Download effect of short time exposure of rats to extreme low temperature on

Bull Vet Inst Pulawy 50, 121-124, 2006
EFFECT OF SHORT TIME EXPOSURE OF RATS
TO EXTREME LOW TEMPERATURE
ON SOME PLASMA AND LIVER ENZYMES
EWA ROMUK1, EWA BIRKNER1, BRONISŁAWA SKRZEP-POLOCZEK1, LESZEK JAGODZIŃSKI2,
AGATA STANEK2, BERNADETA WIŚNIOWSKA3 AND ALEKSANDER SIEROŃ2
1
Department of Biochemistry in Zabrze,
Clinic of Internal Diseases, Angiology and Physical Medicine in Bytom,
Medical University of Silesia, 40-006 Katowice, Poland
3
Center of Cryotherapy, 41-709 Ruda Śląska, Poland
e-mail: [email protected]
2
Received for publication October 10, 2005.
Abstract
The aim of the study was to search the influence of
cryotherapy on liver enzyme activity in experimental rat
model. The first group of rats was exposed 1 min daily to 90°C for 5 d, the second group was exposed 1 min daily to 90°C for 10 d and the control group was not exposed to low
temperature. A statistically significant increase in the activity
of glutamate dehydrogenase, sorbitol dehydrogenase, malate
dehydrogenase, ornithine transcarbamoylase and arginase was
observed in the plasma and liver. The obtained results indicate
the influence of low temperature on liver metabolism.
Key words: rat, cryotherapy, liver enzymes,
metabolism.
There is growing interest in using extreme low
temperature in medical treatment. Cryotherapy makes
the use of low temperature applied for a short time to
stimulate physiological reaction of an organism to this
condition (16). Whole-body cryotherapy results in
analgesic, antiswelling, immune, hormone and
circulatory system reactions (4). The metabolic effects
of cryotherapy are not yet clear (17). One of the
fundamental body functions is an thermoregulation
within a defined temperature range. Thermal
homeostasis is necessary for correct run of every
metabolic
process.
During
cryotherapy,
thermoregulation is affected by an increase in heat
production in the organism caused by an elevated
intensity of metabolism (1). The aim of our study was to
search the influence of cryotherapy on liver enzyme
activity in experimental rat model.
Material and Methods
Studies were carried out on 18 adult (3 monthold) male Wistar FL rats (body weight about 320 g). The
protocol of the studies was reviewed and approved by
the Bioethical Committee of Medical University of
Silesia in Katowice. Animals were supplied by the
Experimental Animal Farm. The rats underwent two
week environmental adaptation cycle. After this period
the animals were randomized into 3 equal groups. Group
I - rats exposed to -90°C for 5 d; group II - rats exposed
to -90°C for 10 d and group III - control rats – without
exposure to cryotherapy. All the groups were fed
standard rat chow ad libitum.
The rats were put individually into cold
chamber in wooden cages. Body weight of the animals
was controlled before and after 5 and 10 d of the
experiment. During ether anaesthesia of the animals
blood was taken from the right ventricle of the heart and
collected into EDTA-containing tubes. Then the animals
were killed by the by spinal cord disruption. Ten per
cent homogenates of the liver were prepared in 0.9%
sodium chloride solution with the use of PotterElvehjem homogenizator. The homogenates were stored
at –20°C for 36 h. After defrosting, the homogenates
were treated by ultrasounds (ultrasonic desintegrator
UD-11). Total protein, glucose, creatinine and urea
concentrations were measured in the plasma. The
activity of glutamate dehydrogenase (GDH), malate
dehydrogenase (MDH), and sorbitol dehydrogenase
(SDH) were determined in the plasma and liver
homogenates, and the activity of ornithine
transcarbamoylase (OTC) and arginase (ARG) was
measured in liver homogenates.
Total protein, glucose, creatinine, and urea
contents were measured with the use of standard
diagnostic kits. The activity of GDH was measured with
the use of spectrophotometric method by Schmidt (11).
