Amino acids are the building blocks of

protein, biomolecules containing at least

one amino group and one carboxyl
group.
There are 20 different amino acids that
are used as building block of protein as
different R chains.
Inherited abnormalities in amino acid
metabolism result in a variety of conditions
most of which are associated with mental
retardation.
Aminoacidopathies are rare inherited
disorder of amino acid metabolism. The
abnormalities exist in either the activity of a
specific enzyme in the metabolic pathway or
in the membrane transport system of amino
acids.
More than 100 diseases have been identified
that result from inborn errors of amino acids
matabolism.
Blood samples for amino acids analysis
should be drawn after at least 6-8 hours fast
to avoid the effect of absorbed amino acids
originating from dietary protein.
The sample is collected in heparin, and the
plasma is promptly removed from the cells,
taking care not to aspirate the layer of
platelets and leukocytes. Deproteinization
should be carried out within 30 minutes of
samples collection.
Thin Layer Chromatography (TLC) with two
dimension is used as preliminary
screening. Two kind of solvent i.e.
Butanol-acetic acid-water and ethanol-
ammonia-water.
The chromatogram is visualized by
staining with ninhydrin which gives blue
colour.
The other choice is to use HPLC reversed
phased system
PKU is inherited disease occurs in
approximately 1 in 10000 birth. Although there
are several variant of this disease, the
biochemical defect in the classic form of PKU
is an almost total absence of activity of the
enzyme Phenylalanine hydroxylase which
catalyzes the conversion of Phenylalanine to
tyrosine.
Phenylalanine accumulates and is metabolized by
an alternate degradative pathway become
phenylpyruvic acid and metabolites. In infant
and children with this inherited defect, retarded
mental development occurs as a result of the
toxic effects on the brain of phenylpyruvic acid
phenylpyruvate and metabolites. Brain damages
can be avoided if the diseases is detected at
birth and the infant is maintained on a diet
containing very low levels of phenyilalanine.
The normal limits of serum phenylalanine is
1,2mg/dL-3,4mg/dL (70 μmol/L-200 μmol/L)
The procedure is based on the fluorescence of a
complex formed of phenylalanine-ninhydrin-
copper in the presence of a dipeptide). The test
required pretreatment of the filter paper
specimen with trichloroecetic acid (TCA). The
extract is then reacted in a micro titer plate
with a mixture of ninhydrin, succinate and
leucynalanine in the presence copper tartrate.
The fluorescence of the complex is measured
using excitation or emission wavelength of 360
nm and 530 nm.
The HPLC methods to analyze phenylalanine in
the serum can also be used
The function protein in the body :
 Tissue nutrition
 Maintenance of water distribution between cells and tissues,
intestitial compartment and the vascular system of the body
 Participation as buffers to maintain pH
 Transportation of metabolic substances
 Part of defense system/antibodies
 Hormones and receptors
 Connective tissue structure
 Biocatalyst/ enzyme
 Participation in the hemostatis and coagulation of blood
Low level of total protein may be due to
excessive loss, decreased synthesis or
accelerated catabolism called by
Hypoproteinemia. Plasma protein can be lost
by excretion in the urine in renal disease,
inflammation of the digestive system, loss of
blood in open wounds, internal bleeding or
extensive burn. Another case of
Hypoproteinemia caused by malnutrition or
intestinal malabsorbtion.
 Elevated levels of total protein may be
associated with dehydration or excessive
production called by Hyperproteinemia. It
is not common disease. When excess of
water is lost from the vascular system,
protein remain within blood vessels.
Dehydration result from a variety
condition including vomiting, diarrhea,
excessive sweating, diabetic acidosis, and
hypoaldosteronism.
Anaysis of total protein
 Kjeldahl : Digestion of protein and mesure
the nitrogen content
 Refractometry : Measurement of refractive
index due to solutes in serum
 Biuret : Formation of violet colored
chelate betwaae Cu ion and peptide bond
in alkaline medium.Used as routine
method
 Dye binding: Protein binds to dye and
cause a spectral shift in the absorbance
maximum of the dye. Bromfenol blue,
Ponceau S, amino black have been used to
stain protein after electrophoresis.
 Serum protein also have been estimated
by UV Spec.
 Salt precipitation : globulin are precipitation in
high salt concentration, albumin in supernatant is
quantitated by biuret reaction
 Dye Binding : Use dye binding such as metil
orange, HABA (2,4, hydroxyazobenzene-benzoic
acid), BCG (Bromcresol green), BCP (Bromcresol
purple)
 Electrophoresis and stained by dye.

Globulin can be calculated by subtracting albumin

from total protein
Enzymes are specific biologic proteins that catalyze
biochemical reactions without altering the
equilibrium point of reaction or being consumed or
undergoing changes in composition. Enzyme found in
all body tissue including serum
Many enzymes are clinically important. Quantitating
serum levels of certain enzymes or isoenzymes can
assist in the diagnosis and prognosis of hepatic
disorder, skeletal muscle disorder, bone disorder,
cardiac disorder, malignancy or acute pancreatitis.
 Measurement of enzymes activity is prefer
than the concentration. A given method
might photometrically measure an
increase in product of concentration,
decrease in substrate concentration or
decrease in coenzyme conc.
 Acid phosphatase : prostatic carcinoma
 Alanine aminotransferase : hepatic disorder
 Alkaline phosphatase : hepatic disorder and bone
disorder
 Amylase : acute pancreatitis
 Aspartate aminotransferase : Myocardial
infarction, hepatic disorder, skeletal muscle
disorder
 Creatine kinase : Myocardial infarction, skeletal
muscle disorder
 GGT : hepatic disorder
 G-6 PD) : drug induced hemolytic anemia