AMINO ACIDS

Overview
Amino acids are the building blocks of proteins.
The precise amino acid content, and the sequence of
those amino acids, of a specific protein is determined
by the sequence of the bases in the gene that
encodes that protein.
The chemical properties of the amino acids of
proteins determine the biologic activity of the
protein.
Growth, repair, and maintenance of all cells are
dependent on amino acids.
Proteins catalyze almost all of the reactions in living
cells, controlling virtually all cellular processes.
Basic Structure
An amino acid contains at least one of
the both amino and carboxylic acid
functional groups.
The N-terminal end amino group (-NH2)
and the C-terminal and carboxyl group
(-COOH) bond to the alpha-carbon with
the amino group of one amino acid
linking with the carboxyl group of
another, forming a peptide bond.
 A chain of amino acids is known as a
polypeptide, and a large polypeptide
constitutes a protein.
In human serum, proteins average
about 100-150 amino acids in the
polypeptide chains.
Amino acids differ from one another
by the chemical composition of their
R group (side chains).
The R groups found on the 20
different amino acids used in building
proteins
Metabolism
About half of the 20 amino acids needed by humans cannot be
synthesized at a rapid enough rate to support growth ; they
must be supplied by food. These nutritionally essential amino
acids must be supplied by the diet in the form of proteins.
The essential amino acids are:
Arginine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Tryptophan
Valine
 The 10 amino acids that the body can produce are:
Alanine
Aspargine
Aspartic Acid
Cysteine
Glutamic Acid
Glutamine
Glycine
Proline
Serine
Tyrosine
Tyrosine is produced from phenylalanine, so if the diet is deficient in
phenylalanine, tyrosine will be required as well.
Humans do not have all the enzymes required for the biosynthesis
of all of the amino acids.
Under normal circumstances, proteolytic enzymes, such as pepsin
and trypsin, completely digest dietary proteins into their
constituent amino acids.
Amino acids are also released by the normal breakdown of body
proteins.
 Protein provides 12-20% total daily body
energy requirement by means of
deamination or transamination.
Ketogenic amino acid
Leucine
Lysine
Glucogenic amino acids
Alanine
Ammonium ion produced during deamination
is converted into urea via the urea cylce in
the liver
Essenti
Function
al

cell wall division, the healing of wounds, stimulation of protein

Arginine synthesis, immune function, release of hormones and urea
generation

basic amino acids involved in immune response, synthesis of

Histidin nucleic acids, repair body tissues, manufacture b, protect the body
e from heavy metal toxicity and stimulates the secretion of the
digestive enzyme gastrin.

isoleuci branched-chain amino acids for healing, for hemoglobin formation,

ne and it helps to regulate blood glucose levels.

second most common amino acid found in protein beside glycine.

Leucine For healing, lowers blood glucose levels, for the optimal growth of
infants and for nitrogen balance in adults.

basic (by charge) amino acids. for production of antibodies and

lowers triglyceride levels, for proper growth and bone development
Lysine
in children and to maintain a proper nitrogen balance in adults, and
calcium absorption
Essentia
Function
l

Methioni initiate translation of messenger RNA, fat metabolism helps to

ne detoxify heavy metals and synthesis of epinephrine and choline.

nonpolar amino acid. promotes alertness and vitality, elevates

Phenylala mood, decreases pain, aids memory and learning, and is used to
nine treat arthritis and depression, part of the composition of
aspartame. biosynthesis of AA.
alcohol-containing AA that is an important component in the
Threonin formation of protein and collagen, maintain proper protein
e balance in the body and it aids liver function, metabolism, and
assimilation.
precursor for serotonin and melatonin, a neurohormone and
powerful antioxidant, a natural relaxant, alleviates insomnia,
Tryptoph
soothes anxiety, and reduces depression, treatment of migraine
an
headaches, aids in weight control by reducing appetite, and helps
control hyperactivity in children
for muscle metabolism and coordination, tissue repair, and
maintenance of nitrogen balance. treatments for muscle, mental,
Valine
and emotional problems; insomnia; anxiety, and liver and
gallbladder disease
 Non
Function
Essential

For CHO metabolism, reduce build-up of toxic substances and

strengthens the immune system through production of
Alanine
antibodies.

involved in the transport of nitrogen, role in the synthesis of

Asparagine
ammonia
Aspartic
participates in gluconeogenesis and in urea cycle
Acid

important structural and functional component of proteins and

Cysteine enzymes.

polar AA, umami, sense of taste, linked to epileptic seizure,

important in the metabolism of sugars and fats, and aids
Glutamic transporting potassium into the spinal fluid, food additive and
Acid flavor enhancer in the form of sodium salt, monosodium
glutamate (MSG)
 Non
Function
Essential

most abundant AA in the body, involved in more metabolic

processes than any other amino acid. 61% of skeletal muscle
tissue is glutamine. converted to glucose when more glucose is
Glutamine
required for energy and aids in immune function. Assists in
maintaining acid-base balance. Involved in urea cycle.

simplest amino acid synthesized in the body w/ no

Glycine stereoisomers.

helps in healing of cartilage and the strengthening of joints,

tendons, and heart muscle, works with vitamin C to promote
Proline
healthy connective tissues.

alcohol based AA . for the proper metabolism of fats and fatty

acids and synthesis of pyrimidines, purines, creatine, and
Serine
porphyrins.

