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Vet.
Pathol.
22:
387-392
(1985)
Experimental Onion-Induced Hemolytic Anemia in
Dogs
J.
W.
HARVEY
and D.
RACKEAR
Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville,
FL
Abstract. Within one day following a single oral dose of dehydrated onions, dogs were found to have large numbers
of
Heinz bodies within erythrocytes. The percentage of erythrocytes that contained Heinz bodies increased slightly to a maximum
on day
3
and then declined. The turbidity index increased more gradually with a maximal value on day 4. Erythrocytes with
hemoglobin contracted to one side of the cell (eccentrocytes) also appeared after onion feeding. Eccentrocytes are believed to
result from a direct injury to the erythrocyte membrane. As with Heinz body-containing cells, the percentages of eccentrocytes
present declined as anemia developed. The packed cell volume began
to
decrease one day after onion administration. A mean
decrease of
19
percentage points was reached by day
5.
The most anemic dogs had evidence
of
intravascular hemolysis.
Reticulocytosis was first observed five days after onion administration.
A
slight increase in methemoglobin content was
measured four hours after onion administration.
No
significant changes in erythrocyte reduced glutathione concentration
were measured. Transient neutrophilia occurred concomitant with the peak reticulocyte response.
Hemolytic anemia that results from the consumption
or oral administration of onions has been reported in
cattle," ~heep,'~ horses," cats," and
dog^.^.^.
12,19,21.22
Onion-induced anemia in dogs was first recognized in
1930,
during research designed to test the potential
value
of
dietary onions in prevention
of
niacin defi-
ciency." The hemolytic nature
of
the anemia was dem-
onstrated in a study the following year.6 Autoclaved
onions and autoclaved onion juice produced effects
similar to raw onions.
Several clinical cases
of
onion-induced hemolytic
anemia have been reDorted in dogs3.
12.21.22
In contrast
female) were fed
5.5
g/kg body weight minced dehydrated
onions (Sauers Dehydrated Minced Onions, C.
F.
Sauer
Co.,
Richmond, VA) mixed with
0.5
kg of raw ground beef. Three
control dogs were fed
0.5
kg raw ground beef with no onions
added. The following day, all dogs were placed back on their
regular diet (Purina Dog Chow, Ralston Purina
Co.,
St. Louis,
MO). Desiccated onions are reported to have less oxidative
potential than fresh
or
cooked onions,' but were used in this
study because of ease
of
administration. Blood samples were
collected, using
ethylenediaminetetraacetic
acid as an antico-
agulant, at time
0
(before feeding onions and/or ground beef),
at two, four, and six hours, and on days
I,
2,
3,
5,
7,
and
9.
Parameters measured in all blood samples included packed
cell volume (determined by the microhematocrit method),
v
to
early
experimental studies where onions were fed
reduced glutathione,I6 reticulocyte count," turbidity index,'
total and differential leukocyte count,'' Heinz body count,
and eccentrocyte count. Heinz bodies were stained by incu-
daily as part of the diet,6.19 severe anemias apparently
occurred in clinical cases after a single ingestion of
bating
one
part
blood with
two
methyl
violet
stain
(
%
onions. One dog consumed several Ounces of dehY-
methyl violet
in
0.85%
NaCI) for
15
minutes prior to blood
drated
onion^,^
and a second dog ate raw onions.22 The
film preparation. The Heinz body count was determined as
remaining dogs were fed table scraps that contained
the percentage
of
cells that had Heinz bodies by examination
of
500
erythrocytes. The eccentrocyte count was determined
cooked onions."."
by examining
500
erythrocytes on each Wright-Giemsa-
sequen-
stained blood film and reported as the percent of total eryth-
tial hematologic changes in dogs given a single oral dose
rocytes that appear as eccentrocytes.' Adjusted reticulocyte
of
onions. In addition to routine hematologic tests,
counts were calculated by multiplying the percentage of retic-
erythrocyte reduced glutathione, methemoglobin, and
ulocytes counted by the measured packed cell volume divided
Heinz bodies were quantified.
