ISRAEL JOURNAL OF
VETERINARY MEDICINE
Vol. 55(4) 2000
SERUM ADENOSINE DEAMINASE ACTIVITY IN DOGS:
ITS IMPORTANCE IN EXPERIMENTAL LIVER
INTOXICATION
N. Altug and Z.T. Agaoglu
Department of Internal Diseases, Faculty of Veterinary Medicine, University of Yuzuncu Yil,
65080 Van, Turkey
Abstract
In the present study, ADA levels were investigated together with clinical, biochemical,
hematological and histopathological findings in dogs experimentally intoxicated with carbon
tetra chloride (CCl4) to determine its role in liver intoxications.
Twenty healthy cross-breed dogs were divided in two groups of ten as acute and chronic
intoxication groups. CCl4 was prepared in olive oil (1:1). Dogs in the acute group received 1.5
ml/kg CCl4 once only. Dogs in the chronic group received 0.5 ml/kg CCl 4 twice a week for 12
was adminisreted by orogastric probe after 12 hours of hunger. Clinical,
haematological and biochemical analysis were made in all dogs 2 days before the
experiment and on days 1, 3, 5, 7 and 9 in the acute group and four times at 21 day
intervals in the chronic group. Liver biopsies were taken on days 4 and 9 from the
acute group and at weeks 6 and 12 from chronic group.
weeks. CCl4
During the experiment signs of inappetance, stillness, watery feces and mild dehydration in
the acute group, and inappetance, weakness, light colour of feces, dark orange colour of
urine and depression in the chronic group were observed. Erythrocyte and haemoglobin
levels increased significantly (p<0.05) on the third day in the acute group, whereas, total
leukocyte counts increased significantly (p<0.001) in the chronic group. Significantly
important increases (p<0.001) were also observed in biochemical parameters (serum AST,
ALT, ALP and ADA) in both groups of dogs. On histopathology, precise hepatocellular
hydropic degeneration, necrosis in the centrilobular and midzonal regions and dissociation in
remarc cordons were observed in the acute group. Fibrosis was observed in the portal
regions and around the vena centralis in the chronic group.
It is concluded that determination of ADA activity can be useful in the assesment of liver
degeneration caused by toxic hepatopathy in dogs and ADA activity can be added to other
routine biochemical tests employed in diagnosis of liver intoxication.
Key Words: Dog, Liver toxication, CCL4, Symptoms, ADA.
Introduction
Adenosine deaminase (ADA) catalyses hydrolytic deamination of either adenosine or
deoxyadenosine to produce inosine and deoxyinosine respectively (1). Several ADA
isozymes exist, with different molecular weights, kinetic properties and tissue distribution (2).
In human (1) and animals (3) ADA consist of three isoenzymes: ADA I, ADAI+ complex
protein (CP) and ADA 2. ADA 2 is the main component of serum ADA activity (2), while the
others (ADA I and ADA I+CP) are major components of tissues (2, 3).
ADA has been found in high levels in the spleen, lungs and lymph nodes, and in low levels in
the myocardium, muscles, kidneys, bladder and liver (4). It has been detected in the cell
cytoplasm and nucleus (5). ADA activity has been reported to be high in lymphoid tissues and
its inadequacy reported to cause immune deficiency (1, 6). On the other hand, ADA activity
increases during hepatitis, tuberculosis, salmonellosis, brucellosis, toxoplasmosis, visceral
leishmaniasis, rickettsiosis, infectious mononucleosis, pneumonia, rheumatoid arthritis and
leukemia in humans (1, 6), enzootic muscular dystrophy in lambs (7), feline infectious
peritonitis (8), hepatopathy in dogs (8) and various hepatic diseases and leukosis in cattle (9).
Although ADA activity reportedly increases in acute and chronic hepatitis, hepatic fibrosis,
liver cirrhosis and hepatoma (10), its importance in veterinary medicine, its state in acute and
chronic liver diseases and its relationship between other routinely used biochemical tests
have not been investigated. In the present study, ADA activities were investigated with
clinical, biochemical, hematological and histopathological findings in dogs experimentally
intoxicated with CCL4 to determine its role in liver intoxication.
Materials and Methods
In this study, 20 healthy cross-bred dogs aged between 1 and 6 years and weighing 15 to 28
kg were divided into two groups of ten as acute and chronic intoxication groups. CCL4 (
Merck) were prepared in olive oil (1:1) and given to the dogs by oro-gastric probe after 12 hours of
hunger. Dogs in acute group received 1.5 ml/kg CCL4 only once, and the chronic group received 0.5
ml/kg twice a week over 12 week.
