Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

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Chapter | 5 Proteins, Proteomics, and the Dysproteinemias
2 . Physical Methods
a . Refractometric
Light is refracted when it passes from air to liquid, and if
the liquid contains dissolved proteins, the degree of refraction
(refractive index) changes in proportion to the concentration
of protein. With appropriate instruments and careful
use, determination of the refractive index of serum can
give an accurate assessment of total protein concentration.
The use of hand-held refractometers allows rapid determination
of protein in serum, plasma , or other body fluid
and is one of the most widespread point-of-care methods
in use. It is important to frequently check the calibration
of the refractometer, as this may be a source of error. Most
refractometers are scaled to read directly both total serum
protein and urine-specific gravity. Studies have shown that
results for protein estimates from hand-held refractometers
correlate well with results from the biuret method, though
there are reports of major discrepancies in samples from
avian species ( George, 2001 ). Because of the dependence
on the transmission of light, it is important that the sample
is clear, nonturbid, nonlipemic, and nonhemolyzed.
In a comparison of refractometry to the biuret method for
the determination of the total protein in plasma from dogs
and cats, the correlation coefficients were high, but there
were differences between the methods of 0.6 g/dl (6 g/liter)
and 0.2 g/dl (2 g/liter) for dog and cat plasma, respectively
( Briend-Marchal et al. , 2005 ). Although the internal scales
on most refractometers limit the measurement of protein to
a minimum of 2.5 g/dl (25 g/liter), it has been shown that a
close correlation to total protein determined by the biuret
method can be obtained to concentrations as low as 0.6 g/dl
(6 g/liter), allowing the use of the method to estimate the
protein content in most body fluid samples ( George and
O’Neill, 2001 ).
b . Fibrinogen
Refractometry can be used to determine the concentration
of fibrinogen in plasma. This large protein (340 kDa)
constitutes about 5% of the total plasma protein, and its
concentration can be estimated from the difference in protein
content before and after heat treatment of plasma at
56°C for 3 minutes, which causes fibrinogen to precipitate.
A refractometer is used to determine the protein concentrations
before and after heating with the fibrinogen being
estimated from the difference between the two readings.
Fibrinogen has also been estimated by measurement of
the height of fibrin clot in microhematocrit tubes or the
assessment of the weight or the protein content of fibrin
clots ( Davey et al. , 1972 ). Monitoring the fibrin formation
spectrophotometrically via enzymic action of thrombin
or the snake venom, batroxobin, on fibrinogen allows
the assay to be automated ( Messmore et al. , 1997 ; Oosting
and Hoffmann, 1997 ) and has been used to monitor canine
fibrinogen ( Mischke et al. , 2005 ). Thrombin time, as
described in Chapter 10 on hemostasis, is also used to estimate
fibrinogen concentration.
B . Fractionation of the Serum Proteins
The total protein content of serum is made up of a large
number of individual proteins, and diagnostic information
can be obtained by detecting changes in the component
proteins or in different fractions of proteins. It has been
suggested ( Anderson and Anderson, 2002 ) that virtually
all diseases affect the proteins found in serum and that
diagnosis would be advanced by using proteomics methods
(Section V.C.4) to monitor change in many serum proteins
at the same time. This is likely to be in the distant
future for routine applications in veterinary clinical pathology
laboratories. Nevertheless, considerable valuable diagnostic
information can be obtained using current methods
to fractionate serum to determine the concentration of its
major proteins or groups of proteins.
Most of these methods require the initial determination
of the total serum protein concentration, which is then used
in calculation of the protein content of different fractions. In
its simplest form, the globulin fraction can be estimated if
the total protein and albumin concentrations are known. The
globulin concentration is the difference between the total protein
and albumin concentrations. For quantitative estimation
of the subdivided globulin fractions ( α-, β -, or γ-globulins),
the percentage of each fraction in a serum sample can be
determined by electrophoresis and the concentration of each
fraction calculated from the total protein by proportion.
1 . Salt, Acid, and Glutaraldehyde Fractionation
The weak bonds that hold together the secondary, tertiary,
and quaternary structures of proteins can be disrupted by
a variety of changes in the aqueous environment leading
to reduced solubility. Because of the different amino acid
composition of proteins, alterations in the environment have
differential effects on individual proteins, and salt fractionation
of serum protein exploits this property. The addition
of salts to serum increases the ionic concentration causing
the flocculation and precipitation of the globulins (particularly
γ -globulins), whereas albumin is more resistant to
increased ionic charge and remains in solution. Precipitation
with ammonium sulphate is a widespread technique used in
the purification of serum (and other) proteins.
For diagnostic test use in animals, the most common
application of salt precipitation is in assessment of the
transfer of antibody ( γ -globulins) from colostrum to the
serum of the neonate ( Weaver et al. , 2000 ). The optimal
concentrations of sodium sulphite ( Pfeiffer and Mcguire,
1977 ) or zinc sulphate ( McEwan et al. , 1970 ) have been
determined, which, when added to a serum sample, will
only precipitate the γ -globulin fraction. Thus, serum from
calves or foals in which passive transfer of immunoglobulin