The effect of exogenous glycerol on the activity of lysosomal enzymes in the blood plasma of young bulls (short communication)

Tlie study was performed on 3 young bulls of the Lowland Black-and-White breed. The animals received daily, during three days about 400 g through the gastric fisrula ofthe glycerol. In Üie period 2, 4 and 8 hours after the glycerol infusion the blood was taken and in the plasma obtained the activities the enzymes BGRD, BGAL, BGLU, NAGL, AP, AAP, LAP, LL and EL were determined. Administration of glycerol had a significant influence on the activity of BGRD, BGAL, NAGL, AP, LAP and EL. The great variability of activities of enzymes has been observed.


Introduction
The problem of biochemical stress in animals is still the focus of interest of biologists and breeders (SPENCER, 1995;KOLATAJ, 1993;DALLMAN, 1991;RABIN et al., 1990).Glycerol is in the ruminants, above all in the cattle the substrate into glucose exchanged.The oscillation of access of this substrate can be directly or indirectly accountable for changes in the glucose metabolism and its concentration in blood can be the expression of homeostase (MONTMINY and GALIGOIS, 1994;LAYCHOCK, 1990;KOLATAJ et al., 1996a;WAPNIR et al., 1996;DE SANCTIS et al., 1996;MÜLLER-WIELAND et al., 1993).In our opinion the artificial introduction of an excess ofthe glycerol to the blood ofthe cow or bull is the biochemical Stressor which disturbs an actual homeostase of animal.In literature we have not found the reports concerning the reactivity of lysosomal enzymes in the cattle, therefore we have taken into account a glycerol infusion as a model of the biochemical Stressor and the activity of these enzymes as the model markers ofthat kind of stress.

Material and Methods
The study was carried out on 3 young bulls, ofthe Lowland Black-and-White breed ofthe body weight 180 -250 kg, at the age of 6 months, which came from the breeding farm of the Research Institute of Animal Produetion in Nitra, Slovakia.The animals were provided with equal conditions of Standard food, nursing and they had a professional veterinary care.The bulls received a food in daily ratio of about 7.5 kg which consisted of 1.5 kg of good quality hay, 4 kg of com grain, 2 kg of barley, and water in unlimited amounts.The experiment was performed from 17-19 April with animals which had been previously starved for 24 hour.The animals received the glycerol in the amount 2 g/kg of body weight (about 400 g/bull) daily, during three days, at 6.30 a.m. through gastric fistula.Before the administration of glycerol the control blood samples were taken from the neck vein of all individuals.In the period 2, 4 and 8 hours after the glycerol administration the blood samples were taken again and in plasma the activities ofthe beta-glucuronidase (BGRD -EC 3.2.1.31);beta-galactosidase (BGAL -EC 3.2.1.23);beta-glucosidase (BGLU -EC 3.2.1.21);N-acetyl-beta-glucosaminidase (NAGL -EC 3.2.1.30);acid Phosphatase (AP -EC 3.1.3.2);alanyl aminopeptidase (AAP -EC 3.4.11.1); leucyl aminopeptidase (LAP -EC 3.4.11.1); lysosomal esterase (EL -EC 3.1.1.2) and lysosomal lipase (LL -EC 3.1.1.3)were determined.The activities BGRD, BGAL, BGLU, NAGL and AP were determined according to the BARRETT 'S method (1972); AAP according to PFLEIDERER et al. (1964); LAP by method of PFLEIDERER and CELLIERS (1963); EL and LL according to the modified method of MAIN (1960).Enzyme activity was expressed in nmol/mg of protein/hour.The results obtained were statistically analysed according to the three way analysis of variance.

Results
From Table 1 we can see that for example during first day after 2, 4 and 8 hours after glycerol administration BGLU activity in blood plasma of first bull decreased significantly to 73%, 76% and 56% suitable.AAP activity for example during first day decreased after 4 hours to 74%.During second day after 4 hours from glycerol administration BGLU activity was lower significantly to 78% of control value.The remained enzymes did not change statistically their activities.
From Table 2 we see that for second bull the differences statistically confirmed after glycerol administration showed during first day BGAL past 2 h (78%) only and AAP during third day past 8 h (78%).Table 3 informs, that during first day in third bull revealed the statistically confirmed changes after glycerol administration for NAGL past 2 h (75%) only.The data of analysis of variance show in the Table 4 the

Discussion
Although glycerol is product of fat metabolism it can be not use by the tissues immediately.It diffuses to the blood plasma and from there it comes to the liver and kidneys.The existent there enzyme, glycerol kinase, catalyzes its conversion to glucose (MAYES and LAKER, 1981;MC GARRY and FOSTER, 1980).The glucose has been transported from these organs to fat tissue and glycerol comes back from it to the liver and kidney again so that it may be able to glucose resynthesis (WATFORD, 1988;NOEL et al., 1997;PILKIS et al., 1988).The changes of activity of lysosome enzymes under the influence of glycerol in the young bulls appear interesting.It was possible to suppose that the excess of glycerol introduced to the blood can be the biochemical Stressor for animal and effects a disturb its normal metabolic homeostasis.Activities of examined enzymes changed statistically significant.The changes of the BGRD, BGAL, NAGL, AP, LAP and EL s-1 for samples: 3; for interaction days x samples: 6: for interaction animals x days: 4; for remainder: 8; activities we have observed eariier during the different aspects of our studies (KOLATAJ et al., 1998(KOLATAJ et al., , 1996;;WITEK et al., 1996WITEK et al., , 1995WITEK et al., , 1994)).In consideration synthesis and degradation of the protein in the cells they seem to be a good model of the stress reactivity after the biochemical excitation.We supposed that according to our eariier results, the excess of exogenous glycerol introduced to blood of the animal initiates the chain of adaptative reactions in lysosomal space of cells probably mainly in the liver and kidneys.Our results presented in this paper are connected with the studies of the activities of these enzymes in the blood serum.On the basis of analysis of variance we have found the differences of these activities in six enzymes in the presence of nine examined after glycerol infusion.There is the statistically confirmed variability between the animals and days of experiment.The studies of this phenomen ought be continued.

Table 1
The activities (in nmol/mg of protein/hour and after the glycerol infusion.Control = 100 % Bullen nach Glyzerininfusion) per cent) ofthe examined enzymes in the blood plasma of I bull (Aktivität der untersuchten Enzyme im Blutplasma von einem

Table 2
The activities (in nmol/mg of protein/hour and per cent) of the examined enzymes in the blood plasma of II bull after the glycerol infusion.Control = 100 % (Aktivität der untersuchten Enzyme im Blutplasma von einem zweiten Bullen nach Glyzerininfusion)