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J Vet Intern Med 2007;21:489494

Serum Iron and Plasma Fibrinogen Concentrations as Indicators of


Systemic Inflammatory Diseases in Horses
Alexandre S. Borges, Thomas J. Divers, Tracy Stokol, and O. Hussni Mohammed
Background: Detection of systemic inflammation, which is important for proper diagnosis and prompt treatment, can be
challenging.
Hypothesis: Measurement of plasma iron concentration is a sensitive method for detecting systemic inflammation in horses
compared with measurements of plasma fibrinogen concentration, a traditional marker for inflammation in the horse.
Animals: Ninety-seven horses hospitalized with diseases causing systemic inflammation, 22 horses with localized
inflammation, and 12 clinically normal horses were included in this study.
Methods: A retrospective study was made on hospitalized horses that had both plasma iron and fibrinogen concentrations
measured on hospital admission.
Results: Plasma iron concentration was lower in horses with systemic inflammation (64 6 45 mg/dL) than the reference
interval minimum (105 mg/dL) and were significantly lower (P 5 .001) than the value in a group of horses with local
inflammation (123 6 45 mg/dL) and in healthy transported horses (143 6 29 mg/dL). Low plasma iron and high fibrinogen
concentrations were both sensitive indicators of systemic inflammation in horses with sensitivity of 90 and 82%, respectively.
There was a similar correlation between either continued decreases in iron concentration (Rsp of 0.239) or increases in
fibrinogen concentration (Rsp of 0.280) during hospitalization and a worse prognosis.
Conclusions and Clinical Importance: Measurement of plasma iron concentration better reflected acute inflammation than
did fibrinogen concentration.
Key words: Acute phase reactant protein; Horse; Inflammation.

ystemic inflammation occurs as part of a multitude


of disease processes in horses and other species.
The early detection of systemic inflammation is essential
in order to provide appropriate treatment. Systemic
inflammation that goes unrecognized can lead to serious
and potentially fatal sequelae for the horse, including
multiple organ failure, disseminated intravascular coagulation and laminitis.1
Inflammation results in an increase in the plasma
concentration of various proteins. Some of these
proteins are released early in the development of
inflammation and are termed acute phase proteins.
Although fibrinogen is considered an acute phase
reactant protein, values increase 24 hours after induction of inflammation and might not peak for 23 days.25
There is also a wide range of values for fibrinogen
concentration in healthy horses and the standard heat
precipitation method used for its measurement is
insensitive to small changes (can only detect changes
of $100 mg/dL).3,6,7 Serum amyloid A (SAA) and Creactive protein, 2 additional acute-phase reactant
proteins, which are commonly used for detecting acute
inflammation in human patients, are not frequently used
in equine clinical cases, although the methodologies are
described and validated for horses.5,8
In contrast to SAA and C-reactive protein determinations, serum or plasma iron concentrations are easily
performed with automated chemistry analyzers. Plasma

From the Department of Veterinary Clinical Science, Sao Paulo


State University, Unesp, FMVZ Botucatu SP, Brazil.
Reprint requests: Alexandre S. Borges, Department of Veterinary
Clinical Science, Sao Paulo State University, Unesp, FMVZ
Botucatu SP, 18618000 Brazil; e-mail: asborges@fmvz.unesp.br.
Submitted July 28, 2006; Revised November 3, 2006; Accepted
December 12, 2006.
Copyright E 2007 by the American College of Veterinary Internal
Medicine
0891-6640/07/2103-0018/$3.00/0

or serum iron concentration rapidly decreases in response to inflammation in both humans and animals.911
This effect is thought to be an important defense
mechanism by the host, because iron is required for
bacterial virulence and replication.12 In horses, a significant decrease in iron occurred within 24 hours of
experimentally induced inflammation.1315 We are aware
of only a single publication that describes iron concentration in hospitalized horses.16 Iron was abnormally low
with a variety of illnesses associated with microbial
invasion, tissue injury, and inflammatory process.16
The objectives of the current study were to determine
the sensitivity and specificity of low iron concentration
in detecting systemic inflammation in horses with
naturally occurring disease, healthy horses, and horses
with localized inflammation; determine if iron or
fibrinogen concentrations are affected by the duration
of systemic inflammation; and determine if the changes
in iron or fibrinogen concentrations over time are
prognostic indicators in horses with naturally occurring
systemic inflammation.

