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Estimation of serum protein Dr. saba
1. Estimation of serum protein,
Albumin: Globulin Ratio
Dr. Saba Amin Khan,
Senior Resident,
Biochemistry dept.
1
2. Objectives:
• To enumerate and state the principle of various
qualitative and quantitative methods of estimation of
protein.
• To understand the clinical significance of estimating
plasma/serum proteins.
• To estimate the total protein and albumin in a given
sample of serum.
• And to calculate the A:G ratio and correlate the findings
with relevant clinical condition.
2
3. Introduction: Protein
• Protein is most abundant
biological-macromolecule.
• Endless diversity of
function.
• polymers built of α-amino
acids, linked end to end.
• Peptide bond.
3
11. Albumin
• Most abundant plasma protein
• Synthesized by hepatocytes
• Single polypeptide 66 kDa
• High no. of charged AA results in ↑ solubility.
• Net negative charge at physiological pH 7.4
• Plasma half life – 15 to 19 days
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Serum
Fructosamine
12. Functions:
• Maintain plasma oncotic pressure.
• Acts as a transporter:
• Negative acute phase reactant.
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Hormones
Bilirubin
Fatty Acids
Ca, Mg
Penicillin
Sulphonamides
13. GLOBULINS
• Heterogeneous family of proteins.
• Less water soluble than albumin.
• Alpha (α):α1 (α1-antitrypsin, α1-acid glycoprotein, α1-FP)
α2 (Haptoglobin, α2-macroglobulin, Ceruloplasmin)
• Beta (β): β1 (Transferrin,C4,LDL)
β2 (C3, β2-microglobulin)
• Gamma (γ): Immunoglobulins, C-reactive protein
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14. REFERENCE LEVELS IN SERUM
Total protein: 6.7 – 8.6 g/dL
Albumin : 3.5 - 5.0 g/dL
Globulins : 2.0 - 3.5 g/dL
A:G ratio : 1.5 to 2.5
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18. Heat coagulation test
Fill half of the test tube with urine
Heat the top 1/3 of the sample
Look for any turbidity at the upper part of the tube
Compare with the lower part of the tube
Add 2 drops of 33% acetic acid
Acidification is necessary because heating in an
alkaline medium may precipitate phosphates 18
24. Biuret Reaction
• Compounds with 2 or more peptide bonds, react with
CuSO4 in alkaline conditions to produce a complex
which is violet colored.
λmax = 540 nm
30. Estimation of Albumin
• Bromo-Cresol Green method
• BCG reagent is negatively charged at pH 4.3
• pI of albumin is 4.7.
• BCG binds with albumin to form green complex with
absorbance max at 670 nm.
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31. Optimum Methods Of Estimation Of
Proteins In Different Samples
Sample Methods
Serum Biuret
Urine Heat test, Turbidimetry
CSF Turbidimetry
Protein extracted by detergent BCA, Lowry, Bradford
Detection of Albumin BCG
Detection of Specific serum
protein fraction
SPEP, Immuno-detection
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32. Protocol for serum total protein
Test Standard Blank
Serum 0.1 mL --- ---
Standard --- 0.1 mL ---
DDW --- --- 0.1 mL
Biuret
Reagent
5 mL 5 mL 5 mL
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Mix well, wait for 10 min and read at 540 nm.
Concentration of standard : 6g/dL
33. Protocol for Serum Albumin
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Test Standard Blank
Serum 0.1 mL --- ---
Standard --- 0.1 mL ---
DDW --- --- 0.1 mL
BCG Solution 5 mL 5 mL 5 mL
Mix well, read immediately at 670 nm
Concentration of standard : 4 g/dL
34. Calculations
Serum Total Protein =
ATest
AStandard
X Conc. of Standard
Serum Albumin =
ATest
AStandard
X Conc. of Standard
Serum globulin = Serum total protein- Serum albumin
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35. Clinical Case:
• 3-year old male child was brought to AIIMS pediatrics OPD by his
parents with chief complaint of generalised swelling of body for last 5
days. Swelling was started from the face and followed by swelling on
abdomen and then involved upper and lower limbs. There is no other
significant past history and family history. On examination swelling was
found to be painless and pitting. Patient was suspected to have
nephrotic syndrome and the serum sample has been sent to you.
• Estimate the serum total protein & A/G ratio and interpret the results.
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Amino acid and peptide bonds. Oligopeptide vs peptide vs protein. Different levels of protein organization
Salt precipitation by Sodium/ Ammonium sulphate The most common method of analysing plasma proteins is by electrophoresis.
Serum Fructosamine Oncotic pressure/ colloid osmotic pressure, is a form of osmotic pressure exerted by proteins, notably albumin, in a blood vessel's plasma that usually tends to pull water into the circulatory system. It is the opposing force to hydrostatic pressure (increases filtration by pushing fluid and solute OUT of the capillaries)
Hepatic failure (Cirrhosis) with Kernicterus Burn patients
Protein present or not, Sample?, Methods-
. Ninhydrin reacts with NH3 and forms a purple coloured complex, which can be measured at 570 nm. All amino acids, except proline, react with ninhydrin at ambient temperature, to form a blue colored complex, which intensifies to purple on heating. Proline forms a yellow coloured complex.
Aromatic side chains
Appropriate standard has to be used
PI ANTOXIDANT
SPT: STABILIZES CU (II) ION
On further inquiry, parent informed that child suffered from fever with rashes few weeks ago.