Synthesized from Phe. aids in the functions of the adrenal,

Two New Amino Acids
Selenocysteine (Sec)
recognized as the 21st amino acid but, unlike other
amino acids present in proteins, it is not coded for
directly in the genetic code.
coded by a UGA codon, which is normally a stop
codon.
has a specialized transfer RNA (tRNA).
has been discovered that HIV-1 encodes a functional
selenoprotein, and patients with HIV infection have
been shown to have a lower-than-average blood
plasma selenium level.
present in several enzymes, formate dehydrogenases,
glycine reductases, and some hydrogenases.
 Pyrrolysine (Pyl)
the 22nd naturally occurring genetically
encoded amino acid used by some
archaea (prokaryotic and single-celled
microorganisms) in enzymes that are
part of their methane-producing
metabolism.
lysine derivative is encoded by the UAG
codon, normally a stop codon.
AMINOACIDOPATHIES

- inherited errors of
metabolism in w/c there is
enzyme defect or in a defect
in membrane transport
system of AA
Phenylketonuria
Inherited as an autosomal recessive
trait.
Absence of activity of the enzyme
Phenylalanine hydroxylase.

Levels:
In the absence of enzyme: 1200 mol/L
In newborn (normal): 120 mol/L (2 mg/dL)
If untreated (blood level): 2.4 mM/L
Phenylketonuria
Phenylalaline metabolites:
Phenyl pyruvic acid
Phenyl pyruvate (phenylketone)
Phenyllactate acid
High levels of phenylalanine and its
metabolites can cause BRAIN
PROBLEMS.
Phenylketonuria
Can found in URINE and BLOOD
URINE: Mousy Odor

Mild PKU: 600-1200 mol/L

Non-PKU mild
Hyperphenylalaninemia: 180-600
mol/L and NO accompanying
accumulation of phenylketones
Phenylketonuria
Infants and Children:
Retarded mental development
Microcephaly

Can be avoided if Infant is maintained on a diet

containing very low levels of phenylalanine
Pregnant women with PKU (untreated)
always have babies who are:
Mentally Retarded
Microcephaly
Phenylketonuria
Hyperphenylalaninemia
Is not a result of the lack of PAH enzyme.
The defect is the Deficiency in the enzymes
needed for the regeneration and synthesis of
tetrahydrobiopterin (BH4).
Results in elevated blood levels of phenylalanine
and deficient production of neurotransmitters
from tyrosine and tryptophan.
Must be identified so that APPROPRIATE treatment
of the active cofactor along with the
neurotransmitter precursors L-DOPA and 5-OH
tryptophan can be initiated.
Phenylketonuria
Every STATE screens newborns from
at about 3 days of age.
Newborn Screening: allows early
identification and implementation of
treatment.
The goal of PKU treatment is to
maintain the blood levels of
phenlyalanine between 2 to 10 mg/dL.
Phenylketonuria
DIET
AVOID:
High protein foods
TAKE:
Calculated amounts of cereals
Starches
Fruits
Vegetables
Milk substitute
Phenylketonuria
KUVAN (sapropterin dihydrochloride)
First drug to help manage PKU.
Help reduce phenylalanine levels by
increasing the activity of the PAH enzyme.
Effective only in PATIENTS:
Who have some PAH activity.
Who continue to follow a phenylalanine-
restricted diet.
Have their phenylalanine levels monitored.
Phenylketonuria
TESTS
Guthrie Test
Semiquantitative, bacterial inhibition assay for
phenylalanine that uses the ability of
phenylalanine to facilitate bacterial growth in a
culture medium with an inhibitor.
Newborn infant blood is collected on a piece of
filter paper.
Small disk of the filter paper is punched out and
placed on an agar gel plate containing Bacillus
subtilis and B-2-thienylalanine.
Phenylketonuria
TESTS
Guthrie Test
The agar gel is able to support bacterial growth but
the B-2-thienylalanine inhibits bacterial growth.
In the presence of extra phenylalanine leached
from the impregnated filter paper disk, the
inhibition is overcome and the bacteria grow.
Sensitive enough to detect serum phenylalanine
levels of 180-240 mol/L (3-4mg/dl).
May provide FALSE-NEGATIVE due to the infant not
being at least 24 hours old.
Phenylketonuria
TESTS
Guthrie Test
Replaced in many areas by newer
techniques.

Tandem Mass Spectrometry

Can detect a wider variety of congenital
diseases.
Phenylketonuria
TESTS
Microfluorometric Assay
For the direct measurement of phenylalanine in
dried blood filter disks.
Yields quantitative results due to infant not being at
least 24 hours old.
High Performance Liquid Chromatography
Reference method for quantitative serum
phenylalanine.
Gas Chromatography/Mass Spectrometry
Can detect wide variety of congenital disorders.
Tyrosinemia
Inborn metabolic disorder of tyrosine catabolism which is
characterized by the excretion of tyrosine and tyrosine
catabolites in urine.
Three types of Tyrosinemia
Type I tyrosinemia
Most severe form of this aminoacidopathy.
Caused by low levels of the enzyme Fumarylacetoacetate
hydrolase.
Symptoms:
Failure to thrive, diarrhea, vomiting, jaundice, cabbage-like odor,
distended abdomen, swelling of legs increased predisposition for
bleeding.
It can lead to liver and kidney failure, problems affecting the nervous
system, and an increased risk of cirrhosis or liver cancer later in life.
Tyrosinemia
Type II tyrosinemia
deficiency of the enzyme Tyrosine
aminotransferase.
Symptoms:
Mental retardation, excessive tearing, photophobia, eye pain,
redness ad painful skin lesions of palms and soles of feet
Type III tyrosinemia
deficiency of the enzyme 4-hydroxyphenylpyruvate
dioxygenase.
Symptoms:
Mild mental retardation, seizures periodic loss of balance and
coordination.
Tyrosinemia
TREATMENT:
Low-protein diet.
Nitisone (NTBC), w/c prevents formation
of maleylacetoacetic acid and
fumarylacetoacetic acid w/c can be
converted to succinyl acetone, a toxin
that damages liver and kidneys.
Full or partial liver transplant.
Alkaptonuria
Inborn metabolic disease transmitted as an
autosomal recessive gene.
lack of the enzyme Homogentisate oxidase.
Urine turns BROWNISH-BLACK when it mixes w/
air.
HGA gradually accumulates in connective
tissue, causing OCHRONOSIS (pigmentation of
tissues), arthritis, dark spots on sclera,
deposition of pigments in the cartilages of ear,
nose and extremities.
Alkaptonuria
TESTS
Urinalysis
When ferric chloride is added to urine, it will
turn black.