Materials and Methods


Animals
This was a retrospective study of 3 different groups of
hospitalized horses: horses with systemic inflammatory response,
horses with local inflammatory process, and clinically healthy
horses. Horses with systemic inflammation were further divided by
historical information on duration of most recent illness into 3
groups: acute, subacute, and chronic inflammation.
The Cornell University Animal Hospital archives were searched
for all horses that were examined between January 2003 and July
2005 with diseases associated with a systemic inflammatory
response and that had both plasma iron and fibrinogen concentrations measured on hospital admission. The clinical history and
medical records of these horses were examined for evidence of
systemic inflammation, defined as the presence of 2 or more of the
following: fever (.102uF), neutrophilia (.6.6 3 103 cells/mL),
neutropenia (,2.7 3 103 cells/mL), left shift in neutrophils (band

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Borges et al

cells noted on laboratory report), and toxic changes in neutrophils.


Foals ,30 days old and horses that had been treated with
corticosteroids before admission were excluded because age and
corticosteroid administration affect plasma iron concentration.16,17
Horses with systemic inflammatory disease were divided into 3
groups based on the duration of clinical signs of inflammation
before examination: Group 1, acute inflammation was defined as
a ,1 day history; Group 2, subacute inflammation with history
consistent with .1 and ,3 days of inflammation; and Group 3,
chronic inflammation with a history consistent with inflammation
.3 days.
Two other groups of horses were used: (1) clinically healthy
horses that had been transported to Cornell University as transport
controls for another study (n 5 12), and (2) horses (n 5 22) with
recurrent airway obstruction based on clinical signs and inflammatory cytology on a transtracheal aspirate, and not meeting
the criteria for systemic inflammation, that were examined between
2003 and 2005, comprising a local inflammatory group.
For horses that had repeated measurements of either iron (n 5
55) or fibrinogen (n 5 53) concentration during hospitalization,
a prognosis of either good, fair, or death based on the hospital
discharge statement was obtained. The changes in iron or
fibrinogen concentration (the hospital admission sample and last
sample taken before discharge or death) in these horses was used to
determine if there was a correlation between the change in these
inflammatory markers with prognosis.

Sample Analysis
Blood samples were collected into ethylenediaminetetraacetic
acid (EDTA) and heparin-anticoagulant tubes from all horses at
admission. CBC results, including a 100-cell differential cell count
and assessment for toxic changes in neutrophils, were obtained
from the EDTA-anticoagulated blood with an automated hematology analyzera and Wrights-stained blood smears. Fibrinogen
concentration was determined on the EDTA-anticoagulated blood
by a heat precipitation method.18 Plasma fibrinogen concentration
was considered high when it was greater than the reference interval
(100200 mg/dL). The heparin samples were centrifuged at 3000 3
g for 10 minutes to obtain heparinized plasma. Full plasma
biochemical profiles, including iron concentration, were measured
on the heparinized plasma samples with an automated chemistry
analyzerb with the manufacturers reagents. The procedure for iron
concentration measurement involved the liberation of iron from
transferrin-iron complexes under acidic conditions. The free iron
was then detected by the formation of a colored complex with
a specific substrate (Ferrozine).c Iron concentration was considered decreased when it was below the reference interval (105
277 mg/dL).

Statistical Analysis
A Students t-test was used to determine if there was any
difference between iron or fibrinogen concentrations in the
clinically healthy transported horses group and the group of horses
with local inflammation. The mean iron and fibrinogen concentrations among the 3 groups (horses with systemic inflammatory
disease, horses with local inflammation, and normal transported
horses) were compared by analysis of variance (ANOVA). Tukey
honest significant difference (HSD) was used post hoc to
determine which group was significantly different from the other.
McNemars test was used to assess concordance (whether there is
an association between the iron and fibrinogen concentrations in
the same horse if the values were categorized based on the reference
intervals) in the horses with systemic and local inflammation.
Spearmans rank correlations (Rsp) for iron and fibrinogen
concentrations were performed in the horses with systemic

inflammation to determine the correlation between these results.


A Tukey HSD was used to compare horses with different durations
of systemic inflammation (acute, subacute, and chronic) and the
local inflammatory control group to determine if iron or fibrinogen
concentration was superior for detecting systemic inflammation in
the individual subgroups (acute, subacute, chronic). A Fishers
exact test with 2 3 2 tables to compare the proportions of horses
with low iron or high fibrinogen concentrations in each of the
subgroups (acute, subacute, chronic inflammation) was done.
Sensitivity was defined as the number of horses with systemic
inflammation that had iron concentration below or fibrinogen
concentration above the reference interval at hospital admission.
Specificity was defined as the number of clinically healthy horses or
horses with local inflammation that had iron or fibrinogen
concentrations within the reference interval. Two Spearman rank
correlations between the change in serum iron or fibrinogen
concentration during hospitalization and outcome were calculated
to determine if changes in these tests were correlated with outcome.
The level of significance was set at P , .05.

