Clinical diagnostic biochemistry - 4
Dr. Maha Al- Sedik
What are Proteins?
Proteins are made of amino acids (CHON)
They contain carbon, hydrogen, oxygen (like carbohydrates
and fats).
In addition, also contain nitrogen
Each amino acid has:
• Acid group (-COOH)
• Amine group (-NH2)
• Side chain (unique)
Amino Acid
Amin
o ac
ids
Essential a.a
Non essential a.a
Amino Acids
Proteins are made up of 20 AAs
o 9 are essential ( can not be formed by the body )
o 11 are nonessential :
Can be made in the body from other amino acids
Proteins are a link of Amino acid with peptide bonds:
Dipeptides: have two amino acids bonded together.
Tripeptides: have three amino acids bonded together.
Polypeptides: have more than two amino acids bonded together.
Amino acid sequence: determines shape and function and what protein is made.
INSULIN
Protein Denaturing
Unfolding of protein shape by heat, acids, bases or salts that
changes its ability to function.
After a certain point, denaturation cannot be reversed.
Stomach acid untangles proteins to aid in digestion.
Denaturation
Protein Digestion and Absorption
Mouth
Crushed and moisten
No digestion of protein in the mouth
Stomach
protein is denatured
Enzymes start to break the peptide bonds
HCl activates pepsinogen to pepsin
Pepsin cleaves proteins into smaller polypeptides.
small intestine:
In the small intestine, polypeptides broken down into tri-,
dipeptides and amino acids by pancreatic protease
(trypsin).
Cells in the small intestine absorb amino acids
to be used by the body.
Protein and the kidney:
Amino acids in blood are filtered through the glomerular
membranes, but normally are reabsorbed in the renal tubules
by transport systems.
When the transport mechanisms become saturated or are
defective, amino acids spill into urine, resulting in a condition
known as aminoaciduria.
Aminoaciduria
Two types of aminoaciduria have been identified:
1. Overflow aminoaciduria: occurs when the plasma concentration
of one or more amino acids exceeds the renal threshold (capacity
for reabsorption).
2. Renal aminoaciduria: occurs when plasma concentrations are
normal, but the renal tubular reabsorption system has a congenital
or acquired defect.
Aminoaciduria
Causes of aminoaciduria:
Primary aminoaciduria ( overflow ):
Due to:
Inherited enzyme defect e.g. defect in the pathway of amino
acid metabolism e.g. ( cysteinuria ).
DNA abnormality.
Secondary amino aciduria ( Renal ):
Due to:
generalized renal tubular dysfunction.
Analysis of amino acids
Many procedures are available to measure amino acids in biological
samples.
To diagnose pathological disorders, the following three groups of
tests for amino acid analysis are important:
1) Screening tests, including thin-layer chromatography (TLC) and
photometric screening test for urine proteins.
2) Quantitative tests to monitor treatment or confirm an initial
diagnosis.
3) Specific tests that identify an unknown amino acid or metabolite.
Anal
ysis
of a
min
oaci
dsScreening tests
Quantitative tests
Specific tests
1- TLC: TLC analysis of amino acids is conducted in three stages:
(1) preparation of the sample,
(2) chromatographic separation,
(3) identification of the separated amino acids.
2- Photometric Screening Tests for Urine
Screening tests
Amino acids are measured quantitatively in body fluids with a variety
of techniques, including:
1. Electrophoresis .
2. Gas chromatography (GC).
3. High-performance liquid chromatography (HPLC).
4. Ion-exchange chromatography.
Quantitative tests
In addition to the general analytical techniques discussed
previously, a variety of simple tests exist that are specific for
individual amino acids.
These tests are used in the diagnosis of specific disorders.
Tests for Specific Amino Acids
Plasma proteins
Functions of plasma proteins:(1) Enzymes are proteins that catalyze biochemical reactions essential
to metabolism.
(2) Hormones regulate body functions.
(3) Immunity: Antibodies and complement system protect against
infection.
(4) Osmotic pressure: Plasma proteins maintain the osmotic pressure
of plasma.
(5) They transport hormones, vitamins, metals, and drugs.
(6) Hemostasis: coagulation system.
Causes of increased plasma protein levels:1. Dehydration: decreased volume of distribution due to relative
water deficiency.
2. Hemoconcentration: due to stasis of blood during venipuncture
and prolonged application of tourniquet.
3. Paraproteinaemia: increase in abnormal protein synthesis.
4. Hypergammaglobulinemia: increase in the antibody concentration.
Causes of decreased plasma protein levels:1. Overhydration: increased volume of distribution ( IV fluid
administration ).
2. Artifactual: blood samples from drip arm.
3. Decrease in protein synthesis: sever liver disease or
immunodiffeciency.
4. Excessive protein loss: in urine e.g. nephrotic syndrom or from skin
e.g. sever burns.
o Normal total plasma protein concentration: 6 – 8.5 gm / dl.
o Electrophoresis separates serum Proteins into 5 distinct zones or
bands at barbital buffer at pH 8.6 :
Prealbumin.
Albumin.
Alpha globulin ( Alpha 1 and Alpha 2 ).
Beta globulin ( Beta 1 and Beta 2 ).
Gamma globulin: IgG, IgA, IgM, IgD, IgE and C-reactive protein.
The width of each band is dependent upon the number
of proteins that are present in that fraction.
Q – WHAT is the difference
between serum proteins and
plasma proteins in electrophoresis ?
Serum is a clear yellowish fluid that remains from blood plasma after
clotting factors (fibrinogen, prothrombin ect.) that have been used in
the formation of a clot.
Plasma is a clear yellowish fluid that still contains all of the clotting
factors and have not been solidified into clot.
If plasma is used in electrophoresis , there will be an extra band
( fibrinogen ) between beta and gama bands.
Fibrinogen
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