This document provides information on the colourimetric method for determining total plasma proteins. It begins with definitions of key terms like colourimetry and blank. It then discusses the principle that absorbance is directly proportional to concentration and path length. The document provides details on the types and functions of plasma proteins like albumins and globulins. It discusses the biuret reaction used to colorimetrically determine total plasma proteins and provides the normal ranges and clinical significance of variations in plasma protein levels.

1. Total Plasma
Proteins
Mostafa Sabry Abdullah, B. Pharm. Sci.
Teaching Assistant, College of Pharmacy, Al-
Azhar University, Assiut, Egypt.

2. Colourimetry
Definition: It is the method that used for
identification and determination of substances
that 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.

3. 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.

4. 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.

5. 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

6. 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 .

7. 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.

8. 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.

9. 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.

10. 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.

11. 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.

12. 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.

13. 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.

14. 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.

15. PROCEDURE
Incubate at room temperature for 30 minutes then measure at 540 nm