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Blood Proteins and Inflammation
Blood Proteins and Inflammation
Blood Proteins and Inflammation
Acute-phase proteins
A primary challenge in medicine involves the detection and monitoring of
inflammation, which results from myriad disease processes. Inflammation is
a complex process involving networks of cellular and humoral events that
are pivotal for the health and survival of all organisms. Early recognition
of systemic inflammation is essential to devise and implement an effective
treatment plan. This is especially critical if the delicate balance between
* Corresponding author.
E-mail address: farmuse@vt.edu (M.V. Crisman).
0749-0739/08/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
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286 CRISMAN et al
during the first 48 hours of clinical signs and then returned to baseline over the
ensuing 11 to 22 days in uncomplicated cases [7]. SAA determinations proved
to be a more sensitive indicator of infection than nasal swabs and correlated
well with disease resolution. A recent study by Cohen and colleagues [6] eval-
uated SAA concentration in foals with R equi pneumonia and its utility to dif-
ferentiate normal from affected foals. Results indicated that bimonthly SAA
determinations in foals less than 1 month of age were not a useful screening
tool for R equi infection. This may have been attributable to the nature of the
disease (insidious with walled off pulmonary abscesses) or the long sampling
interval. Regardless, more research is needed in this area to determine conclu-
sively the usefulness of SAA in foals with R equi pneumonia.
Concentrations of SAA have been determined in horses with colic result-
ing from inflammatory and noninflammatory causes. Horses with colic
attributable to inflammatory causes (enteritis, peritonitis, colitis, or abdom-
inal abscesses) had significantly higher concentrations of SAA than horses
with noninflammatory causes (displacement or obstruction). Additionally,
SAA concentrations were higher in horses that failed to survive the colic ep-
isode compared with survivors; however, the difference was not substantial
enough to be clinically useful at this time [9].
Studies of SAA response to equine joint disease have been recently per-
formed on serum and synovial fluid [21]. SAA concentrations in serum and sy-
novial fluid were lower than assay detection limits in healthy horses. Synovial
fluid and serum SAA concentrations were significantly elevated in horses with
suspected infectious arthritis and tenovaginitis, suggesting that SAA may be
a useful biologic marker for horses with joint disease. This study corroborated
an earlier project using an experimentally (lipopolysaccharide) induced arthri-
tis, in which increases in synovial fluid SAA reflected inflammatory activity
and concentrations decreased during stages of clinical improvement [10].
Recently, an excellent review of equine SAA was published detailing
many of the salient features associated with APPs [22].
Haptoglobin
Hp is classified as a moderate APP, demonstrating an increase of 1 to
10 times greater than the reference interval in horses during the APR (ref-
erence interval: 2–10 g/L). Hp is classified as a major APP in ruminants
and has been proved to be an effective marker for the presence and sever-
ity of such diseases as mastitis, pneumonia, and endocarditis in cattle [24].
Produced primarily by hepatocytes, Hp is an a2-globulin that primarily
functions to prevent the loss of iron by the formation of stable complexes
with free hemoglobin (Hb) in the blood. Hp synthesis is stimulated by the
Hb concentration in plasma, and the resultant Hp-Hb complex provides
an efficient means for collection of free Hb, which prevents external
leak or loss of iron and ameliorates the oxidative damage to tissues asso-
ciated with free Hb (from hemolysis). Additionally, the Hp-Hb complexes
are large enough to reduce renal filtration of free Hb and iron substan-
tially from plasma. These complexes are removed by hepatocytes, allowing
reutilization of iron and amino acids. Although several functions have
been ascribed to Hp, it is believed to have a bacteriostatic effect by lim-
iting the availability of iron, which is essential for bacterial growth. Hp
may also have anti-inflammatory actions by protecting against reactive
oxygen species and inhibiting granulocyte chemotaxis and phagocytosis
[24]. Hp is also reported to aid in wound repair by stimulating angiogen-
esis [25].
Analysis of haptoglobin
Currently, techniques used to determine equine Hp concentrations are
fairly laborious and generally restricted to research laboratories. Techniques
include single radial immunodiffusion (SRID) [30], serum protein electro-
phoresis (SPE; increased a2-globulin fraction) [31], Hb-Hp binding capacity
assay [4], and immunoturbidimetry [32]. A method for estimation of serum
Hp using capillary zone electrophoresis has also been described [33].
