Lab. 3

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:

in the rate of protein synthesis,

the rate of removal,

and in the volume of distribution.
M. Zaharna Clin. Chem. Lab. 2009

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 often with the assumption that:

each of the several hundred individual proteins present in
serum reacts similarly in chemical reactions.
M. Zaharna Clin. Chem. Lab. 2009

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.

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.
M. Zaharna Clin. Chem. Lab. 2009
 Method
1: Kjeldahl; quantitative, protein
nitrogen determination
 Method
2: Ultraviolet absorption;
quantitative, absorption at 210 nm
 Method
3: refractometry; quantitative, RI
 Method 4: Biuret; quantitative, increased
absorption at 540 nm;
M. Zaharna Clin. Chem. Lab. 2009
 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
M. Zaharna Clin. Chem. Lab. 2009
The concentration of proteins in plasma is affected by
posture: an increase in concentration of 10-20% occurs
within 30 minutes of becoming upright after a period
of recumbency.
 Also, if a tourniquet is applied before veinpuncture, a
significant rise in protein concentration can occur
within a few minutes.
 In both cases, the change in protein concentration is
caused by increased diffusion of fluid from the
vascular into the interstitial compartment.
 These effects must be borne in mind when blood is
being drawn for the determination of protein
concentration.

M. Zaharna Clin. Chem. Lab. 2009
 Hypoproteinemia
 Malnutrition and/or malabsorption
 Excessive loss as in renal disease, GI leakage,
 excessive bleeding, severe burns
 Excessive catabolism
 Liver disease
 Hyperproteinemia
• Dehydration
• Monoclonal increase
• 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.
M. Zaharna Clin. Chem. Lab. 2009
 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.
M. Zaharna Clin. Chem. Lab. 2009

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.

It is possible to use the Biuret reaction to determine the
concentration of proteins because (for most proteins)
peptide bonds occur with approximately the same
frequency per gram of material.

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.
M. Zaharna Clin. Chem. Lab. 2009
M. Zaharna Clin. Chem. Lab. 2009
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.

M. Zaharna Clin. Chem. Lab. 2009
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.
 These properties have been exploited in the
estimation of albumin in body fluids.

M. Zaharna Clin. Chem. Lab. 2009

Method 1: Precipitation; quantitative



Method 2: Tryptophan content; quantitative



Salt fractionation, Solvent fractionation, Acid
fractionation
Principle of analysis: Changes of net charge of protein
result in precipitation
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
M. Zaharna Clin. Chem. Lab. 2009
 Method

Radial immunodiffusion; Turbidimetry;
Nephelometry; Radioimmunoassay; Enzyme
immunoassay;
 Method

5: Dye binding, quantitative
Methyl orange; BCG (bromcresol green); BCP
(bromcresol purple);
 Method

4: Immunochemical
6: 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
M. Zaharna Clin. Chem. Lab. 2009
 Serum
is the specimen of choice
 Fasting is not required, although it may
be desirable since marked lipemia
interferes in the BCG assay.
 Venostasis should be avoided when
collecting samples since
hemoconcentration increases the
apparent concentrations of albumin and
other plasma proteins.
M. Zaharna Clin. Chem. Lab. 2009
 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
M. Zaharna Clin. Chem. Lab. 2009
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,
 and increased catabolism observed in fevers,
untreated diabetes mellitus, and hyperthyroidism.
M. Zaharna Clin. Chem. Lab. 2009
 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
M. Zaharna Clin. Chem. Lab. 2009
 Principle


BCG complexes with albumin, resulting in the dye
having a spectral shift
The presence of albumin increases the absorbance
at 546 nm which is determined
spectrophotometrically
pH 4.3
BCG + Albumin
BCG-albumin complex
M. Zaharna Clin. Chem. Lab. 2009