Before and during the experiment, clinical examinations were made and the findings
recorded. Ten ml of blood without anticoagulant and 5 ml blood with anticoagulant (EDTA)
were taken from the animals 2 days before experiment and on days 1, 3, 5, 7 and 9 in the
acute group and four times over 21 days intervals in the chronic group. Whole blood was
used to assess hematological changes. For this purpose. Coulter-Maxim cell counter was
used. Blood without anticoagulant was used to prepare serum samples to determine
aspartate aminotransferase (AST) (Randox AS-483), alanine aminotransferase (ALT)
(Randox AL- 485) and alkaline phosphatase (ALP) (Randox AP-502) levels using commercial
kits. ADA activity was determined colorimetrically by a modified Martinek method (11). Liver
biopsies with the guide of ultrasonography were taken on days 4 and 9 from the acute group
and on weeks 6 and 12 from the chronic group for examining histopathological changes.
For statistical analysies. Student's t test was used to determine the effect of CCl4 on several
biochemical and histopathological parameters (12).
This study was approved by the ethics committee. The dogs used in the experiment were
hospitalized after the experiment at the Animal Hospital. They were fed with a controlled diet
(low in protein, fat and carbohydrates) containing a balanced vitamin and mineral supplement.
Diuretic and sodium restricted diet were given to one dog with submandibular oedema. All the
dogs also received antibiotic and glycocorticoids for their antibacterial, antifibrotic properties
respectively. The dogs were kept one month in the hospital then were donated to a dog
rehabilitation center when they were clinically healthy.
Results
After CCL4 adminstration, non-specific liver toxicity symptoms such as anorexia, stillness and
watery feces were seen in the acute group of dogs. These signs continued for 3 days and
disappeared by 9 days post-inoculation. In the chronic group anorexia, weakness, light colour
and muddy feces, dark orange urine and depression were observed as the number of doses
increase. Furthermore, in 4 dogs low level of icterus, in 2 dogs polydypsia and in one dog with
submandibular oedema were observed.
On hematological examination, total leukocyte (WBC), thrombocyte and hematocrit values
(PCV) were not significantly changed during the experiment compared with the preexperiment values of the acute group. However, statistically significant increases(p<0.05)
were observed in erythrocyte (RBC) and hemoglobin (Hb) values only on day 3. Furthermore,
RBC, Hb, PCV and thrombocyte values were not significantly different from the preexperinantal values in the chronic group. The WBC values, however, significantly increased
during experiment (p<O.OOl).
Serum AST, ALT, ALP and ADA levels in the acute group before and after experiment are
given in Table 1. These enzymes increased significantly (p<0.001) as seen in Table 1 except
for AST on day 9. During the experiment, serum enzyme activities peaked on day 1 for AST,
ALT and ADA, on day 3 for ALP. Serum AST, ALT, ALP and ADA activities before and after
experiment (weeks 3, 6, 9 and 12) in the chronic group are given in Table 2. All the enzyme
levels increased significantly (p<0.001). The experiment was terminated at 12 weeks, when
the values were still increasing.
Table I :
Biochemical levels in the acute intoxication group
DAYS
AST (U/L) X±Sx
ALT (U/L) X±Sx
ALP (U/L) X±Sx
ADA (U/L) X±Sx
29.86±1.7
32.57±1.4
113.0±4.5
2.34±0.24
1
387.9±19*
425.4±14.0*
180.7±4.0*
5.41±0.32*
3
315.4±13*
321.6±10.6*
455.2±6.9*
5.09±0.09*
5
175.0±5.7*
222.5±7.32*
399.9±11.0*
4.83±0.10*
7
63.14±3.2*
124.4±5.0*
311.4±11.0*
3.40±0.15*
9
32.43±2.0
68.0±1.6*
268.5±6.7*
2.94±0.19*
Before experient
* p<0.001, n=10
Table 2 :
Biochemical levels in the chronic intoxication group
Weeks
AST (U/L) X±Sx
ALT (U/L) X±Sx
ALP (U/L) X±Sx
ADA (U/L) X±Sx
Before experient
37.88±1.0
39.63±1.77
98.87±4.23
2.83±0.20
3
238.37±6.55*
456.5±10.0*
647.4±11.7*
13.02±0.20*
6
298.30±11.8*
490.9±11.4*
791.75±9.55*
17.56±0.33*
9
320.7±10.3*
952.9±11.3*
22.12±0.48*
12
370.8±11.9*
575.3±11.1*
626.3±11.0*
1093.1±10.4*
24.18±0.43*
* p<0.001, n=10
Liver biopsies were taken on days 4 and 9 from the acute group and on weeks 6 and 12
from the chronic group. Precise hepatocellular hydropic degeneration, necrosis in
centrilobular and midzonal regions, and dissociation in remarc cordons were observed in the
acute group (Figure 1). Fibrosis was observed in the portal regions, around the vena
centralis and paranchimous tissues in the chronic group (Figure 2).
Picture
1.
Histopathological
appearance of a liver
section
from
the
acute
intoxication
group (HE x 80).
Hepatocellular
degeneration
and
necrosis
(1)
in
centrilobular (2) and
midzonal (3) regions.
Picture
2.
Histopathological
appearance of a liver
section
from
the
chronic
toxication
group (HE x 80).
Fibrotic
areas
stretching from one
hepatic lobule (1) to
another (2).