BLOOD PROTEINS AND INFLAMMATION IN THE HORSE 291
Fibrinogen
Fb was one of the earliest recognized APPs. Fb, a soluble plasma glyco-
protein synthesized by the liver, is considered a moderate APP with concen-
trations increasing 1- to 10-fold over 24 to 72 hours after the induction of
inflammation. The relatively wide reference interval for Fb concentrations
in healthy horses (200–400 mg/dL, 2–4 g/L) and lengthy response period af-
ter an inflammatory stimulus have rendered Fb a fairly insensitive APP. Sev-
eral functions have been ascribed to Fb, including providing a substrate for
fibrin formation in tissue repair and providing a matrix for migration of in-
flammatory-related cells. Fb binds to cell surface integrins (CD11/CD18) of
phagocytes, initiating a cascade of intracellular signals promoting the en-
hancement of degranulation, phagocytosis, and antibody-dependent cyto-
toxicity. Over the past several decades, Fb has been used to diagnose and
monitor various inflammatory conditions in horses. A recent study evaluat-
ing serum iron and plasma Fb concentrations in systemic inflammatory dis-
eases in horses concluded that an increase in Fb concentration was
associated with a poor prognosis. Hypoferremia was a more accurate reflec-
tion of acute, subacute, and chronic inflammation in sick horses older than
2 months of age, however [34]. Plasma Fb concentrations have been used to
detect and monitor R equi pneumonia in foals. Measurement of Fb concen-
trations and leukocyte counts proved useful for early identification of
R equi–infected foals, although leukocyte counts proved superior under field
conditions [35]. Another study evaluated SAA and Fb concentrations in
healthy horses experimentally infected with Streptococcus zooepidemicus
and monitored the progression of pneumonia. Results indicated that SAA
responded more rapidly than Fb to changes in clinical signs of pneumonia
[36]. Together, these studies suggest that an alteration in Fb concentration
is not necessarily in agreement with actual disease detection or progression.
Although determination of plasma Fb concentration has long been used for
detecting inflammatory diseases in horses, its relatively slow APR after an
inflammatory insult seriously hampers its clinical utility. Nevertheless, Fb
measurements are relatively easy and inexpensive, and this fact has likely se-
cured its continued wide use in veterinary medicine.
Analysis of fibrinogen
A heat precipitation method is used as a quick estimate of Fb concentration
[37]. More accurate methods include modifications of the Ratnoff-
Menzie assay, measurement of clot weight, and quantification of immunopre-
cipitate formed with specific anti-Fb antiserum.
a1-acid glycoprotein
AGP is a highly glycosylated protein synthesized and secreted primarily
by hepatocytes. It is considered a moderate APP in most species and is
292 CRISMAN et al
more likely to be associated with chronic conditions rather than acute in-
flammation. Local (extrahepatic) AGP production has been confirmed
and is believed to contribute to the general maintenance of homeostasis
by reducing tissue damage associated with inflammation, particularly in ep-
ithelial and endothelial cells [38]. Two major functions have been attributed
to AGP, namely, drug binding and immunomodulation. Similar to albumin,
AGP is capable of binding to endogenous or exogenous substances, such as
heparin, histamine, serotonin, and steroids [38]. This critical function may
keep total drug-binding levels constant during the APR, whereas albumin,
a negative APP, decreases in total concentration. AGP has been reported
to inhibit neutrophil activation, increase secretion of IL-1 receptor antago-
nist by macrophages, and enhance clearance of lipopolysaccharide by di-
rectly binding and neutralizing the latter [38,39].
Although AGP has been proved to be a useful APP in other species, in-
cluding pigs [40] and cattle [41,42], little work has been done in horses. One
study reported increased concentrations of AGP (as determined by SRID)
in colts 2 to 3 days after castration and in adult horses after jejunojejunos-
tomy and return to baseline values 14 to 28 days later [43]. Another study
evaluating a carbohydrate overload model of laminitis in ponies reported in-
creased concentrations of AGP 4 hours after administration of carbohy-
drate (24 hours before the onset of clinical lameness) [27].
C-reactive protein
CRP has been well documented as an APP in human beings, ruminants,
dogs, and, to a lesser degree, horses [1,24]. It is considered to be a moderate
APP in horses, with a two- to threefold increase over several days. CRP has
several proinflammatory effects, including activation of the complement cas-
cade, induction of inflammatory cytokines, and phagocytosis. CRP also has
significant anti-inflammatory effects, such as inhibiting chemotaxis and the
generation of superoxide by neutrophils and preventing the adhesion of neu-
trophils to endothelial cells. Studies conducted in the early 1990s suggested
that high CRP concentrations occurred in horses with pneumonia, enteritis,
and arthritis [44].
proteins, and the results can be a useful diagnostic aid to the clinician. There
are, however, only a few diseases for which the pattern of SPE is pathogno-
monic [31].
The principle of the electrophoretic separation of serum proteins is based
on the migration of charged proteins in an electric field [31]. The direction
and rate of migration of a protein are based on the type of charge (anion
or cation) and size of the protein. A ‘‘normal’’ equine SPE consists of six
fractions, including albumin, a1-globulin, a2-globulin, b1-globulin, b2-glob-
ulin, and g-globulin.