by 45 (mean normal packed cell volume for dogs). The
turbidity index was determined by measuring the difference
in absorbance of cyanomethemoglobin solutions
(1
to
501
dilution of blood) at 540 nm in 1-cm-pathway cuvettes before
and after centrifugation at
1000
g for three minutes, and
multiplying the difference times
1000
divided by the packed
The
present study
was
conducted
to
Materials and Methods
Adult, clinically normal mixed-breed dogs were utilized in
this study. After a 24-hour fast, six test dogs (five males, one
387
388
Harvey
and
Rackear
cell
volume
(%).8
Blood methemoglobin content was deter-
mined on
all
samples
up
to and including the
day
3
sample
and expressed as percentage of
total
hemoglobin.'
Statistical analyses of results were done utilizing the Stu-
dent's
f-test
for comparisons of
test
group
and
control group
means
and
the analysis
of
variance
and
least significant
dif-
ference tests for comparisons within a group.
All
differences
mentioned
in
the
text
are
significant
(p
<
0.05)
unless other-
wise indicated.
Results
Initial packed cell volume ranged form 39
to
55%
in
test dogs. The change in packed cell volume relative to
initial
0
time values is given (fig.
1).
Packed cell volumes
began
to
decrease on day
1,
with the lowest mean value
creases in packed cell volume varied considerably.
Three dogs had maximal decreases of
25
to 30 units,
while the remaining 3 dogs had maximal decreases of
only
12
to 13 units. The packed cell volume in control
dogs was unchanged during the experimental period.
Intravascular hemolysis, observed as red plasma in
centrifuged microhematocrit tubes, was present
on
days
1
and
2,
in two of the most severely
affected
dogs.
Although urine was not collected for evaluation in this
study, prominent hemoglobinuria was observed in one
of these dogs on day
2.
Significant Heinz body formation was observed on
day
1,
with maximal percentages seen on day 3 (fig.
1).
No
Heinz bodies were observed in control animals.
reached on day
5.
Packed cell volumes had not in-
creased significantly by day 9. The magnitude of de- Heinz bodies varied in appearance from a single large
one to several small ones per erythrocyte. Heinz body
I
.81
X
w
z
a
>
L
-
e
m
LL
3
I
.5
I
.2
0.9
0.6
0.3
k
0.1
J
90
-
8
n
m
Y
cn
60
0
N
z
I
Li
30
Fig.
1:
Mean
(+
standard error) values
for turbidity index, percentage
of
erythro-
cytes
that
contained Heinz bodies, and
changes
in
packed cell volume
(A
PCV)
from test dogs (open triangles,
n
=
6)
fed
ground beef
that
contained dehydrated on-
ions
and control dogs (closed circles,
n
=
3)
fed
only
ground beef
at
time
0.
Changes
in
packed cell volume are given
as
percent-
age points above
(+)
or below
(-)
the pre-
feeding (time
0)
packed cell volume value.
T-
i
-2
5-1
I
I
I
I
I
I
I
6
I
2
34
5
6
+
b
9
DAYS
Onion-Indu
numbers decreased after day 3, but were still present
on the last day examined (day 9).
A
significant increase in turbidity index was observed
on day
1
(fig.
1).
The turbidity index reached a maxi-
mum on day
5
and then declined. The mean turbidity
index in control dogs remained low throughout the
experimental period.
Various degrees of poikilocytosis was observed on
routinely stained blood films after onion consumption.
In addition to nipple-like projections, that resulted from
membrane-bound Heinz bodies, erythrocytes were fre-
quently observed to have hemoglobin that appeared
dense and contracted to one side of the cell and left a
pale area (fig.
2).
These cells have previously been called
eccentrocytes in dogs.' The percentage of erythrocytes
classified as eccentrocytes increased significantly, being
maximal one to two days after onion consumption
(fig.
3). Considerable individual variability was observed,
with maximal eccentrocyte counts that ranged from
3
to
20%.