The activity of MDH was measured with the use of
colorymetric method by Więckowski (4). The activity of
SDH was measured with the use of spectrophotometric
method by Gerlach (6). The activity of OTC was
determined with the use of colorimetric method by Żak
122
changes between the studied groups. However, there
was statistically significant increase in the activity of
plasma and liver GDH as compared to the control group
(Figs 1 and 4). MDH activity was significantly increased
in the liver of animals kept at low temperature
comparing to the control (Fig. 5). There was also
statistically significant increase in plasma and liver
activity of SDH in comparison to the control group (Figs
2 and 3). Liver OTC and ARG activity showed
statistically significant increase in groups exposed to
low temperature as compared with the control (Figs 6
and7).
There were no statistically significant changes
in all the examined parameters between groups exposed
for 5 d and 10 d to cryotherapy.
et al. (18). The activity of ARG was measured with the
use of colorimetric method by Tamir et al. (13).
Statistical analysis was performed using
STATISTICA 6.0 PL. The differences between groups
were analysed using U-Mann-Whitney test. To be
considered statistically significant, we required P<0.05
(confidence limits).
Results
A comparative assesment of plasma protein,
glucose, creatinine, and urea concentrations in rats
subjected to different conditions of cryotherapy are
shown in Table 1. There were no statistically significant
Table1
Protein, glucose, creatinine and urea concentrations in the plasma
protein
g/l
glucose
mmol/l
creatinine
umol/
urea
mmol/l
-90/I
-90/II
control
65.8±16.5
65.7±18.5
66.4±18.2
5.90±1.25
5.33±0.96
4.52±1.02
39.8±19.6
37.1±16.3
43.5±11.9
6.17±1.29
6.30±0.99
6.31±1.17
10.8±2.13
12
20
14.8±3.21
15.7±4.11
10
16
9.62±1.25
8
P=0.006
12
P=0.004
-90/I
control
5.16±1.0
8
-90/II
0
0
-90/I
control
-90/II
4
2
25
P= 0.006
2.75±0.21
4
Fig. 1. Plasma GDH activity in rats exposed
to -90°C compared to the control.
P=0.05
6
Fig. 2. Plasma SDH activity in rats exposed
to -90°C compared to the control.
21.9±5.24
69.7±23.6
80
20
70
15.5±3.62
48.7±12.3
60
15
9.62±2.2
10
5
-90/I
control
-90/II
P=0.004
P=0.004
0
Fig. 3. Liver SDH activity in rats exposed
to -90°C compared to the control.
50
P=0.004
-90/I
40
30
control
19.2±3.69
20
10
P=0.05
0
Fig. 4. Liver GDH activity in rats exposed
to -90°C compared to the control.
-90/II
123
200
158±23.6
5
120
P=0.006
-90/I
4
control
3
-90/II
-90/I
P=0.05
control
-90/II
0
Fig. 5. Liver MDH activity in rats exposed
to -90°C compared to the control.
100
P=0.02
1
0
120
5.07±2.03
4.41±1.12
2
P=0.01
40
5.98±1.39
6
116±19.5
160
80
7
145±24.3
Fig. 6. Liver ARG activity in rats exposed
to -90°C compared to the control.
98.5±23.6
73.2±19.8
87.9±30.1
80
-90/I
60
control
P=0.004
-90/II
40
20
P=0.01
0
Fig. 7. Liver OTC activity in rats exposed
to -90°C compared to the control.
Discussion
A constant level of body temperature can only
be preserved if the heat production by the organism is
balanced by its heat loss, which is achieved by
physiological mechanism of thermoregulation. One of
the factors determining the intensity of metabolism and
consequently the rate of heat production, is the
temperature of surroundings.
In
biochemical
thermoregulation the liver plays a crucial role. The
temperature of the blood in the hepatic vein is higher
than that in the hepatic artery, which points to active
heat production in the liver. This process is intensified
when the body is cooled during cryotherapy. A very
important question is the influence of cryotherapy on
individual metabolic pathways. In our study we focused
on liver enzyme activity.