TREATMENT
High dose of vitamin C
shown to decrease buildup of brown pigment
in cartilages and slow the development of
arthritis
Maple Syrup Urine Disease (MSUD)

Results from an absence or greatly reduced

activity of the enzyme branched-chain a-
ketoacid decarboxylase.
autosomal recessive genetic inherited
disorder
Has Maple Syrup or Burnt Sugar Odor of
the urine, breath, and skin.
The result of enzyme defect is accumulation
of branched-chain AA and their corresponding
ketoacids in the blood, urine and CSF.
Maple Syrup Urine Disease (MSUD)

Infants:
Seem normal at birth but will develop
within a week lethargy, vomiting, lack of
appetite and signs of failure to thrive.

CNS symptoms
stupor, muscle rigidity, respiratory
irregularity and can progress to severe
mental retardation and death.
Maple Syrup Urine Disease (MSUD)

TESTS
Modified Gutrie test: B. subtilis and 4-
azaleucine as inhibitor.
Microfluormetric assay.
Isovaleric Acidemia
An autosomal recessive metabolic
disorder from a deficiency of the enzyme
Isovaleryl-CoA dehydrogenase.
Urine: Sweaty Feet Odor
SYMPTOMS: Apparent after few days of
birth
Failure to thrive
Vomiting
Lethargy seizure
Coma and Death
Some people are asymptomatic
Isovaleric Acidemia
TREATMENT:
Protein-restrictive diet.
Oral administration of Glycine.
Carnitine Supplement w/c can interact
w/ isovaleric acid to form nontoxic,
readily excreted products.
TESTS:
Chromatography.
MS/MS
Isovaleric Acidemia
Laboratory results reveal
Metabolic acidosis
Mild to moderate ketonuria
Hyperammonemia
Thrompocytopenia
Neutropenia
Homocystinuria
Inherited autosomal recessive disorder of amino
acid metabolism.
Lack of the enzyme Cystathionine-B
synthetase.
Symptoms:
Osteoporosis
Dislocated lenses in the eye resulting from lack of
cysteine synthesis and mental retardation.
Multisystemic disorders of the connective tissue,
muscles, CNS and bone.
Thrombosis resulting from toxicity of homocysteine to
the vascular endothelium is it goes untreated.
Homocystinuria
TREATMENT:
Dietary restriction of methionine as well as high
doses of vitamin B6.
Trimethylglycine w/ folic acid.

TESTS:
Neonatal Screening: Guthrie test w/ L-methionine
sulfoximine.
High-Performance liquid Chromatography as
confirmatory test: methionine level > than mg/dL.
LC-MS/MS to test for urinary total homocysteine.
Citrullinemia
Inherited in an autosomal recessive pattern.
Belongs to a class of genetic diseases called urea cycle
disorders.

Type I Citrullinemia:
metabolic defect caused by the lack of the enzyme
Argininosuccinic acid synthetase.
SYMPTOMS:
Lack of appetite
Failure to thrive
Vomiting
Lethargy
Seizure
Coma and Death.
Citrullinemia
Type II Citrullinemia:
Caused by a mutation of the gene that would
otherwise provide instructions for making the
protein citrin.
TREATMENT:
High-caloric,
Protein-restrictive diet
Arginine supplementation
Sodium benzoate
Sodium phenylacetate
Argininosuccinic Aciduria (ASA)
Is inherited in an autosomal recessive
pattern that also belongs to a class of
genetic diseases, the urea cycle disorders.
Lack the enzyme arginosuccinic lyase,
w/c prevents the conversion of
arginosuccinic acid to arginine.
SYMPTOMS:
Lethargy
Unwillingness to eat
Argininosuccinic Aciduria (ASA)

