprotein that has enzyme activities
( copper oxidase, histaminase and ferrous oxidase)
• It is synthesized in the liver, where 6-8 atoms of copper, half as Cu+ and
half as Cu++, are attached to an apoceruloplasmin. 90% or more of the
total serum copper is found in ceruloplasmin
• Most assays today use immunochemical methods including radial
immunodiffusion and nephelometry
• Low concentrations of ceruloplasmin at birth gradually increase to adult
levels and slowly continue to rise with age thereafter

1
Chapter 1:Amino acids and proteins
• Adult females have higher concentrations than do males, and
pregnancy, inflammatory processes, malignancies, oral estrogen,
and contraceptives cause an increased serum concentration
• Certain diseases are associated with low serum concentration. In
Wilson disease ( autosomal recessive disease, the copper is
deposited in the skin, liver, and brain resulting in degenerative
cirrhosis and neurologic damage).
• Low ceruloplasmin is also seen in malnutrition, malabsorption,
severe liver disease, nephrotic syndrome and Menkes’ kinky hair
syndrome, in which a decreased absorption of copper results in a
decrease in ceruloplasmin
Chapter 1:Amino acids and proteins
E. α2-macroglobulin
• It is a dimeric, large protein synthesized in hepatocytes. It is
found principally in the intravascular spaces because its
movement is restricted due to its size. However, much lower
concentrations can be found in other body fluids such as CSF
• Upon binding with and inhibiting proteases, it is removed by the
reticuloendothelial tissues.
• This protein reaches a maximum serum concentration at the
age of 2-4 years and then decreases to about one-third of that
at about 45 years. Later in life, a moderate increase is seen.
• This change with age is more pronounced in males than in
females. There is a distinct difference in the reference values
for males and females, adult females having higher values than
males
3
Chapter 1:Amino acids and proteins
• It inhibits proteases such as trypsin, pepsin, and plasmin. It also
contributes more than one-fourth of the thrombin inhibition normally
present in the blood
• In nephrosis, its serum levels may increase as much as 10 times
because its large size aids in its retention.
• The protein is also increased in diabetes and liver diseases. Use of
contraceptive medications and pregnancy increase the serum
levels by 20%
• The analytic methods that have been used for the assay of this
protein are radial immunodiffusion, immunonephelometry, enzyme-
linked immunosorbent assay (ELISA), and latex agglutination assay
Chapter 1:Amino acids and proteins
F. Transferrin ( siderophilin)
• It is a glycoprotein, synthesized primarily by the liver. 2 molecules of ferric iron
can bind to each molecule of transferrin. Normally, only about 33% of the iron
binding sites on transferrin are occupied. It is the major component of the β-
globulin fraction and appears as a distinct band on high resolution serum
protein electrophoresis
• The analytical methods used for the quantitation of transferrin are
immunodiffusion and immunonephelometry
• The major functions of transferrin are the transport of iron and the prevention
of loss of iron through the kidney. Its binding of iron prevents iron deposition in
the tissues during temporary increases in absorbed iron or free iron
• Transferrin transports iron to its storage sites where it is incorporated into
another protein, apoferritin, to form ferritin. It also carries iron to cells such as
bone marrow that synthesize Hb and other iron-containing compounds
• In iron deficiency anemia, transferrin in serum is normal or increased
5
Chapter 1:Amino acids and proteins
• A decreased transferrin level generally reflects an overall decrease in
synthesis of protein, such as seen in liver disease or malnutrition, or it
may be seen in protein-losing disorders such as nephrotic syndrome
• A deficiency of plasma transferrin may result in the accumulation of iron
in apoferritin or in histiocytes, or it may precipitate in tissues as
hemosiderin
• An increase of iron bound to transferrin is found in a hereditary disorder
of iron metabolism, hemochromatosis, in which excess iron is
deposited in the tissues, especially the liver and the pancreas. This
disorder is associated with bronze skin, cirrhosis, DM, and low plasma
transferrin levels
• Atransferrinemia is inherited as an autosomal recessive trait due to
mutation of both transferrin genes with a resulting absence of
transferrin. It is characterized by anemia and hemosiderosis (iron
deposition) in the heart and liver. The iron damage to the heart can lead
to heart failure. This disease can be effectively treated by plasma
infusions of transferrin. 6
Chapter 1:Amino acids and proteins
G. β2 microglobulin
• It is the light chain component of HLA. This protein is found on the surface
of most nucleated cells and is present in high concentrations on
lymphocytes
• Due to its small size, it is filtered by the renal glomerulus but most is
reabsorbed and catabolized in the proximal tubules. Elevated serum levels
are the result of impaired clearance by the kidney or over production of the
protein, as occurs in a number of inflammatory diseases such as RA and
SLE
• In patients with HIV, a high level of this protein in the absence of renal failure
indicates a large LC turnover rate which suggests the virus is killing LC
• It is usually measured by immunoassay
Chapter 1:Amino acids and proteins
H. Fibrinogen
• It is one of the largest proteins in plasma. It is synthesized in the liver,
and because it has considerable carbohydrate content, it is classified as
glycoprotein. On plasma electrophoresis, it is seen as a distinct band
between the β and γ globulins. Its function is to form fibrin clot when
activated by thrombin. Thus, fibrinogen is virtually all removed in the
clotting process and is not seen in the serum
• Fibrin split products ( degradation products of fibrinogen and fibrin) are
determined by immunoassay methods such as radial immunodiffusion,
nephelometry and radioimmunoassay
• It is one of the acute-phase reactants. Fibrinogen levels also rise with
pregnancy and the use of birth control pills. Decreased values generally
reflect extensive coagulation during which the fibrinogen is consumed

