1. Estimation of Serum Total Protein
BIO-3
Estimation of Serum Total Protein

2. 【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.

3. Question: 1. Concentration of serum total protein
2. Albumin/globulin ( A / G )
1. concentration of albumin
2. total protein – albumin = globulin
3. A/G
blood
plasma
serum

4. Centrifuged Blood Sample
Add anticoagulants (heparin, potassium oxalate)
Centrifuged Blood Sample

5. Separation of Components
Plasma = Less Dense
Platelets / WBCs
RBCs
More Dense

6. Plasma vs. serum Anticoagulated Clotted serum= plasma - fibrinogen
Plasma is the liquid, cell-free part of blood, that has been treated with anti-coagulants.
Serum is the liquid part of blood AFTER coagulation, therefore devoid of clotting factors as fibrinogen.
Anticoagulated
Clotted
serum= plasma - fibrinogen

7. Estimation of serum Total Protein （biuret method）

8. Plasma proteins

9. 【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.

10. Methods for estimating protain:
Kjeldahl method
Absorbance Assay (ultraviolet light)
Bradford method
(Coomassie Brilliant Blue )
Lowry’s method
Biuret method

11. Biuret reaction (urea) (biuret) (violet colored complex)
When biuret is treated with dilute copper sulfate in alkaline medium, a purple color is obtained.

12. Biuret reaction 【PRINCIPLE】
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。

13. 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.

14. 【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.

15. 【Clinic significant】 A/G normal range：1.5-2.5
Liver disease, nephrotic syndromes, A/G decrease, even converse.

16. 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.

17. 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 nm.

18. Estimation of serum Total Protein （biuret method）

19. 【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)

20. Method---Total ProteinB S T
Biuret reagent
5.0ml
NS (0.9% NaCl)
1.0ml -
Standard
Serum
Mix well, incubate for 10mins at 37C, measure the absorbance of T and S setting zero with B, λ=540nm.

21. 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.

22. 【Calculation 】 Serum total protein（g/L） = 【 Normal value 】 60~80 g/LAS
× CS
×10
【 Normal value 】
60~80 g/L

23. Discussion
Comparing the results to the nomal value and clinical significants, discuss the results.

24. Operating steps of Spectrophotometry
Switch on , for 20 min before using.
Select Wave length of Maximal Absorption
Prepare test sample, blank sample, standard sample . put them into Spectrophotometry.
To “Blank”, mode “T” , press “100%T/0A”, Set T =100 or A=0.
Pull the pole once time, press “0%T”, Set T =0.
Repeat step “4” to “5”.
Change mode to “A”.
pull the pole second time, record A1; Third time ,record A2; Forth time ,record A3.