Total proteins &

Albumin Analysis

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 1, 2, , and
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 (4060 % 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)
2144

33.361.2

27.856.5

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