Bull Vet Inst Pulawy 50, 121-124, 2006

EFFECT OF SHORT TIME EXPOSURE OF RATS TO EXTREME LOW TEMPERATURE ON SOME PLASMA AND LIVER

EWA ROMUK1, EWA BIRKNER1, BRONISŁAWA SKRZEP-POLOCZEK1, LESZEK JAGODZIŃSKI2, AGATA STANEK2, BERNADETA WIŚNIOWSKA3 AND ALEKSANDER SIEROŃ2

1Department of Biochemistry in Zabrze, 2Clinic of Internal Diseases, Angiology and Physical Medicine in Bytom, Medical University of Silesia, 40-006 Katowice, Poland 3Center of Cryotherapy, 41-709 Ruda Śląska, Poland e-mail: [email protected]

Received for publication October 10, 2005.

Abstract protocol of the studies was reviewed and approved by the Bioethical Committee of Medical University of The aim of the study was to search the influence of Silesia in Katowice. Animals were supplied by the cryotherapy on liver activity in experimental rat Experimental Animal Farm. The rats underwent two model. The first group of rats was exposed 1 min daily to - week environmental adaptation cycle. After this period 90°C for 5 d, the second group was exposed 1 min daily to - the animals were randomized into 3 equal groups. Group 90°C for 10 d and the control group was not exposed to low I - rats exposed to -90°C for 5 d; group II - rats exposed temperature. A statistically significant increase in the activity to -90°C for 10 d and group III - control rats – without of , sorbitol dehydrogenase, malate exposure to cryotherapy. All the groups were fed dehydrogenase, ornithine transcarbamoylase and arginase was observed in the plasma and liver. The obtained results indicate standard rat chow ad libitum. the influence of low temperature on liver metabolism. The rats were put individually into cold chamber in wooden cages. Body weight of the animals Key words: rat, cryotherapy, liver enzymes, was controlled before and after 5 and 10 d of the metabolism. 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 There is growing interest in using extreme low were killed by the by spinal cord disruption. Ten per temperature in medical treatment. Cryotherapy makes cent homogenates of the liver were prepared in 0.9% the use of low temperature applied for a short time to sodium chloride solution with the use of Potter- stimulate physiological reaction of an organism to this Elvehjem homogenizator. The homogenates were stored condition (16). Whole-body cryotherapy results in at –20°C for 36 h. After defrosting, the homogenates analgesic, antiswelling, immune, hormone and were treated by ultrasounds (ultrasonic desintegrator circulatory system reactions (4). The metabolic effects UD-11). Total protein, glucose, creatinine and urea of cryotherapy are not yet clear (17). One of the concentrations were measured in the plasma. The fundamental body functions is an thermoregulation activity of glutamate dehydrogenase (GDH), malate within a defined temperature range. Thermal dehydrogenase (MDH), and sorbitol dehydrogenase homeostasis is necessary for correct run of every (SDH) were determined in the plasma and liver metabolic process. During cryotherapy, homogenates, and the activity of ornithine thermoregulation is affected by an increase in heat transcarbamoylase (OTC) and arginase (ARG) was production in the organism caused by an elevated measured in liver homogenates. intensity of metabolism (1). The aim of our study was to Total protein, glucose, creatinine, and urea search the influence of cryotherapy on liver enzyme contents were measured with the use of standard activity in experimental rat model. 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 Material and Methods colorymetric method by Więckowski (4). The activity of SDH was measured with the use of spectrophotometric Studies were carried out on 18 adult (3 month- method by Gerlach (6). The activity of OTC was old) male Wistar FL rats (body weight about 320 g). The determined with the use of colorimetric method by Żak 122 et al. (18). The activity of ARG was measured with the changes between the studied groups. However, there use of colorimetric method by Tamir et al. (13). was statistically significant increase in the activity of Statistical analysis was performed using plasma and liver GDH as compared to the control group STATISTICA 6.0 PL. The differences between groups (Figs 1 and 4). MDH activity was significantly increased were analysed using U-Mann-Whitney test. To be in the liver of animals kept at low temperature considered statistically significant, we required P<0.05 comparing to the control (Fig. 5). There was also (confidence limits). 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 Results statistically significant increase in groups exposed to low temperature as compared with the control (Figs 6 and7). A comparative assesment of plasma protein, There were no statistically significant changes glucose, creatinine, and urea concentrations in rats in all the examined parameters between groups exposed subjected to different conditions of cryotherapy are for 5 d and 10 d to cryotherapy. shown in Table 1. There were no statistically significant