Results
Ninety-seven horses met the criteria for systemic
inflammatory response and were included in this study.
These 97 horses were diagnosed with a variety of
inflammatory diseases with infectious intestinal and
respiratory diseases being the most common. The
plasma iron concentration was significantly lower in
horses with systemic inflammation than in both the
clinically healthy transported horses and the group of
horses with local inflammation (Table 1). The plasma
iron and fibrinogen concentrations of the horses with
systemic inflammation were well below and above,
respectively, the reference range for the laboratory
(Table 1). There were no significant differences in
plasma iron concentration between clinically healthy
transported horses and horses with local inflammation
(Table 1). The plasma fibrinogen concentration was
significantly higher in the horses with systemic inflammation than clinically healthy transported horses
and horses with local inflammation. However, horses
with local inflammation had significantly higher fibrinogen concentration than clinically healthy transported
horses (P 5 .01) (Table 1).
Results for iron and fibrinogen concentrations were
concordant in 74% (72/97) and 59% (13/22) of the horses
with systemic and local inflammation, respectively
(Table 2). There was a negative correlation between
plasma iron and fibrinogen concentrations (R of
20.240) among the 97 horses with systemic inflammation.
Of the 97 horses with systemic inflammation, 28 had
acute, 19 had subacute; and 50 had chronic inflammation. The iron concentration was not significantly
different among these groups, although all 3 groups
had significantly lower iron concentration than the
horses with local inflammation. The fibrinogen concentration was not significantly different among the 3
groups of horses with acute, subacute, or chronic
systemic inflammation. Horses with subacute or chronic
systemic inflammation had significantly higher fibrinogen concentrations than that of horses with local
inflammation, but the mean fibrinogen concentration

Iron and Systemic Inflammation

491

Table 1. Iron and fibrinogen concentrations in plasma of horses with systemic or local inflammation (recurrent
airway obstruction) or of healthy transported horses.
Variable

Horses with Systemic


Inflammation (n 5 97)

Horses with Local


Inflammation (n 5 22)

Clinically Healthy
Transported Horses (n 5 12)

Reference Interval

Iron (ug/dL)
Fibrinogen (mg/dL)

64 6 45
445 6 213a

123 6 45
277 6 138b

152 6 33
158 6 67c

105277
100200

a,b,c

Data are shown as mean 6 SD. Means with different superscripts within each row are significantly different (P,.05).

Table 2. Concordance between plasma iron and fibrinogen concentrations in horses with systemic and local
inflammation (recurrent airway obstruction).
Systemic Inflammation

Local Inflammation

High Fibrinogen

Normal Fibrinogen

High Fibrinogen

Normal Fibrinogen

71
9

16
1

5
7

2
8

Low iron
Normal iron

in horses with acute systemic inflammation was not


significantly different from that of the local inflammatory group (Table 3).
When the proportions of horses with low iron
concentration and high fibrinogen concentration were
compared within each of the 3 systemic inflammatory
subgroups (acute, subacute, or chronic systemic inflammation), significantly more horses with acute inflammation had low iron concentration than high
fibrinogen concentration (P 5 .039). These proportions
were not significantly different in the other 2 inflammatory subgroups (data not shown).

Low iron concentration was a sensitive test for


systemic inflammation and was highly specific when
the clinically healthy transported horses were used to
establish the specificity (Table 4). Only 1 of the 12
transported horses had an iron concentration (100 mg/
dL) below the reference interval (105 mg/dL). Specificity
of low iron concentration as a test for systemic
inflammation was lower when horses with local inflammation were used as a comparison group (Table 4).
High fibrinogen concentration was also a sensitive test
for detecting systemic inflammation, although it was less
sensitive than a low iron concentration (Table 4). The

Table 3. Iron and fibrinogen concentrations in plasma of subgroups of horses with systemic inflammation based on
the duration of inflammation before referral (acute inflammation ,1-day history; subacute inflammation .1- and
,3-day history; and chronic inflammation .3-day history) and those with local inflammation (recurrent
airway obstruction).
Horse Groups

Acute Systemic Inflammation


(n 5 28)

Subacute Systemic
Inflammation (n 5 19)

Chronic Systemic
Inflammation (n 5 50)

Horses with Local


Inflammation (n 5 22)

51 6 38a
368 6 177a,b

75 6 57a
463 6 227a

67 6 43a
482 6 21a

123 6 45b
277 6 138b

Iron (mg/dL)
Fibrinogen (mg/
dL)
a,b

Data are shown as mean 6 SD. Means with different superscripts within each row are significantly different (P , .05).

