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