8
Chapter 1:Amino acids and proteins
I. C-reactive protein ( CRP)
• It appears in the blood of patients with diverse inflammatory diseases
but is undetectable in healthy individuals. It is synthesized in the liver
• CRP was so named because it precipitates with the C substance, a
polysaccharide of pneumococci.
• CRP bound to bacteria promotes the binding of complement which
facilitates their uptake by phagocytes. This process of protein coating to
enhance phagocytosis is known as opsonization
• It is generally measured by immunological methods including latex slide
precipitation test, nephelometry, and EIA
• CRP is elevated in acute rheumatic fever, bacterial infections,
myocardial infarcts, RA, gout and viral infections

9
Chapter 1:Amino acids and proteins
• Elevated levels of CRP stimulate the production of tissue factor that
initiates coagulation, activates complement, and binds to LDL in the
atherosclerotic plaque—evidence that points to a causal relationship
between CRP levels and cardiovascular disease.

• new research shows that having CRP in the high-normal range may also
be associated with other diseases such as colon cancer, complications of
diabetes, obesity, and the risk of developing type 2 diabetes.

10
 Chapter 1:Amino acids and proteins
J - Inter-α-trypsin inhibitor ( ITI):

• Inter-trypsin inhibitors (ITIs) are a family of serine protease inhibitors, assembled from two
precursor proteins: a light chain (bikunin) and either one or two heavy chains.
• On high resolution serum electrophoresis, ITI migrates in the zone between α1 and α2
fractions
• The light chain is responsible for the inhibition of the proteases trypsin, plasmin and
chemotrypsin
• While there is only one type of light chain, there are five different homologous heavy chains
(ITIHs). ITIH molecules have been shown to play a particularly important role in inflammation
and carcinogenesis.
• Elevations are seen in inflammatory disorders.

11
 Chapter 1:Amino acids and proteins
K. Hemopexin
• It is synthesized in the parenchymal cells of the liver that migrates
electrophoretically in the β-globulin region. It can be determined by radial
immunodiffusion. Its function is to remove circulating heme
• The heme-hemopexin complex is carried to the liver where the complex is
destroyed. Hemopexin also removes ferriheme and porphyrins
• Its level is very low at birth but reaches adult values within the first year of
life. Pregnant mothers have increased plasma hemopexin levels. Increased
concentrations are also found in DM, Duchenne muscular dystrophy, and
some malignancies, especially melanomas
• In hemolytic disorders, serum hemopexin concentrations decrease.
Administration of diphenylhydantoin also results in decreased concentration