All dogs had eccentrocytes on days
1
through
7,
but two dogs lacked eccentrocytes by day 9. Eccen-
trocytes were not present in control dogs.
Adjusted reticulocyte counts in dogs fed onions were
significantly increased above
0
time and control dog
values on days 5 through 9 (fig. 3). Although not
statistically significant, dogs with the greatest decreases
in packed cell volume tended to have lower reticulocyte
responses than did mildly affected dogs.
Fig. 2: Eccentrocytes (arrows) in blood film from a dog
two days after feeding ground beef that contained dehydrated
onions.
Wright-Giemsa.
ced Anemia
389
Methemoglobin content in erythrocytes increased
slightly (but significantly) from 0.10
+
0.04%
(k
SE)
at
0
time to a maximum of 1.9
+
0.3% (not shown) four
hours after onions were fed. Methemoglobin content
was no longer significantly increased by day
2.
Methe-
moglobin content in control dogs remained low (mean
daily values between
0.3%
and
0.7%)
during the ex-
amination period.
Although a substantial decrease in reduced glutathi-
one from
2.9
pmoles/ml erythrocytes to 1.2 pmoles/ml
erythrocytes was observed in the most severely affected
dog after onion feeding, most test dogs had no decrease
in reduced glutathione content. Consequently, no sig-
nificant differences
in
erythrocyte reduced glutathione
content were found following oral onion administra-
tion.
Mean absolute neutrophil numbers increased signifi-
cantly from 9,890
f
1,05O/pl
(+
SE)
at
0
time to a
maximum of 19,840
f
3,82O/pl on day
7
(not shown).
This maximal value was also higher than the control
group on day
7
(9,540
f
1,44O/pl), but the difference
was not statistically significant. Two of the six onion-
treated dogs had moderate left shifts associated with
their neutrophilias.
No
significant changes were ob-
served in absolute lymphocyte, monocyte, or eosinophil
counts. Low numbers of nucleated erythrocytes (gen-
erally less than
6/
100 leukocytes) were consistently
observed on blood films from test dogs on days 3
through 9. Nucleated erythrocytes were not present in
control dog blood samples.
Discussion
Onion ingestion causes oxidative erythrocyte injury
as evidenced by the production of Heinz bodies. Heinz
bodies are erythrocyte inclusions that form as a conse-
quence of irreversible oxidative denaturation of hemo-
globin.
l3
In the present study, the denaturation and precipita-
tion of hemoglobin was measured by determining a
turbidity index as well as Heinz body count. Following
a single oral dose of desiccated onions, Heinz body
numbers reached near-maximal values more rapidly
than did turbidity indexes. In a study of oxidant-in-
duced Heinz body formation in cats,' Heinz bodies
were observed to increase in size for three days after
oxidant administration. It is likely that the increase in
turbidity index during the first four days in the present
study represents a continued aggregation and precipi-
tation of denatured hemoglobin. Changes in Heinz
body size could not be accurately measured in these
Harvey
and
Rackear
+5
0
r
Fig.3:
Mean
(+
standard error) values
for percentage
of
eccentrocytes, percentage
of
adjusted reticulocytes (adj. retic.) and
percentage points change in packed cell
volume
(A
PCV)
from test dogs (open
tri-
angles, n
=
6)
fed ground beef that con-
tained dehydrated onions and control dogs
(closed circles, n
=
3)
fed only ground beef
at time
0.
-25
J
I
1
1
I
I
I
I
1
I
1
0
I
2
34
56
7
89
DAYS
dogs, due to the frequent occurrence of multiple, rather
than single, Heinz bodies within erythrocytes.