TREATMENT:
High-calorie.
Protein-restrictive diet; Arginine
supplementation.
Administration of Sodium benzoate and
Sodium phenylacetate.
Cystinuria
Inherited autosomal recessive defect.
Caused by a defect in the amino acid transport
system rather than a metabolic enzyme deficiency.
Characterized by inadequate reabsorption of cystine
during the filtering process in the kidneys, resulting
in an excessive concentration of this amino acid.
Cystine precipitates out of the urine and forms
stones in the kidneys, ureters, or bladder.
SYMPTOMS:
Hematuria
Pain in the side due to kidney pain
UTI
Cystinuria
TREATMENT:
Prevent formation of cystine stones by increasing
volume of urine (high fluid intake 4L/day).
Penicillamine to form solule complex w/ cysteine.
Percutaneous nephrolithotripsy(PNL) to remove
the kidney stone.
TESTS:
Testing urine w/ cyanide nitroprusside (produces a
red-purple color on reaction w/ sulhydryl groups).
Ion exchange chromatography: quantitative
analysis of amino acids in plasma or urine.
PROTEINS
 Proteins
Importance:
All biochemical reactions are catalyzed by enzymes, which
contain proteins.
The structure of cells and the extracellular matrix that
surrounds all cells is largely made of protein group
collagens. Collagens are the most abundant protein in the
human body.
The transport materials in body fluids depends on proteins
such as transferrin, receptors for hormones are
transmembrane proteins, and transcription factors,
needed to initiate the transcription of a gene, are proteins.
Proteins make up antibodies, which are a major
component of immune system.
 Molecular Size
Macromolecules
Are polymers of one or more
unbranched chains of amino acids.
Contains 200-300 amino acids.
Molecular mass of approximately
6000 for insulin.
 Synthesis
They are synthesized in the Liver and
secreted by the hepatocyte into the
circulation.
TRANSCRIPTION double-stranded DNA
unfolds in the nucleus, and one strand
of complementary messenger RNA.
TRANSLATION process of synthesizing
a protein from an mRNA template.
 Catabolism and Nitrogen Balance
Unlike fats and carbohydrates, Nitrogen has no
designated storage depots in the body
Nitrogen Balance
Negative Nitrogen Balance
Positive Nitrogen Balance
The disintegration of CHON occurs in the digestive
tract, kidneys and Liver
Two main routes for converting intracellular proteins
to free amino acids:
a. LYSOSOMAL PATHWAYS degrades extracellular
and some intracellular proteins.
b. CYSTOLIC PATHWAYS important in degrading
intracellular proteins.
 Catabolism and Nitrogen Balance
TRANSAMINATIONS central reactions that
remove amino acid nitrogen from the body.
TRANSAMINASES reversible reactions are
catalyzed by a group of intracellular enzymes.
These reactions move nitrogen from free AA into
smaller compounds into ammonia and ketoacid.
Ammonia is converted to urea by the urea cycle
in hepatocytes and excreted in the urine
The ketoacids are oxidized by means of the citric
acid cycle and converted to glucose or fats
 Structure: Four Distinct Levels of
Proteins
PRIMARY STRUCTURE represents the number and types
of amino acids in the specific amino acid sequence.
SECONDARY STRUCTURE regularly repeating structures
stabilized by hydrogen bonds between the amino acids
within the protein.
TERTIARY STRUCTURE overall shape, or confirmation of
the protein molecule.
Confirmation is the fold.
QUATERNARY STRUCTURE shape or structure that results
from the interaction of more than one protein molecule, pr
protein subunits, held together by noncovalent forces.
Hydrogen bonds
Electrostatic interactions
 Nitrogen Content
Carbon
Oxygen
Hydrogen
Nitrogen
Sulfur
The nitrogen content of serum protein is
approximately 16%
Measurement of nitrogen content is used in
one method of evaluating for total protein
Charge and Isoelectric point
Proteins can be positively or negatively
charged.
ISOLECTRIC POINT pH at which the
amino acid or protein has no net
charge.
NET NEGATIVE CHARGE pH > pI
NET POSITIVE CHARGE pH < pI
pI the point at which the number of
positively charges groups in a protein.
 Solubility
Soluble proteins have a charge on
their surfaces.
Protein has its lowest solubility at its
isoelectric point.
Classification by Protein Functions
ENZYMES proteins that catalyze chemical reactions.
HORMONES proteins that are chemical messengers
that control the actions of specific cells or organs.
TRANSPORT PROTEINS proteins that transport
movement of ions, small molecules or
macromolecules, such as hormones, vitamins,
minerals and lipids.
IMMUNOGLOBULINS (antibodies) proteins produced
by B-cells in the bone marrow that mediate the
humoral immune response to identify and neutalize
foreign objects.
 STRUCTURAL PROTEINS fibrous proteins are
the structures of cells and tissues such as
muscle, tendons and bone matrix.
STORAGE PROTEINS proteins that serve as
reserves of metal ions and amino acids that
can be released and used later without harm
occurring to cells during the time of storage.
ENERGY SOURCE plasma proteins serve as a
reserve source of energy for tissues and
muscles.
OSMOTIC FORCE plasma proteins function in
the destruction of water throughout the
compartments of the body.
Classification by Protein Structure

DATABASE (manual) created by manual

inspection and aided by a battery of
automated methods.
DATABASE (automated) based on three-
dimensional structure comparison of
protein structures in the PDB.
SIMPLE PROTEINS contain peptide chains
composed of only amino acids.
CONJUGATED PROTEINS consist of protein
and a nonprotein group.
 PLASMA PROTEINS
The plasma proteins are the most frequently
analyzed of the proteins.
The major measured plasma proteins are
divided into 2 groups albumins and globulins.
There are four major types of globulins, each
with specific properties and actions.
Prealbumin (Transthyretin)
Prealbumin is so named because it
migrates of albumin in the classic
electrophoresis of serum or plasma
proteins.
Prealbumin is transport protein for
thyroxine and triiodothyronine (thyroid
hormone)
It binds w/ retinol binding protein to
form complex w/ retinol/Vit A and is rich
in tryptophan
Decreased in prehepatic damage, acute
phase inflammatory response and tissue
necrosis.
Low prealbumin level is a sensitive
marker of poor nutritional status because
of its short half life (2 days)
Increased in patients receiving steroids,
alcoholism and chronic renal failure
Albumin
Albumin is synthesized in the liver from 585
amino acids at the rate of 9-12/day with no
reserve or storage
Albumin is responsible for nearly 80% of the
colloid osmotic pressure of the intravascular
fluid, which maintains the appropriate fluid
balance in the tissue.
Albumin also buffers pH and is a negative
acute-phase reactant protein.
Also functions to bind various substance in
blood. (4 binding sites)
Glycated albumin is More sensitive indicator
than glycated hemoglobin for DM.
Decreased levels in:
malnutrition/malabsorption, liver disease,
protein losing enteropathy, kidney loss skin
loss hypothyroidism, dilution by excess
mutation and redistribution by dilution.
 Globulins
The globulins group of protein consists of
a1,a2,b and y fractions

