estimation of total protein, albumin

The good physician treats the disease; the great physician treats the patient who has the disease.

-- Sir William Osler

ESTIMATION OF TOTAL PROTEIN, ALBUMIN, A/G RATIO

Dr. Gangadhar ChatterjeeMBBS;MD

Assistant ProfessorRCSM Govt. Medical college, Kolhapur, MH, India

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.

Function of plasma proteins.flv

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,

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

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

1- Ultraviolet Absorption: 270 –290 nm 200 -225 nm

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.

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.

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)

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.

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.

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.

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

• 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

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.

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.

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.

• 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

Clinic significance of A/G ratio

Liver disease, nephrotic syndromes, A/G decrease, even converse.

A/G normal range： 1.5-2.5

Reach me at Ganga.chatterjee@gmail.com

Drgangadhar.chatterjee@bsnl.in

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