Important indicatory enzyme involved in
protein metabolism is GDH. On the basis of the
performed experiment we observed increased activity in
GDH in groups subjected to cryotherapy at -90ºC. This
increase was observed in the plasma as well as in the
liver of examined animals.
GDH is situated in the mitochondrium. This is
the regulatory enzyme which is under influence of an
activator (ADP) and inhibitors (ATP, GTP, NADH).
Intensification of heat production during cryotherapy is
connected with some ATP degradation in thermogenesis
processes. As the effect, we have more ADP.
Conversion of α-amino groups of amino acids is
catalyzed by GDH in the process called
transdeamination (10). It is a cross reversible reaction
both in catabolism and glutamate anabolism.
Transdeamination leads to the occurrence of ammonia.
This is a very toxic product even in the minimal
concentration. The main mechanism of ammonia
detoxication in the liver is production of urea in the urea
cycle (2).
In our experiment an increase in the GDH
activity was accompanied by the increase in liver OTC
activity. OTC is the key enzyme of urea cycle. This
enzyme, just as the GDH, is present in the liver
mitochondrium and catalyses conversion of carbamoyl
phosphate to ornithine (9).
Other important urea cycle enzyme, arginase,
was also increased during the cryotherapy. The low
temperature exert a great influence on metabolic
pathways through changes in the activity of hormones.
Concentration of amino acids in blood is the main factor
adjusting conversion of nitrogen in the liver. Anabolic
hormones lower the concentration of amino acid in
blood through their stimulation of catching by skeleton
muscles. As the result, liver amino acids uptake falls off.
Then, catabolic hormones increase the supply of amino
acid in the liver and utilization in the process of protein
resynthesis, as well as they intensify the process of
ureapoiesis. We supposed that intensification of protein
catabolism during cryotherapy is connected with
modification of hormonal regulation of this process (7).
We have not observed any changes in serum urea and
creatinine concentration in rats exposed to -90˚C. There
124
were no changes in serum protein concentration during
the cryotherapy.
An increased MDH activity in the liver during
the cryotherapy was observed. There were no
statistically significant changes in serum MDH activity.
Increase in liver MDH activity may indicate the
enhanced transport of reducing equivalent by
mitochondrial membrane in shuttle system (5). This
transport gets through the couple of enzymes – malate
dehydrogenase and aspartate dehydrogenase. Increased
activity of tissue oxidation processes in low temperature
condition leads to the increase in heat production at the
cost of oxidative phosphorylation process. MDH takes
part in supplying acetyl-CoA and NADPH for fatty acid
biosynthesis and in suupplying substrats for
gluconeogenesis (12).
A statistically significant increase in SDH
activity during the cryotherapy was observed. Increased
SDH activity was found in the plasma and liver of the
examined animals. This is NAD dependent enzyme
which catalyzes conversion of sorbitol to fructose. In the
glycolysis process fructose is metabolized more rapidly
than glucose. It is due to the fact that fructose passes
over the reaction catalyzed by phosphofructokinase.
Fructose intensifies fatty acid biosynthesis. It is
supossed that at low temperature increased activity of
sorbitol pathway makes possible glucose utilization in
spite of saturate glycolyse pathways (3). SDH is the
cytoplasm enzyme. Simultaneous increase in the plasma
and liver SDH activity points at migration of this
enzyme outside the hepatocytes. Concentration of
glucose did not differ between animals subjected to the
cryotherapy and the control group. Activation of glucose
metabolism pathways in the liver assures contstant
glucose concentration in plasma in low temperature
condition (8).
It is known that starvation, as well as physical
effort, leads to energy deficiency and, as a result, to the
increased activity of some enzymes. It is supossed that
temperature -90˚C causes increased activity of some
enzymes to compensate energy deficiency and
maintanains the constant body temperature. There may
be some different causes of the increased enzyme
activity during cryotherapy. It may be increase in the
concentration of enzymatic protein, it may be due to
blockade of inhibitors of some enzymes in low
temperature condition, or it may be the consequence of
changes in condition of enzyme transport between the
liver and plasma (15).
