Transcript Estimation of serum Total Protein （biuret method）

BIO-3
Estimation of Serum Total Protein
【PURPOSE】
1. To understand the principle of biuret
method.
2. To understand the principle of
bromcresol green method.
3. To understand the clinic significance of
serum total protein, albumin and A/G.
• Question:
1. Concentration of serum total protein
blood
plasma
serum
2. Albumin/globulin ( A / G )
1. concentration of albumin
2. total protein – albumin = globulin
3. A/G
Centrifuged Blood Sample
Add anticoagulants
(heparin, potassium
oxalate)
Separation of Components
Plasma = Less Dense
Platelets / WBCs
RBCs
More Dense
Plasma vs. serum
Serum is the liquid
part of blood AFTER
coagulation,
therefore devoid of
clotting factors as
fibrinogen.
•Plasma is the
liquid, cell-free
part of blood, that
has been treated
with anticoagulants.
Anticoagulated
Clotted
•serum= plasma - fibrinogen
Estimation of serum Total Protein
（biuret method）
Plasma proteins
【Clinic
significant】
• Total protain normal range：60-80g/L
Decreased in different clinical conditions
associated with nephrotic syndromes, malnutrition,
Kwashiorkor syndrome, cirrhosis of liver and other
liver disorders.
Increased total protein values may be found in
multiple myeloma, and conditions associated with high
globulin concentration.
Methods for estimating protain:
Kjeldahl method
Absorbance Assay (ultraviolet light)
Bradford method
(Coomassie Brilliant Blue )
Lowry’s method
Biuret method
Biuret reaction
(urea)
(biuret)
(violet colored complex)
• When biuret is treated with dilute copper sulfate in
alkaline medium, a purple color is obtained.
【PRINCIPLE】 Biuret reaction
• Many peptide bonds(-CO-NH-)conjoint each
other in protein molecule ,can react with Cu2+
in alkali medium，forming violet colored
complex .
• The absorbance of the violet complex is
proportional to concentration of protein in
solution。
Estimation of Serum Albumin
（ bromcresol green, BCG method ）
• Albumin is the most abundant plasma protein in
human. It accounts for about 60% of the total
serum protein.
• It is synthesized in the liver.
• The main biological functions of albumin are to
maintain the water balance in serum and plasma
and to transport and store a wide variety of
ligands e.g. fatty acids, calcium, bilirubin and
hormones such as thyroxine.
• Albumin also provides an endogenous source of
amino acids.
【Clinic
significant】
• Hypoalbuminemia is very common in many diseases and
stems from various factors:
– impaired synthesis, either primary as a result of liver
disease or secondary due to diminished protein intake;
– increased catabolism because of tissue damage (severe
burns) or inflammation;
– malabsorption of amino acids (Crohn’s disease);
– proteinuria due to nephrotic syndrome;
– protein loss by way of feces .
– In severe hypoalbuminemia plasma albumin levels are
below 25 g/L. The low plasma oncotic pressure allows water
to move out of the blood capillaries into the tissues (edema).
• Hyperalbuminaemia has little diagnostic
relevance except, perhaps in dehydration.
【Clinic

significant】
A/G normal range：1.5-2.5
Liver disease, nephrotic syndromes, A/G decrease,
even converse.

Methods for estimating albumin
• For a number of years the standard method for
albumin determination was measurement of
protein remaining in solution following salt
precipitation of globulin fractions.
• Electrophoresis has also been widely used.
• Measurement of albumin has been greatly
simplified by the introduction of dye binding
methods.
– Bromcresol green (BCG) albumin assay is designed
to measure albumin directly in biological samples
without any pretreatment.
Principle
• Albumin (pI 4.9) at pH 4.2 is sufficiently cationic
to bind the anionic dye bromcresol green (BCG)
to form a blue-green colored complex.
pH 4.2
Albumin + BCG
BCG complex
• The intensity of the blue-green color is directly
proportional to albumin concentration in the
specimen.
• It is determined by measuring the increase in
absorbance at 620 - 630 nm.
Estimation of serum Total Protein
（biuret method）
【Reagent solution】
1．Normal saline(NS): 0.9% NaCl
2．Biuret reagent
dissolve 3.0g CuSO4·5H2O in 500ml distilled
H2O，add potassium sodium tartrate 9.0g，KI
5.0g, 24% NaOH 100ml ，add dH2O to 1L 。
Potassium sodium tartrate: maintains cupric ions in solution at an
alkaline pH.
3. Standard solution(10mg/ml): bovine serum
albumin(BSA)powder dissolve into dH2O。
4.Diluted serum(1:10)
Method---Total Protein
Biuret reagent
NS (0.9% NaCl)
Standard
Serum
B
5.0ml
1.0ml
-
S
5.0ml
1.0ml
-
T
5.0ml
1.0ml
•Mix well, incubate for 10mins at 37C,
measure the absorbance of T and S setting
zero with B, λ=540nm.
When absorptiometric determinations are made,
one must be sure that the absorption produced is
due to the particular substances, not by the
solvent and compounds in the reagents. The batch
of analysis must include the following solutions.
•Blank: This will help to exclude the
absorption due to reagents.
•Standard: it includes a solution of known
concentration of the substance which is going
to be determined in the test container.
•Test: it contains an unknown quantity of the
substance.
【Calculation 】
Serum total
=
protein（g/L）
【 Normal value 】
60~80 g/L
AT
AS
× CS ×10
Discussion
• Comparing the results to the nomal value
and clinical significants, discuss the results.
Operating steps of Spectrophotometry
1. Switch on , for 20 min before using.
2. Select Wave length of Maximal Absorption
3. Prepare test sample, blank sample, standard sample .
put them into Spectrophotometry.
4. To “Blank”, mode “T” , press “100%T/0A”, Set T =100
or A=0.
5. Pull the pole once time, press “0%T”, Set T =0.
6. Repeat step “4” to “5”.
7. Change mode to “A”.
8. pull the pole second time, record A1; Third
time ,record A2; Forth time ,record A3.