a1-Antitrypsin
AAT a glycoprotein mainly synthesized in the
liver, has the most important function the
inhibition of the protease neutrophil elastase.
a-antitrypsin protein is an abnormal form of
the protein that cannot control neutrophil
elastase and can accumulate in the liver and
cause cirrhosis.
Increased: inflammatory reaction, pregnancy
and contraceptive use.
Phenotype: MM (normal) and ZZ (associated w/
liver and lung disease
Assay: immunodiffusion, automated
immunonephelometric assays
a1-Fetoprotein
AFP is synthesized in the developing
embryo and fetus and then by the
parechymal of the liver.
AFP gradually deceases after birth
reaching adult levels by 8-12 months.
No known function in normal adult
It binds w/ stradiol
Functions to protect the fetus from
immunologic attack by the mother.
Increased in spina bifida, neural tube
defects, abdominal wall defects
anecephaly and general fetus
distress and twins
 Low levels of maternal AFP indicates
increase risk of Down syndrome and
trisomy 18
Screening is done 15 to 20 weeks
gestational age
Levels can be affected by maternal
weight, race, and diabetes
Values must be adjusted using Multiple of
median
2.0MoM upper limit and 0.5 MoM as the
lower limit.
Methods: RIA, EIA
Fractions: L1, L2 and L3
AFP L3 is considered as tumor marker
for screening chronic liver disease
1- Acid Glycoprotein
(Orosomucoid)
Acid Glycroprotein a major plasma
glycoprotein, is negatively charged even in
acid solutions.
This proteins is produced by the liver and
is an acute phase reactant.
Increased in stress, inflammation, tissue
damage AMI, trauma, pregnancy cancer,
pneumonia, rheumatoid arthritis and
surgery.

3 methods for determination

(AAG)
Radial immunodiffusion
Immunoturbidity
Antichymotrypsin

It is alpha globulin glycoprotein that is a

member of the serine proteinase
inhibitor(serpin) family
Produce in the liver and is an acute phase
reactant
Deficiency of this protein been associated
with the liver disease.
Mutations identified in the patient with
Parkinson disease and chronic obstruction
pulmonary.
Associated w/ alzheimer disease
Inter- a- trypsin
Inhibitor
ITIs are family of serine
protease inhibitors,
assembled from 2 precursor
proteins a Light chain
(bikunin) and either one or
two are heavy chains and
only 1 is light chain.
Elevations are seen in
inflammation and
Component: Vit D-Binding
Protein)

Gc-globulin, synthesized mainly by

hepatocytes, is the major carrier protein of vit.
D and its metabolites in the circulation and
also transport components such as fatty acid
and endotoxin.
Elevations are seen in pregnancy and in
patients taking estrogen oral contraceptives
Low levels are seen in liver disease and
protein losing syndromes
Important for bone formation and in immune
system.
Gc may act as a co-chemotactic factor in
facilitating chemotaxis of neutrophils and
monocytes inflammation.
Method: Immunonephelometry
Haptoglobin
Hp is an alpha2 glycoprotein is
synthesized by the hepatocytes.
Mature haptoglobin molecule is a
tetramer, consisting of 2 alpha
and 2 beta chains.
Can seen in patient with
ulcerative colitis, acute rheumatic
disease, heart attack and severe
infection and burns
Independent risk factor for CVD
in individuals w/ type 2 DM
It functions to bind free hemoglobin to
prevent the loss of hemoglobin and its
constituent, iron, into the kidney
Used to detect and evaluate hemolytic
anemia and to distinguish it from other
anemias.
Decrease haptoglobin, increase
reticulocyte count and decrease RBC count
is the profile for hemolytic anemia
Used to evaluate the degree of
intravascular hemolysis that has occured in
transfusion reaction or hemolytic disease
of the new born.
Quantitative tests: RIA and
Immunonephelometric methods
Ceruloplasmin
Ceruloplasmin is a copper-containing alpha2-
glycoprotein enzyme that is synthesized in the
liver
It is acute phase reactant.
Can seen in inflammation, severe infection, and
tissue damage and may increased with some
cancers.
Also increased in pregnancy and use of estrogen
and medications (antiepileptic drugs)
90% of copper is bounded to ceruloplasmin and
10% is w/ albumin.
Used to diagnosis Wilsons disease and Menkes
syndrome
Enzymatic method: copper oxidase activity,
immunochemical methods, immunodiffusion and
Macroglobulin (A2M)
Macroglobulin a large protein is
synthesized by the liver and is a
major component 2 band in protein
electrophoresis.
It is a tetramer of 4 identical
subunits that inhibits protease such
as trypsin, thrombin, kallikrein, and
plasmin
Increased in nephrosis, diabetes, liver
disease, pregnancy and contraceptive
medications
Immunodiffusion, Immunonephelometry,
ELISA latex agglutination immunoassay
Transferrin (Siderophilin)
Transferrin is a glycoprotein that is negative
acute-phase protein synthesized primarily by
the liver.
The major function of transferrin are the
transport of iron and prevent of loss of iron
through the kidney.
Its binding of iron prevents iron deposition in
the tissue during temporary increases in
absorbed iron or free iron.
It transports Iron to its storage sites
Transferrin levels are tested for determine
the cause of anemia, to gauge iron
metabolism
Decreased levels: liver disease or
malnutrition , protein losing disorders,
Hemopexin
Hemopexin migrates electrophoretically in
the b-globulin region and is an acute-
phase reactant.

