Bull Vet Inst Pulawy 50, 221-223, 2006
EFFECT OF IONIZING RADIATION ON TISSUE ENZYMES
IN PIG LIVER AND KIDNEYS
PETR DVOŘÁK, JAN ŠALPLACHTA1, STANISLAV ZIMA,
MAGDALENA GROLICHOVÁ AND HANA MUSILOVÁ
Department of Biochemistry, Chemistry and Biophysics, Faculty of Veterinary Hygiene and Ecology,
University of Veterinary and Pharmaceutical Sciences, 612 42 Brno, Czech Republic
1
Department of Analytical Biotechnology, Veterinary Research Institute, 612 00 Brno, Czech Republic.
e-mail: [email protected]
Received for publication December 07, 2005.
Abstract
The activity of alkaline phosphatase (ALP), acid
phosphatase (ACP), alanine aminotransferase (ALT),
aspartate
aminotransferase
(AST)
and
L-lactate
dehydrogenase (LD) was determined in pig liver and kidneys
that differ in their isoenzyme patterns Tissues were taken
from slaughterhouse, cut and sorted into three groups (10
samples/group). Samples were kept at 5oC. The samples of
two groups were irradiated with doses of 2.5 and 5 kGy,
respectively, 24 h post mortem. The 60Co γ-iradiation was
applied at room temperature, dose-rate was 47.7 Gy/min. Our
data indicates, that pig liver and kidneys were significantly
different only in LD activity between irradiated and control
samples (89% or 78% of mean LD activity of control group,
P < 0.05). ALT, AST, ALP, and ACP activities in the organs
were found resistant to the applied irradiation. This fact
should be taken into account during radiation treatment of
animal tissue as it can affect storage and processing of the
foodstuffs. The effect of ionizing radiation on LD, ALP,
ACP, ALP, and AST in tissues of cadaver is not substantial
and does not explain the changes of activity of these enzymes
in living organisms after γ-irradiation.
Key words: swine, liver, kidneys, enzymes,
γ-radiation.
Ionizing radiation may, by change of
molecular structure, affect properties of target
molecules. It is well known the direct damage of
nucleic acids, carbohydrates and fragmentation of
lipids as well proteins by γ-radiation. The radiation also
induces radical products of water and oxygen that
diffuse and react with macromolecules. This indirect
damage is more substantial than the direct one (1-3).
Effect of radiolytic products of water and oxygen is
different in liquid or frozen solution. Therefore,
proteins in frozen solutions are 10-3 to 10-4 as sensitive
to radiation as in the liquid state (7). The result of
radiation effect on macromolecules is their reduced
biological function, as was shown on the toxicity of
polypeptide crotamine (8), and enzymes (6). However,
ionizing radiation is widely used for the sterilization of
human tissue allografts (15) and the preservation of
foods (13).
Enzymes are catalysts and make faster an
inter-conversion of substrates changing the properties
of sample. In this study, the effect of the ionizing
radiation on enzyme activity of pig alkaline
phosphatase (ALP), acid phosphatase (ACP), alanine
aminotransferase (ALT), aspartate aminotransferase
(AST) and L-lactate dehydrogenase (LD) was
investigated in pig liver and kidneys. Considering that
both organs differ in isoenzyme patterns of the
mentioned enzymes (3, 13), we explored them to find
the isoenzyme most sensible to ionizing radiation.
Material and Methods
The liver and kidneys were taken from
slaughterhouse, cut and sorted into three groups of
samples (10 samples in each group) and kept at 5oC.
The samples of two groups were irradiated with doses
of 2.5 and 5 kGy, respectively, 24 h post mortem.
Industrial radiation source of 60Co (BIOSTER,
Veverská Bitýška, Czech Republic) at a dose output of
47.7 Gy/min was applied at room temperature.
The samples of 2.5 g were processed in a
blender with 25 ml of 5 mM TRIS-HCl-buffered
saline, pH 7.4. The homogenate was centrifuged at 2
700x g and 20oC for 15 min and the supernatant was
used for analysis.
