Total Proteins & Albumin Analysis Lab. 4

Introduction Proteins are polymers of amino acids produced by living cells in all forms of life 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. M. Zaharna Clin. Chem. Lab. 2009

Introduction In very general terms, variations in plasma protein concentrations can be due to any of three changes: in the rate of protein synthesis, the rate of removal, and in the volume of distribution.

Proteins: Common properties In 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.

Proteins: Common properties Based on these properties, laboratory methods have been developed to determine the concentration of proteins in serum often with the assumption that: each of the several hundred individual proteins present in serum reacts similarly in chemical reactions. M. Zaharna Clin. Chem. Lab. 2009

Serum Total Protein Serum total protein, also called plasma total protein or total protein, is a biochemical test for measuring the total amount of protein in blood plasma or serum. Total protein measurements can reflect: nutritional status kidney disease liver disease

Serum Total Protein Protein in the plasma is made up of albumin and globulins. The globulin in turn is made up of α1, α2, β, and γ globulins. These fractions can be quantitated using protein electrophoresis, but the total protein test is a faster and cheaper test that estimates the total of all fractions together. The traditional method for measuring total protein uses the biuret reagent, but other chemical methods are also available. M. Zaharna Clin. Chem. Lab. 2009

Methods of Total Protein Analysis Kjeldahl: quantitative, protein nitrogen determination Ultraviolet absorption: quantitative, absorption at 210 nm Refractometry: quantitative, RI Biuret: quantitative, increased absorption at 540 nm Dye-binding Total nitrogen

The Biuret Method The Biuret reagent is made of (NaOH) and copper (II) sulfate (CuSO4), together with potassium sodium tartrate (KNaC4H4O6). A blue reagent which turns violet in the presence of proteins, The Sodium hydroxide does not participate in the reaction at all, but is merely there to provide an alkaline medium so that the reaction can take place.

Principle: Biuret Method Peptide bonds of proteins react with tartrate- complexed cupric ions in alkaline solutions to form a colored product. In a positive test, a copper(II) ion is reduced to copper(I), which forms a complex with the nitrogens and carbons of the peptide bonds in an alkaline solution. A violet color indicates the presence of proteins. The intensity of the color, and hence the absorption at 546(520-570) nm, is directly proportional to the protein concentration.

Specimen Total protein is stable in serum and plasma for 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 at least 3 days at 2-8° C, and 6 months at –20° C

Procedure Mix and incubate for 10 minutes at room temperature . Sample Standard Blank 1000ul Reagent A - 20ul Mix and incubate for 10 minutes at room temperature . Read the absorbance at 546 (520-570)nm against blank. Color is stable for 60 minutes. M. Zaharna Clin. Chem. Lab. 2009

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.

Hypoproteinemia Hyperproteinemia Malnutrition and/or malabsorption Excessive loss as in renal disease, GI leakage, excessive bleeding, severe burns Excessive catabolism Liver disease Hyperproteinemia Dehydration Monoclonal increase Polyclonal increase M. Zaharna Clin. Chem. Lab. 2009

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. M. Zaharna Clin. Chem. Lab. 2009

note To detect small amount of protein we can use TCA(trichloroacetic acid)/3% SSA (sulfosalicylic acid) TCA/SSA Principle: Protein denatured by acid, precipitates and renders the specimen more turbid as its concentration increased M. Zaharna Clin. Chem. Lab. 2009

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. These properties have been exploited in the estimation of albumin in body fluids.

Analysis Methods Precipitation: quantitative Salt fractionation, Solvent fractionation, Acid fractionation Principle of analysis: Changes of net charge of protein result in precipitation Tryptophan content: quantitative Principle of analysis: Glyoxylic acid + tryptophan in globulin Purple chromogen (Amax, 540 nm); Total protein – globulin = albumin. Electrophoresis: quantitative Principle of analysis: Albumin is separated from other proteins in electrical field; percent staining of albumin fraction multiplied by total protein value

Analysis Methods Immunochemical Dye binding: quantitative Radial immunodiffusion; Turbidimetry; Nephelometry; Radioimmunoassay; Enzyme immunoassay; Dye binding: quantitative Methyl orange; BCG (bromcresol green); BCP (bromcresol purple); Dye binding: semi-quantitative Bromphenol blue in test strip changes color from yellow to blue in presence of albumin most commonly used test for urine protein

Dye-binding Techniques Serum albumin is most often assayed using dye- binding techniques. Albumin preferentially binds to anionic dyes that do not attract globulins Bromcresol purple (BCP) and bromcresol green (BCG) are most commonly used The amount of light absorbed by the albumin – dye complex is proportional to the amount of albumin present M. Zaharna Clin. Chem. Lab. 2009

Clinical Significance Plasma albumin levels, although important for management and follow-up, have very little value in clinical diagnosis. Hyperalbuminemia is usually attributable to: dehydration or hemoconcentration. Hypoalbuminemia is usually the result of hemodilution, a rate of synthesis less than the albumin loss, diseases that cause a large albumin loss from urine, skin, or intestine, and increased catabolism observed in fevers, untreated diabetes mellitus, and hyperthyroidism.

Bromcresol Green principle In citrate buffer albumin forms with bromocresol green(BCG) a colored compound with a color intensity proportional to the albumin concentration present in the sample. The presence of albumin increases the absorbance at 546 (520-570) nm which is determined spectrophotometrically BCG + Albumin BCG-albumin complex pH 4.3 M. Zaharna Clin. Chem. Lab. 2009

Specimen Serum is the specimen of choice Fasting is not required, although it may be desirable since marked lipemia interferes in the BCG assay. Venostasis should be avoided when collecting samples since hemoconcentration increases the apparent concentrations of albumin and other plasma proteins.

Procedure Mix and incubate for 5 minutes at room temperature . Sample Standard Blank 1000ul Reagent A - 5ul Mix and incubate for 5 minutes at room temperature . Read the absorbance at 546 (520-570)nm against blank. Color is stable for 60 minutes. M. Zaharna Clin. Chem. Lab. 2009 M. Zaharna Clin. Chem. Lab. 2009

Albumin Reference Interval for Serum Reference range: Albumin Reference Interval for Serum Age Men (g/L) Women (g/L) 21–44 33.3–61.2 27.8–56.5 M. Zaharna Clin. Chem. Lab. 2009

Interfering Factors Albumin 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, overhydration

note A urine microalbumin test is a test to detect very small levels of a blood protein (albumin) in your urine. A microalbumin test is used to detect early signs of kidney damage in people who are at risk of developing kidney disease. M. Zaharna Clin. Chem. Lab. 2009