Lipoproteins
Lipoproteins are complex of proteins and
lipids whose function is to transport
cholesterol, triglycerides, and phospholipids
in the blood.
Microglobulin
Microglobulin (B2M) is the light chain
component of the major
histocompatibility complex (HLA)
This protein is found on the surface of
most nucleated cells and present in high
concentrations
Complement
Its is one of the natural defense mechanisms
that protects the human body from
infections,
This protein are synthesized in the liver as
single polypeptide chains and circulate in the
blood as non-functional precursors.
Complement is increased in inflammatory
states and decreased in malnutrition and
hemolytic anemia.
Fibrinogen
Fibrinogen is one of the largest proteins in
blood plasma, synthesized by the liver, and it
is classified as a glycoprotein because it has
considerable carbohydrate content.
Fibrinogen customarily has been determined
as clottable protein.
C-reactive protein
CRP is synthesized in the liver and one of
the acute-phase proteins to rise in response
to inflammatory.

High-sensitivity C-reactive
protein
*hsCRP is same protein but is named for the
newer, monoclonal antibody-body-based test
methodologies that can detect CRP at level
below.
Immunoglobulins

This are the IGs glycoproteins composed

of 82%-96% protein and 4%-18%
carbohydrate produced by WBC.

5 Classes of Immunoglobulins

IgG
IgA
IgM
IgE
IgD
IgG
is the most abundant class of antibodies found in
blood plasma and lymph.
Act on bacteria, fungi, viruses and foreign particles.
IgA
Is the main immunoglobulin that found in the mucous
secretions, including tears, saliva, colostrums vaginal
fluid.
IgM
First antibody that appears in response to antigenic
stimulation. IgM is present on b cells.
IgD
Present on the surface of most but not all, B cells
early in their development, but little IgD is ever
released into circulation.
It help to regulate B cell function.
IgE
Produced by B cells and plasma
Associated with allergic and anaphylactic reaction.
Myoglobin
It is located in the muscle tissue and is responsible for its
brown color.
Its function is to store and transport oxygen in the skeletal
muscles.
It is a relatively small protein made up of a single
polypeptide chain that contains 153 amino acid residues .
It contains a heme group (which is a prosthetic group
consisting of a protoporphyrin organic ring and a central
iron atom).
It is the heme group which is responsible for the oxygen
binding capacity of Myoglobin.
Myoglobin is very similar to Hemoglobin in both its
function and structure ( since both are capable of
oxygenation and deoxygenation).
Myoglobin is an extremely compact molecule(in its interior
there is room for only 4 H2O molecules)
 Myoglobin
A cardiac biomarker, used in conjunction w/
troponin to help diagnose or rule out a heart attack
In AMI, the increase is is seen w/in 2-3 hrs of onset
and reaches peak concentration in 8-12 hours.
Myoglobin is freely filtered by the kidneys allowing
levels to return to normal in 18-30 hours after AMI
Methodology: Latex agglutination, ELISA,
immunonephelometry. ECLIA and
fluoroimmunoassays and SPOT test.
 Causes of Myoglobin Elevation
AMI
Angina w/o infarction
Rhabdomyolysis
Muscle trauma
Renal failure
Myopathies
Vigorous exercise
Open heart surgery
Seizures
Electric shock
Arterial thrombosis
Certain tumors
Brain Natriuretic Peptide and N-
Terminal-Brain Natriuretic
Peptide
BNP is a good marker for congestive
heart failure
NT affects blood fluid homeostasis
Methods: Immunoradiometric assay,
microparticle enzyme immunoassay.
 Fibronectin
It is synthesized in the liver
hepatocytes, endothealial cells,
macrophages and fibroblast.
It is used as nutritional marker
Fetal fibronectin is used to predict
the short term risk of premature
delivery.
 Adinopectin
Indicator of obesity
Inverse correlation b/n BMI and
adinopectin values
Lower levels correlate w/ increased
risk of heart disease. Type 2 DM and
metabolic syndrome
 Trace Protein
An accurate marker of CSF leakage
Marker for impaired renal function
Also a marker for perilymphatic
fistulas
Cross-Linked C-Telopeptides
CTX is a biomarker for bone
resorption and it can be detected in
serum and urine.
CTX test is most useful for
monitoring the response for
antiresorptive therapy.
ECLIA technology is used to measure
CTX
 Cystatin C
New marker to assess glomerular filtration
rate
Increased levels in cases of decreased GFR
Not affected by physiologic variations
Also a marker for cardiovascular disease
and heart failure in the elderly
Immunoturbidimetry and
immunonephelometric methods are used to
measure Cystatin C levels
 Amyloids
An insoluble fibrous protein aggregates
It characteristically stains w/ Congo red.
Amyloidosis refers to a variety of
conditions in w/c amyloid proteins are
deposited in organs/tissues causing
localized or widespread organ failure
Amyloid 42 and Tau proteins are used
to assess and diagnose Alzheimer
disease from other forms of demenia
HYPOPROTEINEMIA
A total protein level less than reference
interval. Occurs in any condition where
negative nitrogen balance exist.
One cause is of level of protein in the plasma is
excessive loss.
Plasma protein can be lost by excretion in the
urine in renal disease (nephrotic syndrome).
Another circumstances producing
hypoproteinemia is decreased intake
-deficiency of protein in diet (malnutrition).
-intestinal malabsorption due to structural
damage(sprue)
 Without adequate intake of proteins,
there is a deficiency of certain essential
amino acids and protein synthesis is
impaired.
a decreased in serum protein due to
decreased synthesis is also seen in liver
disease (site of all non-immune protein
synthesis).
hypoproteinemia may also result form
accelerated catabolism of proteins, such
as burns, trauma, or injuries.
 TOTAL PROTEIN ABNORMALITIES