Table1 Protein, glucose, creatinine and urea concentrations in the plasma

-90/I -90/II control

protein 65.8±16.5 65.7±18.5 66.4±18.2 g/l glucose 5.90±1.25 5.33±0.96 4.52±1.02 mmol/l creatinine 39.8±19.6 37.1±16.3 43.5±11.9 umol/ urea 6.17±1.29 6.30±0.99 6.31±1.17 mmol/l

20 12 10.8±2.13 14.8±3.21 15.7±4.11 9.62±1.25 10 16 8 P=0.05 12 P=0.006 P=0.004 -90/I P= 0.006 -90/I 6 control control 2.75±0.21 8 5.16±1.0 -90/II 4 -90/II

4 2

0 0

Fig. 1. Plasma GDH activity in rats exposed Fig. 2. Plasma SDH activity in rats exposed to -90°C compared to the control. to -90°C compared to the control.

25 21.9±5.24 69.7±23.6 80 20 15.5±3.62 70 48.7±12.3 60 15 -90/I 50 P=0.004 -90/I 9.62±2.2 control 40 control 10 -90/II 30 19.2±3.69 -90/II P=0.004 20 5 P=0.004 10 P=0.05

0 0

Fig. 3. Liver SDH activity in rats exposed Fig. 4. Liver GDH activity in rats exposed to -90°C compared to the control. to -90°C compared to the control. 123

7 5.98±1.39 200 158±23.6 145±24.3 6 5.07±2.03 160 116±19.5 5 4.41±1.12

120 -90/I 4 P=0.02 -90/I control control 3 80 P=0.05 -90/II P=0.006 -90/II 2 40 P=0.01 1

0 0

Fig. 5. Liver MDH activity in rats exposed Fig. 6. Liver ARG activity in rats exposed to -90°C compared to the control. to -90°C compared to the control.

120 98.5±23.6

100 73.2±19.8 87.9±30.1 80 -90/I 60 P=0.004 control -90/II 40 P=0.01 20

0

Fig. 7. Liver OTC activity in rats exposed to -90°C compared to the control.

Discussion transdeamination (10). It is a cross reversible reaction both in catabolism and glutamate anabolism. A constant level of body temperature can only Transdeamination leads to the occurrence of . be preserved if the heat production by the organism is This is a very toxic even in the minimal balanced by its heat loss, which is achieved by concentration. The main mechanism of ammonia physiological mechanism of thermoregulation. One of detoxication in the liver is production of urea in the urea the factors determining the intensity of metabolism and cycle (2). consequently the rate of heat production, is the In our experiment an increase in the GDH temperature of surroundings. In biochemical activity was accompanied by the increase in liver OTC thermoregulation the liver plays a crucial role. The activity. OTC is the key enzyme of urea cycle. This temperature of the blood in the hepatic vein is higher enzyme, just as the GDH, is present in the liver than that in the hepatic artery, which points to active mitochondrium and catalyses conversion of carbamoyl heat production in the liver. This process is intensified phosphate to ornithine (9). when the body is cooled during cryotherapy. A very Other important urea cycle enzyme, arginase, important question is the influence of cryotherapy on was also increased during the cryotherapy. The low individual metabolic pathways. In our study we focused temperature exert a great influence on metabolic on liver enzyme activity. pathways through changes in the activity of hormones. Important indicatory enzyme involved in Concentration of amino acids in blood is the main factor protein metabolism is GDH. On the basis of the adjusting conversion of nitrogen in the liver. Anabolic performed experiment we observed increased activity in hormones lower the concentration of amino acid in GDH in groups subjected to cryotherapy at -90ºC. This blood through their stimulation of catching by skeleton increase was observed in the plasma as well as in the muscles. As the result, liver amino acids uptake falls off. liver of examined animals. Then, catabolic hormones increase the supply of amino GDH is situated in the mitochondrium. This is acid in the liver and utilization in the process of protein the regulatory enzyme which is under influence of an resynthesis, as well as they intensify the process of activator (ADP) and inhibitors (ATP, GTP, NADH). ureapoiesis. We supposed that intensification of protein Intensification of heat production during cryotherapy is catabolism during cryotherapy is connected with connected with some ATP degradation in thermogenesis modification of hormonal regulation of this process (7). processes. As the effect, we have more ADP. 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