estimation of total protein, albumin
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The good physician treats the disease; the great physician treats the patient who has the disease.
-- Sir William Osler
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ESTIMATION OF TOTAL PROTEIN, ALBUMIN, A/G RATIO
Dr. Gangadhar ChatterjeeMBBS;MD
Assistant ProfessorRCSM Govt. Medical college, Kolhapur, MH, India
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Introduction
• The key roles which plasma proteins play in bodily function, together with the relative ease of assaying them, makes their determination a valuable diagnostic tool as well as a way to monitor clinical progress.
• In very general terms, variations in plasma protein concentrations can be due to any of three changes:
--rate of protein synthesis, --rate of removal, --the volume of distribution.
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Function of plasma proteins.flv
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Plasma Protein : Common PropertiesIn spite of functional differences between the various serum proteins, they have certain common biophysical and biochemical properties. These include:• a basic composition of carbon, hydrogen, nitrogen and oxygen; • a backbone of covalent peptide bonds which join the amino
acid units together; and • absorption maxima in the ultraviolet region.
Based on these properties, laboratory methods have been developed to determine the concentration of proteins in serum,
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Specimen
• Serum and plasma may be used, and all usually yield comparable results, though, because of the presence of fibrinogen, plasma levels for total protein are 2 to 4 g/L higher than serum levels.
• A fasting specimen is not required but may be desirable to decrease lipemia.
• Total protein is stable in serum and plasma for 1 week
at room temperature, and for at least 2 months at –20° C
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Methods available for estimation of TP
1-Ultraviolet absorption method2-Specific gravity methods for T.P. a)Phillips or b)Lowry &Hunter 3-Refractrometry. 4-Kjeldahl nitrogen detection method a)Titration or b) kinetic 5-CuSO4 (Cu-Pr complex) Methods a)Lowry or b) Biuret
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1- Ultraviolet Absorption: 270 –290 nm 200 -225 nm
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4-Kjeldahl Method: *The reference method *using protein free filtrate. *Depend on estimation of protein nitrogen.
Protein
H2SO4+Catalyst +Na-Molybdate
NH4+
Alkaline PH
NH3 HCl(standard Sol.) NADPH +2oxoglutarate ( either) (Titration) Neutral PH NADP + glutamate (Nisselerization) (Monitor abs.change at 340 nm) Concentration of total protein= detected nitrogen X100/16 = detected nitrogen X 6.25 *factor 6.25 is the result of 100/16 as each 100 g prt.16 g Nitrogen.
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5-Alkaline CUSO4 soln.Methods: Sample NaOH + CuSO4. Copper 6-peptide bond protein complex Folin(Fenol)+ K&NaTartarate(=color stabilizer) Cio-Calteau(PTA+Ph-Molbdic a.) Molybdinum blue + Violet color of Cu-Pr.Complex Tungesten blue(at 650- 750nm) (at 546nm) LOWRY’s Method BIURET’s Method
Sensitivity: 100 times > Biuret’s good for Pr. 2-12 g
Specificity: Less specific specific
No. of reagents: 2 Reagents One reagent Drug Interference Dependence on Tryptophan&Tyrosine (salicylates,sulfa&tetracyclines) e.g Alb.=0.2 % Tryptophan by wt. Glob.=2% Tryptophan by wt.
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Principle of BIURET Reaction• Many peptide bonds(-CO-NH-)conjoint each other in
protein molecule ,can react with Cu2+ in alkali medium, forming violet colored complex .
• The absorbance of the violet complex is proportional to concentration of protein in solution。 ( Read at 540nm)
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Reference Range• Reference range for total proteins is 66.6 to 81.4 g/L• Results for males are approximately 1 g/L higher
than results for females; this difference is probably not of clinical significance.
• In newborns, the mean serum protein concentration is 57 g/L, increasing to 60 g/L by 6 months and to adult levels by about 3 years of age.
• Serum protein levels of premature infants can be much lower than that of full term infants, ranging from 36 to 60 g/L.
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Hypoproteinemia• Malnutrition and/or malabsorption• Excessive loss as in renal disease, GI leakage,• excessive bleeding, severe burns• Excessive catabolism• Liver disease
Hyperproteinemia• Dehydration• Monoclonal increases• Polyclonal increase
-- Only disorders affecting the concentration of albumin and/or the immunoglobulins will give rise to abnormal total protein levels. -- Other serum proteins are never present in high enough concentrations for changes to have a significant overall effect.
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ALBUMIN
• Albumin is the most abundant circulating plasma protein (40–60 % of the total)
• Playing important roles in the maintenance of the colloid osmotic pressure of the blood, in transport of various ions, acids, and hormones.
• It is a globular protein with a molecular weight of approximately 66,000 D and is unique among major plasma proteins in containing no carbohydrate.
• It has a relatively low content of tryptophan and is an anion at pH 7.4.
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Analytical Methods available• Method 1: Precipitation; quantitative Salt fractionation, Acid fractionation Principle of analysis: Changes of net charge of protein result in precipitation• Method 2: Tryptophan content; quantitative Principle of analysis: Glyoxylic acid + tryptophan in globulin Purple chromogen (Amax, 540 nm); Total protein – globulin = albumin.• Method 3: Electrophoresis; quantitative Principle of analysis: Albumin is separated from other proteins in electrical field; percent staining of albumin fraction multiplied by total protein value
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• Method 4: Dye binding, quantitative Methyl orange; BCG (bromcresol green); BCP (bromcresol purple);
• Method 5: Dye binding; semiquantitative Bromphenol blue in test strip changes color from yellow to blue in presence of albumin most commonly used test for urine protein
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Estimation of Albumin by Dye-binding Method
• Measurement of albumin has been greatly simplified by the introduction of dye binding methods.
• Bromcresol green (BCG) albumin assay is designed to measure albumin directly in biological samples without any pretreatment.
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Principle• Albumin (pI 4.9) at pH 4.2 is sufficiently cationic to
bind the anionic dye bromcresol green (BCG) to form a blue-green colored complex.
pH 4.2 Albumin + BCG BCG complex
• The intensity of the blue-green color is directly proportional to albumin concentration in the specimen.
• It is determined by measuring the increase in absorbance at 620 - 630 nm.
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Interfering FactorsAlbumin is decreased in:• Pregnancy (last trimester, owing to increased plasma
volume)• Oral birth control (estrogens) and other drugs. • Prolonged bed rest.• IV fluids, rapid hydration, over hydration.
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• Hypoalbuminemia is very common in many diseases and stems from various factors: – impaired synthesis, either primary as a result of liver disease or
secondary due to diminished protein intake; – increased catabolism because of tissue damage (severe burns) or
inflammation; surgery, sepsis(SIRS), stress response– malabsorption of amino acids (Crohn’s disease); – proteinuria due to nephrotic syndrome; – protein loss by way of feces . – In severe hypoalbuminemia plasma albumin levels are below 25
g/L. The low plasma oncotic pressure allows water to move out of the blood capillaries into the tissues (edema).
• Hyperalbuminaemia has little diagnostic relevance except, perhaps in dehydration.
Interpretation
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Clinic significance of A/G ratio
Liver disease, nephrotic syndromes, A/G decrease, even converse.
A/G normal range: 1.5-2.5