Total Plasma Proteins

Mostafa Sabry Abdullah, B. Pharm. Sci.

Teaching Assistant, College of Pharmacy, Al-Azhar University, Assiut, Egypt.

Colourimetry

Definition: It is the method that used for identification and determination of substancesthat present in low concentrations or that of difficult isolation by measurement of their absorbance after the reaction with chromogene (coloring reagent) .

Blank: It is the solution that fanned from the chromogene (reagent) and distilled water instead of the substance to be identified. why????

Aim: It used to remove the interference of the reagent colour and the measured colour.

so color become only that produced from the reaction between the substance to be identified and the chromogene.

Principle: The absorbance of the substance solution (A) is directly proportional to

I) the light path length (b) that pass through the solution. And,

2) The concentration of the substance (a)

A = Kba Where K is a constant.

When there are more than one concentration will be measured by the same cuvette (i.e. the same path length)

A1/ K1C1b1=A2/K2C2b2

K 1 = K2 because it is a constant depend on the wave length that the same

b1=b2 because we will use the same cuvette i.e. the same bath length.

So, the equation will be:

A1/C1 = A2/C2.

Standard Solution

Au / Cu=As /Cs

Cu =Au x Cs/As

Where, Au = Absorbance of unknown. Cu = Concentration of unknown.

As = Absorbance of stander. Au = Concentration of stander.

Estimation of Total Plasma Proteins

Introduction:

Plasma is the liquid portion of blood after addition of anti coagulant. It is composed of:-

1. Water about 90%

2. Solids about 10% (dissolved substances) including:-

a) organic substances: Proteins + Lipids "Lipoproteins", carbohydrates, NPN , e.g. urea & NH3, hormones and enzymes.

b) Inorganic substances: - e.g. minerals as Ca2+ & Na~ & K+ and cl.

Normal value of total plasma proteins:-

total plasma proteins concentration about 6.3 -8.3g/dl.

Site of synthesis:-

• All types of plasma proteins are synthesized in the liver,

except gamma-globulins that are synthesized in plasma cells.

• Liver synthesizes about 12gm albumin per day.

• Nearly all types of plasma proteins are synthesized in the pre-protein form then it is subjected to modifications such as "phosphorylation & glycosylation to become mature protein .

Types of plasma protein

Plasma is reported to contain over than 100 individual proteins but mainly classified ;into:-

I - Albumins.

II- Globulins.

III - Fibrinogen

I-Albumins

a- Pre- albumin:-

It is present in very low concentration about 25mg/dl.

It helps in the transport of T3, T4 and Vitamin A.

b- Albumin- It is the major type of plasma protein in concentration of 3.4-4.7g/dl.

It is synthesized in the liver (about 12 g/day),

It has low Mwt about 68 KD acting about 60% of plasma proteins.

40% of albumin is present in the plasma and the remaining 60% is present in extracellur space.

Mature human albumin consists of one polypeptide chain of 585 amino acid with 17 disulphide bonds .

Albumin has a long plasma half life (t0.5) about 20 days , So that its plasma level is normal in" acute liver diseases” but give clinical significance in chronic liver disease

Due to low Mwt and high concentration albumin is responsible for about 75- 80 % of plasma osmotic (oncotic) pressure .

II - Globulins:

Its normal plasma concentration is about 1.5 - 3 g/dl and of high Mwt about 90 - 1000 kDa.

It is classified into:-

A - Alpha-I globulins:-

e.g.:- prothrombin and αl- antitrypsin.

B - Alpha-II globulinss-

e.g.:- ceruloplasmin (Cu2+ transporter) .

C - Beta globulins:-

e.g.:- transferrin (Fe2+ transporter).

D - Gamma globulins

Including all antibodies, which are IgG, IgM, IgD, IgA

and IgE.

1- IgG:

The major type (about 80%) of serum antibodies.

It is responsible for second immune response.

It is the only type which can cross the placental barrier.

