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
Essential Function

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 nucleic

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

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

e and it helps to regulate blood glucose levels.

second most common amino acid found in protein beside glycine. For
Leucine 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 in
Lysine
children and to maintain a proper nitrogen balance in adults, and
calcium absorption
Essential Function

Methionin initiate translation of messenger RNA, fat metabolism helps to

e 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

Threonine formation of protein and collagen, maintain proper protein 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, soothes
Tryptoph
anxiety, and reduces depression, treatment of migraine headaches,
an
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

Alanine strengthens the immune system through production of 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 required
Glutamine
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 stereoisomers.

Glycine

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, thyroid,

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.
High levels are seen in dehydration or after
excessive albumin infusion.
 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
Nephelometry
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 carcinogenesis
Gc- globulin (Group-Specific
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
nephelometry.
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, infection and malignancy.
Increase in iron deficiency anemia
methods>: Immmunodiffusion, immunonephelometry
and measurement of TIBC
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
fundamental physical properties) in order
to, for example, assess their composition
or purity.
A refractometer 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 a chemical test used for detecting the presence
of peptide bonds.
In the presence of peptides, a copper(II) ion forms violet-
colored coordination complexes in an alkaline solution.
The Biuret reaction can be used to assess
the concentration of proteins because peptide bonds
occur with the same frequency per amino acid in the
peptide.
Despite its name, the reagent does not in fact
contain biuret((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.