13th balkan biochemical biophysical days & meeting on metabolic disorders’ programme & abstracts

M. Betül YERER, Sami AYDOGAN

Free radicals attacking biomembranes can lead to the oxidative damage of the membrane lipids an proteins. Furthermore, reactive oxygen species avidly reacts with nitric oxide (NO) producing cytotoxic reactive nitrogen species capable of nitrating proteins and damaging other molecules which leads to the reduction of erythrocyte deformability. The aim of this investigation was to assess the importance of - tocopherol (Vit-E) in the total antioxidant status of the erythrocytes in Sodium nitroprussid (SNP), a nitric oxide donor, induced oxidative stress and its relation to erythrocyte deformability.

Male Swiss Albino rats were used in 4 groups, comprising of 10 animals in each group. The first group was the control, and the other groups were administered SNP (10mg/kg, i.p ), Vit E (10mg/kg, i.p) + SNP, and SNP + L-NAME (10mg/kg, i.p), respectively. Relative filtration rate (RFR), Relative filtration time (RFT) and relative resistance (Rrel) were determined as the indexes of erythrocyte deformability. In addition, Malondialdehyde (MDA, as an index of lipid peroxidation) and nitric oxide levels and the antioxidant activities of Glutathione peroxidase (GSH-Px), Superoxide dismutase (SOD) and Catalase (CAT) were also determined in the red blood cells of all groups revealing the oxidant-antioxidant activity.

RFT and the Rrel of the erythrocytes of the SNP-treated rats increased significantly (p<0.05) whereas the RFR of the erythrocytes decreased (p<0.05) in comparison to all groups reflecting the impaired deformability. Lipid peroxidation was suppressed by Vit-E and L-NAME significantly, where the red blood cell deformability was improved. Furthermore, SOD and CAT activities were significantly stimulated with SNP treatment (p<0.05), where as GSH-Px remained unchanged. In the contrary, GSH-Px activity was triggered significantly by Vit-E administration, whereas the SOD and CAT activities were reduced (p<0.05).

As a result, these data reveal that Vit-E improves the erythrocyte deformability in SNP-induced oxidative stress by its antioxidant effects on the lipid peroxidation and antioxidant enzyme activities.

P261

INVITRO EFFECTS OF MELATONIN ON THE FILTRABILITY OF ERYTHROCYTES IN OXIDATIVE STRESS

Sami AYDOGAN, M. Betul YERER

University of Erciyes, Medical Faculty, Dept. Of Physiology, 38039, Kayseri, TURKEY.

eczbetul@yahoo.com

Erythrocyte deformability is one of the most important charactheristics of erythrocytes for an effective microcirculatory function and is affected from a number of factors, including the oxidative-damage-induced by nitric oxide (NO). This study was performed to investigate the effects of in vitro melatonin incubation on the antioxidant status and deformability of erythrocytes in sodiumnitroprusside (SNP), a nitric oxide donor, induced oxidative stress.

40 blood samples taken from the adult healthy people were divided into 4 groups randomly and incubated with saline, SNP (1mM), Melatonin (MEL, 1mM), MEL+SNP and SNP+L-NAME (5mM) respectively. Relative filtration rate (RFR), Relative filtration time (RFT) and relative resistance (Rrel) were determined as the indexes of erythrocyte filterability. In addition, Malondialdehyde (MDA, as an index of lipid peroxidation) and the antioxidant activities of Glutathione Peroxidase (GSH-Px), Superoxide dismutase (SOD) and catalase (CAT) were also determined in the red blood cells of all groups revealing the oxidant-antioxidant activity.

RFT and the Rrel of the erythrocytes incubated with SNP increased significantly (p<0.05) whereas the RFR of the erythrocytes decreased (p<0.05) in comparision to all groups. This reduction in RFR was prevented with both L-NAME or MEL incubation. Furthermore, MEL was found to be significantly efficient in preventing the erythrocytes from lipid peroxidation in these groups. In addition, GSH-Px and SOD activities were elevated with SNP incubation reflecting the oxidative stress in erythrocytes, whereas the CAT activity remained unchanged. Melatonin has no significant effect on the GSH-Px and Cat activity but, it caused a significant decrease in SOD activity (p<0.05).

These results reveal that, Melatonin can protect the erythrocytes from impaired deformability in SNP-induced oxidative stress due to antioxidant effects as revealed by lipid peroxidation and antioxidant enzyme activities.

P262

PURIFICATION AND CHARACTERIZATION OF BUTYRYLCHOLINESTERASE FROM RAT SMALL INTESTINE

Yıldız Ö., Bodur E., Çokuğraş A.N., Özer N.

Butyrylcholinesterase (BChE; E.C. 3.1.1.8) plays an important function in toxicology and pharmacology as a detoxification enzyme. In toxicity assesment investigations, rat is the most commonly used animal. It is clear that orally introduced toxic compounds will first be faced with intestinal BChE so, we purified and characterized the soluble isoform of BChE from the rat small intestine. In this study, small intestines were obtained from female Wistar rats killed for students' laboratory coursework at Hacettepe University Medical School. BChE was purified from soluble fraction of rat intestine by chromatography on Sephadex G-25 following repeated chromatography on affinity gel, procainamide-Sepharose 4B with a purification fold of 260. Purity and purified molecular form(s) were controlled by nonreducing polyacrylamide gel electrophoresis. Consecutive protein staining with Commassie Brillant Blue R-250 followed by AgNO₃ gave two bands; a major band corresponding to tetrameric globular form of rat BChE and a minor faster moving protein band; presumably an impurity or degraded BChE. In the activity staining gel with BTCh as substrate only the major protein band was stained for BChE activity. The migration of rat BChE activity band was slower than tetrameric globular forms of both human and horse serum butyrylcholinesterases. In the activity staining gel, in human BChE lane, activity bands corresponding to monomeric, dimeric and tetrameric forms of BChE were observed.

Acetylcholinesterase (E.C. 3.1.1.7) contamination was controlled by using ethopropazine, the specific inhibitor of BChE. It is found that purified soluble isoform of the enzyme from rat intestine was completely pure BChE. The optimum pH value was determined as 7.2 after eliminating the ion strenght effect on activity by zero-buffer extrapolation. The optimum temperature of the enzyme was examined as 37°C after eliminating the effect of time on activity by zero-time extrapolation.

P263