12
 Chapter 1:Amino acids and proteins
L. Lipoproteins
• Are complexes of proteins and lipids whose function is to transport
cholesterol, TG and phospholipids in the blood. They are subclassified
according to the apoprotein and specific lipid content. On high resolution
serum protein electrophoresis, HDL migrate between the albumin and α1
zone, VLDL migrate at the beginning of the β-fraction ( pre- β) and LDL
appear as a separate band in the β region

13
 Chapter 1:Amino acids and proteins
M. Complement:
• It is a collective term for several proteins that participate in the
immune reaction and serve as a link to the inflammatory
response
• These proteins circulate in the blood as nonfunctional
precursors. In the classic pathway, activation of these proteins
begins when the first complement factor, C1q, binds to an
antigen-antibody complex. An alternate pathway ( properdin
pathway) for complement activation exists in which the early
components are bypassed and the process begins with C3

14
 Chapter 1:Amino acids and proteins
• Immunochemical methods such as radial immunodiffusion and
nephelometry are the ones used for the measurement of
complement
• Complement is increased in inflammatory states and decreased
in malnutrition, lupus erythematosus, and disseminated
intravascular coagulopathies.
• Inherited deficiencies of individual complement proteins also
have been described. In most cases, the deficiencies are
associated with recurrent infections

15
Chapter 1:Amino acids and proteins
N. Gc-globulin ( Group specific component; Vitamin D-binding protein)
• It is a protein that migrates in the α1-α2 interzone. This protein exhibits a high
binding affinity for vit D compounds. It is the major carrier protein of vitamin D and
its metabolites in the circulation and also transports components such as fatty
acids and endotoxin.
• Elevations of Gc-globulin are seen in the third trimester of pregnancy and in
patients taking estrogen oral contraceptives.
• Severe liver disease and protein-losing-syndromes are associated with low
levels.
• Gc binds actin released from cells upon injury, and the Gc–actin complexes are
rapidly cleared from the circulation, thereby preventing the harmful effects of actin
filaments in blood vessels.
• Gc may be of importance for bone formation and in the immune system. Gc may
act as a co-chemotactic factor in facilitating chemotaxis of neutrophils and
monocytes in inflammation. Immunonephelometry, which can be automated, is
the method of choice for measurement. 16
 Chapter 1:Amino acids and proteins
O- Immunoglobulins
• There are five major groups of immunoglobulins in the serum: IgA, IgG,
IgM, IgD, and IgE. They are synthesized in plasma cells. Their synthesis
is stimulated by an immune response to foreign particles and
microorganisms

• Polyclonal antibodies (pAbs) are mixture of heterogeneous which are

usually produced by different B cell clones in the body. They can recognize
and bind to many different epitopes of a single antigen.

• Monoclonal antibodies (mAbs) are generated by identical B cells which are

clones from a single parent cell. This means that the monoclonal antibodies
have monovalent affinity and only recognize the same epitope of an
antigen.

17
Chapter 1:Amino acids and proteins
• IgG crosses the placenta, and the IgG present in the newborn’s serum is that
synthesized by the mother.
• IgM does not cross the placenta and initially is 0.21g/L, but this increases rapidly to
adult levels by about 6 months.
• IgA is virtually lacking at birth ( 0.003g/L), increases slowly to reach adult values at
puberty, and continues to increase during the lifetime.
• IgD and IgE levels are undetectable at birth by customary methods and increase
slowly until adulthood.
• IgA is generally higher in males than in females, IgM and IgG levels are somewhat
higher in females. IgE levels vary with the allergic condition of the individual
• IgG is increased in liver disease, infections, IgG myeloma, parasitic disease and many
rheumatic diseases
Chapter 1:Amino acids and proteins

• Decreased IgG levels are associated with acquired immunodeficiency, protein-losing

states, and non-IgG myeloma.