Total protein test is a rough measure

of all proteins in the plasma
It can reflect nutritional status,
kidney disease, liver disease and
many other conditions
If total protein is abnormal, further
test must be performed to identify
the abnormal fraction for specific
diagnosi
 Hypoproteinemia
A total protein level less than the
reference interval
Occurs in condition where there is a
negative nitrogen balance.
Causes: renal disease, GIT
inflammation, open wounds, internal
bleeding, malnutrition, liver disease
 Hyperproteinemia
It is an increase in total plasma proteins.
One condition in which an elevation of all
the protein fraction is observed is
dehydration.
Dehydration results from variety of
condition, vomiting, diarrhea, excessive
sweating, diabetic acidosis and
hypoaldosteronism
In addition is due to excessive production
primarily of y-globulins.
 Some disorders are characterized by the
appearance of a monoclonal protein or
paraprotein in the serum and often in the urine as
well.
the paraprotein in this case is usually IgG and
IgA. IgE and IgD occurs very rarely.
Not all paraprotein is associated with multiple
myeloma.
IgM is paraprotein is often found in patients with
Walden-stroms macroglobulinemia, a more
benign condition.
Other disorder associated in hyperprotenemia
include chronic inflammatory states, collagen
vascular disorder, and other neoplasm
METHODS
OF
ANALYSIS
 TOTAL NITROGEN
A total nitrogen determination measures all
chemically bound nitrogen in the sample.
The method can be applied to various
biologic samples, including plasma and
urine.
In plasma, both the total protein and
nonprotein nitrogenous compounds, such as
urea and creatinine, are measured.
The analysis of total nitrogen level is useful
in assessing nitrogen balance.
 TOTAL PROTEINS
The specimen often used to
determine the total protein is serum
rather than plasma. A fasting
specimen is not needed.
Interferences in some of the
methods occur in the presence of
lipemia, hemolysis falsely elevates
the total protein result because of
the release of RBC proteins into the
serum.
 Total Protein Methods
Kjeldahl
Refractometry
Biuret
Dye Binding
 KJELDAHL
This method is not used in the clinical
laboratory because it is time
consuming and too tedious for routine
use. In this method, an average of 16
% nitrogen mass in protein is assumed
to calculate the protein concentration.
The actual nitrogen content of serum
proteins varies from 15.1% to 16.8%.
Reference method
 REFRACTOMETRY
is the method of measuring
substances'refractive index(one of their
fundamentalphysical properties) in order to,
for example, assess their composition or
purity.
Arefractometer is the instrument used to
measure refractive index ("RI"). Although
refractometers are best known for measuring
liquids, they are also used to measure gases
and solids; such as glass and gemstones.
 BIURET
is achemical testused for detecting the presence
ofpeptide bonds.
In the presence of peptides, acopper(II)ionformsviolet-
colored coordination complexesin analkaline solution.
The Biuret reaction can be used to assess
theconcentrationof proteins because peptide bonds
occur with the same frequency per amino acid in the
peptide.
Despite its name, the reagent does not in fact
containbiuret((H2N-CO-)2NH).
The test is so named because it also gives a positive
reaction to the peptide-like bonds in the biuret molecule.
 DYE BINDING
are based on the ability of most
proteins in serum to bind dyes,
although the affinity with which they
bind may vary.
Simple and fast but non specific
 Methods for Measuring Protein
Fractions
Salt fractionation
Dye Binding Methods for albumin
Electrophoresis
Total globulin methods
Electrophoresis
 FRACTIONATION, IDENTIFICATION,
AND QUANTITATON OF SPECIFIC
PROTEINS
In the assay of total serum proteins, useful diagnostic information
can be obtained by determining the albumin fraction and the
globulins.
A reversal or significant change in the ratio of albumin and total
globulin was first noticed in diseases of the kidney and liver.
To determine the albumin/globulin (A/G) ratio, it is common to
determine total protein and albumin.
an abnormality is found in the total protein or albumin, an
electrophoretic analysis is usually performed.
Serum proteins are separable into five or more fractions by the
customary electrophoretic methods.
If an abnormality is seen on the electrophoretic pattern, an analysis
of the individual proteins within the area of abnormality is made.
 SALT FRACTIONATION
Fractionation of proteins has been accomplished
by various procedures using precipitation.
Globulins can be separated from albumin by
salting out using sodium salts.
The albumin that remains in solution in the
supernatant can then be measured by any of the
routine total protein methods.
Salting out is not used to separate the albumin
fraction in most laboratories today because
direct methods are available that react
specifically with albumin in a mixture of proteins.
 ELECTROPHORESIS
is separates proteins on the basis of their electric charge
densities, Protein, when placed in an electric current, will
move according to their charge density, which is
determined by the pH of a surrounding buffer.
The speed of the migration largely depends on the degree
of ionization of the protein at the pH of the buffer.
The more the pH of the buffer differs from the pi, the
greater the magnitude of the net charge of that protein
and the faster it will move in the electric field.
In addition to the charge density, the velocity of the
movement also depends on the electric field strength,
size, and shape of the molecule; temperature; and the
characteristics of the buffer (i.e., pH, qualitative
composition, and ionic strength).
SERUM PROTEIN ELECTROPHORESIS
In the standard method for serum protein
electrophoresis (SPE), serum samples are applied
close to the cathode end of a support medium strip
that is saturated with an alkaline buffer (pH, 8.6).
The support strip is connected to two electrodes and
a current is passed through the strip to separate the
proteins. All major serum proteins carry a net
negative charge at pH 8.6 and will migrate toward
the anode.
Using the standard methods, the serum proteins
arrange themselves into five bands: albumin travels
farthest to the anode followed by 1-globulins, 2-
globulins, -globulins, and -globulins, in that order.
SERUM PROTEIN ELECTROPHORESIS

