For MBBS Biochemistry Practical. Explains various methods of protein estimation and estimation of AG ratio, conditions leading to alterations in AG ratio etc.
1. Estimation of Serum Protein
and
Albumin-Globulin Ratio
ASHIKH SEETHY
SENIOR RESIDENT
DEPT OF BIOCHEMISTRY
AIIMS- NEW DELHI
2. Objectives
• To enumerate & state principles of the methods
used for quantitative estimation of proteins.
• To estimate total protein and albumin in a given
sample of serum, calculate the A:G ratio and
correlate the findings to relevant clinical
condition.
4. Plasma
• Plasma refers to the fluid portion of blood after
cells are removed by centrifugation.
• Anticoagulant needed
Heparin
EDTA
Citrate
5. Serum
• Serum is the fluid component of blood after it is
allowed to clot.
• Approximate 4% decrease in total protein
content compared to plasma (removal of
fibrinogen during coagulation)
• Less viscosity; improves specimen delivery
• Preferred for protein electrophoresis
7. Albumin
• L. albus = white
• Most abundant plasma protein (~50%)
• Synthesized by hepatocytes
• Single polypeptide - 66 kDa
• Net negative charge at physiological pH 7.4
• High no. of charged AA results in ↑ solubility
8. Albumin
• Catabolism occurs by pinocytosis followed by
lysosomal degradation to AA
• Plasma half life – 15 to 19 days
• Acts as a transporter and major component of
colloid osmotic pressure (oncotic pressure)
• Reference Interval of Serum Albumin: 3.5 - 5.5 g/dL
Hormones
Bilirubin
Fatty Acids
Ca, Cu
Penicillin
Sulphonamides
9. Globulins
• Heterogeneous family of proteins
• Mol weight: 12-900 kDa
• Less water soluble than albumin
• Types
Alpha (α) :
α1 (α1-antitrypsin, α1-acid glycoprotein, HDL)
α2 (Haptoglobin, α2-macroglobulin, Ceruloplasmin)
Beta (β) :
β1 (Transferrin,C4,LDL)
β2 (C3, β2-microglobulin)
Gamma (γ) : Immunoglobulins, C-reactive protein
10. Normal Values in Serum
• Total protein : 6.7 – 8.6 g/dL
• Albumin : 3.5 - 5.5 g/dL
• Globulins : 2.0 - 3.5 g/dL
• A:G ratio : 1.5 to 2.5 : 1
Ref: Harrison’s Principles of Internal Medicine. 19th Ed.
11. Hypoproteinemia
Hypoproteinemia is serum protein level < 6.7 g/dL
↓ (A/G)
Due to decrease in albumin
↑ Loss
Renal disorders
(e.g. nephrotic syndrome)
↓ Synthesis
Chronic liver disease
(cirrhosis)
Chronic malnutrition- PEM
↑ (A/G)
Due to decrease in globulin
Agammaglobulinemia/
hypogammaglobulinemia
12. Hyperproteinemia
Hyperproteinemia is serum protein level > 8.3 g/dL
Normal (A/G)
Relative hyperproteinemia
Dehydration
↓ (A/G)
Due to increase in globulin
Chronic infections -
e.g. kala-azar
Malignant proliferation of
plasma cells -
e.g. multiple myeloma
13. Methods of Protein Estimation
Biuret assay
Lowry assay
BCA assay
Quantitative
Copper based Dye based
Bradford assay
14. 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
18. Method Advantage Disadvantage
Biuret Most commonly used in Clinical
Chemistry
Amino acids and dipeptides don’t
give the reaction
Sensitivity = 100 mg/dL (1 mg/mL)
Lowry Sensitivity = 10 μg/mL
Fast
Interference by detergents and
chelating agents.
Proteins containing tyrosine and
tryptophan give a biased reaction
BCA Sensitivity = 0.5 μg/mL
(Most sensitive method)
It has minimum interference
Interference by chelating agents
Bradford Sensitivity = 20 μg/mL
Fastest & easiest
Proteins containing basic amino acids
give a biased reaction
19. A-G Ratio
• Direct:
Albumin (A) & Globulins (G) in the serum sample are
separated & determined separately
• Indirect:
Serum total protein: Biuret reaction
Serum albumin: Bromocresol Green (BCG)
Serum globulin = Serum total protein - Serum albumin
20. Bromo-Cresol Green Method
• BCG reagent at pH 4.3 is negatively charged
• pI of Albumin is 4.7
Bromocresol green binds quantitatively with albumin to from an intense blue-
green complex with an absorbance max at 670 nm.
21. 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
Mix well, wait for 10 min and read at 540 nm
Concentration of standard : 8 g/dL
23. Protocol for Serum Albumin
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
24. Calculations
Serum Total Protein
Serum albumin
Serum globulin
Conc. of Standard
Conc. of Standard
ATest
AStandard ABlank
ABlank
ATest
AStandard ABlank
ABlank
Serum total protein Serum albumin
25. Precautions
• Standard precautions should be maintained.
• In BCG method readings should be taken
immediately to avoid reaction of BCG with other
fractions of protein.
26. Case
• A 60 year old male presented with complaints of bone pain,
fatigue and weakness.
• Routine investigations:
Hb 8 g/dL (13 - 16 g/dL)
Serum Calcium 14 mg/dL (8.7 -10.2 mg/dL)
Serum Urea 60 mg/dL (15 - 40 mg/dL)
Serum Creatinine 2 mg/dL (0.6 - 1.2 mg/dL)
• On X-ray, osteolytic bony lesions were found on the skull.
Patient was suspected to have multiple myeloma and the
serum sample has been sent to you.
• Estimate the serum total protein & A/G ratio and interpret
the results.