• Polyclonal increase in the serum IgA are found in liver disease, infections and
autoimmune diseases. Decreased serum concentrations are found in depressed protein
synthesis, ataxia-telangiectasia and hereditary immunodeficiency disorders

• IgM is the first antibody that appears in response to antigenic stimulation. Increased IgM
concentration is found in toxoplasmosis, cytomegalovirus, rubella, herpes, syphilis, and
various bacterial and fungal diseases.

19
Chapter 1:Amino acids and proteins
• A monoclonal increase is seen in Waldenstrom’s macroglobulinemia. This increase is
seen as a spike in the vicinity of the late β zone of a protein electrophoretic pattern.
Decreases are seen in protein-losing conditions and immunodeficiency disorders
Chapter 1:Amino acids and proteins

• IgD is the immunoglobulin with the fourth highest

concentration in normal serum. Its concentration is increased
in infections, liver disease, and connective tissue disorders
• IgE is the immunoglobulin present in the respiratory and
gastrointestinal mucosa. Polyclonal increases are seen in
allergies, including asthma and hay fever. Monoclonal
increases are seen in IgE myeloma, a rare disease
• The immunoglobulins have been determined using radial
immunodiffusion, nephelometry, turbidimetry, and RIA.
Fluorescent immunoassay techniques and
immunonephelometric assays also have been used

21
Chapter 1:Amino acids and proteins
 Miscellaneous proteins
1. Myoglobin
• Myoglobin is a single-chain globular protein of 153 amino acids, containing a heme (iron-
containing) prosthetic group. Myoglobin is the primary oxygen-carrying protein
(approximately 2% of total muscle protein) found in striated skeletal and cardiac muscle
• As a cardiac biomarker, myoglobin is used in conjunction with troponin to help diagnose or
rule out a heart attack.
• When striated muscle is damaged, myoglobin is released, elevating the blood levels.
• In an acute myocardial infarction (AMI), this increase is seen within 2–3 hours of onset
and reaches peak concentration in 8–12 hours. For the diagnosis of AMI, serum
myoglobin should be measured serially. If a repeated myoglobin level doubles within 1–2
hours after the initial value, it is highly diagnostic of an AMI.
• Myoglobin is a small molecule freely filtered by the kidneys allowing level to return to
normal in 18–30 hours after the AMI.

22
Chapter 1:Amino acids and proteins
• Myoglobin is toxic to the kidneys and in severe muscle injury, levels of
myoglobin may rise very quickly and the kidneys may be damaged by the
increased amounts.
• Latex agglutination, ELISA, immunonephelometry, and
fluoroimmunoassays for myoglobin are used in the clinical laboratory for
myoglobin measurement

23
Chapter 1:Amino acids and proteins
2. Troponin
• It is a complex of 3 proteins that bind to the thin filaments of striated muscle ( cardiac
and skeletal) but are not present in smooth muscle.
• The complex consists of troponin T (TnT), troponin I ( TnI), and troponin C (TnC).
Together they function to regulate muscle contraction
• 3 genes code for TnT, one each in cardiac muscle and fast and slow skeletal muscle.
TnI, also encoded by 3 genes, has similar isoforms while TnC encoded by 2 genes has
only a cardiac and slow skeletal muscle form
• Of particular interest is the cardiac isoform of troponin T. Cardiac troponin T levels in
serum begin to rise within 4h following the onset of myocardial damage and remain
elevated for 10-14 days following an AMI.
• Measurement of this protein is a valuable aid in the diagnosis of an acute myocardial
infarction. It is also useful in monitoring the effectiveness of thrombolytic therapy in
myocardial infarction patients
• Cardiac TnT is measured by Elisa assay using 2 monoclonal antibodies directed
against different epitopes on the protein. Its reference interval is 0-0.1 ϻg/L
24
Chapter 1:Amino acids and proteins

• TnI or a TnT can be performed by itself or along with other cardiac biomarkers, such as
creatine kinase (CK), CK-MB, and myoglobin.

• TnI and TnT tests have begun to replace CK and CK-MB tests because they are more
specific for heart injury (versus skeletal muscle injury) and are elevated for a longer
period of time.

25
These enzymes and proteins are used as diagnostic biomarkers for MI