Soluble protein and activity of ALP, ACP,
ALP and AST were determined by the colorimetric
methods using spectrophotometry kits (Lachema-Pliva
Brno, Czech Republic). LD was determined by the
kinetic method in solution of 50 mM sodium lactate, 5
mM NAD, and 0.1 M TRIS-HCl, pH 8.6, at 30 oC.
222
The obtained data were processed
ANOVA using the EXCEL (Microsoft).
by
20.6% or 36.3% in pig liver, respectively. The relative
activity of the LD4 and LD5 isoenzymes is 9.2% or
4.4% in pig liver and is absent in kidneys whereas in
muscle no LD1 and LD2 activity was detected by
electrophoretic method (13). A decrease in LD activity
after irradiation we determined in our experiments was
low taking into account the findings of Uzunov et al.
(14). They identified the LD4 and LD5 isoenzymes of
bovine muscles more resistant to γ-irradiation than
LD1 and LD2, which activity disappeared by γ irradiation with the dose of 2.5 Mrad.
This discrepancy could be explained either
with the effect of radicals and their different diffusion
in the kidneys, liver and muscles of pig or cattle and/or
non-linear detection of LD isoenzymes as for the
activity of single LD isoenzyme in gel and different
analytical sensitivity of both electrophoretic methods.
The works of Donnadieu et al. (4) and Jamdar and
Harikumar (6) support the former, and the study of
Šalplachta and Nečas (12) supports the latter
conception.
Comparing the results of our study and the
similar studies dealing with living organisms (4, 5, 9),
it is evident that the effect of ionizing radiation on LD,
ALP, ACP, ALP, ALT, and AST is not substantial and
does not explain the changes of activity of these
enzymes in living organisms after γ-irradiation.
Results
The activity of LD, ACP, ALP, and ALT in
control and irradiated organs are presented in Table 1.
Significant difference of LD activity between
irradiated and control samples of pig liver or kidneys
was found at both radiation doses (P < 0.05). Mean LD
activity of irradiated kidneys or liver represented 89%
or 78% of mean LD activity of control group,
respectively.
Considering the above data and LD
isoenzyme pattern in the liver and kidneys (12) we
suppose the LD isoenzymes could be resistant to γradiation in the same way. The difference of ALP,
ACP, ALP, and AST activity between irradiated and
control kidneys or liver was found insignificant (P <
0.05).
This
finding
means,
that
the
enzymes/isoenzymes are resistant to 60Co γ -radiation
applied in doses of 2.5 or 5 kGy.
Discussion
The relative activity of LD1 or LD2
isoenzyme is 51.0% or 36.6% in pig kidneys and
Table 1
The effect of ionizing radiation on enzyme activity in pig liver and kidneys (mean ± SE)
Control
LD (microkat /g of soluble protein)
Liver
20.5 ± 5.6
Kidneys
30.7 ± 3.6
ACP (microkat /g of soluble protein)
Liver
0.20 ± 0.05
Kidneys
0.53 ± 0.10
ALP (microkat /g of soluble protein)
Liver
0.26 ± 0.08
Kidneys
5.75 ± 1.11
AST (microkat /g of soluble protein)
Liver
2.38 ± 0.56
Kidneys
2.29 ± 0.61
ALT (microkat /g of soluble protein)
Liver
0.57 ± 0.37
2.5 kGy
5 kGy
16.1 ± 2.9*
16.0 ± 1.9**
27.4 ± 3.0**
27.3 ± 3.5**
0.18 ± 0.03
0.18 ± 0.03
0.51 ± 0.07
0.54 ± 0.07
0.27 ± 0.13
0.25 ± 0.11
5.46 ± 0.14
5.57 ± 1.13
2.14 ± 0.35
2.12 ± 0.34
2.19 ± 0.38
2.06 ± 0.28
0.48 ± 0.25
0.47 ± 0.23
Kidneys
1.22 ± 0.27
1.13 ± 0.24
1.11 ± 0.21
Values with asterix differ significantly from control (*P < 0.05, **P < 0.01).
2
Acknowledgments: This study was funded
by the grant No. MSM6215712402 of the Ministry of
Education, Youth and Sports of the Czech Republic.
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