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