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