Download Serum Total Protein

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