In addition to Heinz body formation, other potential
forms of oxidant-induced damage to erythrocytes in-
clude oxidation of membrane lipid and sulfhydryl
groups and methemoglobin formation. Eccentrocytes
are believed to form as a result
of
direct oxidative injury
to the erythrocyte membranes.’ They have previously
been reported to occur in dogs after the subcutaneous
injection of acetylphenylhydrazine.’ Eccentrocytes are
erythrocytes with hemoglobin that appears dense and
contracted to one side of the cell and leaves a pale area
that still contains a small amount of hemoglobin. Sim-
ilar abnormal erythrocytes, recognized in people given
a variety of oxidant drugs,’ have also been described as
pyknocytes2 and erythrocyte hemighosts.’ Eccentro-
cytes are not spherocytes, although they were mistak-
enly classified as such in a case of methylene blue-
induced hemolytic anemia in a dog.17 The observation
of eccentrocytes on blood films from clinical cases
should prompt a search for Heinz bodies if they have
not already
been
recognized.
Eccentrocytes are more rigid and less deformable
than normal erythrocytes. In experimental studies of
acetylphenylhydrazine-induced
hemolytic anemia in
dogs, erythrocyte destruction was more clearly related
to eccentrocyte formation than to Heinz body forma-
ti~n.~
Most drugs that induce Heinz body formation also
induce methemoglobin f0rmati0n.l~ Although in-
creased methemoglobin content was measured in eryth-
rocytes following onion administration, the increase
was small and of
no
clinical significance.
Reduced glutathione is a sulfhydryl-containing tri-
peptide that is considered to be important in the pro-
tection of erythrocytes against oxidative injury. It func-
tions as a substrate for both the glutathione peroxidase
enzyme and for an additional uncharacterized pro-
tein(s) that protects against lipid per~xidation.~
Al-
though a dramatic decrease in erythrocyte-reduced glu-
tathione was measured in the most severely affected
dog, no change was observed in most other dogs given
onions. Erythrocytes can reduce oxidized glutathione,
and some oxidant drugs actually increase glutathione
Onion-Induced Anemia
39
1
synthesis in
erythrocyte^'^;
consequently, glutathione
concentration may be normal or even increased in
association with oxidant stress.
Ally1 propyl disulfide has been considered to be the
main constituent in onion oil responsible for the oxi-
dative damage to
erythrocyte^.^
This compound has not
been studied experimentally, but a closely related com-
pound, di-n-propyl disulfide, has been shown to have a
hemolytic effect in dogs similar to onion administra-
Onions, garlic and
Brussicu
spp are also re-
ported to contain a rare amino acid S-methylcysteine
sulphoxide.20 This compound is relatively inactive as
an oxidant, but can be converted to the oxidant di-
methyl disulfide by rumen microorganisms in ruminant
animals.20 It is not known whether this amino acid can
be metabolized to a substance with oxidative potential
in dogs.
Dogs appear to vary considerably in susceptibility to
orally administered onions. Three dogs had dramatic
decreases in packed cell volume and three were mildly
affected. Anemia developed gradually over several days.
Consequently, in the absence of substantial methemo-
globinemia, clinical signs of onion toxicity may not be
recognized until a number
of
days after onion con-
sumption. Delayed hemolysis after oxidant drug expo-
sure has previously been recognized in animals and
man.8 The only early clinical sign in the present study
was the occurrence of diarrhea for one to two days after
oral onion administration.
The reticulocyte response to the anemias in this study
was dramatic. The peak response occurred several days
after substantial decreases in packed cell volume had
occurred. This delayed response is expected inasmuch
as several days are required for increased reticulocyte
production to occur in bone marrow.18 Based on the
decreasing Heinz body counts and increased reticulo-
cyte counts, the plateau
of
packed cell volume between
days
5
and
9
probably results from new erythrocyte
production matched with continued destruction
of
ox-
idant injured cells.
The only significant leukocyte change was a neutro-
philia. Mean neutrophil numbers were highest during
days when mean reticulocyte counts were highest. Neu-
trophilias with left shifts have been reported in two
clinical cases of onion-induced hemolytic anemia in
dog^.^,^
'
Acknowledgement
The authors thank Patricia McNulty for technical assist-
References
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VAN KAMPEN, K.R.; JAMES, L.F.; JOHNSON, A.E.: He-
Request reprints from
Dr.
John
w.
Harvey, Box
5-144,
College
of
Veterinary Medicine. University
of
Florida, Gainesville,
FL
32610
(USA).