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
References
1.
Brengelmann G.L.: Body temperature regulation. In:
Texbook of Physiology. Patton H.D., Fuchs A.F., Hill B.,
Scher R S. Saunders Comp. Philadelphia 1989, pp. 15841596.
18.
Brosnan J.T.: Glutamate, at the interface between amino
acid and carbohydrate metabolism. J Nutr 2000, 130,
988-990.
El-Kabani O., Darmanin C., Chung R.P.: Sorbitol
dehydrogenase: structure, function and ligand design.
Curr Med Chem 2004, 11, 465-476.
Ernst E., Fialka V.: Ice freezes pain? A review of the
clinical effectiveness of analgesic cold therapy. J Pain
Symptom Manage 1994, 9, 56-59.
Fernandez C.A., Des-Rosiers C.: Modeling of liver citric
acid cycle and gluconeogenesis based on 13C mass
isotopomer distribution analysis of intermediates. J Biol
Chem 1995, 270, 10037-10042.
Gerlach U.: Pathological occurrence of sorbitol
dehydrogenase in blood in liver disease. Klin
Wochenschr 1957, 35, 1144-1145.
Iossa S., Lionetti L., Mollica M.P., Crescenzo R.,
Barletta A., Liverini G.: Effects of cold exposure on
energy balance and liver respiratory capacity in postweaning rats fed a high-fat diet. Br J Nutr 2001, 85, 8996.
Iossa S., Liverini G., Barleta H.: Physiological changes
due to cold adaptation in rat liver. Cell Physiol Biochem
1991, 1, 226-239.
Morris S.M. Jr.: Regulation of enzymes of the urea
cycles and arginine metabolism. Ann Rev Nutr 2002, 22,
87-105.
Nissim I., Brosnan M.E., Yudkoff M., Brosnan J.T.:
Studies of glutamine metabolism in the perfused rat liver
with (15)N-labeled glutamine. J Biol Chem 1999, 274,
28958-28965.
Schmidt E., Schmidt F.: Methods and value of
determination of glutamic acid dehydrogenase activity in
the serum. A contribution to the importance of
examination of enzyme relations in the serum. Klin
Wochenschr 1962, 40, 962-969.
Shiota M., Hiramatsu M., Fujimoto Y., Moriyama M.,
Kimura K., Ohta M., Sugano T.: The capacity of the
malate-aspartate shuttle differs between periportal and
perivenous hepatocytes from rats. Arch Biochem
Biophys 1994, 308, 349-356.
Tamir H., Ratner S.: Enzymes of arginine metabolism in
chicks. Arch Biochem Biophys 1963, 102, 249-254.
Taschini L., Radice L., Bernelli-Zazzera A.: Differential
activation of some transcription factors during rat liver
ischemia, reperfusion and heat shock. J Cell Physiol
1999, 180, 355-362.
Więckowska B., Gregorczyk J.: Ocena przydatności
diagnostycznej
izoenzymogramów
surowiczych
dehydrogenazy jabłczanowej. Diag Labor 1972, 8, 247251.
Yamauchi T., Nogami S., Miura K.: Whole body cryotherapy is method of extreme cold –175°C treatment,
initially used for reumathoid artritis. Z Phys Diat Ther
Einschl Balneol Klimatol 1986, 15, 311-315.
Yamauchi T., Nogami S., Miura K.: Various applications
of extreme cryotherapy and extremous exercise
programme – focusing on chronic rheumatoid arthritis.
Physiother Rehabil 1981, 5, 35-39.
Żak T., Jóźkiewicz S., Tarnawski R.: Hormonalna
regulacja aktywności niektórych enzymów cyklu
mocznikowego. Endokrynol Pol 1971, 22, 49-54.