Serum Protein and Albumin-Globulin Ratio
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Transcript of Serum Protein and Albumin-Globulin Ratio
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Estimation of Serum Protein and
Albumin-Globulin RatioASHIKH SEETHY
SENIOR RESIDENT
DEPT OF BIOCHEMISTRY
AIIMS- NEW DELHI
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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.
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Plasma vs Serum
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Plasma• Plasma refers to the fluid portion of blood after
cells are removed by centrifugation.
•Anticoagulant neededHeparin
EDTA
Citrate
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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
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Serum Protein Electrophoresis
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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
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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
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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
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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.
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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
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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
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Methods of Protein Estimation
Biuret assay
Lowry assay
BCA assay
Quantitative
Copper based Dye based
Bradford assay
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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
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Lowry assay• Folin-Ciocaltau reagent
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BCA assay• Bi-Cinchoninic Acid reagent
Protein + Cu2+OH
-
Cu1+
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Bradford assay•Dye based assay
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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
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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
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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.
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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
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Biuret Reagent Composition• Sodium hydroxide
• Copper sulphate
• Sodium potassium tartrate
• Potassium iodide
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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
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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
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Precautions• Standard precautions should be maintained.
• In BCG method readings should be takenimmediately to avoid reaction of BCG with otherfractions of protein.
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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.
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THANK YOU