2- IgA

Present in the body secretions as milk and saliva.

3- IgM:-

Responsible for primary immune response.

4- IgE:-

Responsible for immunity against parasites.

5-IgD

Present in B-lymphocyte surface acting

as specific antigen receptors.

Function of plasma protein: -

1- Maintenance of plasma osmotic pressure (mainly albumin).

In arterioles:

In arterioles ,the hydrostatic pressure is about 37mmHg forcing plasma fluids to the interstiacial spaces. The osmotic and tissue pressure are about 25 mmHg and 1 mmHg respectively. Both of them force the plasma fluids to the blood vessels. So, in the arterioles the net force is about + 11 mmHg forcing plasma fluids to extra cellular spaces.

In venules:

In venules ,the hydrostatic pressure is about 17 mmHg, the osmotic pressure and the tissue pressure are the same as in arterioles, so the net force is about -9 mmHg forcing plasma fluids to venules. So that if the osmotic pressure decreased (due to decreased plasma proteins), plasma fluids will accumulate in extracellular spaces leading to edema.

2- Transport functions-

Plasma proteins act as carrier for many components in blood such as lipids (lipoproteins), hormones (T3 & T4), minerals (ceruloplasmin & Transferrin)

3- Defense mechanism: - such as gamma-globulins.

4- Coagulation: - such as fibrinogen.

5- Buffering action: - Due to zwittet ion of amino acid.

6- Nutritive function: - amino acids resulting from metabolized plasma proteins inter in;-

Amine acid pool.

Synthesis of new proteins.

Synthesis of other nitrogenous compounds.

All of the above is required for energy production.

Clinical Significance:-

Variations in plasma proteins concentration occur due to one or more of the following changes:-

1- Change in the rate of synthesis.

2- Chang in the rate of excretion.

3- Change in the volume of distribution.

I-hypo-albuminaemia: or hypo-proteinaemia:-Due to:-

a- Decrease in the rate of protein synthesis: -

as in liver disease, and malabsorption (diarrhea), malnutrition

b- Increase in the rate of protein excretion (inc. protein loss)

as in: kidney disease e.g. nephrotic syndrome, sever burns, hemorrhage, fever, cancer and preeclampsia.

c- Increase the volume of distribution

as in: over hydration, hypoxia and septicemia.

II-hyper-albuminaemia. Mainly due to decreased volume of distribution as in: dehydration.

III- Hyper -gamma-globulinaemia:-Due to increase the rate of synthesis of gamma-globulins as in cancer.

V-Hypo -gamma-globulinaemia:-Either congenital or acquired leading to immune deficiency and recurrent

infections.

Albumin / Globulin ratio (A/G ratio)

Normally A/G ratio it is about 1.6 - 2.5.

Clinical Significance of A/G ratio:

1- Decreased A/G ratio: as in

a- Chronic liver diseases due to decrease in the rate of albumin synthesis.

b- Kidney diseases due to loss of albumin in urine due to its low Mwt and high concentration.

2- Increased A/G ratio: as in

a- Hypo gamma-globulinaemia as in immune deficiency.

b- After IV albumin infusion.

Principle of assay:-Colourimetric determination of total plasma proteins based on the principle of

biuret reaction (cupper salt in alkaline medium).

Cu +OH +PROTEIN-----------)

Peptide bonds of protein react with CU2+ in alkaline medium to form a colored Complex measured at 540 nm,( violet complex).

Biuret reagent formed of:-1-CUSO4. 5 H2O: as chromogene (Complexing agent).

2- NaOH: For alkaline medium

3-Sodium potassium tartarate: To help complex formation and maintain its stability

4- Potassium iodide: as antioxidant.

N.B:- Complex formation needs at least two peptide bonds. So, individual amino acids and dipeptides give -ve test and

this test is used for detection of complete protein hydrolysis.

PROCEDURE

Incubate at room temperature for 30 minutes then measure at 540 nm