Total proteins & Albumin Analysis

1. Total proteins & Albumin Analysis

2. 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.

3. 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. • Based on these properties, laboratory methods have been developed to determine the concentration of proteins in serum,

4. 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. • Protein in the plasma is made up of albumin and globulins. • Note: 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.

5. Methods of Total Protein Analysis • Method 1: Kjeldahl; quantitative, protein nitrogen determination • Method 2:Biuret; quantitative, increased absorption at 540 nm;

6. 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

7. 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.

8. 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.

9. 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 540 nm, is directly proportional to the protein concentration, and can be determined spectrophotometrically at 540 nm.

10. 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.

11. 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.

12. Analysis Methods • 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

13. 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

14. Specimen: Serum is the specimen of choice, but heparinized plasma can also be used if precautions are taken to prevent heparin interferences. • 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.

15. 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, • increased catabolism observed in fevers, untreated diabetes mellitus, and hyperthyroidism.

16. 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