The electrophoretogram is stained, and a densitometer is used to deter-
mine the proportion of proteins in these fractions, which are then used in
conjunction with the total serum protein concentration to determine specific
concentrations of the fractions. Reference values for these fractions on SPE
of the adult horse are albumin (26–37 g/L), a1-globulin (0.6–7 g/L), a2-glob-
ulin (3–13 g/L), b1-globulin (4–16 g/L), b2-globulin (3–9 g/L), g-globulin
(6–19 g/L), and total serum protein (52–79 g/L) [31].
Albumin is the most prominent of the normal serum proteins on SPE and
constitutes approximately 50% of the total serum protein [31]. The albumin
fraction migrates closest to the anode and is the most homogeneous fraction
on SPE [31,45]. Equine serum often has a minor postalbumin fraction,
which appears as a shoulder on the cathodal side of the albumin peak.
This shoulder often becomes more prominent with hypoalbuminemia [31].
The a-globulin fraction is the most rapidly migrating fraction of the glob-
ulins and migrates as a1- (fast) and a2- (slow) globulin fractions [31]. The a1-
and a2-globulin fractions are identified as the first two peaks after albumin
on SPE. Important a1- and a2-globulins include antitrypsin, high-density li-
poprotein, very-low-density lipoprotein, macroglobulin, ceruloplasmin, and
Hp [31].
The b-globulin fraction trails the a-globulin fraction on SPE and mi-
grates as b1- (fast) and b2- (slow) globulin fractions [31]. The b1- and b2-
globulin fractions are identified as the third and fourth peaks after albumin
on SPE. Important b-globulins include complement (C3, C4), transferrin,
ferritin, and CRP. Some of the immunoglobulins (IgM and IgA) can mi-
grate in the b-globulin region [31].
The g-globulin fraction trails the b-globulin fraction on SPE and includes
IgG, IgA, IgM, and IgG subclass T (IgG [T]). The concentrations of these
immunoglobulins in horses have also been quantitated by SRID [46].
hyperthermia, and inflammation also can influence the profile of SPE [31].
Common abnormalities identified on SPE include hypoalbuminemia, hyper-
globulinemia, and hypoglobulinemia.
Hypoalbuminemia is caused by a decreased synthesis or an increased loss
of albumin. Albumin is synthesized in the liver, and hypoalbuminemia is
a feature of chronic diffuse liver disease [31,45]. A prominent postalbumin
fraction on SPE, with or without hypoalbuminemia, has been considered
pathognomonic for liver disease in the horse. An increased loss of albumin
may be caused by renal or gastrointestinal disease and accumulation within
the thoracic or abdominal cavity [31,45].
Hyperglobulinemia is caused by an increase in the a-, b-, or g-globulin
fractions and occurs in a variety of disorders. An increase in the a-globulin
fraction occurs in acute inflammatory disorders, because the APPs, includ-
ing SAA and macroglobulin, migrate in the a-globulin fraction [31]. An in-
crease in the b-globulin fraction occurs in active liver disease, because
transferrin and IgM migrate in the b-globulin fraction [31]. An increase in
the beta and gamma globulin fractions (beta-gamma bridging) on SPE is
noted when there is no clear separation between the beta-2 and gamma glob-
ulin fractions. Beta-gamma bridging may be caused by an increase in IgM or
IgA, chronic active hepatitis, or lymphosarcoma [31,45]. Experimental infec-
tions of the intestinal tract with Strongylus vulgaris larvae have been asso-
ciated with an increased concentration of IgG (T) [47,48].
Hypergammaglobulinemia may be caused by a broad increase (poly-
clonal gammopathy) or a sharp increase (monoclonal gammopathy) in
gamma globulins. The broad increase in gamma globulins that characterizes
a polyclonal gammopathy is caused by the heterogeneity of clones of plasma
cells, which produce a heterogeneous mix of immunoglobulins. Any or all of
the immunoglobulin groups can be increased. A polyclonal gammopathy
often is associated with a chronic inflammatory disease, such as hepatitis,
pleuropneumonia, immune-mediated disease, neoplasia, or a chronic suppu-
rative disorder [31,45].
A monoclonal gammopathy is characterized by a sharp increase (or
spike) in one of the immunoglobulins. The monoclonal spike is caused
by a single clone of plasma cells that produces a single class of immuno-
globulin or an immunoglobulin fragment (referred to as a paraprotein, M
protein, or M component), which can be identified by the results of
electrophoresis, immunoelectrophoresis, or immunodiffusion [31,49,50].
Monoclonal gammopathy occurs infrequently in the horse and has been
associated with plasma cell myeloma [49], malignant lymphoma [51] and
idiopathic causes [52].
The diagnostic and prognostic value of SPE in horses with chronic diar-
rhea was reported [49]. Horses with larval cyathostomiasis had significantly
higher levels of beta-1 globulin. A normal concentration of beta-1 globulin
was not a reliable indicator of the absence of larval cyathostomiasis, how-
ever. Horses with chronic diarrhea that did not survive were more likely
BLOOD PROTEINS AND INFLAMMATION IN THE HORSE 295
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