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Estimation of Serum Protein and
Albumin-Globulin RatioASHIKH SEETHY
SENIOR RESIDENT
DEPT OF BIOCHEMISTRY
AIIMS- NEW DELHI
Objectives• To enumerate & state principles of the methods
used for quantitative estimation of proteins.
• To estimate total protein and albumin in a givensample of serum, calculate the A:G ratio andcorrelate the findings to relevant clinicalcondition.
Plasma vs Serum
Plasma• Plasma refers to the fluid portion of blood after
cells are removed by centrifugation.
•Anticoagulant neededHeparin
EDTA
Citrate
Serum• Serum is the fluid component of blood after it is
allowed to clot.
•Approximate 4% decrease in total proteincontent compared to plasma (removal offibrinogen during coagulation)
• Less viscosity; improves specimen delivery
• Preferred for protein electrophoresis
Serum Protein Electrophoresis
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
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 ofcolloid osmotic pressure (oncotic pressure)
• Reference Interval of Serum Albumin: 3.5 - 5.5 g/dL
HormonesBilirubin
Fatty AcidsCa, Cu
Penicillin Sulphonamides
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
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.
HypoproteinemiaHypoproteinemia 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
HyperproteinemiaHyperproteinemia 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
Methods of Protein Estimation
Biuret assay
Lowry assay
BCA assay
Quantitative
Copper based Dye based
Bradford assay
Biuret Reaction• Compounds with 2 or more peptide bonds, react
with CuSO4 in alkaline conditions to produce acomplex which is violet colored.
λmax = 540 nm
Lowry assay• Folin-Ciocaltau reagent
BCA assay• Bi-Cinchoninic Acid reagent
Protein + Cu2+OH
-
Cu1+
Bradford assay•Dye based assay
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 acidsgive a biased reaction
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
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.
Protocol for Serum Total ProteinTest 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
Biuret Reagent Composition• Sodium hydroxide
• Copper sulphate
• Sodium potassium tartrate
• Potassium iodide
Protocol for Serum AlbuminTest 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
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
Precautions• Standard precautions should be maintained.
• In BCG method readings should be takenimmediately to avoid reaction of BCG with otherfractions of protein.
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 theserum sample has been sent to you.
• Estimate the serum total protein & A/G ratio and interpretthe results.
THANK YOU