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Total Protein

Total Protein. Measurement methods. Human proteins More than 50,000 Within one cell 3000 to 5000 Serum More than 1400 different proteins. Interim Consensus Reference Intervals for 14 Plasma Proteins In Human Serum. *Values are slightly lower in fresh samples (assayed <8 hr after draw).

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Total Protein

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  1. Total Protein Measurement methods

  2. Human proteins • More than 50,000 • Within one cell 3000 to 5000 • Serum • More than 1400 different proteins

  3. Interim Consensus Reference Intervals for 14 Plasma Proteins In Human Serum *Values are slightly lower in fresh samples (assayed <8 hr after draw)

  4. Distribution • Vascular,extravascular space • Kind and proportions of individual proteins • Molecular size • Specificity of some of their transport mechanisms • Disease

  5. Plasma proteins • Alterations of plasma proteins • Genetic origin • Physiological • Pathological • Clinical findings • Technical

  6. Plasma proteins • Patient ’s endocrine status • Rates of hepatic synthesis • Steroid hormones • Inflammatory acute-phase reaction • Mask effect

  7. Increased Plasma Levels • acute dehydration • no clinical utility • Synthetic rate and intravascular—extravascular shifts • Decreased Plasma Levels • Decreased synthesis • Primary or genetic • Analbuminemia • Acquired • Inflammatory processes • Increased catabolism • Utilization or loss

  8. ANALYSIS OF PROTEINS Methods • Specific quantitative assays (Individual proteins) • Immunochemical methods • Nephelometry • Turbidimetry • RID • Electroimmunoassay • RIA or enzyme immunoassay (EIA) • very low concentrations • Detection and identification • Electrophoresis • Quantitative measurements of total protein

  9. ANALYSIS OF PROTEINS Methods • Nephelometric and turbidimetric • Speed and ease • Formation of Ag-Ab complex • Light absorption & scattering

  10. Assay Characteristics • Limit of Detection • nephelometric methods • 10 µg/mL • turbidimetric methods • 20 to 30 µg/mL • RID methods • 10 to 20 µg/mL • RIA methods • nanograms per milliliter

  11. Assay Characteristics • Precision • Nephelometry and turbidimetry • within-run coefficients of variation (CVs) of less than 5% • equilibrium methods • less precise than the kinetic methods • RID and EIA systems • 5 to 1 5% • RIA • 5 to 10%

  12. Assay Characteristics • Turnaround Time • Nephelometric and turbidimetric • Kinetic methods • Fast,within minutes • Equilibrium methods • Up to 1 h • RID • 24 to 48 h of incubation • RIA • Several hours

  13. Electrophoresis,Laboratory Considerations • Buffer • Barbital • ionic strength of 0.05 • pH 8.6 • Sample is 3 to 5 µL • Support medium • 1 .5 mA per 2-cm width of cellulose acetate • 10 mA per 1-cm width of agarose medium

  14. Usually five bands (albumin, α1, α2, β and y) • a sixth band • Serum is fresh • buffer containing Ca2+ ions • Densitometry • quantification of individual bands • Stain poorly • high proportions of lipid • Carbohydrate

  15. Mask effect • Too low concentrations • over- shadowed • Visualize bands • Amido black and Ponceau S • Coomassie brilliant blue • Dried, record • Lipoproteins • Migrate variable • Normal control serum

  16. Hyperproteinemia • Dehydration • Inadequate water intake • Excessive water loss • Vomiting, diarrhea, Addison’s disease, or diabetic acidosis • Hypoproteinemia • Hemodilution • Recumbent position • Decreases total protein concentration by 0.3 to 0.5 gIdL • All the individual plasma proteins to the same degree • Intravenous infusions

  17. Mild hyperproteinemia • Increases in APR and polyclonal immunoglobulins • Marked hyperproteinemia • high levels of the monoclonal immunoglobulins • Multiple myeloma

  18. Methods (total protein) • Biuret Method • Peptide bonds react with Cu2+ ions in alkaline solutions to form a colored product • Presence of peptide bonds • Tri-, oligo-, and polypeptides react • By spectrophotometry at 540 nm • The intensity of the color produced is proportional to the number of peptide bonds • Interference • Small peptides • Ammonium ions

  19. MethodsBiuret Method • Detection limit • 1 and 15mg of protein • Simple, sufficiently precise • A fasting serum or plasma • to decrease lipemia • Hemolysis should be avoided

  20. Direct Photometric Methods • Absorption of ultraviolet (UV) light • at 200 to 225 nm • at 270 to 290 nm • Limitations • Uneven distribution aromatic ring • Free tyrosine and tryptophan • Uric acid • Bilirubin

  21. At 200 to 225 nm • Peptide bonds are chiefly responsible for UV absorption • Removal of small interfering molecules • by dilution • gel filtration

  22. Dye-Binding Methods • CBB binds to protonated amine groups of amino acid simple • Fast, & linear up to 150 mg/dl • Absorbance at 595 nm • Limitation • Unequal affinities for dyes • Binding capacities of individual proteins • Inability to define a consistent material for use as a calibrator

  23. Positive interferences • Tolbutamide , high concentrations of urea • Negative interferences • Very high concentrations of NaCl • Hydrogen chloride (HCl)

  24. FoIin-Ciocalteu (Lowry) Method • Reaction with Cu2+ in alkaline solution to form copper—peptide bond protein complexes • Folin-Ciocalteu reagent • Phosphotungstic-phosphomolybdic acid • Tyrosine or tryptophan Reduce Cu2+ • Reduced Cu2+ form complex with Folin-Ciocalteu reagent • Measurement at 650 - 750 nm • Detection limit • 1 0—60 mg/mL

  25. FoIin-Ciocalteu (Lowry) Method • Measuring total protein in urine or CSF • Limitation • Positive interference • Drugs such as salicylates, chlorpromazine, tetracyclines, and some sulfa drugs • Removal of interferences • Gel filtration

  26. KjeldahI’s Method • Acid digestion • Convert nitrogen in the protein to ammonium ion • Concentration of ammonia nitrogen • Double iodides (potassium and mercuric) form a colored complex with ammonia • Limitation • Time consuming • Impractical for wide spread routine use

  27. Precipitation methods • Precipitation of protein • Sulfosalicylic acid • Trichloroacetic acid (TCA) • Scatter incident Light • TCA precipitation (Another approach) • Addition of biuret reagent to the precipitate • Suitable for a fairly large volume of specimen(urine)

  28. Comments • Specimen Collection and Storage • Test specimens must be • Nonhemolyzed • Cell-free • Lipemic sera should not be assayed • Test tubes must remain covered • Dust and dirt particle contamination • Storage conditions • Use of outdated reagents

  29. Calibration of Total Protein Methods • Reference material • Bovine or human albumin • Biuret method • Serum (or serum pool) with a normal albumin/globulin ratio • Precipitation methods • Dye-binding methods • Calculations • Calibration curve consisting of 8 to 15 points

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