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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. 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, 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. Methods of Total Protein Analysis • Method 1: Kjeldahl; quantitative, protein nitrogen determination • Method 2: Biuret; quantitative, increased absorption at 540 nm; 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 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. 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 tartratecomplexed 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. 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. 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. 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 • 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 • 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. Albumin Reference Interval for Serum Age Men (g/L) Women (g/L) 21–44 33.3–61.2 27.8–56.5 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. 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