For MBBS Biochemistry Practical. Explains various methods of protein estimation and estimation of AG ratio, conditions leading to alterations in AG ratio etc.
2025 Inpatient Prospective Payment System (IPPS) Proposed Rule
Serum Protein and Albumin-Globulin Ratio
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.