Discussion
Diagnosis of liver disease is based upon clinical history, clinical signs, biochemical assays,
serum
protein
electrophoresis,
radiography,
ultrasonography
and,
eventually,
histopathological examination (13).
In the present study, clinical signs seen in the acute group were anorexia, stillness, watery
defecation and mild dehydration, and for the chronic group anorexia, depression, pain in
abdominal palpation (in four dogs), mild icterus (in two dogs), polydypsia (in two dogs) and
submandibular oedema in one dog 6 weeks after toxication (in one dog). Similar clinical signs
have been reported by other workers (14, 15).
Significant increases in RBC and Hb levels could be seen only on the third day in the acute
group (p<0.05). In contrast, a significant increase in WBCs could be seen throughout the
experiment in the chronic group (p<0.001). These results agree with the findings of others
(15, 16), but disagree with the results of another group (17). Other hematological parameters
were not significantly changed in either group.
Increases in RBC and Hb levels observed in the present study could be due to mild
dehydration as a result of watery defecation in the acute group. Ogawa et al. (16) reported an
increase in WBC intoxication group and tubuler changes caused by CCl 4 claimed to be the
reason for leucocytosis. These workers also reported liver damages after CCl4 intoxication.
Therefore, the leucocytosis reported in the present study could well be due to liver damage.
Several experimental studies have shown that chronic administration of CCl 4 leads to hepatic
cirrhosis as well as renal damage (16). The target site of the renal nephrotoxicity has been postulated
as the mitochondria of the renal tubular cells (16). The dark orange colour of urine observed in the
chronic toxication group could well be the result of nephrotoxicosis.
All enzymes studied increased significantly (p<0.001) in the acute group (Table 1), except the
value for AST obtained on day 9. During the experiment, serum enzyme reached peak levels
on day I for AST, ALT and ADA,and on day 3 for ALP. These findinds are similar to those
reported by others (5, 14, 18, 19) in acute hepatic liver damage.
Serum AST, ALT and ALP activities increased up to 12 weeks, when the experiment was
terminated (Table 2). These findings agree with other studies on chronic liver damage (15, 17,
20). Serum AST, ALT and ALP were reported to increase in chronic CCl4 toxication (20, 21),
and this increase has been suggested to be positively correlate with increased liver damage.
The highest increase was also observed at the end of chronic toxication by some workers
(20, 21). Similarly, a simultaneous increase in these serum enzymes in the chronic group
was also observed in this study.
Increase in ADA levels has been reported in animals (8) and patients (22) with liver
disease. Determination of ADA isoenzymes was reported to be an important clue in
diagnosing liver damage (6, 23). Furthermore, determination of ADA reported to be a usefill
tool especially in the differentiation of paranchymal and obstructive liver damage (24, 25).
Some have also suggested that determination of serum ADA isoenzyme activities may be a
new marker for liver disease (10). Ellis et al. (25) also suggested that determination of ADA
activity is especially valuable in late hepatitis, since serum ADA activity does not return to
normal as rapidly as that of other liver enzymes, while higher than normal activity is frequent
in cirrhosis at times when other enzyme levels are normal. Serum ADA activities in the acute
group increased significantly similar to AST, ALT and ALP activities (p<0.001). It reached a
peak level on the first day and decreased up to the ninth day post-experiment. Similar to other
enzymes, ADA also increased significantly in the chronic group (p<0.001). Similar findings
were reported by several workers in serum samples obtained from animals (8, 26), and
patients (1, 25) with liver damages. ADA activities has been reported to increase in acute and
chronic liver damages caused by CCl4 toxication. In addition to cell damage, detection of ADA
activities has been reported to be a useful clue to early diagnosis of liver diseases (5, 27).
Increased ADA levlels with liver cell damage were also observed in the present study. Low
levels of ADA have also been found in the kidney (4). A possible nephrotoxicity may have
been developed in the chronic group in the present study. Histopathology of kidneys was not
investigated in the present study, but abnormal urination in the chronic group indicates some
kind of nephrotoxicity.
Kurata (23) reported an increase in ADA in patients with liver damage. ADA 1 is
increased in acute liver damages which arise from damaged tissues (23). Furthermore,
increased ADA 2 arise from stimulated T lymphocytes (23). Although determination of ADA
isoenzymes are important parameters in diagnosing liver damage, they could not be detected
in the present study.
Increased ADA paralleled the liver damage demonstrated histopathologically in the acute
group. This result is in agreement with those of some workers (10, 23) but disagree with those
of Chicuma (9). Furthermore, in the chronic group ADA increased in parallel with the severity
of fibrosis and degeneration in the liver determined histopathologically. Similar findings were
reported by Kobayashi et al.(10).
It is concluded that, determination of ADA can be usefill in the assessment of liver
degeneration caused by toxic hepatopathy. The fact that ADA levels remained high suggests
a continuous liver intoxication. Furthermore, determination of ADA isoenzymes may be helpful
in differentiating acute and chronic liver disease. Thus, determination of ADA activity can be
added to the other routine biochemical tests that are used in the diagnosis of liver disease.
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