Estimation of Serum Total Protein

BIO-3

【【 PURPOSEPURPOSE 】】

1. To understand the principle of 1. To understand the principle of biuret method.

2. To understand the principle of bromcreso2. To understand the principle of bromcresol green method.l green method.

3. 3. To understandTo understand the clinic significance of serum total protein, albumin and A/G.

• 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 serumplasma

Centrifuged Blood SampleAdd anticoagulants

(heparin, potassium oxalate)

Separation of Components

Plasma = Less Dense

RBCsMore Dense

Platelets / WBCs

Plasma vs. serum•Plasma is the liquid, cell-free part of blood, that has been treated with anti-coagulants.

Anticoagulated

Serum is the liquid part of blood AFTER coagulation, therefore devoid of clotting factors as fibrinogen.

•serum= plasma - fibrinogen

Clotted

Estimation of serum Total Protein

（ biuret method ）

Plasma proteins

• Total protain normal range ： 60-80g/L【【 Clinic Clinic significantsignificant 】】

DecreasedDecreased in different clinical conditions associa in different clinical conditions associated with nephrotic syndromes, malnutrition, Kwashiorkoted with nephrotic syndromes, malnutrition, Kwashiorkor syndrome, cirrhosis of liver and other liver disorder syndrome, cirrhosis of liver and other liver disorders. rs. IncreasedIncreased total protein values may be found in mul total protein values may be found in multiple myeloma, and conditions associated with high glotiple myeloma, and conditions associated with high globulin concentration.bulin concentration.

Methods for estimating protain:

Lowry’s method

Absorbance Assay (ultraviolet light)

Bradford method (Coomassie Brilliant Blue )

Kjeldahl 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.

• 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 。

Biuret reaction【【 PRINCIPLEPRINCIPLE】】

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.

• Hypoalbuminemia is very common in many diseases and stems from various factors: – impaired synthesis, either primary as a result of liver diseas

e or secondary due to diminished protein intake; – increased catabolism because of tissue damage (severe bur

ns) 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 belo

w 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 Clinic significantsignificant 】】

【【 Clinic Clinic significantsignificant 】】

Liver disease, nephrotic syndromes, A/G deLiver disease, nephrotic syndromes, A/G decrease, even converse.crease, even converse.

A/G normal rangeA/G normal range ：： 1.5-2.51.5-2.5

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 simpli

fied by the introduction of dye binding methods.– Bromcresol green (BCG) albumin assay is designed t

o 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 ）

1 ． Normal saline(NS): 0.9% 0.9% NaCl

2 ． Biuret reagentBiuret reagent　　 dissolve 3.0g CuSOdissolve 3.0g CuSO44··5H5H22O in 500ml distilled HO in 500ml distilled H2OO ，， add potassium sodium tartrateadd potassium sodium tartrate 9.0g9.0g ，， KI 5.KI 5.0g, 24% NaOH 100ml 0g, 24% NaOH 100ml ，， add dHadd dH2O to 1L O to 1L 。。

Potassium sodium tartrate:Potassium sodium tartrate: maintains cupric ions in solution at an alkalimaintains cupric ions in solution at an alkaline pHne pH..

3. Standard solution(10mg/mlStandard solution(10mg/ml)): : bovine serum albbovine serum albumin(BSA)powder dissolveumin(BSA)powder dissolve into dH2O 。。

4.4.Diluted serum(1:10)Diluted serum(1:10)

【 Reagent solution 】】

Method---Total Protein

B S T

Biuret reagent 5.0ml 5.0ml 5.0ml

NS (0.9% NS (0.9% NaCl) 1.0ml - -

Standard - 1.0ml -

Serum - - 1.0ml

•Mix well, incubate for 10mins at 37C, measure the absorbance of T and S setting zero with B, λ=540nm.

•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.

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.

【 Calculation 】】

Serum total protein （ g/L ）

=AT

AS

× CS ×10

【 Normal value 】】

60~80 g/L

Discussion

• Comparing the results to the nomal value

and clinical significants, discuss the results.

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.

Operating steps of Spectrophotometry