The width of the band of proteins in a fraction

depends on the number of proteins with slightly
different molecular characteristics that are
present in that fraction.
Homogenous protein gives a narrow band. After
separation, the protein fractions are fixed by
immersing the support strip in an acid solution
(e.g., acetic acid) to denature the proteins and
immobilize them on the support medium. The
proteins are then stained. The protein appears
as bands on the support medium.
 HIGH RESOLUTION PROTEIN
ELECTROPHORESIS

Standard SPE separates the protein into

five distinct zones, which comprise many
individual proteins. By modifying the
electrophoretic parameters, these fractions
may be further resolved into as many as
12 zones.
This modification, known as high-resolution
electrophoresis (HRE),is accomplished by
use of a higher voltage coupled with a
cooling system in the electrophoretic
apparatus and a more concentrated buffer.
 HIGH RESOLUTION PROTEIN
ELECTROPHORESIS
Each zone is compared with the same zone on a
reference pattern for color density, appearance,
migration rates, and appearance of abnormal bands
or regions of density HRE is useful in detecting small
monoclonal bands and differentiating unusual bands
or prominent increases of normal bands that can
masquerade as a monoclonal gammopathy.
For example, in patients with nephrotic syndrome,
an increased 2-macroglobulin band in the 2
region may be confused with a migrating
monoclonal protein such as an IgA monoclonal
protein gammopathy.
 CAPILLARY ELECTROPHORESIS
is a collection of techniques in which the
separation of molecules takes place in small-bore
fused silica capillaries. The capillaries are
typically 30-50 centimeters long, with an internal
diameter between 25 and 100 .m. In capillary
zone electrophoresis, the capillaries are filled with
a conducting solution, usually an aqueous buffer.
One end of the capillary is grounded (detection
end) and the other-the sample injection end-is
connected to a high voltage power supply. When
a positive voltage is applied, the positively
charged buffer molecules flow to the detection
end, which is grounded and therefore negative
relative to the injection end. The net flow of
buffer is called electroosmotic flow (EOF).
 CAPILLARY
ELECTROPHORESIS
This is referred to as electrophoretic mobility. EOF is
usually stronger than electrophoretic mobility and all
ions (positively charged, neutral, and negatively
charged) will migrate to the detector end but with
different net mobilities based on size and charge
differences. The separated molecules are detected by
their absorbance as they pass through a small window
near the detection end of the capillary. Use of the
small-bore capillaries allows heat to be effectively
dissipated, which means that higher operating voltages
can be used and, therefore, analysis times are faster.
Additionally, the sample size required is small
(nanoliters).
 IMMUNOCHEMICAL METHODS

Specific protein may be identified by

immunochemical assays in which the
reaction of the protein (antigen) and its
antibody is measured.
Methods using various modifications of
this principle include radial
immunodifixation,immunelectrophoresis,i
mmunofixationelectrophoresis,
electroimmunodiffussion,immunoturbidim
etry, and immunonephelometry
PROTEINS IN OTHER BODY
FLUIDS
 Body fluids now being studied for
their include peritoneal, pleural,
seminal and vaginal fluids and tears.
It includes the two fluids( urine and
CSF)
URINARY PROTEINS
Proteins found in the urine are from
the blood;
Originate from the kidney and
urinary tract and from extraneous
sources such as vagina and prostate.
Plasma proteins
Passed through the renal glomerulus
and have not been reabsorbed by the
renal tubules
Qualitative test for
proteinuria
Performed by a reagent strip.
-are based on the change of indicator dye
in the presence of protein.
In an acid pH, the indicator that is yellow in
the absence of protein progresses through
various shades of green and finally blue as
the concentration of protein increases.
6mg/dL or greater produces a color
change.
Quantitative assays
Are performed on urine specimens of 12 or
24 hours.
24 hr timing allows for circadian rhythmic
changes in excretion at certain times of
day.
Collected from that time for the next 24hrs.
Results are reported generally in terms of
weight of protein per 24hrs by calculating
the amount of protein present in the total
volume of urine collected during that time.
Precipitation methods
Measurement of turbidity when
urinary proteins are mixed with an
anionic acid such as sulfosalicylic
acid, TCA, or benzethenium chloride.
Dye- binding methods
Coomassie blue and Ponceau S, used to
determined the quantitative total protein
content of urine.
Too insensitive for urine micralbumin(MAU)
testing, making immunochemical assays
the most widely used MAU methods.
ELISA,immunoturbidimetry
immunofluorescence, RIA, and zone
immunoelectrophoresis.(10-8 table)
 Reference values or intervals for
urinary proteins, ranging from 100 to
250 mg every 24 hours.
Cerebrospinal fluid proteins
Formed in the choroid plexus of the
ventricles of the brain by ultrafiltration of
the blood plasma
Reference interval for patients between 10
to 40 years of age is 15-45 mg/dL of CSF
protein.
Increased total CSF proteins may be found
in the conditions include bacterial, viral,
and fungal meningitis, traumatic tap,
multiple sclerosis; obstruction; neoplasm;
disk herniation; and cerebral infarction.
 Low CSF protein values are found in
hyperthyroidism and when fluid is
leaking from the CNS.
Procedures for determining CSF
protein are turbidimetric using TCA,
sulfosalicylic acid with sodium sulfate
or benzethonium cholride.