Table 4. Estimates of sensitivity in horses with systemic inflammation and specificity in horses with local (recurrent
airway obstruction) or no inflammation (healthy transported horses) for low iron, high fibrinogen, or either low iron
or high fibrinogen concentrations combined.
Sensitivity Systemic
Inflammation [97]

Specificity Local
Inflammation [22]

Specificity Healthy
Horses [12]

Low iron Concentration


(,105 ug/dL)

90% (87)

68% (7)

92% (1)

High fibrinogen concentration


(.200 mg/d)

82% (80)

45% (12)

92% (1)

Low iron or high fibrinogen


concentrations

99% (96)

36% (14)

83% (2)

Parentheses ( ), number of horses in each group positive for the test. Square brackets [ ], total number of horses in group.

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Borges et al

specificity of a high fibrinogen concentration as a measure of systemic inflammation was similar to a low iron
concentration when clinically healthy horses were used
to determine specificity. Only 1 of the latter 12 horses
had a fibrinogen concentration (300 mg/dL) above the
reference interval. High fibrinogen concentration had
a lower specificity than a low iron concentration when
horses with local inflammation were used to determine
specificity (Table 4). Using either a low iron or high
fibrinogen concentration together improved sensitivity
for detecting horses with systemic inflammation, but
specificity declined.
There was a similar correlation between either
continued decreases in iron concentration (Rsp of
0.239) or increases in fibrinogen concentration (Rsp of
0.280) with hospitalization, and a worse prognosis.

Discussion
The results of this study indicate that low plasma
iron concentration is a sensitive test for detecting
systemic inflammation in horses. Values were significantly lower in horses with systemic inflammation,
regardless of duration of inflammation. This is not
surprising considering that iron concentration decreases
as soon as 5 hours after induction of inflammation by
turpentine injection in rats.19 In a study in Shetland
ponies, iron concentration decreased to less than 25%
of that in control ponies 24 hours after the administration of turpentine intramuscularly.14 Also, in a
recent study comparing SAA and other acute phase
reactants to monitor the inflammatory response after
castration in horses, it was concluded that only SAA and
serum iron concentrations were useful for monitoring
severity of postoperative inflammation.5 Serum iron
concentrations predict the severity of clinical signs and
decreased before the onset of clinical signs (within
24 hours) and pyrexia produced in an experimental
disease model of Streptococcus zooepidemicus infection
in horses.13
Inflammatory processes, including infections, the
presence of endotoxins, or both can cause a rapid
(,24 hours) lowering of iron concentration and this is
thought to be important for enhancing the nonspecific
resistance to bacterial infection.12,20,21 Observations of
iron exchanges during inflammation in rats suggests that
there is a decrease in both iron absorption from gut and
iron release by reticuloendothelial cells, causing a drop
in plasma iron concentration.19 This occurs rapidly after
an inflammatory process is initiated and is thought to be
mediated by release of a recently described hepatic
peptide, hepcidin.22,23 Inflammation causes an increases
in systemic concentrations of interleukin (IL)-6, that
directly stimulates hepatocytes to release hepcidin,
which then blocks the efflux of iron from macrophages
(by binding to the iron efflux channel ferroportin and
causing its internalization), resulting in a rapid development of hypoferremia.2225 This host response is
particularly valuable during the earliest phases of
infection, before other components of innate and
adaptive immunity are mobilized.

