Serum Protein and Albumin-Globulin Ratio
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For MBBS Biochemistry Practical. Explains various methods of protein estimation and estimation of AG ratio, conditions leading to alterations in AG ratio etc.
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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
- 15. Lowry assay • Folin-Ciocaltau reagent
- 16. BCA assay • Bi-Cinchoninic Acid reagent Protein + Cu2+ OH - Cu1+
- 17. Bradford assay • Dye based assay
- 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
- 22. Biuret Reagent Composition • Sodium hydroxide • Copper sulphate • Sodium potassium tartrate • Potassium iodide
- 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.
- 27. THANK YOU
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