Fibrinogen, a well-accepted acute-phase reactant protein in the horse, was not as sensitive as iron in detecting
systemic inflammation in this cohort of horses. Determination of the plasma fibrinogen concentration has previously been found to be particularly useful in detecting
inflammatory diseases26 but has the disadvantage of
being a slow-reacting acute-phase protein, as its concentration increases slowly in response to tissue injury.5
Similar to iron concentrations, fibrinogen concentrations were not significantly different in the groups of
horses with different durations of systemic inflammation. However, when iron and fibrinogen concentrations
in these subgroups were compared with normal values,
iron concentration was a better marker of acute (horses
with a history of inflammation ,24 hours) systemic
inflammation than fibrinogen concentration. This finding is probably because iron concentration falls more
rapidly than fibrinogen concentration rises with systemic
inflammation. Indeed, fibrinogen concentration changes
relatively slowly in response to disease, sometimes
requiring 3648 hours to maximally increase after
a single inflammatory insult.3,7 Both an increase in
fibrinogen concentration and a decrease in iron concentration were observed in an acute-phase reaction elicited
in 4 horses in which Freuds adjuvant was administered
intramuscularly.15 In the latter study, iron concentration
decreased to less than 30% of the pre-inflamed value on
day 1, whereas fibrinogen concentration was only
increased on day 3. The effect of surgery on the acutephase response in clinically normal and diseased horses
was studied, and it was observed that fibrinogen
concentration increased more slowly than either iron
or SAA after surgery; similar results were obtained in
castrated horses.5,7 Hence, iron concentration appears to
be a more sensitive test than fibrinogen concentration
for detecting acute systemic inflammation in horses.
Although we cannot be certain of the duration of
inflammation in the horses in this study, the higher
sensitivity of low iron than high fibrinogen for detecting
acute inflammation in the study is consistent with
experimental models and would suggest that horses
assigned to the subacute systemic inflammatory group in
this study were, as a whole, properly assigned. If either
low iron or high fibrinogen concentration was used as
a test for systemic inflammation, only 1 of the 97 horses
would have been misclassified.
When compared with clinically healthy horses transported to the clinic, iron and fibrinogen concentrations
were specific indicators of inflammation. However,
specificity of both tests decreased in horses with
recurrent airway obstruction, the model of localized
inflammation used in this study. Although none of these
latter horses met the predetermined criteria for systemic
inflammation, it might be that the classification of local
versus systemic inflammation based on these criteria is
not accurate. Indeed, the changes in iron and fibrinogen
concentrations in some of these horses suggest they had
some degree of systemic inflammation. A negative
correlation between plasma iron and fibrinogen concentrations in individual horses with systemic inflammation in this study was also observed in inflammation

Iron and Systemic Inflammation

induced in horses.15 This finding was expected because


inflammation causes a decrease in iron concentration
and increase in fibrinogen concentration. This relatively
weak correlation in this study might be explained by
differences in the duration, type of inflammation, or
both in these horses.
In this study, iron concentration was similarly low
in horses with systemic inflammation, regardless of
the duration of the process, suggesting that iron
would not return to normal until the inflammatory
process was resolved. After inflammation was induced
experimentally with a single event in horses, iron
concentration decreased within 24 hours and normalized within 6 days.14 Changes in iron and SAA
concentrations were also good indicators of resolving
inflammation after castration in horses and reflected
this process better than fibrinogen concentration.5 In
this study, a decline in serum iron (and an increase
in fibrinogen) during hospitalization was associated
with a poor prognosis. There was no attempt to show
the opposite trend in horses assigned a good prognosis
because a large number of those horses were discharged after the diagnostic workup and most of the
treatments were administered at the farm, preventing
sufficient repeat iron or fibrinogen data capture on those
horses.
There are some factors that might affect the use of
iron concentration as a marker of systemic inflammation. These include age, corticosteroids, hemolysis (in
vitro or in vivo), liver disease, and iron supplementation.17,27 Horses with liver disease or hemolysis frequently have high iron concentration in spite of systemic
inflammation (Divers and Stokol, unpublished observations), and iron concentration should not be used as
a predictor of inflammation in horses with these
conditions. Absolute iron deficiency is extremely rare
in adult horses, so low iron concentration is most likely
a result of inflammation in the horse.16
In summary, the results of this study show that
hypoferremia is a sensitive indicator of acute, subacute,
and chronic systemic inflammation in sick horses .2
months of age. Iron concentration may be preferable
to fibrinogen concentration for detecting acute inflammation in horses. The use of both iron and
fibrinogen concentrations will further increase the
sensitivity of these tests for diagnosing systemic inflammatory disease in horses. In this study, a continued
decrease in iron concentration and increase in fibrinogen
concentration with hospitalization was associated with
a poor prognosis, suggesting that changes in these
parameters might be useful for monitoring response to
therapy.

Footnotes
a

Advia 120, Bayer Healthcare, Tarrytown, NY


Hitachi 917, Roche Diagnostics, Indianapolis, IN
c
Ferrozine, Roche Diagnostics, Indianapolis, IN
b

493

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